U.S. patent application number 14/491245 was filed with the patent office on 2015-04-02 for nitrile hydratase variant.
This patent application is currently assigned to Mitsui Chemicals, Inc.. The applicant listed for this patent is Mitsui Chemicals, Inc.. Invention is credited to Yasushi Kazuno, Kazuya Matsumoto, Daisuke Mochizuki, Junko Tokuda.
Application Number | 20150093803 14/491245 |
Document ID | / |
Family ID | 42169811 |
Filed Date | 2015-04-02 |
United States Patent
Application |
20150093803 |
Kind Code |
A1 |
Matsumoto; Kazuya ; et
al. |
April 2, 2015 |
NITRILE HYDRATASE VARIANT
Abstract
A nitrile hydratase variant of the present invention comprises
substitution of at least one amino acid with another amino acid to
improve two or more properties of nitrile hydratase by substitution
of one amino acid.
Inventors: |
Matsumoto; Kazuya;
(Mobara-shi, JP) ; Kazuno; Yasushi; (Varsity Park,
SG) ; Mochizuki; Daisuke; (Mobara-shi, JP) ;
Tokuda; Junko; (Chiba-shi, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Mitsui Chemicals, Inc. |
Minato-ku |
|
JP |
|
|
Assignee: |
Mitsui Chemicals, Inc.
Tokyo
JP
|
Family ID: |
42169811 |
Appl. No.: |
14/491245 |
Filed: |
September 19, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13128323 |
May 9, 2011 |
8871484 |
|
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PCT/JP2009/006055 |
Nov 12, 2009 |
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14491245 |
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Current U.S.
Class: |
435/232 ;
435/252.33; 435/320.1; 536/23.2 |
Current CPC
Class: |
C12N 9/88 20130101; C12Y
402/01084 20130101 |
Class at
Publication: |
435/232 ;
435/320.1; 435/252.33; 536/23.2 |
International
Class: |
C12N 9/88 20060101
C12N009/88 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 14, 2008 |
JP |
2008-292819 |
Claims
1. A nitrile hydratase variant comprising substitution of at least
one amino acid with another amino acid to improve two or more
properties of nitrile hydratase by substitution of 1, 2 or 3 amino
acids, wherein said properties to be improved are the initial
reaction rate and thermal stability, and wherein the nitrile
hydratase variant comprises an .alpha.-subunit defined in SEQ ID
No: 1 in the Sequence Listing and a .beta.-subunit defined in SEQ
ID No: 2 in the Sequence Listing, and substitution of at least one
amino acid with another amino acid selected from substitution sites
of the amino acid consisting of the following (b) to (l): (a) 92nd
of .alpha.-subunit; (b) 94th of .alpha.-subunit; (c) 197th of
.alpha.-subunit; (d) 4th of .beta.-subunit; (e) 24th of
.beta.-subunit; (f) 79th of .beta.-subunit; (g) 96th of
.beta.-subunit; (h) 107th of .beta.-subunit; (i) 226th of
.beta.-subunit; (j) 110th of .beta.-subunit and 231st of
.beta.-subunit; (k) 206th of .beta.-subunit and 230th of
.beta.-subunit; and (l) 13th of .alpha.-subunit, 27th of
.alpha.-subunit and 110th of .beta.-subunit, wherein Ile is
substituted by Leu when 13th amino acid of the .alpha.-subunit is
substituted, Met is substituted by Ile when the 27th amino acid of
the .alpha.-subunit is substituted, Asp is substituted by Glu when
the 92nd amino acid of the .alpha.-subunit is substituted, Met is
substituted by Ile when the 94th amino acid of the .alpha.-subunit
is substituted, Gly is substituted by Cys when the 197th amino acid
of the .alpha.-subunit is substituted, Val is substituted by Met
when the 4th amino acid of the .beta.-subunit is substituted, Val
is substituted by Ile when the 24th amino acid of the
.beta.-subunit is substituted, His is substituted by Asn when the
79th amino acid of the .beta.-subunit is substituted, Gln is
substituted by Arg when the 96th amino acid of the .beta.-subunit
is substituted, Pro is substituted by Met when the 107th amino acid
of the .beta.-subunit is substituted, Glu is substituted by Asn
when the 110th amino acid of the .beta.-subunit is substituted, Pro
is substituted by Leu when the 206th amino acid of the
.beta.-subunit is substituted, Val is substituted by Ile when the
226th amino acid of the .beta.-subunit is substituted, Ala is
substituted by Glu when the 230th amino acid of the .beta.-subunit
is substituted, and Ala is substituted by Val when the 231st amino
acid of the .beta.-subunit is substituted.
2. The nitrile hydratase variant according to claim 1, comprising
substitution of at least one amino acid with another amino acid
selected from substitution sites of the amino acid consisting of
the following (m) to (x): (m) in case of (b) or (g), 13th Ile in
the .alpha.-subunit is substituted by Leu; (n) in case of (b) or
(h), 27th Met in the .alpha.-subunit is substituted by Ile; (o) (d)
and (f); (p) in case of (f), 230th Ala in the .beta.-subunit is
substituted by Glu; (q) (a) and (i); (r) in case of (i), 13th Ile
in the .alpha.-subunit is substituted by Leu and 206th Pro in the
.beta.-subunit is substituted by Leu; (s) in case of (a) and (d),
206th Pro in the .beta.-subunit is substituted by Leu; (t) in case
of (c) and (h), 230th Ala in the .beta.-subunit is substituted by
Glu; (u) in case of (f), 230th Ala in the .beta.-subunit is
substituted by Glu and 231st Ala in the .beta.-subunit is
substituted by Val; (v) (a) and (e) and (i); (w) in case of (c) and
(e) and (h), 230th Ala in the .beta.-subunit is substituted by Glu;
and (x) in case of (e) and (f), 230th Ala in the .beta.-subunit is
substituted by Glu and 231st Ala in the .beta.-subunit is
substituted by Val.
3. The nitrile hydratase variant according to claim 1, further
comprising substitution of at least one amino acid with another
amino acid selected from the group consisting of (a), (c), (f),
(i), (h), 230th of the .beta.-subunit and 231st of the
.beta.-subunit in case of (e) is substituted with another amino
acid.
4. The nitrile hydratase variant according to claim 1, further
comprising substitution of at least one amino acid selected from
substitutions of the amino acid consisting of the following (aa) to
(br): (aa) 36th Thr in the .alpha.-subunit is substituted by Met
and 126th Phe in the .alpha.-subunit is substituted by Tyr; (ab)
148th Gly in the .alpha.-subunit is substituted by Asp and 204th
Val in the .alpha.-subunit is substituted by Arg; (ac) 51st Phe in
the .beta.-subunit is substituted by Val and 108th Glu in the
.beta.-subunit is substituted by Asp; (ad) 118th Phe in the
.beta.-subunit is substituted by Val and 200th Ala in the
.beta.-subunit is substituted by Glu; (ae) 160th Arg in the
.beta.-subunit is substituted by Trp and 186th Leu in the
.beta.-subunit is substituted by Arg; (af) 6th Leu in the
.alpha.-subunit is substituted by Thr, 36th Thr in the
.alpha.-subunit is substituted by Met, and 126th Phe in the
.alpha.-subunit is substituted by Tyr; (ag) 19th Ala in the
.alpha.-subunit is substituted by Val, 71st Arg in the
.alpha.-subunit is substituted by His, and 126th Phe in the
.alpha.-subunit is substituted by Tyr; (ah) 36th Thr in the
.alpha.-subunit is substituted by Met, 148th Gly in the
.alpha.-subunit is substituted by Asp, and 204th Val in the
.alpha.-subunit is substituted by Arg; (ai) 10th Thr in the
.beta.-subunit is substituted by Asp, 118th Phe in the
.beta.-subunit is substituted by Val, and 200th Ala in the
.beta.-subunit is substituted by Glu; (aj) 37th Phe in the
.beta.-subunit is substituted by Leu, 108th Glu in the
.beta.-subunit is substituted by Asp, and 200th Ala in the
.beta.-subunit is substituted by Glu; (ak) 37th Phe in the
.beta.-subunit is substituted by Val, 108th Glu in the
.beta.-subunit is substituted by Asp, and 200th Ala in the
.beta.-subunit is substituted by Glu; (al) 41st Phe in the
.beta.-subunit is substituted by Ile, 51st Phe in the
.beta.-subunit is substituted by Val, and 108th Glu in the
.beta.-subunit is substituted by Asp; (am) 46th Met in the
.beta.-subunit is substituted by Lys, 108th Glu in the
.beta.-subunit is substituted by Arg, and 212th Ser in the
.beta.-subunit is substituted by Tyr; (an) 48th Leu in the
.beta.-subunit is substituted by Val, 108th Glu in the
.beta.-subunit is substituted by Arg, and 212th Ser in the
.beta.-subunit is substituted by Tyr; (ao) 127th Leu in the
.beta.-subunit is substituted by Ser, 160th Arg in the
.beta.-subunit is substituted by Trp, and 186th Leu in the
.beta.-subunit is substituted by Arg; (ap) 6th Leu in the
.alpha.-subunit is substituted by Thr, 19th Ala in the
.alpha.-subunit is substituted by Val, 126th Phe in the
.alpha.-subunit is substituted by Tyr, 46th Met in the
.beta.-subunit is substituted by Lys, 108th Glu in the
.beta.-subunit is substituted by Arg, and 212th Ser in the
.beta.-subunit is substituted by Tyr; (aq) 6th Leu in the
.alpha.-subunit is substituted by Thr, 19th Ala in the
.alpha.-subunit is substituted by Val, 126th Phe in the
.alpha.-subunit is substituted by Tyr, 48th Leu in the
.beta.-subunit is substituted by Val, 108th Glu in the
.beta.-subunit is substituted by Arg, and 212th Ser in the
.beta.-subunit is substituted by Tyr; (ar) 6th Leu in the
.alpha.-subunit is substituted by Ala, 19th Ala in the
.alpha.-subunit is substituted by Val, 126th Phe in the
.alpha.-subunit is substituted by Tyr, 127th Leu in the
.beta.-subunit is substituted by Ser, 160th Arg in the
.beta.-subunit is substituted by Trp, and 186th Leu in the
.beta.-subunit is substituted by Arg; (as) 6th Leu in the
.alpha.-subunit is substituted by Thr, 36th Thr in the
.alpha.-subunit is substituted by Met, 126th Phe in the
.alpha.-subunit is substituted by Tyr, 10th Thr in the
.beta.-subunit is substituted by Asp, 118th Phe in the
.beta.-subunit is substituted by Val, and 200th Ala in the
.beta.-subunit is substituted by Glu; (at) 19th Ala in the
.alpha.-subunit is substituted by Val, 71st Arg in the
.alpha.-subunit is substituted by His, 126th Phe in the
.alpha.-subunit is substituted by Tyr, 37th Phe in the
.beta.-subunit is substituted by Leu, 108th Glu in the
.beta.-subunit is substituted by Asp, and 200th Ala in the
.beta.-subunit is substituted by Glu; (au) 19th Ala in the
.alpha.-subunit is substituted by Val, 71st Arg in the
.alpha.-subunit is substituted by His, 126th Phe in the
.alpha.-subunit is substituted by Tyr, 37th Phe in the
.beta.-subunit is substituted by Val, 108th Glu in the
.beta.-subunit is substituted by Asp, and 200th Ala in the
.beta.-subunit is substituted by Glu; (av) 36th Thr in the
.alpha.-subunit is substituted by Met, 148th Gly in the
.alpha.-subunit is substituted by Asp, 204th Val in the
.alpha.-subunit is substituted by Arg, 41st Phe in the
.beta.-subunit is substituted by Ile, 51st Phe in the
.beta.-subunit is substituted by Val, and 108th Glu in the
.beta.-subunit is substituted by Asp; (aw) 148th Gly in the
.alpha.-subunit is substituted by Asp, 204th Val in the
.alpha.-subunit is substituted by Arg, 108th Glu in the
.beta.-subunit is substituted by Asp, and 200th Ala in the
.beta.-subunit is substituted by Glu; (ax) 36th Thr in the
.alpha.-subunit is substituted by Gly and 188th Thr in the
.alpha.-subunit is substituted by Gly; (ay) 36th Thr in the
.alpha.-subunit is substituted by Ala and 48th Asn in the
.alpha.-subunit is substituted by Gln; (az) 48th Asn in the
.alpha.-subunit is substituted by Glu and 146th Arg in the
.beta.-subunit is substituted by Gly; (ba) 36th Thr in the
.alpha.-subunit is substituted by Trp and 176th Tyr in the
.beta.-subunit is substituted by Cys; (bb) 176th Tyr in the
.beta.-subunit is substituted by Met and 217th Asp in the
.beta.-subunit is substituted by Gly; (bc) 36th Thr in the
.alpha.-subunit is substituted by Ser, and 33rd Ala in the
.beta.-subunit is substituted by Val; (bd) 176th Tyr in the
.beta.-subunit is substituted by Ala and 217th Asp in the
.beta.-subunit is substituted by Val; (be) 40th Thr in the
.beta.-subunit is substituted by Val and 218th Cys in the
.beta.-subunit is substituted by Met; (bf) 33rd Ala in the
.beta.-subunit is substituted by Met and 176th Tyr in the
.beta.-subunit is substituted by Thr; (bg) 40th Thr in the
.beta.-subunit is substituted by Leu and 217th Asp in the
.beta.-subunit is substituted by Leu; (bh) 40th Thr in the
.beta.-subunit is substituted by Ile and 61st Ala in the
.beta.-subunit is substituted by Val; (bi) 61st Ala in the
.beta.-subunit is substituted by Thr and 218th Cys in the
.beta.-subunit is substituted by Ser; (bj) 112th Lys in the
.beta.-subunit is substituted by Val and 217th Asp in the
.beta.-subunit is substituted by Met; (bk) 61st Ala in the
.beta.-subunit is substituted by Trp and 217th Asp in the
.beta.-subunit is substituted by His; (bl) 61st Ala in the
.beta.-subunit is substituted by Leu and 112th Lys in the
.beta.-subunit is substituted by Ile; (bm) 146th Arg in the
.beta.-subunit is substituted by Gly and 217th Asp in the
.beta.-subunit is substituted by Ser; (bn) 171st Lys in the
.beta.-subunit is substituted by Ala and 217th Asp in the
.beta.-subunit is substituted by Thr; (bo) 150th Ala in the
.beta.-subunit is substituted by Ser and 217th Asp in the
.beta.-subunit is substituted by Cys; (bp) 61st Ala in the
.beta.-subunit is substituted by Gly and 150th Ala in the
.beta.-subunit is substituted by Asn; (bq) 61st Ala in the
.beta.-subunit is substituted by Ser and 160th Arg in the
.beta.-subunit is substituted by Met; and (br) 160th Arg in the
.beta.-subunit is substituted by Cys and 168th Thr in the
.beta.-subunit is substituted by Glu.
5. The nitrile hydratase variant according to claim 1, comprising
substitutions at the following substitution sites (I), (II), (III),
(IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII), (XIII) or
(XIV) with another amino acids: (I) 36th of .alpha.-subunit, 92nd
of .alpha.-subunit, 148th of .alpha.-subunit, 204th of
.alpha.-subunit, 41st of .beta.-subunit, 51st of .beta.-subunit and
108th of .beta.-subunit; (II) 6th of .alpha.-subunit, 19th of
.alpha.-subunit, 94th of .alpha.-subunit, 126th of .alpha.-subunit,
46th of .beta.-subunit, 108th of .beta.-subunit and 212th of
.beta.-subunit; (III) 6th of .alpha.-subunit, 19th of
.alpha.-subunit, 126th of .alpha.-subunit, 4th of .beta.-subunit,
127th of .beta.-subunit, 160th of .beta.-subunit and 186th of
.beta.-subunit; (IV) 19th of .alpha.-subunit, 71st of
.alpha.-subunit, 126th of .alpha.-subunit, 37th of .beta.-subunit,
79th of .beta.-subunit, 108th of .beta.-subunit and 200th of
.beta.-subunit; (V) 6th of .alpha.-subunit, 19th of
.alpha.-subunit, 126th of .alpha.-subunit, 48th of .beta.-subunit,
96th of .beta.-subunit, 108th of .beta.-subunit and 212th of
.beta.-subunit; (VI) 19th of .alpha.-subunit, 71st of
.alpha.-subunit, 126th of .alpha.-subunit, 37th of .beta.-subunit,
107th of .beta.-subunit, 108th of .beta.-subunit and 200th of
.beta.-subunit; (VII) 13th of .alpha.-subunit, 19th of
.alpha.-subunit, 71st of .alpha.-subunit, 126th of .alpha.-subunit,
37th of .beta.-subunit, 96th of .beta.-subunit, 108th of
.beta.-subunit and 200th of .beta.-subunit; (VIII) 6th of
.alpha.-subunit, 27th of .alpha.-subunit, 36th of .alpha.-subunit,
126th of .alpha.-subunit, 10th of .beta.-subunit, 107th of
.beta.-subunit, 118th of .beta.-subunit and 200th of
.beta.-subunit; (IX) 6th of .alpha.-subunit, 19th of
.alpha.-subunit, 126th of .alpha.-subunit, 48th of .beta.-subunit,
79th of .beta.-subunit, 108th of .beta.-subunit, 212th of
.beta.-subunit and 230th of .beta.-subunit; (X) 6th of
.alpha.-subunit, 36th of .alpha.-subunit, 126th of .alpha.-subunit,
10th of .beta.-subunit, 118th of .beta.-subunit, 200th of
.beta.-subunit, 206th of .beta.-subunit and 230th of
.beta.-subunit; (XI) 36th of .alpha.-subunit, 148th of
.alpha.-subunit, 204th of .alpha.-subunit, 41st of .beta.-subunit,
51st of .beta.-subunit, 108th of .beta.-subunit, 206th of
.beta.-subunit and 230th of .beta.-subunit; (XII) 6th of
.alpha.-subunit, 19th of .alpha.-subunit, 126th of .alpha.-subunit,
48th of .beta.-subunit, 79th of .beta.-subunit, 108th of
.beta.-subunit, 212th of .beta.-subunit, 230th of .beta.-subunit
and 231st of .beta.-subunit; (XIII) 6th of .alpha.-subunit, 19th of
.alpha.-subunit, 126th of .alpha.-subunit, 46th of .beta.-subunit,
79th of .beta.-subunit, 108th of .beta.-subunit, 212th of
.beta.-subunit, 230th of .beta.-subunit and 231st of
.beta.-subunit; (XIV) 6th of .alpha.-subunit, 19th of
.alpha.-subunit, 126th of .alpha.-subunit, 24th of .beta.-subunit,
48th of .beta.-subunit, 79th of .beta.-subunit, 108th of
.beta.-subunit, 212th of .beta.-subunit, 230th of .beta.-subunit
and 231st of .beta.-subunit.
6. The nitrile hydratase variant according to claim 5, wherein: in
case of (I), 36th of .alpha.-subunit is substituted with Met, 92nd
of .alpha.-subunit is substituted with Glu, 148th of
.alpha.-subunit is substituted with Asp, 204th of .alpha.-subunit
is substituted with Arg, 41st of .beta.-subunit is substituted with
Ile, 51st of .beta.-subunit is substituted with Val, and 108th of
.beta.-subunit is substituted with Asp; in case of (II), 6th of
.alpha.-subunit is substituted with Thr, 19th of .alpha.-subunit is
substituted with Val, 94th of .alpha.-subunit is substituted with
Ile, 126th of .alpha.-subunit is substituted with Tyr, 46th of
.beta.-subunit is substituted with Lys, 108th of .beta.-subunit is
substituted with Arg, and 212th of .beta.-subunit is substituted
with Tyr; in case of (III), 6th of .alpha.-subunit is substituted
with Ala, 19th of .alpha.-subunit is substituted with Val, 126th of
.alpha.-subunit is substituted with Tyr, 4th of .beta.-subunit is
substituted with Met, 127th of .beta.-subunit is substituted with
Ser, 160th of .beta.-subunit is substituted with Trp, and 186th of
.beta.-subunit is substituted with Arg; in case of (IV), 19th of
.alpha.-subunit is substituted with Val, 71st of .alpha.-subunit is
substituted with His, 126th of .alpha.-subunit is substituted with
Tyr, 37th of .beta.-subunit is substituted with Val, 79th of
.beta.-subunit is substituted with Asn, 108th of .beta.-subunit is
substituted with Asp, and 200th of .beta.-subunit is substituted
with Glu; in case of (V), 6th of .alpha.-subunit is substituted
with Thr, 19th of .alpha.-subunit is substituted with Val, 126th of
.alpha.-subunit is substituted with Tyr, 48th of .beta.-subunit is
substituted with Val, 96th of .beta.-subunit is substituted with
Arg, 108th of .beta.-subunit is substituted with Arg, and 212th of
.beta.-subunit is substituted with Tyr; in case of (VI), 19th of
.alpha.-subunit is substituted with Val, 71st of .alpha.-subunit is
substituted with His, 126th of .alpha.-subunit is substituted with
Tyr, 37th of .beta.-subunit is substituted with Val, 107th of
.beta.-subunit is substituted with Met, 108th of .beta.-subunit is
substituted with Asp, and 200th of .beta.-subunit is substituted
with Glu; in case of (VII), 13th of .alpha.-subunit is substituted
with Leu, 19th of .alpha.-subunit is substituted with Val, 71st of
.alpha.-subunit is substituted with His, 126th of .alpha.-subunit
is substituted with Tyr, 37th of .beta.-subunit is substituted with
Leu, 96th of .beta.-subunit is substituted with Arg, 108th of
.beta.-subunit is substituted with Asp, and 200th of .beta.-subunit
is substituted with Glu; in case of (VIII), 6th of .alpha.-subunit
is substituted with Thr, 27th of .alpha.-subunit is substituted
with Ile, 36th of .alpha.-subunit is substituted with Met, 126th of
.alpha.-subunit is substituted with Tyr, 10th of .beta.-subunit is
substituted with Asp, 107th of .beta.-subunit is substituted with
Met, 118th of .beta.-subunit is substituted with Val, and 200th of
.beta.-subunit is substituted with Glu; in case of (IX), 6th of
.alpha.-subunit is substituted with Thr, 19th of .alpha.-subunit is
substituted with Val, 126th of .alpha.-subunit is substituted with
Tyr, 48th of .beta.-subunit is substituted with Val, 79th of
.beta.-subunit is substituted with Asn, 108th of .beta.-subunit is
substituted with Arg, 212th of .beta.-subunit is substituted with
Tyr, and 230th of .beta.-subunit is substituted with Glu; in case
of (X), 6th of .alpha.-subunit is substituted with Thr, 36th of
.alpha.-subunit is substituted with Met, 126th of .alpha.-subunit
is substituted with Tyr, 10th of .beta.-subunit is substituted with
Asp, 118th of .beta.-subunit is substituted with Val, 200th of
.beta.-subunit is substituted with Glu, 206th of .beta.-subunit is
substituted with Leu, and 230th of .beta.-subunit is substituted
with Glu; in case of (XI), 36th of .alpha.-subunit is substituted
with Met, 148th of .alpha.-subunit is substituted with Asp, 204th
of .alpha.-subunit is substituted with Arg, 41st of .beta.-subunit
is substituted with Ile, 51st of .beta.-subunit is substituted with
Val, 108th of .beta.-subunit is substituted with Asp, 206th of
.beta.-subunit is substituted with Leu, and 230th of .beta.-subunit
is substituted with Glu; in case of (XII), 6th of .alpha.-subunit
is substituted with Thr, 19th of .alpha.-subunit is substituted
with Val, 126th of .alpha.-subunit is substituted with Tyr, 48th of
.beta.-subunit is substituted with Val, 79th of .beta.-subunit is
substituted with Asn, 108th of .beta.-subunit is substituted with
Arg, 212th of .beta.-subunit is substituted with Tyr, 230th of
.beta.-subunit is substituted with Glu, and 231st of .beta.-subunit
is substituted with Val; in case of (XIII), 6th of .alpha.-subunit
is substituted with Thr, 19th of .alpha.-subunit is substituted
with Val, 126th of .alpha.-subunit is substituted with Tyr, 46th of
.beta.-subunit is substituted with Lys, 79th of .beta.-subunit is
substituted with Asn, 108th of .beta.-subunit is substituted with
Arg, 212th of .beta.-subunit is substituted with Tyr, 230th of
.beta.-subunit is substituted with Glu, and 231st of .beta.-subunit
is substituted with Val; in case of (XIV), 6th of .alpha.-subunit
is substituted with Thr, 19th of .alpha.-subunit is substituted
with Val, 126th of .alpha.-subunit is substituted with Tyr, 24th of
.beta.-subunit is substituted with Ile, 48th of .beta.-subunit is
substituted with Val, 79th of .beta.-subunit is substituted with
Asn, 108th of .beta.-subunit is substituted with Arg, 212th of
.beta.-subunit is substituted with Tyr, 230th of .beta.-subunit is
substituted with Glu, and 231st of .beta.-subunit is substituted
with Val.
7. A gene encoding the nitrile hydratase variant according to claim
1.
8. A gene encoding a nitrile hydratase variant having a gene
encoding the .alpha.-subunit defined in SEQ ID No: 3 in the
Sequence Listing and a gene encoding the .beta.-subunit defined in
SEQ ID No: 4 in the Sequence Listing, comprising substitution of at
least one base selected from substitution sites of the base
consisting of the following (b) to (l): (a) 274th to 276th of the
base sequence of SEQ ID No: 3; (b) 280th to 282nd of the base
sequence of SEQ ID No: 3; (c) 589th to 591st of the base sequence
of SEQ ID No: 3; (d) 10th to 12th of the base sequence of SEQ ID
No: 4; (e) 70th to 72st of the base sequence of SEQ ID No: 4; (f)
235th to 237th of the base sequence of SEQ ID No: 4; (g) 286th to
288th of the base sequence of SEQ ID No: 4; (h) 319th to 321st of
the base sequence of SEQ ID No: 4; (i) 676th to 678th of the base
sequence of SEQ ID No: 4; (j) 328th to 330th of the base sequence
of SEQ ID No: 4 and 691st to 693rd of the base sequence of SEQ ID
No: 4; (k) 616th to 618th of the base sequence of SEQ ID No: 4, and
688th to 690th of the base sequence of SEQ ID No: 4; and (l) 37th
to 39th of the base sequence of SEQ ID No: 3, 79th to 81st of the
base sequence of SEQ ID No: 3, and 328th to 330th of the base
sequence of SEQ ID No: 4, wherein ATC is substituted by CTC when
37th to 39th of the base sequence of SEQ ID No: 3 are substituted
by another base, ATG is substituted by ATC when 79th to 81th of the
base sequence of SEQ ID No: 3 are substituted by another base, GAC
is substituted by GAG when 274th to 276th of the base sequence of
SEQ ID No: 3 are substituted by another base, ATG is substituted by
ATC when 280th to 282th of the base sequence of SEQ ID No: 3 are
substituted by another base, GGC is substituted by TGC when 589th
to 591th of the base sequence of SEQ ID No: 3 are substituted by
another base, GTG is substituted by ATG when 10th to 12th of the
base sequence of SEQ ID No: 4 are substituted by another base, GTC
is substituted by ATC when 70th to 72st of the base sequence of SEQ
ID No: 4 are substituted by another base, CAC is substituted by AAC
when 235th to 237th of the base sequence of SEQ ID No: 4 are
substituted by another base, CAG is substituted by CGT when 286th
to 288th of the base sequence of SEQ ID No: 4 are substituted by
another base, CCC is substituted by ATG when 319th to 321st of the
base sequence of SEQ ID No: 4 are substituted by another base, GAG
is substituted by AAC when 328th to 330th of the base sequence of
SEQ ID No: 4 are substituted by another base, CCG is substituted by
CTG when 616th to 618th of the base sequence of SEQ ID No: 4 are
substituted by another base, GTC is substituted by ATC when 676th
to 678th of the base sequence of SEQ ID No: 4 are substituted by
another base, GCG is substituted by GAG when 688th to 690th of the
base sequence of SEQ ID No: 4 are substituted by another base, and
GCC is substituted by GTC when 691th to 693th of the base sequence
of SEQ ID No: 4 are substituted by another base.
9. The gene encoding a nitrile hydratase variant according to claim
8, further comprising substitution of at least one base selected
from substitution sites of the base consisting of the following (m)
to (x): (m) in case of (b) or (g), 37th to 39th ATC of the base
sequence of SEQ ID No: 3 are substituted by CTC; (n) in case of (b)
or (h), 79th to 81st ATG of the base sequence of SEQ ID No: 3 are
substituted by ATC; (o) (d) and (f); (p) in case of (f), 688th to
690th GCG of the base sequence of SEQ ID No: 4 are substituted by
GAG; (q) (a) and (i); (r) in case of (i), 37th to 39th ATC of the
base sequence of SEQ ID No: 3 are substituted by CTC and 616th to
618th CCG of the base sequence of SEQ ID No: 4 are substituted by
CTG; (s) in case of (a) and (d), 616th to 618th CCG of the base
sequence of SEQ ID No: 4 are substituted by CTG; (t) in case of (c)
and (h), 688th to 690th GCG of the base sequence of SEQ ID No: 4
are substituted by GAG; (u) in case of (f), 688th to 690th GCG of
the base sequence of SEQ ID No: 4 are substituted by GAG and 691st
to 693rd GCC of the base sequence of SEQ ID No: 4 are substituted
by GTC; (v) (a) and (e) and (i); (w) in case of (c) and (e) and
(h), 688th to 690th GCG of the base sequence of SEQ ID No: 4 are
substituted by GAG; and (x) in case of (e) and (f), 688th to 690th
GCG of the base sequence of SEQ ID No: 4 are substituted by GAG and
691st to 693rd GCC of the base sequence of SEQ ID No: 4 are
substituted by GTC.
10. The gene encoding a nitrile hydratase variant according to
claim 8, further comprising substitution of at least one base with
another base selected from substitution sites of the base
consisting of (a), (c), (f), (i), (h), 688th to 690th of the base
sequence of SEQ ID No: 4, and 691st to 693rd of the base sequence
of SEQ ID No: 4, in case of (e), are substituted with another
base.
11. The gene encoding a nitrile hydratase variant according to
claim 8, comprising substitution of at least one base selected from
substitution sites of the base consisting of the following (aa) to
(br): (aa) 106th to 108th ACG of the base sequence of SEQ ID No: 3
are substituted by ATG, and 376th to 378th TTC of the base sequence
of SEQ ID No: 3 are substituted by TAC; (ab) 442nd to 444th GGC of
the base sequence of SEQ ID No: 3 are substituted by GAC, and 610th
to 612th GTC of the base sequence of SEQ ID No: 3 are substituted
by CGC; (ac) 151st to 153rd TTC of the base sequence of SEQ ID No:
4 are substituted by GTC, and 322nd to 324th GAG of the base
sequence of SEQ ID No: 4 are substituted by GAT; (ad) 352nd to
354th TTC of the base sequence of SEQ ID No: 4 are substituted by
GTC, and 598th to 600th GCC of the base sequence of SEQ ID No: 4
are substituted by GAG; (ae) 478th to 480th CGG of the base
sequence of SEQ ID No: 4 are substituted by TGG, and 556th to 558th
CTG of the base sequence of SEQ ID No: 4 are substituted by CGG;
(af) 16th to 18th CTG of the base sequence of SEQ ID No: 3 are
substituted by ACG, 106th to 108th ACG of the base sequence of SEQ
ID No: 3 are substituted by ATG, and 376th to 378th TTC of the base
sequence of SEQ ID No: 3 are substituted by TAC; (ag) 55th to 57th
GCG of the base sequence of SEQ ID No: 3 are substituted by GTG,
211th to 213th CGT of the base sequence of SEQ ID No: 3 are
substituted by CAT, and 376th to 378th TTC of the base sequence of
SEQ ID No: 3 are substituted by TAC; (ah) 106th to 108th ACG of the
base sequence of SEQ ID No: 3 are substituted by ATG, 442nd to
444th GGC of the base sequence of SEQ ID No: 3 are substituted by
GAC, and 610th to 612th GTC of the base sequence of SEQ ID No: 3
are substituted by CGC; (ai) 28th to 30th ACC of the base sequence
of SEQ ID No: 4 are substituted by GAC, 352nd to 354th TTC of the
base sequence of SEQ ID No: 4 are substituted by GTC, and 598th to
600th GCC of the base sequence of SEQ ID No: 4 are substituted by
GAG; (aj) 109th to 111th TTC of the base sequence of SEQ ID No: 4
are substituted by CTC, 322nd to 324th GAG of the base sequence of
SEQ ID No: 4 are substituted by GAT, and 598th to 600th GCC of the
base sequence of SEQ ID No: 4 are substituted by GAG; (ak) 109th to
111th TTC of the base sequence of SEQ ID No: 4 are substituted by
GTC, 322nd to 324th GAG of the base sequence of SEQ ID No: 4 are
substituted by GAT, and 598th to 600th GCC of the base sequence of
SEQ ID No: 4 are substituted by GAG; (al) 121st to 123rd TTC of the
base sequence of SEQ ID No: 4 are substituted by ATC, 151st to
153rd TTC of the base sequence of SEQ ID No: 4 are substituted by
GTC, and 322nd to 324th GAG of the base sequence of SEQ ID No: 4
are substituted by GAT; (am) 136th to 138th ATG of the base
sequence of SEQ ID No: 4 are substituted by AAG, 322nd to 324th GAG
of the base sequence of SEQ ID No: 4 are substituted by CGG, and
634th to 636th TCC of the base sequence of SEQ ID No: 4 are
substituted by TAC; (an) 142nd to 144th CTG of the base sequence of
SEQ ID No: 4 are substituted by GTG, 322nd to 324th GAG of the base
sequence of SEQ ID No: 4 are substituted by CGG, and 634th to 636th
TCC of the base sequence of SEQ ID No: 4 are substituted by TAC;
(ao) 379th to 381st CTG of the base sequence of SEQ ID No: 4 are
substituted by TCG, 478th to 480th CGG of the base sequence of SEQ
ID No: 4 are substituted by TGG, and 556th to 558th CTG of the base
sequence of SEQ ID No: 4 are substituted by CGG; (ap) 16th to 18th
CTG of the base sequence of SEQ ID No: 3 are substituted by ACG,
55th to 57th GCG of the base sequence of SEQ ID No: 3 are
substituted by GTG, 376th to 378th TTC of the base sequence of SEQ
ID No: 3 are substituted by TAC, 136th to 138th ATG of the base
sequence of SEQ ID No: 4 are substituted by AAG, 322nd to 324th GAG
of the base sequence of SEQ ID No: 4 are substituted by CGG, and
634th to 636th TCC of the base sequence of SEQ ID No: 4 are
substituted by TAC; (aq) 16th to 18th CTG of the base sequence of
SEQ ID No: 3 are substituted by ACG, 55th to 57th GCG of the base
sequence of SEQ ID No: 3 are substituted by GTG, 376th to 378th TTC
of the base sequence of SEQ ID No: 3 are substituted by TAC, 142nd
to 144th CTG of the base sequence of SEQ ID No: 4 are substituted
by GTG, 322nd to 324th GAG of the base sequence of SEQ ID No: 4 are
substituted by CGG, and 634th to 636th TCC of the base sequence of
SEQ ID No: 4 are substituted by TAC; (ar) 16th to 18th CTG of the
base sequence of SEQ ID No: 3 are substituted by GCG, 55th to 57th
GCG of the base sequence of SEQ ID No: 3 are substituted by GTG,
376th to 378th TTC of the base sequence of SEQ ID No: 3 are
substituted by TAC, 379th to 381st CTG of the base sequence of SEQ
ID No: 4 are substituted by TCG, 478th to 480th CGG of the base
sequence of SEQ ID No: 4 are substituted by TGG, and 556th to 558th
CTG of the base sequence of SEQ ID No: 4 are substituted by CGG;
(as) 16th to 18th CTG of the base sequence of SEQ ID No: 3 are
substituted by ACG, 106th to 108th ACG of the base sequence of SEQ
ID No: 3 are substituted by ATG, 376th to 378th TTC of the base
sequence of SEQ ID No: 3 are substituted by TAC, 28th to 30th ACC
of the base sequence of SEQ ID No: 4 are substituted by GAC, 352nd
to 354th TTC of the base sequence of SEQ ID No: 4 are substituted
by GTC, and 598th to 600th GCC of the base sequence of SEQ ID No: 4
are substituted by GAG; (at) 55th to 57th GCG of the base sequence
of SEQ ID No: 3 are substituted by GTG, 211th to 213th CGT of the
base sequence of SEQ ID No: 3 are substituted by CAT, 376th to
378th TTC of the base sequence of SEQ ID No: 3 are substituted by
TAC, 109th to 111th TTC of the base sequence of SEQ ID No: 4 are
substituted by CTC, 322nd to 324th GAG of the base sequence of SEQ
ID No: 4 are substituted by GAT, and 598th to 600th GCC of the base
sequence of SEQ ID No: 4 are substituted by GAG; (au) 55th to 57th
GCG of the base sequence of SEQ ID No: 3 are substituted by GTG,
211th to 213th CGT of the base sequence of SEQ ID No: 3 are
substituted by CAT, 376th to 378th TTC of the base sequence of SEQ
ID No: 3 are substituted by TAC, 109th to 111th TTC of the base
sequence of SEQ ID No: 4 are substituted by GTC, 322nd to 324th GAG
of the base sequence of SEQ ID No: 4 are substituted by GAT, and
598th to 600th GCC of the base sequence of SEQ ID No: 4 are
substituted by GAG; (av) 106th to 108th ACG of the base sequence of
SEQ ID No: 3 are substituted by ATG, 442nd to 444th GGC of the base
sequence of SEQ ID No: 3 are substituted by GAC, 610th to 612th GTC
of the base sequence of SEQ ID No: 3 are substituted by CGC, 121st
to 123rd TTC of the base sequence of SEQ ID No: 4 are substituted
by ATC, 151st to 153rd TTC of the base sequence of SEQ ID No: 4 are
substituted by GTC, and 322nd to 324th GAG of the base sequence of
SEQ ID No: 4 are substituted by GAT; (aw) 442nd to 444th GGC of the
base sequence of SEQ ID No: 3 are substituted by GAC, 610th to
612th GTC of the base sequence of SEQ ID No: 3 are substituted by
CGC, 322nd to 324th GAG of the base sequence of SEQ ID No: 4 are
substituted by GAT, and 598th to 600th GCC of the base sequence of
SEQ ID No: 4 are substituted by GAG; (ax) 106th to 108th ACG of the
base sequence of SEQ ID No: 3 are substituted by GGG, and 562nd to
564th ACC of the base sequence of SEQ ID No: 3 are substituted by
GGC; (ay) 106th to 108th ACG of the base sequence of SEQ ID No: 3
are substituted by GCG, and 142nd to 144th AAC of the base sequence
of SEQ ID No: 3 are substituted by CAA; (az) 142nd to 144th AAC of
the base sequence of SEQ ID No: 3 are substituted by GAA, and 436th
to 438th CGG of the base sequence of SEQ ID No: 4 are substituted
by GGG; (ba) 106th to 108th ACG of the base sequence of SEQ ID No:
3 are substituted by TGG, and 526th to 528th TAC of the base
sequence of SEQ ID No: 4 are substituted by TGC; (bb) 526th to
528th TAC of the base sequence of SEQ ID No: 4 are substituted by
ATG, and 649th to 651st GAC of the base sequence of SEQ ID No: 4
are substituted by GGC; (bc) 106th to 108th ACG of the base
sequence of SEQ ID No: 3 are substituted by TCG, and 97th to 99th
GCG of the base sequence of SEQ ID No: 4 are substituted by GTG;
(bd) 526th to 528th TAC of the base sequence of SEQ ID No: 4 are
substituted by GCC, and 649th to 651st GAC of the base sequence of
SEQ ID No: 4 are substituted by GTC; (be) 118th to 120th ACG of the
base sequence of SEQ ID No: 4 are substituted by GTG, and 652nd to
654th TGC of the base sequence of SEQ ID No: 4 are substituted by
ATG; (bf) 97th to 99th GCG of the base sequence of SEQ ID No: 4 are
substituted by ATG, and 526th to 528th TAC of the base sequence of
SEQ ID No: 4 are substituted by ACC; (bg) 118th to 120th ACG of the
base sequence of SEQ ID No: 4 are substituted by CTG, and 649th to
651st GAC of the base sequence of SEQ ID No: 4 are substituted by
CTC; (bh) 118th to 120th ACG of the base sequence of SEQ ID No: 4
are substituted by ATT, and 181st to 183rd GCC of the base sequence
of SEQ ID No: 4 are substituted by GTC; (bi) 181st to 183rd GCC of
the base sequence of SEQ ID No: 4 are substituted by ACG, and 652nd
to 654th TGC of the base sequence of SEQ ID No: 4 are substituted
by TCC; (bj) 334th to 336th AAG of the base sequence of SEQ ID No:
4 are substituted by GTG, and 649th to 651st GAC of the base
sequence of SEQ ID No: 4 are substituted by ATG; (bk) 181st to
183rd GCC of the base sequence of SEQ ID No: 4 are substituted by
TGG, and 649th to 651st GAC of the base sequence of SEQ ID No: 4
are substituted by CAC; (bl) 181st to 183rd GCC of the base
sequence of SEQ ID No: 4 are substituted by CTC, and 334th to 336th
AAG of the base sequence of SEQ ID No: 4 are substituted by ATT;
(bm) 436th to 438th CGG of the base sequence of SEQ ID No: 4 are
substituted by GGG, and 649th to 651st GAC of the base sequence of
SEQ ID No: 4 are substituted by AGC; (bn) 511th to 513th AAG of the
base sequence of SEQ ID No: 4 are substituted by GCG, and 649th to
651st GAC of the base sequence of SEQ ID No: 4 are substituted by
ACC; (bo) 448th to 450th GCG of the base sequence of SEQ ID No: 4
are substituted by TCG, and 649th to 651st GAC of the base sequence
of SEQ ID No: 4 are substituted by TGT; (bp) 181st to 183rd GCC of
the base sequence of SEQ ID No: 4 are substituted by GGC, and 448th
to 450th GCG of the base sequence of SEQ ID No: 4 are substituted
by AAT; (bq) 181st to 183rd GCC of the base sequence of SEQ ID No:
4 are substituted by TCG, and 478th to 480th CGG of the base
sequence of SEQ ID No: 4 are substituted by ATG; and (br) 478th to
480th CGG of the base sequence of SEQ ID No: 4 are substituted by
TGT, and 502nd to 504th ACG of the base sequence of SEQ ID No: 4
are substituted by GAG.
12. The gene encoding a nitrile hydratase variant according to
claim 8, comprising substitutions of the following (I), (II),
(III), (IV), (V), (VI), (VII), (VIII), (IX), (X), (XI), (XII),
(XIII) or (XIV): (I) the base sequences of 106th to 108th, 274th to
276th, 442nd to 444th and 610th to 612th in SEQ ID No: 3, and the
base sequences of 121st to 123rd, 151st to 153rd, and 322nd to
324th in SEQ ID No: 4; (II) the base sequences of 16th to 18th,
55th to 57th, 280th to 282nd and 376th to 378th in SEQ ID No: 3,
and the base sequences of 136th to 138th, 322nd to 324th and 634th
to 636th in SEQ ID No: 4; (III) the base sequences of 16th to 18th,
55th to 57th, and 376th to 378th in SEQ ID No: 3, and the base
sequences of 10th to 12th, 379th to 381st, 478th to 480th and 556th
to 558th in SEQ ID No: 4; (IV) the base sequences of 55th to 57th,
211th to 213th and 376th to 378th in SEQ ID No: 3, and the base
sequences of 109th to 111th, 235th to 237th, 322nd to 324th and
598th to 600th in SEQ ID No: 4; (V) the base sequences of 16th to
18th, 55th to 57th, and 376th to 378th in SEQ ID No: 3, and the
base sequences of 142nd to 144th, 286th to 288th, 322nd to 324th
and 634th to 636th in SEQ ID No: 4; (VI) the base sequences of 55th
to 57th, 211th to 213th, and 376th to 378th in SEQ ID No: 3, and
the base sequences of 109th to 111th, 319th to 321st, 322nd to
324th and 598th to 600th in SEQ ID No: 4; (VII) the base sequences
of 37th to 39th, 55th to 57th, 211th to 213th and 376th to 378th in
SEQ ID No: 3, and the base sequences of 109th to 111th, 286th to
288th, 322nd to 324th and 598th to 600th in SEQ ID No: 4; (VIII)
the base sequences of 16th to 18th, 79th to 81st, 106th to 108th
and 376th to 378th in SEQ ID No: 3, and the base sequences of 28th
to 30th, 319th to 321st, 352nd to 354th and 598th to 600th in SEQ
ID No: 4; (IX) the base sequences of 16th to 18th, 55th to 57th and
376th to 378th in SEQ ID No: 3, and the base sequences of 142nd to
144th, 235th to 237th, 322nd to 324th, 634th to 636th and 688th to
690th in SEQ ID No: 4; (X) the base sequences of 16th to 18th,
106th to 108th and 376th to 378th in SEQ ID No: 3, and the base
sequences of 28th to 30th, 352nd to 354th, 598th to 600th, 616th to
618th and 688th to 690th in SEQ ID No: 4; (XI) the base sequences
of 106th to 108th, 422nd to 444th and 610th to 612th in SEQ ID No:
3, and the base sequences of 121st to 123rd, 151st to 153rd, 322nd
to 324th, 616th to 618th and 688th to 690th in SEQ ID No: 4; (XII)
the base sequences of 16th to 18th, 55th to 57th and 376th to 378th
in SEQ ID No: 3, and the base sequences of 142nd to 144th, 235th to
237th, 322nd to 324th, 634th to 636th, 688th to 690th and 691st to
693rd in SEQ ID No: 4; (XIII) the base sequences of 16th to 18th,
55th to 57th and 376th to 378th in SEQ ID No: 3, and the base
sequences of 136th to 138th, 235th to 237th, 322nd to 324th, 634th
to 636th, 688th to 690th and 691st to 693rd in SEQ ID No: 4; (XIV)
the base sequences of 16th to 18th, 55th to 57th and 376th to 378th
in SEQ ID No: 3, and the base sequences of 70th to 72st, 142nd to
148th, 235th to 237th, 322nd to 324th, 634th to 636th, 688th to
690th and 691st to 693rd in SEQ ID No: 4;
13. The gene encoding a nitrile hydratase variant according to
claim 12, wherein in case of substitutions of (I): 106th to 108th
of the base sequence of SEQ ID No: 3 are substituted by ATG; 274th
to 276th of the base sequence of SEQ ID No: 3 are substituted by
GAG; 442nd to 444th of the base sequence of SEQ ID No: 3 are
substituted by GAC; 610th to 612th of the base sequence of SEQ ID
No: 3 are substituted by CGC; 121st to 123rd of the base sequence
of SEQ ID No: 4 are substituted by ATC; 151st to 153rd of the base
sequence of SEQ ID No: 4 are substituted by GTC; and 322nd to 324th
of the base sequence of SEQ ID No: 4 are substituted by GAT;
wherein in case of substitutions of (II): 16th to 18th of the base
sequence of SEQ ID No: 3 are substituted by ACG; 55th to 57th of
the base sequence of SEQ ID No: 3 are substituted by GTG; 280th to
282nd of the base sequence of SEQ ID No: 3 are substituted by ATC;
376th to 378th of the base sequence of SEQ ID No: 3 are substituted
by TAC; 136th to 138th of the base sequence of SEQ ID No: 4 are
substituted by AAG; 322nd to 324th of the base sequence of SEQ ID
No: 4 are substituted by CGG; and 634th to 636th of the base
sequence of SEQ ID No: 4 are substituted by TAC; wherein in case of
substitutions of (III): 16th to 18th of the base sequence of SEQ ID
No: 3 are substituted by GCG; 55th to 57th of the base sequence of
SEQ ID No: 3 are substituted by GTG; 376th to 378th of the base
sequence of SEQ ID No: 3 are substituted by TAC; 10th to 12th of
the base sequence of SEQ ID No: 4 are substituted by ATG; 379th to
381st of the base sequence of SEQ ID No: 4 are substituted by TCG;
478th to 480th of the base sequence of SEQ ID No: 4 are substituted
by TGG; and 556th to 558th of the base sequence of SEQ ID No: 4 are
substituted by CGG; wherein in case of substitutions of (IV): 55th
to 57th of the base sequence of SEQ ID No: 3 are substituted by
GTG; 211th to 213th of the base sequence of SEQ ID No: 3 are
substituted by CAT; 376th to 378th of the base sequence of SEQ ID
No: 3 are substituted by TAC; 109th to 111th of the base sequence
of SEQ ID No: 4 are substituted by GTC; 235th to 237th of the base
sequence of SEQ ID No: 4 are substituted by AAC; 322nd to 324th of
the base sequence of SEQ ID No: 4 are substituted by GAT; and 598th
to 600th of the base sequence of SEQ ID No: 4 are substituted by
GAG; wherein in case of substitutions of (V): 16th to 18th of the
base sequence of SEQ ID No: 3 are substituted by ACG; 55th to 57th
of the base sequence of SEQ ID No: 3 are substituted by GTG; 376th
to 378th of the base sequence of SEQ ID No: 3 are substituted by
TAC; 142nd to 144th of the base sequence of SEQ ID No: 4 are
substituted by GTG; 286th to 288th of the base sequence of SEQ ID
No: 4 are substituted by CGT; 322nd to 324th of the base sequence
of SEQ ID No: 4 are substituted by CGG; and 634th to 636th of the
base sequence of SEQ ID No: 4 are substituted by TAC; wherein in
case of substitutions of (VI): 55th to 57th of the base sequence of
SEQ ID No: 3 are substituted by GTG; 211th to 213th of the base
sequence of SEQ ID No: 3 are substituted by CAT; 376th to 378th of
the base sequence of SEQ ID No: 3 are substituted by TAC; 109th to
111th of the base sequence of SEQ ID No: 4 are substituted by GTC;
319th to 321st of the base sequence of SEQ ID No: 4 are substituted
by ATG; 322nd to 324th of the base sequence of SEQ ID No: 4 are
substituted by GAT; and 598th to 600th of the base sequence of SEQ
ID No: 4 are substituted by GAG; wherein in case of substitutions
of (VII): 37th to 39th of the base sequence of SEQ ID No: 3 are
substituted by CTC; 55th to 57th of the base sequence of SEQ ID No:
3 are substituted by GTG; 211th to 213th of the base sequence of
SEQ ID No: 3 are substituted by CAT; 376th to 378th of the base
sequence of SEQ ID No: 3 are substituted by TAC; 109th to 111th of
the base sequence of SEQ ID No: 4 are substituted by CTC; 286th to
288th of the base sequence of SEQ ID No: 4 are substituted by CGT;
322th to 324th of the base sequence of SEQ ID No: 4 are substituted
by GAT; and 598th to 600th of the base sequence of SEQ ID No: 4 are
substituted by GAG; wherein in case of substitutions of (VIII):
16th to 18th of the base sequence of SEQ ID No: 3 are substituted
by ACG; 79th to 81st of the base sequence of SEQ ID No: 3 are
substituted by ATC; 106th to 108th of the base sequence of SEQ ID
No: 3 are substituted by ATG; 376th to 378th of the base sequence
of SEQ ID No: 3 are substituted by TAC; 28th to 30th of the base
sequence of SEQ ID No: 4 are substituted by GAC; 319th to 321st of
the base sequence of SEQ ID No: 4 are substituted by ATG; 352nd to
354th of the base sequence of SEQ ID No: 4 are substituted by GTC;
and 598th to 600th of the base sequence of SEQ ID No: 4 are
substituted by GAG; wherein in case of substitutions of (IX): 16th
to 18th of the base sequence of SEQ ID No: 3 are substituted by
ACG; 55th to 57th of the base sequence of SEQ ID No: 3 are
substituted by GTG; 376th to 378th of the base sequence of SEQ ID
No: 3 are substituted by TAC; 142nd to 144th of the base sequence
of SEQ ID No: 4 are substituted by GTG; 235th to 237th of the base
sequence of SEQ ID No: 4 are substituted by AAC; 322nd to 324th of
the base sequence of SEQ ID No: 4 are substituted by CGG; 634th to
636th of the base sequence of SEQ ID No: 4 are substituted by TAC;
and 688th to 690th of the base sequence of SEQ ID No: 4 are
substituted by GAG; wherein in case of substitutions of (X): 16th
to 18th of the base sequence of SEQ ID No: 3 are substituted by
ACG; 106th to 108th of the base sequence of SEQ ID No: 3 are
substituted by ATG; 376th to 378th of the base sequence of SEQ ID
No: 3 are substituted by TAC; 28th to 30th of the base sequence of
SEQ ID No: 4 are substituted by GAC; 352nd to 354th of the base
sequence of SEQ ID No: 4 are substituted by GTC; 598th to 600th of
the base sequence of SEQ ID No: 4 are substituted by GAG; 616th to
618th of the base sequence of SEQ ID No: 4 are substituted by CTG;
and 688th to 690th of the base sequence of SEQ ID No: 4 are
substituted by GAG; wherein in case of substitutions of (XI): 106th
to 108th of the base sequence of SEQ ID No: 3 are substituted by
ATG; 442nd to 444th of the base sequence of SEQ ID No: 3 are
substituted by GAC; 610th to 612th of the base sequence of SEQ ID
No: 3 are substituted by CGC; 121st to 123rd of the base sequence
of SEQ ID No: 4 are substituted by ATC; 151st to 153rd of the base
sequence of SEQ ID No: 4 are substituted by GTC; 322nd to 324th of
the base sequence of SEQ ID No: 4 are substituted by GAT; 616th to
618th of the base sequence of SEQ ID No: 4 are substituted by CTG;
and 688th to 690th of the base sequence of SEQ ID No: 4 are
substituted by GAG; wherein in case of substitutions of (XII): 16th
to 18th of the base sequence of SEQ ID No: 3 are substituted by
ACG; 55th to 57th of the base sequence of SEQ ID No: 3 are
substituted by GTG; 376th to 378th of the base sequence of SEQ ID
No: 3 are substituted by TAC; 142nd to 144th of the base sequence
of SEQ ID No: 4 are substituted by GTG; 235th to 237th of the base
sequence of SEQ ID No: 4 are substituted by AAC; 322nd to 324th of
the base sequence of SEQ ID No: 4 are substituted by CGG; 634th to
636th of the base sequence of SEQ ID No: 4 are substituted by TAC;
688th to 690th of the base sequence of SEQ ID No: 4 are substituted
by GAG; and 691st to 693rd of the base sequence of SEQ ID No: 4 are
substituted by GTC; wherein in case of substitutions of (XIII):
16th to 18th of the base sequence of SEQ ID No: 3 are substituted
by ACG; 55th to 57th of the base sequence of SEQ ID No: 3 are
substituted by GTG; 376th to 378th of the base sequence of SEQ ID
No: 3 are substituted by TAC; 136th to 138th of the base sequence
of SEQ ID No: 4 are substituted by AAG; 235th to 237th of the base
sequence of SEQ ID No: 4 are substituted by AAC; 322nd to 324th of
the base sequence of SEQ ID No: 4 are substituted by CGG; 634th to
636th of the base sequence of SEQ ID No: 4 are substituted by TAC;
688th to 690th of the base sequence of SEQ ID No: 4 are substituted
by GAG; and 691st to 693rd of the base sequence of SEQ ID No: 4 are
substituted by GTC; wherein in case of substitutions of (XIV): 16th
to 18th of the base sequence of SEQ ID No: 3 are substituted by
ACG; 55th to 57th of the base sequence of SEQ ID No: 3 are
substituted by GTG; 376th to 378th of the base sequence of SEQ ID
No: 3 are substituted by TAC; 70th to 72st of the base sequence of
SEQ ID No: 4 are substituted by ATC; 142nd to 148th of the base
sequence of SEQ ID No: 4 are substituted by GTG; 235th to 237th of
the base sequence of SEQ ID No: 4 are substituted by AAC; 322nd to
324th of the base sequence of SEQ ID No: 4 are substituted by CGG;
634th to 636th of the base sequence of SEQ ID No: 4 are substituted
by TAC; 688th to 690th of the base sequence of SEQ ID No: 4 are
substituted by GAG; and 691st to 693rd of the base sequence of SEQ
ID No: 4 are substituted by GTC.
14. A linked DNA comprising further DNA containing a promoter
sequence necessary for the expression of the gene in the upstream
region of the 5'-terminal of the gene encoding a nitrile hydratase
variant according to claim 8, and a ribosome binding sequence
contained in SEQ ID No: 7 in the downstream region of the
3'-terminal of the promoter.
15. A plasmid comprising the DNA according to claim 14.
16. A transformant obtained by transformation of a host cell using
the plasmid according to claim 15.
17. A method for producing a nitrile hydratase variant, comprising
cultivating the transformant according to claim 16 in a culture
medium and producing a nitrile hydratase variant based on the
nitrile hydratase gene carried by the plasmid in the transformant.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a Continuation of U.S.
application Ser. No. 13/128,323, filed May 9, 2011, which is a U.S.
National Stage Application of PCT/JP2009/006055, filed Nov. 12,
2009, which claims priority to Japanese application number
2008-292819, filed Nov. 14, 2008, the entire contents of which are
incorporated by reference herein in their entirety.
TECHNICAL FIELD
[0002] The present invention relates to a nitrile hydratase
variant, a gene encoding the nitrile hydratase variant, a DNA
containing the gene, a plasmid containing the gene, a transformant
by means of the plasmid, and a method for producing a nitrile
hydratase variant using the transformant.
BACKGROUND ART
[0003] In recent years, a nitrile hydratase has been discovered
which is an enzyme having the nitrile-hydrating activity to convert
a nitrile group of various compounds to an amide group by
hydration, and a number of microorganism strains producing the
above-mentioned enzyme have been disclosed. In order to produce an
amide compound from a nitrile compound using a nitrile hydratase on
an industrial scale, it is important to reduce the production costs
for this enzyme in the total production costs for producing the
amide compound. More specifically, it is necessary to increase the
activity value in a unit weight of the preparation obtained from
the enzyme production.
[0004] As a method for increasing the activity value by increasing
the amount of the enzyme in the enzyme preparation, attempts have
already been made to clone the gene encoding the above-mentioned
enzyme for the purpose of expressing a large amount of the enzyme
through genetic engineering methods.
[0005] For example, there are produced a plasmid expressing a large
number of the Pseudonocardia thermophila-derived nitrile hydratase
in the transformant and a transformant strain transformed with the
plasmid. In addition, it has been made possible to produce a
nitrile hydratase by means of these transformant strains, and to
produce a corresponding amide compound by bringing the transformant
strain or the nitrile hydratase obtained therefrom into contact
with the nitrile compound (see Patent Document 1).
[0006] On the other hand, when high activation of enzyme molecule
itself can be achieved, the activity value of the enzyme
preparation can be further enhanced.
[0007] Attempts have heretofore been made to search for a nitrile
hydratase variant with improved substrate specificity, enzyme
stability or the like by introducing mutation into a specific amino
acid residue in the amino acid sequence of the nitrile hydratase
without damaging its activity (see Patent Document 2 to 4).
[0008] Furthermore, a nitrile hydratase variant derived from
Rhodococcus rhodochrous has been disclosed in Patent Documents 5
and 6.
RELATED DOCUMENT
Patent Document
[0009] Patent Document 1: Japanese Patent Laid-open No. H9
(1997)-275978
[0010] Patent Document 2: Japanese Patent Laid-open No.
2004-194588
[0011] Patent Document 3: Japanese Patent Laid-open No.
2005-160403
[0012] Patent Document 4: WO 2004/056990
[0013] Patent Document 5: Japanese Patent Laid-open No.
2007-143409
[0014] Patent Document 6: Japanese Patent Laid-open No.
2008-253182
DISCLOSURE OF THE INVENTION
[0015] However, as compared to a wild nitrile hydratase disclosed
in Patent Document 1, specific mutants in which both of the initial
reaction rate and enzyme stability are improved have not been
known. It is expected that the production costs for producing the
amide compound can be reduced by improving the initial reaction
rate and enzyme stability at the same time.
[0016] An object of the present invention is to provide a nitrile
hydratase having high initial reaction rate and enzyme
stability.
[0017] That is, the present invention is specified by matters
described in below.
[0018] [1] A nitrile hydratase variant comprising substitution of
at least one amino acid with another amino acid to improve two or
more properties of nitrile hydratase by substitution of one or more
and three or less amino acids.
[0019] [2] The nitrile hydratase variant according to [1], wherein
the properties to be improved are the initial reaction rate and
thermal stability.
[0020] [3] The nitrile hydratase variant according to [1] or [2],
comprising an .alpha.-subunit defined in SEQ ID No: 1 in the
Sequence Listing and a .beta.-subunit defined in SEQ ID No: 2 in
the Sequence Listing, and substitution of at least one amino acid
with another amino acid selected from substitution sites of the
amino acid consisting of the following (a) to (l):
[0021] (a) 92nd of .alpha.-subunit;
[0022] (b) 94th of .alpha.-subunit;
[0023] (c) 197th of .alpha.-subunit;
[0024] (d) 4th of .beta.-subunit;
[0025] (e) 24th of .beta.-subunit;
[0026] (f) 79th of .beta.-subunit;
[0027] (g) 96th of .beta.-subunit;
[0028] (h) 107th of .beta.-subunit;
[0029] (i) 226th of .beta.-subunit;
[0030] (j) 110th of .beta.-subunit and 231st of .beta.-subunit;
[0031] (k) 206th of .beta.-subunit and 230th of .beta.-subunit;
and
[0032] (l) 13th of .alpha.-subunit, 27th of .alpha.-subunit and
110th of .beta.-subunit.
[0033] [4] The nitrile hydratase variant according to [3],
comprising substitution of at least one amino acid with another
amino acid selected from substitution sites of the amino acid
consisting of the following (m) to (u):
[0034] (m) in case of (b) or (g), 13th of .alpha.-subunit;
[0035] (n) in case of (b) or (h), 27th of .alpha.-subunit;
[0036] (o) (d) and (f);
[0037] (p) in case of (f), 230th of .beta.-subunit;
[0038] (q) (a) and (i);
[0039] (r) in case of (i), 13th of .alpha.-subunit and 206th of
.beta.-subunit;
[0040] (s) in case of (a) and (d), 206th of .beta.-subunit;
[0041] (t) in case of (c) and (h), 230th of .beta.-subunit; and
[0042] (u) in case of (f), 230th of .beta.-subunit and 231st of
.beta.-subunit.
[0043] [5] The nitrile hydratase variant according to [3], further
comprising substitution of at least one amino acid with another
amino acid selected from the group consisting of (a), (c), (f),
(i), (h), 230th of the .beta.-subunit and 231st of the
.beta.-subunit in case of (e) is substituted with another amino
acid.
[0044] [6] The nitrile hydratase variant according to any one of
[1] to [5], wherein Ile is substituted by Leu when 13th amino acid
of the .alpha.-subunit is substituted,
[0045] Met is substituted by Ile when the 27th amino acid of the
.alpha.-subunit is substituted,
[0046] Asp is substituted by Glu when the 92nd amino acid of the
.alpha.-subunit is substituted,
[0047] Met is substituted by Ile when the 94th amino acid of the
.alpha.-subunit is substituted,
[0048] Gly is substituted by Cys when the 197th amino acid of the
.alpha.-subunit is substituted,
[0049] Val is substituted by Met when the 4th amino acid of the
.beta.-subunit is substituted,
[0050] Val is substituted by Ile when the 24th amino acid of the
.beta.-subunit is substituted,
[0051] His is substituted by Asn when the 79th amino acid of the
.beta.-subunit is substituted,
[0052] Gln is substituted by Arg when the 96th amino acid of the
.beta.-subunit is substituted,
[0053] Pro is substituted by Met when the 107th amino acid of the
.beta.-subunit is substituted,
[0054] Glu is substituted by Asn when the 110th amino acid of the
.beta.-subunit is substituted,
[0055] Pro is substituted by Leu when the 206th amino acid of the
.beta.-subunit is substituted,
[0056] Val is substituted by Ile when the 226th amino acid of the
.beta.-subunit is substituted,
[0057] Ala is substituted by Glu when the 230th amino acid of the
.beta.-subunit is substituted, and
[0058] Ala is substituted by Val when the 231st amino acid of the
.beta.-subunit is substituted.
[0059] [7] The nitrile hydratase variant according to any one of
[3] to [6], further comprising substitution of at least one amino
acid selected from substitutions of the amino acid consisting of
the following (aa) to (br):
[0060] (aa) 36th Thr in the .alpha.-subunit is substituted by Met
and 126th Phe in the .alpha.-subunit is substituted by Tyr;
[0061] (ab) 148th Gly in the .alpha.-subunit is substituted by Asp
and 204th Val in the .alpha.-subunit is substituted by Arg;
[0062] (ac) 51st Phe in the .beta.-subunit is substituted by Val
and 108th Glu in the .beta.-subunit is substituted by Asp;
[0063] (ad) 118th Phe in the .beta.-subunit is substituted by Val
and 200th Ala in the .beta.-subunit is substituted by Glu;
[0064] (ae) 160th Arg in the .beta.-subunit is substituted by Trp
and 186th Leu in the .beta.-subunit is substituted by Arg;
[0065] (af) 6th Leu in the .alpha.-subunit is substituted by Thr,
36th Thr in the .alpha.-subunit is substituted by Met, and 126th
Phe in the .alpha.-subunit is substituted by Tyr;
[0066] (ag) 19th Ala in the .alpha.-subunit is substituted by Val,
71st Arg in the .alpha.-subunit is substituted by His, and 126th
Phe in the .alpha.-subunit is substituted by Tyr;
[0067] (ah) 36th Thr in the .alpha.-subunit is substituted by Met,
148th Gly in the .alpha.-subunit is substituted by Asp, and 204th
Val in the .alpha.-subunit is substituted by Arg;
[0068] (ai) 10th Thr in the .beta.-subunit is substituted by Asp,
118th Phe in the .beta.-subunit is substituted by Val, and 200th
Ala in the .beta.-subunit is substituted by Glu;
[0069] (aj) 37th Phe in the .beta.-subunit is substituted by Leu,
108th Glu in the .beta.-subunit is substituted by Asp, and 200th
Ala in the .beta.-subunit is substituted by Glu;
[0070] (ak) 37th Phe in the .beta.-subunit is substituted by Val,
108th Glu in the .beta.-subunit is substituted by Asp, and 200th
Ala in the .beta.-subunit is substituted by Glu;
[0071] (al) 41st Phe in the .beta.-subunit is substituted by Ile,
51st Phe in the .beta.-subunit is substituted by Val, and 108th Glu
in the .beta.-subunit is substituted by Asp;
[0072] (am) 46th Met in the .beta.-subunit is substituted by Lys,
108th Glu in the .beta.-subunit is substituted by Arg, and 212th
Ser in the .beta.-subunit is substituted by Tyr;
[0073] (an) 48th Leu in the .beta.-subunit is substituted by Val,
108th Glu in the .beta.-subunit is substituted by Arg, and 212th
Ser in the .beta.-subunit is substituted by Tyr;
[0074] (ao) 127th Leu in the .beta.-subunit is substituted by Ser,
160th Arg in the .beta.-subunit is substituted by Trp, and 186th
Leu in the .beta.-subunit is substituted by Arg;
[0075] (ap) 6th Leu in the .alpha.-subunit is substituted by Thr,
19th Ala in the .alpha.-subunit is substituted by Val, 126th Phe in
the .alpha.-subunit is substituted by Tyr, 46th Met in the
.beta.-subunit is substituted by Lys, 108th Glu in the
.beta.-subunit is substituted by Arg, and 212th Ser in the
.beta.-subunit is substituted by Tyr;
[0076] (aq) 6th Leu in the .alpha.-subunit is substituted by Thr,
19th Ala in the .alpha.-subunit is substituted by Val, 126th Phe in
the .alpha.-subunit is substituted by Tyr, 48th Leu in the
.beta.-subunit is substituted by Val, 108th Glu in the
.beta.-subunit is substituted by Arg, and 212th Ser in the
.beta.-subunit is substituted by Tyr;
[0077] (ar) 6th Leu in the .alpha.-subunit is substituted by Ala,
19th Ala in the .alpha.-subunit is substituted by Val, 126th Phe in
the .alpha.-subunit is substituted by Tyr, 127th Leu in the
.beta.-subunit is substituted by Ser, 160th Arg in the
.beta.-subunit is substituted by Trp, and 186th Leu in the
.beta.-subunit is substituted by Arg;
[0078] (as) 6th Leu in the .alpha.-subunit is substituted by Thr,
36th Thr in the .alpha.-subunit is substituted by Met, 126th Phe in
the .alpha.-subunit is substituted by Tyr, 10th Thr in the
.beta.-subunit is substituted by Asp, 118th Phe in the
.beta.-subunit is substituted by Val, and 200th Ala in the
.beta.-subunit is substituted by Glu;
[0079] (at) 19th Ala in the .alpha.-subunit is substituted by Val,
71st Arg in the .alpha.-subunit is substituted by His, 126th Phe in
the .alpha.-subunit is substituted by Tyr, 37th Phe in the
.beta.-subunit is substituted by Leu, 108th Glu in the
.beta.-subunit is substituted by Asp, and 200th Ala in the
.beta.-subunit is substituted by Glu;
[0080] (au) 19th Ala in the .alpha.-subunit is substituted by Val,
71st Arg in the .alpha.-subunit is substituted by His, 126th Phe in
the .alpha.-subunit is substituted by Tyr, 37th Phe in the
.beta.-subunit is substituted by Val, 108th Glu in the
.beta.-subunit is substituted by Asp, and 200th Ala in the
.beta.-subunit is substituted by Glu;
[0081] (av) 36th Thr in the .alpha.-subunit is substituted by Met,
148th Gly in the .alpha.-subunit is substituted by Asp, 204th Val
in the .alpha.-subunit is substituted by Arg, 41st Phe in the
.beta.-subunit is substituted by Ile, 51st Phe in the
.beta.-subunit is substituted by Val, and 108th Glu in the
.beta.-subunit is substituted by Asp;
[0082] (aw) 148th Gly in the .alpha.-subunit is substituted by Asp,
204th Val in the .alpha.-subunit is substituted by Arg, 108th Glu
in the .beta.-subunit is substituted by Asp, and 200th Ala in the
.beta.-subunit is substituted by Glu;
[0083] (ax) 36th Thr in the .alpha.-subunit is substituted by Gly
and 188th Thr in the .alpha.-subunit is substituted by Gly;
[0084] (ay) 36th Thr in the .alpha.-subunit is substituted by Ala
and 48th Asn in the .alpha.-subunit is substituted by Gln;
[0085] (az) 48th Asn in the .alpha.-subunit is substituted by Glu
and 146th Arg in the .beta.-subunit is substituted by Gly;
[0086] (ba) 36th Thr in the .alpha.-subunit is substituted by Trp
and 176th Tyr in the .beta.-subunit is substituted by Cys;
[0087] (bb) 176th Tyr in the .beta.-subunit is substituted by Met
and 217th Asp in the .beta.-subunit is substituted by Gly;
[0088] (bc) 36th Thr in the .alpha.-subunit is substituted by Ser,
and 33rd Ala in the .beta.-subunit is substituted by Val;
[0089] (bd) 176th Tyr in the .beta.-subunit is substituted by Ala
and 217th Asp in the .beta.-subunit is substituted by Val;
[0090] (be) 40th Thr in the .beta.-subunit is substituted by Val
and 218th Cys in the .beta.-subunit is substituted by Met;
[0091] (bf) 33rd Ala in the .beta.-subunit is substituted by Met
and 176th Tyr in the .beta.-subunit is substituted by Thr;
[0092] (bg) 40th Thr in the .beta.-subunit is substituted by Leu
and 217th Asp in the .beta.-subunit is substituted by Leu;
[0093] (bh) 40th Thr in the .beta.-subunit is substituted by Ile
and 61st Ala in the .beta.-subunit is substituted by Val;
[0094] (bi) 61st Ala in the .beta.-subunit is substituted by Thr
and 218th Cys in the .beta.-subunit is substituted by Ser;
[0095] (bj) 112th Lys in the .beta.-subunit is substituted by Val
and 217th Asp in the .beta.-subunit is substituted by Met;
[0096] (bk) 61st Ala in the .beta.-subunit is substituted by Trp
and 217th Asp in the .beta.-subunit is substituted by His;
[0097] (bl) 61st Ala in the .beta.-subunit is substituted by Leu
and 112th Lys in the .beta.-subunit is substituted by Ile;
[0098] (bm) 146th Arg in the .beta.-subunit is substituted by Gly
and 217th Asp in the .beta.-subunit is substituted by Ser;
[0099] (bn) 171st Lys in the .beta.-subunit is substituted by Ala
and 217th Asp in the .beta.-subunit is substituted by Thr;
[0100] (bo) 150th Ala in the .beta.-subunit is substituted by Ser
and 217th Asp in the .beta.-subunit is substituted by Cys;
[0101] (bp) 61st Ala in the .beta.-subunit is substituted by Gly
and 150th Ala in the .beta.-subunit is substituted by Asn;
[0102] (bq) 61st Ala in the .beta.-subunit is substituted by Ser
and 160th Arg in the .beta.-subunit is substituted by Met; and
[0103] (br) 160th Arg in the .beta.-subunit is substituted by Cys
and 168th Thr in the .beta.-subunit is substituted by Glu.
[0104] [8] A gene encoding the nitrile hydratase variant according
to any one of [1] to [7].
[0105] [9] A gene encoding a nitrile hydratase variant having a
gene encoding the .alpha.-subunit defined in SEQ ID No: 3 in the
Sequence Listing and a gene encoding the .beta.-subunit defined in
SEQ ID No: 4 in the Sequence Listing, comprising substitution of at
least one base selected from substitution sites of the base
consisting of the following (a) to (l):
[0106] (a) 274th to 276th of the base sequence of SEQ ID No: 3;
[0107] (b) 280th to 282nd of the base sequence of SEQ ID No: 3;
[0108] (c) 589th to 591st of the base sequence of SEQ ID No: 3;
[0109] (d) 10th to 12th of the base sequence of SEQ ID No: 4;
[0110] (e) 69th to 71st of the base sequence of SEQ ID No: 4;
[0111] (f) 235th to 237th of the base sequence of SEQ ID No: 4;
[0112] (g) 286th to 288th of the base sequence of SEQ ID No: 4;
[0113] (h) 319th to 321st of the base sequence of SEQ ID No: 4;
[0114] (i) 676th to 678th of the base sequence of SEQ ID No: 4;
[0115] (j) 328th to 330th of the base sequence of SEQ ID No: 4 and
691st to 693rd of the base sequence of SEQ ID No: 4;
[0116] (k) 616th to 618th of the base sequence of SEQ ID No: 4, and
688th to 690th of the base sequence of SEQ ID No: 4; and
[0117] (l) 37th to 39th of the base sequence of SEQ ID No: 3, 79th
to 81st of the base sequence of SEQ ID No: 3, and 328th to 330th of
the base sequence of SEQ ID No: 4.
[0118] [10] The gene encoding a nitrile hydratase variant according
to [9], further comprising substitution of at least one base
selected from substitution sites of the base consisting of the
following (m) to (u):
[0119] (m) in case of (b) or (g), 37th to 39th of the base sequence
of SEQ ID No: 3;
[0120] (n) in case of (b) or (h), 79th to 81st of the base sequence
of SEQ ID No: 3;
[0121] (o) (d) and (f);
[0122] (p) in case of (f), 688th to 690th of the base sequence of
SEQ ID No: 4;
[0123] (q) (a) and (i);
[0124] (r) in case of (i), 37th to 39th of the base sequence of SEQ
ID No: 3 and 616th to 618th of the base sequence of SEQ ID No:
4;
[0125] (s) in case of (a) and (d), 616th to 618th of the base
sequence of SEQ ID No: 4;
[0126] (t) in case of (c) and (h), 688th to 690th of the base
sequence of SEQ ID No: 4; and
[0127] (u) in case of (f), 688th to 690th of the base sequence of
SEQ ID No: 4 and 691st to 693rd of the base sequence of SEQ ID No:
4.
[0128] [11] The gene encoding a nitrile hydratase variant according
to [9], further comprising substitution of at least one base with
another base selected from substitution sites of the base
consisting of (a), (c), (f), (i), (h), 688th to 690th of the base
sequence of SEQ ID No: 4, and 691st to 693rd of the base sequence
of SEQ ID No: 4, in case of (e), are substituted with another
base.
[0129] [12] The gene encoding a nitrile hydratase variant according
to any one of [9] to [11], wherein ATC is substituted by CTC when
37th to 39th of the base sequence of SEQ ID No: 3 are substituted
by another base,
[0130] ATG is substituted by ATC when 79th to 81th of the base
sequence of SEQ ID No: 3 are substituted by another base,
[0131] GAC is substituted by GAG when 274th to 276th of the base
sequence of SEQ ID No: 3 are substituted by another base,
[0132] ATG is substituted by ATC when 280th to 282th of the base
sequence of SEQ ID No: 3 are substituted by another base,
[0133] GGC is substituted by TGC when 589th to 591th of the base
sequence of SEQ ID No: 3 are substituted by another base,
[0134] GTG is substituted by ATG when 10th to 12th of the base
sequence of SEQ ID No: 4 are substituted by another base,
[0135] GTC is substituted by ATC when 69th to 71th of the base
sequence of SEQ ID No: 4 are substituted by another base,
[0136] CAC is substituted by AAC when 235th to 237th of the base
sequence of SEQ ID No: 4 are substituted by another base,
[0137] CAG is substituted by CGT when 286th to 288th of the base
sequence of SEQ ID No: 4 are substituted by another base,
[0138] CCC is substituted by ATG when 319th to 321st of the base
sequence of SEQ ID No: 4 are substituted by another base,
[0139] GAG is substituted by AAC when 328th to 330th of the base
sequence of SEQ ID No: 4 are substituted by another base,
[0140] CCG is substituted by CTG when 616th to 618th of the base
sequence of SEQ ID No: 4 are substituted by another base,
[0141] GTC is substituted by ATC when 676th to 678th of the base
sequence of SEQ ID No: 4 are substituted by another base,
[0142] GCG is substituted by GAG when 688th to 690th of the base
sequence of SEQ ID No: 4 are substituted by another base, and
[0143] GCC is substituted by GTC when 691th to 693th of the base
sequence of SEQ ID No: 4 are substituted by another base.
[0144] [13] The gene encoding a nitrile hydratase variant according
to any one of [9] to [12], comprising substitution of at least one
base selected from substitution sites of the base consisting of the
following (aa) to (br), and having the nitrile hydratase
activity:
[0145] (aa) 106th to 108th ACG of the base sequence of SEQ ID No: 3
are substituted by ATG, and 376th to 378th TTC of the base sequence
of SEQ ID No: 3 are substituted by TAC;
[0146] (ab) 442nd to 444th GGC of the base sequence of SEQ ID No: 3
are substituted by GAC, and 610th to 612th GTC of the base sequence
of SEQ ID No: 3 are substituted by CGC;
[0147] (ac) 151st to 153rd TTC of the base sequence of SEQ ID No: 4
are substituted by GTC, and 322nd to 324th GAG of the base sequence
of SEQ ID No: 4 are substituted by GAT;
[0148] (ad) 352nd to 354th TTC of the base sequence of SEQ ID No: 4
are substituted by GTC, and 598th to 600th GCC of the base sequence
of SEQ ID No: 4 are substituted by GAG;
[0149] (ae) 478th to 480th CGG of the base sequence of SEQ ID No: 4
are substituted by TGG, and 556th to 558th CTG of the base sequence
of SEQ ID No: 4 are substituted by CGG;
[0150] (af) 16th to 18th CTG of the base sequence of SEQ ID No: 3
are substituted by ACG, 106th to 108th ACG of the base sequence of
SEQ ID No: 3 are substituted by ATG, and 376th to 378th TTC of the
base sequence of SEQ ID No: 3 are substituted by TAC;
[0151] (ag) 55th to 57th GCG of the base sequence of SEQ ID No: 3
are substituted by GTG, 211th to 213th CGT of the base sequence of
SEQ ID No: 3 are substituted by CAT, and 376th to 378th TTC of the
base sequence of SEQ ID No: 3 are substituted by TAC;
[0152] (ah) 106th to 108th ACG of the base sequence of SEQ ID No: 3
are substituted by ATG, 442nd to 444th GGC of the base sequence of
SEQ ID No: 3 are substituted by GAC, and 610th to 612th GTC of the
base sequence of SEQ ID No: 3 are substituted by CGC;
[0153] (ai) 28th to 30th ACC of the base sequence of SEQ ID No: 4
are substituted by GAC, 352nd to 354th TTC of the base sequence of
SEQ ID No: 4 are substituted by GTC, and 598th to 600th GCC of the
base sequence of SEQ ID No: 4 are substituted by GAG;
[0154] (aj) 109th to 111th TTC of the base sequence of SEQ ID No: 4
are substituted by CTC, 322nd to 324th GAG of the base sequence of
SEQ ID No: 4 are substituted by GAT, and 598th to 600th GCC of the
base sequence of SEQ ID No: 4 are substituted by GAG;
[0155] (ak) 109th to 111th TTC of the base sequence of SEQ ID No: 4
are substituted by GTC, 322nd to 324th GAG of the base sequence of
SEQ ID No: 4 are substituted by GAT, and 598th to 600th GCC of the
base sequence of SEQ ID No: 4 are substituted by GAG;
[0156] (al) 121st to 123rd TTC of the base sequence of SEQ ID No: 4
are substituted by ATC, 151st to 153rd TTC of the base sequence of
SEQ ID No: 4 are substituted by GTC, and 322nd to 324th GAG of the
base sequence of SEQ ID No: 4 are substituted by GAT;
[0157] (am) 136th to 138th ATG of the base sequence of SEQ ID No: 4
are substituted by AAG, 322nd to 324th GAG of the base sequence of
SEQ ID No: 4 are substituted by CGG, and 634th to 636th TCC of the
base sequence of SEQ ID No: 4 are substituted by TAC;
[0158] (an) 142nd to 144th CTG of the base sequence of SEQ ID No: 4
are substituted by GTG, 322nd to 324th GAG of the base sequence of
SEQ ID No: 4 are substituted by CGG, and 634th to 636th TCC of the
base sequence of SEQ ID No: 4 are substituted by TAC;
[0159] (ao) 379th to 381st CTG of the base sequence of SEQ ID No: 4
are substituted by TCG, 478th to 480th CGG of the base sequence of
SEQ ID No: 4 are substituted by TGG, and 556th to 558th CTG of the
base sequence of SEQ ID No: 4 are substituted by CGG;
[0160] (ap) 16th to 18th CTG of the base sequence of SEQ ID No: 3
are substituted by ACG, 55th to 57th GCG of the base sequence of
SEQ ID No: 3 are substituted by GTG, 376th to 378th TTC of the base
sequence of SEQ ID No: 3 are substituted by TAC, 136th to 138th ATG
of the base sequence of SEQ ID No: 4 are substituted by AAG, 322nd
to 324th GAG of the base sequence of SEQ ID No: 4 are substituted
by CGG, and 634th to 636th TCC of the base sequence of SEQ ID No: 4
are substituted by TAC;
[0161] (aq) 16th to 18th CTG of the base sequence of SEQ ID No: 3
are substituted by ACG, 55th to 57th GCG of the base sequence of
SEQ ID No: 3 are substituted by GTG, 376th to 378th TTC of the base
sequence of SEQ ID No: 3 are substituted by TAC, 142nd to 144th CTG
of the base sequence of SEQ ID No: 4 are substituted by GTG, 322nd
to 324th GAG of the base sequence of SEQ ID No: 4 are substituted
by CGG, and 634th to 636th TCC of the base sequence of SEQ ID No: 4
are substituted by TAC;
[0162] (ar) 16th to 18th CTG of the base sequence of SEQ ID No: 3
are substituted by GCG, 55th to 57th GCG of the base sequence of
SEQ ID No: 3 are substituted by GTG, 376th to 378th TTC of the base
sequence of SEQ ID No: 3 are substituted by TAC, 379th to 381st CTG
of the base sequence of SEQ ID No: 4 are substituted by TCG, 478th
to 480th CGG of the base sequence of SEQ ID No: 4 are substituted
by TGG, and 556th to 558th CTG of the base sequence of SEQ ID No: 4
are substituted by CGG;
[0163] (as) 16th to 18th CTG of the base sequence of SEQ ID No: 3
are substituted by ACG, 106th to 108th ACG of the base sequence of
SEQ ID No: 3 are substituted by ATG, 376th to 378th TTC of the base
sequence of SEQ ID No: 3 are substituted by TAC, 28th to 30th ACC
of the base sequence of SEQ ID No: 4 are substituted by GAC, 352nd
to 354th TTC of the base sequence of SEQ ID No: 4 are substituted
by GTC, and 598th to 600th GCC of the base sequence of SEQ ID No: 4
are substituted by GAG;
[0164] (at) 55th to 57th GCG of the base sequence of SEQ ID No: 3
are substituted by GTG, 211th to 213th CGT of the base sequence of
SEQ ID No: 3 are substituted by CAT, 376th to 378th TTC of the base
sequence of SEQ ID No: 3 are substituted by TAC, 109th to 111th TTC
of the base sequence of SEQ ID No: 4 are substituted by CTC, 322nd
to 324th GAG of the base sequence of SEQ ID No: 4 are substituted
by GAT, and 598th to 600th GCC of the base sequence of SEQ ID No: 4
are substituted by GAG;
[0165] (au) 55th to 57th GCG of the base sequence of SEQ ID No: 3
are substituted by GTG, 211th to 213th CGT of the base sequence of
SEQ ID No: 3 are substituted by CAT, 376th to 378th TTC of the base
sequence of SEQ ID No: 3 are substituted by TAC, 109th to 111th TTC
of the base sequence of SEQ ID No: 4 are substituted by GTC, 322nd
to 324th GAG of the base sequence of SEQ ID No: 4 are substituted
by GAT, and 598th to 600th GCC of the base sequence of SEQ ID No: 4
are substituted by GAG;
[0166] (av) 106th to 108th ACG of the base sequence of SEQ ID No: 3
are substituted by ATG, 442nd to 444th GGC of the base sequence of
SEQ ID No: 3 are substituted by GAC, 610th to 612th GTC of the base
sequence of SEQ ID No: 3 are substituted by CGC, 121st to 123rd TTC
of the base sequence of SEQ ID No: 4 are substituted by ATC, 151st
to 153rd TTC of the base sequence of SEQ ID No: 4 are substituted
by GTC, and 322nd to 324th GAG of the base sequence of SEQ ID No: 4
are substituted by GAT;
[0167] (aw) 442nd to 444th GGC of the base sequence of SEQ ID No: 3
are substituted by GAC, 610th to 612th GTC of the base sequence of
SEQ ID No: 3 are substituted by CGC, 322nd to 324th GAG of the base
sequence of SEQ ID No: 4 are substituted by GAT, and 598th to 600th
GCC of the base sequence of SEQ ID No: 4 are substituted by
GAG;
[0168] (ax) 106th to 108th ACG of the base sequence of SEQ ID No: 3
are substituted by GGG, and 562nd to 564th ACC of the base sequence
of SEQ ID No: 3 are substituted by GGC;
[0169] (ay) 106th to 108th ACG of the base sequence of SEQ ID No: 3
are substituted by GCG, and 142nd to 144th AAC of the base sequence
of SEQ ID No: 3 are substituted by CAA;
[0170] (az) 142nd to 144th AAC of the base sequence of SEQ ID No: 3
are substituted by GAA, and 436th to 438th CGG of the base sequence
of SEQ ID No: 4 are substituted by GGG;
[0171] (ba) 106th to 108th ACG of the base sequence of SEQ ID No: 3
are substituted by TGG, and 526th to 528th TAC of the base sequence
of SEQ ID No: 4 are substituted by TGC;
[0172] (bb) 526th to 528th TAC of the base sequence of SEQ ID No: 4
are substituted by ATG, and 649th to 651st GAC of the base sequence
of SEQ ID No: 4 are substituted by GGC;
[0173] (bc) 106th to 108th ACG of the base sequence of SEQ ID No: 3
are substituted by TCG, and 97th to 99th GCG of the base sequence
of SEQ ID No: 4 are substituted by GTG;
[0174] (bd) 526th to 528th TAC of the base sequence of SEQ ID No: 4
are substituted by GCC, and 649th to 651st GAC of the base sequence
of SEQ ID No: 4 are substituted by GTC;
[0175] (be) 118th to 120th ACG of the base sequence of SEQ ID No: 4
are substituted by GTG, and 652nd to 654th TGC of the base sequence
of SEQ ID No: 4 are substituted by ATG;
[0176] (bf) 97th to 99th GCG of the base sequence of SEQ ID No: 4
are substituted by ATG, and 526th to 528th TAC of the base sequence
of SEQ ID No: 4 are substituted by ACC;
[0177] (bg) 118th to 120th ACG of the base sequence of SEQ ID No: 4
are substituted by CTG, and 649th to 651st GAC of the base sequence
of SEQ ID No: 4 are substituted by CTC;
[0178] (bh) 118th to 120th ACG of the base sequence of SEQ ID No: 4
are substituted by ATT, and 181st to 183rd GCC of the base sequence
of SEQ ID No: 4 are substituted by GTC;
[0179] (bi) 181st to 183rd GCC of the base sequence of SEQ ID No: 4
are substituted by ACG, and 652nd to 654th TGC of the base sequence
of SEQ ID No: 4 are substituted by TCC;
[0180] (bj) 334th to 336th AAG of the base sequence of SEQ ID No: 4
are substituted by GTG, and 649th to 651st GAC of the base sequence
of SEQ ID No: 4 are substituted by ATG;
[0181] (bk) 181st to 183rd GCC of the base sequence of SEQ ID No: 4
are substituted by TGG, and 649th to 651st GAC of the base sequence
of SEQ ID No: 4 are substituted by CAC;
[0182] (bl) 181st to 183rd GCC of the base sequence of SEQ ID No: 4
are substituted by CTC, and 334th to 336th AAG of the base sequence
of SEQ ID No: 4 are substituted by ATT;
[0183] (bm) 436th to 438th CGG of the base sequence of SEQ ID No: 4
are substituted by GGG, and 649th to 651st GAC of the base sequence
of SEQ ID No: 4 are substituted by AGC;
[0184] (bn) 511th to 513th AAG of the base sequence of SEQ ID No: 4
are substituted by GCG, and 649th to 651st GAC of the base sequence
of SEQ ID No: 4 are substituted by ACC;
[0185] (bo) 448th to 450th GCG of the base sequence of SEQ ID No: 4
are substituted by TCG, and 649th to 651st GAC of the base sequence
of SEQ ID No: 4 are substituted by TGT;
[0186] (bp) 181st to 183rd GCC of the base sequence of SEQ ID No: 4
are substituted by GGC, and 448th to 450th GCG of the base sequence
of SEQ ID No: 4 are substituted by AAT;
[0187] (bq) 181st to 183rd GCC of the base sequence of SEQ ID No: 4
are substituted by TCG, and 478th to 480th CGG of the base sequence
of SEQ ID No: 4 are substituted by ATG; and
[0188] (br) 478th to 480th CGG of the base sequence of SEQ ID No: 4
are substituted by TGT, and 502nd to 504th ACG of the base sequence
of SEQ ID No: 4 are substituted by GAG.
[0189] [14] A linked DNA comprising further DNA containing a
promoter sequence necessary for the expression of the gene in the
upstream region of the 5'-terminal of the gene encoding a nitrile
hydratase variant according to any one of [9] to [13], and a
ribosome binding sequence contained in SEQ ID No: 7 in the
downstream region of the 3'-terminal of the promoter.
[0190] [15] A plasmid comprising the DNA according to [14].
[0191] [16] A transformant obtained by transformation of a host
cell using the plasmid according to [15].
[0192] [17] A method for producing a nitrile hydratase variant,
comprising cultivating the transformant according to [16] in a
culture medium and producing a nitrile hydratase variant based on
the nitrile hydratase gene carried by the plasmid in the
transformant.
[0193] According to the present invention, a nitrile hydratase
composed of an .alpha.-subunit defined in SEQ ID No: 1 in the
Sequence Listing and a .beta.-subunit defined in SEQ ID No: 2 in
the Sequence Listing comprises substitution of at least one amino
acid with another amino acid, selected from substitution sites of
the amino acid consisting of the above (a) to (l). Thus, both of
the initial reaction rate and enzyme stability of the nitrile
hydratase are improved, so that the activity value in a unit weight
of the enzyme preparation can be increased, and at the same time
the risk of enzyme deactivation due to temperature variation or the
like for the industrial use can be reduced. Accordingly, the amide
compound can be stably produced with a smaller amount of the
enzyme, so that the production costs for producing the amide
compound can be reduced.
[0194] According to the present invention, it is possible to
provide a novel nitrile hydratase variant in which the initial
reaction rate and enzyme stability are improved than those of the
wild nitrile hydratase, and to reduce the production costs for the
enzyme in the total production costs for producing the amide
compound.
DESCRIPTION OF EMBODIMENTS
[0195] The above and other objects, features and advantages will be
more apparent from the following description of the preferred
embodiments. The present invention will be described in more detail
below.
[0196] The nitrile hydratase variant of the present invention
comprises substitution of at least one amino acid with another
amino acid to improve two or more properties of the nitrile
hydratase by the substitution of one or more three or less amino
acids.
[0197] The term "properties" to be improved in the nitrile
hydratase variant of the present invention refer to properties
relating to the reaction itself for hydrating a nitrile group to
convert it into an amide group, and enzyme stability. The term
"properties" relating to the reaction itself refer to the activity
of the enzyme, the substrate specificity, Vmax, Km, and the initial
reaction rate. The enzyme stability includes thermal stability,
stability against the substrate, and stability against the
product.
[0198] The nitrile hydratase variant of the present invention
preferably comprises substitution of at least one amino acid with
another amino acid to improve properties of the thermophilic
bacteria-derived nitrile hydratase. As the thermophilic bacteria,
suitably used are those belonging to the genus Psuedonocardia. A
specific example includes Psuedonocardia thermophila.
[0199] More specifically, the nitlile hydratase variant includes at
least one amino acid substituted with another amino acid, selected
from substitution sites of (a) to (l) as shown in Table I, in the
nitrile hydratase consisting of the .alpha.-subunit defined in SEQ
ID No: 1 in the Sequence Listing and the .beta.-subunit defined in
SEQ ID No: 2 in the Sequence Listing. Thus, the nitrile hydratase
variant of the present invention is provided with higher initial
reaction rate and enzyme stability than the wild nitrile hydratase
as described in Patent Document 1.
TABLE-US-00001 TABLE I Before After SEQ ID No. No. Substitution
Substitution (a) 1 92 Asp Glu (b) 1 94 Met Ile (c) 1 197 Gly Cys
(d) 2 4 Val Met (e) 2 24 Val Ile (f) 2 79 His Asn (g) 2 96 Gln Arg
(h) 2 107 Pro Met (i) 2 226 Val Ile (j) 2 110 Glu Asn 2 231 Ala Val
(k) 2 206 Pro Leu 2 230 Ala Glu (l) 1 13 Ile Leu 1 27 Met Ile 2 110
Glu Asn
[0200] A plurality of substitutions of the amino acid of (a) to (l)
shown in Table I may be combined, or may be combined with
substitutions of the amino acid at the different sites other than
(a) to (l). For example, in case of (e), at least one amino acid
selected from the group consisting of (a), (c), (f), (i), (h),
230th of the .beta.-subunit and 231st of the .beta.-subunit may be
substituted with another amino acid. Examples of substitution of
the amino acid which can be combined with (a) to (l) include those
in Table II.
TABLE-US-00002 TABLE II Before After SEQ ID No. No. Substitution
Substitution (m-1) 1 13 Ile Leu 1 94 Met Ile (m-2) 1 13 Ile Leu 2
96 Gln Arg (n-1) 1 27 Met Ile 1 94 Met Ile (n-2) 1 27 Met Ile 2 107
Pro Met (o) 2 4 Val Met 2 79 His Asn (p) 2 79 His Asn 2 230 Ala Glu
(q) 1 92 Asp Glu 2 226 Val Ile (r) 1 13 Ile Leu 2 206 Pro Leu 2 226
Val Ile (s) 1 92 Asp Glu 2 4 Val Met 2 206 Pro Leu (t) 1 197 Gly
Cys 2 107 Pro Met 2 230 Ala Glu (u) 2 79 His Asn 2 230 Ala Glu 2
231 Ala Val (v) 1 92 Asp Glu 2 24 Val Ile 2 226 Val Ile (w) 1 197
Gly Cys 2 24 Val Ile 2 107 Pro Met 2 230 Ala Glu (x) 2 24 Val Ile 2
79 His Asn 2 230 Ala Glu 2 231 Ala Val
[0201] The nitrile hydratase variant of the present invention may
further comprise mutation in any one nitrile hydratase variant of
the above (a) to (x) at sites (aa) to (br) of the amino acid of the
nitrile hydratase of SEQ ID Nos: 1 and 2 as shown in Table III.
TABLE-US-00003 TABLE III Before After SEQ ID No. No. Substitution
Substitution (aa) 1 36 Thr Met 1 126 Phe Tyr (ab) 1 148 Gly Asp 1
204 Val Arg (ac) 2 51 Phe Val 2 108 Glu Asp (ad) 2 118 Phe Val 2
200 Ala Glu (ae) 2 160 Arg Trp 2 186 Leu Arg (af) 1 6 Leu Thr 1 36
Thr Met 1 126 Phe Tyr (ag) 1 19 Ala Val 1 71 Arg His 1 126 Phe Tyr
(ah) 1 36 Thr Met 1 148 Gly Asp 1 204 Val Arg (ai) 2 10 Thr Asp 2
118 Phe Val 2 200 Ala Glu (aj) 2 37 Phe Leu 2 108 Glu Asp 2 200 Ala
Glu (ak) 2 37 Phe Val 2 108 Glu Asp 2 200 Ala Glu (al) 2 41 Phe Ile
2 51 Phe Val 2 108 Glu Asp
TABLE-US-00004 TABLE III-1 (am) 2 46 Met Lys 2 108 Glu Arg 2 212
Ser Tyr (an) 2 48 Leu Val 2 108 Glu Arg 2 212 Ser Tyr (ao) 2 127
Leu Ser 2 160 Arg Trp 2 186 Leu Arg (ap) 1 6 Leu Thr 1 19 Ala Val 1
126 Phe Tyr 2 46 Met Lys 2 108 Glu Arg 2 212 Ser Tyr (aq) 1 6 Leu
Thr 1 19 Ala Val 1 126 Phe Tyr 2 48 Leu Val 2 108 Glu Arg 2 212 Ser
Tyr (ar) 1 6 Leu Ala 1 19 Ala Val 1 126 Phe Tyr 2 127 Leu Ser 2 160
Arg Trp 2 186 Leu Arg (as) 1 6 Leu Thr 1 36 Thr Met 1 126 Phe Tyr 2
10 Thr Asp 2 118 Phe Val 2 200 Ala Glu
TABLE-US-00005 TABLE III-2 (at) 1 19 Ala Val 1 71 Arg His 1 126 Phe
Tyr 2 37 Phe Leu 2 108 Glu Asp 2 200 Ala Glu (au) 1 19 Ala Val 1 71
Arg His 1 126 Phe Tyr 2 37 Phe Val 2 108 Glu Asp 2 200 Ala Glu (av)
1 36 Thr Met 1 148 Gly Asp 1 204 Val Arg 2 41 Phe Ile 2 51 Phe Val
2 108 Glu Asp (aw) 1 148 Gly Asp 1 204 Val Arg 2 108 Glu Asp 2 200
Ala Glu (ax) 1 36 Thr Gly 1 188 Thr Gly (ay) 1 36 Thr Ala 1 48 Asn
Gln (az) 1 48 Asn Glu 2 146 Arg Gly (ba) 1 36 Thr Trp 2 176 Tyr Cys
(bb) 2 176 Tyr Met 2 217 Asp Gly (bc) 1 36 Thr Ser 2 33 Ala Val
TABLE-US-00006 TABLE III-3 (bd) 2 176 Tyr Ala 2 217 Asp Val (be) 2
40 Thr Val 2 218 Cys Met (bf) 2 33 Ala Met 2 176 Tyr Thr (bg) 2 40
Thr Leu 2 217 Asp Leu (bh) 2 40 Thr Ile 2 61 Ala Val (bi) 2 61 Ala
Thr 2 218 Cys Ser (bj) 2 112 Lys Val 2 217 Asp Met (bk) 2 61 Ala
Trp 2 217 Asp His (bl) 2 61 Ala Leu 2 112 Lys Ile (bm) 2 146 Arg
Gly 2 217 Asp Ser (bn) 2 171 Lys Ala 2 217 Asp Thr (bo) 2 150 Ala
Ser 2 217 Asp Cys (bp) 2 61 Ala Gly 2 150 Ala Asn (bq) 2 61 Ala Ser
2 160 Arg Met (br) 2 160 Arg Cys 2 168 Thr Glu
[0202] In the present invention, the term "nitrile hydratase
activity" refers to the nitrile-hydrating activity to convert a
nitrile group of various compounds to an amide group by hydration,
and more preferably refers to the activity to convert acrylonitrile
to acrylamide.
[0203] In the present invention, the term "improved nitrile
hydratase activity" refers to improvement of the initial reaction
rate. The "initial reaction rate" in the present invention may be
confirmed in the following manner. First, a nitrile hydratase
preparation is added to a 50 mM Tris-HCl aqueous solution (pH 8.0)
containing 2.5% (v/v) of acrylonitrile as a substrate. In place of
the nitrile hydratase preparation, a microorganism cell, a culture
or a crude purification product of the nitrile hydratase may be
used. After the addition of the nitrile hydratase, the reaction is
carried out at 20 degrees centigrade for 15 minutes. 1M phosphoric
acid is added to the reaction solution to stop the reaction, and
the produced acrylamide is quantitatively analyzed. The amount of
acrylamide may be measured through HPLC analysis.
[0204] The term "improvement of the initial reaction rate" in the
present invention refers to significant improvement of the initial
reaction rate as compared to the wild nitrile hydratase and
conventionally known nitrile hydratase variant, and specifically
refers to improvement of not less than 1.2 times.
[0205] The term "improvement of enzyme stability" in the present
invention refers to improvement of thermal stability of the nitrile
hydratase. The nitrile hydratase with improved thermal stability is
expected to increase stability against stress other than heating,
i.e., stability against an organic solvent, a high-concentration
substrate or a product as well, because structural stability of a
protein is considered to be strengthened.
[0206] The term "thermal stability of the enzyme" in the present
invention may be confirmed in the following manner. First, a
nitrile hydratase preparation is heated at 60 degrees centigrade
for 2 hours, and then the temperature is returned to 20 degrees
centigrade, and a 50 mM Tris-HCl aqueous solution (pH 8.0)
containing 2.5% (v/v) of acrylonitrile as a substrate is added
thereto. In place of the nitrile hydratase preparation, a
microorganism cell, a culture or a crude purification product of
the nitrile hydratase may be used. The heated nitrile hydratase and
substrate are mixed together, and then reacted at 20 degrees
centigrade for 15 minutes to measure the initial reaction rate.
[0207] The term "improvement of thermal stability of the enzyme" in
the present invention refers to significant improvement of the
initial reaction rate after heating as compared to the wild nitrile
hydratase and conventionally known nitrile hydratase variant heated
in the same manner, and specifically refers to improvement of not
less than 1.2 times.
[0208] As the wild nitrile hydratase in the present invention,
preferably used is Pseudonocardia thermophila-derived nitrile
hydratase as disclosed in Patent Document 1. As the plasmid
expressing a large number of the wild nitrile hydratase in the
transformant and a transformant strain transformed with the
plasmid, there may be cited MT-10822 (deposited with the
International Patent Organism Depositary at the National Institute
of Advanced Industrial Science and Technology, 1-1-1 Higashi,
Tsukuba-shi, Ibaraki-ken, Japan, under the deposit number FERM
BP-5785, as of Feb. 7, 1996). As the conventionally known nitrile
hydratase variant in the present invention, there may be cited the
nitrile hydratase variant described in Patent Documents 1 to 4.
[0209] The nitrile hydratase variant of the present invention has
the following properties in addition to improvement of the nitrile
hydratase activity. The enzyme comprises a dimer having the
.alpha.-subunit and the .beta.-subunit which are in association as
the fundamental structural unit, and the dimers are further
associated to form tetramers. 111th cysteine residue of the
.alpha.-subunit undergoes a post-translational modification in a
cysteine sulfinic acid (Cys-SOOH), while 113th cysteine residue
undergoes a post-translational modification in a cysteine sulfenic
acid (Cys-SOH). A polypeptide chain of the .alpha.-subunit is
bonded to a cobalt atom via the modified amino acid residue to form
an active center. The reaction may be preferably carried out in the
temperature range of 0 to 60 degrees centigrade, while pH during
the reaction is usually selected in the range of 4 to 10 and
preferably in the range of 6 to 9.
[0210] The nitrile hydratase variant of the present invention may
be produced in the following manner.
[0211] First, a plasmid containing DNA encoding the nitrile
hydratase variant is prepared, and a transformant or a transformant
strain is obtained by transforming an arbitrary host cell using the
plasmid. Subsequently, the nitrile hydratase variant is produced by
cultivating the above-mentioned transformant or transformant
strain.
[0212] The gene encoding a wild nitrile hydratase comprises a base
sequence defined in SEQ ID No: 3 in the Sequence Listing and a base
sequence defined in SEQ ID No: 4 in the Sequence Listing. The base
sequence defined in SEQ ID No: 3 in the Sequence Listing
corresponds to the amino acid sequence consisting of SEQ ID No: 1
in the Sequence Listing, while the base sequence defined in SEQ ID
No: 4 in the Sequence Listing corresponds to the amino acid
sequence consisting of SEQ ID No: 2 in the Sequence Listing. DNA
encoding the nitrile hydratase variant can be obtained by
performing base substitution of the base sequence defined in SEQ ID
No: 3 and/or SEQ ID No: 4. Specifically, substitutions of the amino
acid of (a) to (l) shown in Table I may be realized by base
substitution, as shown in Table IV-1.
TABLE-US-00007 TABLE IV-1 Sequence ID Before No. No. Substitution
After Substitution (a) 3 274~276 GAC GAA, GAG (b) 3 280~282 ATG
ATT, ATC, ATA (c) 3 589~591 GGC TGT, TGC (d) 4 10~12 GTG ATG (e) 4
69~71 GTC ATT, ATC, ATA (f) 4 235~237 CAC AAT, AAC (g) 4 286~288
CAG CGT, CGG, CGA, CGG, AGA, AGG (h) 4 319~321 CCC ATG (i) 4
676~678 GTC ATT, ATC, ATA (j) 4 328~330 GAG AAT, AAC 4 691~693 GCC
GTT, GTG, GTA, GTG (k) 4 616~618 CCG TTA, TTG, CTT, CTC, CTA, CTG 4
688~690 GCG GAA, GAG (l) 3 37~39 ATC TTA, TTG, CTT, CTC, CTA, GTG 3
79~81 ATG ATT, ATC, ATA 4 328~330 GAG AAT, AAC
[0213] Furthermore, substitutions of the amino acid illustrated in
Table II may be realized by base substitution, as shown in Table
IV-2.
TABLE-US-00008 TABLE IV-2 Sequence Before ID No. No. Substitution
After Substitution (m-1) 3 37~39 ATC TTA TTG, CTT, CTC, CTA, CTG 3
280~282 ATG ATT, ATC, ATA (m-2) 3 37~39 ATC TTA, TTG, CTT, CTC,
CTA, CTG 4 286~288 CAG CGT, CGC, CGA, CGG, AGA AGG (n-1) 3 79~81
ATG ATT, ATC, ATA 4 280~282 ATG ATT, ATC, ATA (n-2) 3 79~81 ATG
ATT, ATC, ATA 4 319~321 CCC ATG (o) 4 10~12 GTG ATG 4 235~237 CAC
AAT, AAC (p) 4 235~237 CAC AAT, AAC 4 688~690 GCG GAA, GAG (q) 3
274~276 GAC GAA, GAG 4 676~678 GTC ATT, ATC, ATA (r) 3 37~39 ATC
TTA, TTG, CTT, CTC, CTA CTG 4 616~618 CCG TTA, TTG, CTT, CTC, CTA,
CTG 4 676~678 GTC ATT, ATC, ATA (s) 3 274~276 GAC GAA, GAG 4 10~12
GTG ATG 4 616~618 CCG TTA, TTG, CTT, CTC, CTA CTG (t) 3 589~591 GGC
TGT, TGC 4 319~321 CCC ATG 4 688~690 GCG GAA GAG (u) 4 235~237 CAC
AAT, AAC 4 688~690 GCG GAA, GAG 4 691~693 GCC GTT, GTC, GTA, GTG
(v) 3 274~276 GAC GAA, GAG 4 69~71 GTC ATT, ATC, ATA 4 676~678 GTC
ATT, ATC, ATA (w) 3 589~591 GGC TGT, TGC 4 69~71 GTC ATT, ATC, ATA
4 319~321 CCC ATG 4 688~690 GCG GAA, GAG (x) 4 69~71 GTC ATT, ATC,
ATA 4 235~237 CAC AAT, AAC 4 688~690 GCG GAA, GAG 4 691~693 GCC
GTT, GTC, GTA, GTG
[0214] Furthermore, substitutions of the amino acid illustrated in
Table III may be realized by base substitution, as shown in Table
V.
TABLE-US-00009 TABLE V Sequence Before ID No. No. Substitution
After Substitution (aa) 3 106~108 ACG ATG 3 376~378 TTC TAT, TAC
(ab) 3 442~444 GGC GAU, GAC 3 610~612 GTC CGT, CGC, CGA, CGG, AGA,
AGG (ac) 4 151~153 TTC GTT, GTC, GTA, GTG 4 322~324 GAG GAT, GAC
(ad) 4 352~354 TTC GTT, GTC, GTA, GTG 4 598~600 GCC GAA, GAG (ae) 4
478~480 CGG TGG 4 556~558 CTG CGT, CGC, CGA, CGG, AGA, AGG (af) 3
16~18 CTG ACT, ACC, ACA, ACG 3 106~108 ACG ATG 3 376~378 TTC TAT,
TAC (ag) 3 55~57 GCG GTT, GTC, GTA, GTG 3 211~213 CGT CAT, CAC 3
376~378 TTC TAT, TAC (ah) 3 106~108 ACG ATG 3 442~444 GGC GAT, GAC
3 610~612 GTC CGT, CGC, CGA, CGG, AGA, AGG (ai) 4 28~30 ACC GAT,
GAC 4 352~354 TTC GTT, GTC, GTA, GTG 4 598~600 GCC GAA, GAG (aj) 4
109~111 TTC TTA, TTG, CTT, CTC, CTA, CTG 4 322~324 GAG GAT, GAC 4
598~600 GCC GAA, GAG (ak) 4 109~111 TTC GTT, GTC, GTA, GTG 4
322~324 GAG GAT, GAC 4 598~600 GCC GAA, GAG
TABLE-US-00010 TABLE V-1 (al) 4 121~123 TTC ATT, ATC, ATA 4 151~153
TTC GTT, GTC, GTA, GTG 4 322~324 GAG GAT, GAC (am) 4 136~138 ATG
AAA, AAG 4 322~324 GAG CGT, CGC, CGA, CGG, AGA, AGG 4 634~636 TCC
TAT, TAC (an) 4 142~144 CTG GTT, GTC, GTA, GTG 4 322~324 GAG CGT,
CGC, CGA, CGG, AGA, AGG 4 634~636 TCC TAT, TAC (ao) 4 379~381 CTG
TCT, TCC, TCA, TCG, AGT, AGC 4 478~480 CGG TGG 4 556~558 CTG CGT,
CGC, CGA, CGG, AGA, AGG (ap) 3 16~18 CTG ACT, ACC, ACA, ACG 3 55~57
GCG GTT, GTC, GTA, GTG 3 376~378 TTC TAT, TAC 4 136~138 ATG AAA,
AAG 4 322~324 GAG CGT, CGC, CGA, CGG, AGA, AGG 4 634~636 TCC TAT,
TAC (aq) 3 16~18 CTG ACT, ACC, ACA, ACG 3 55~57 GCG GTT, GTC, GTA,
GTG 3 376~378 TTC TAT, TAC 4 142~144 CTG GTT, GTC, GTA, GTG 4
322~324 GAG CGT, CGC, CGA, CGG, AGA, AGG 4 634~636 TCC TAT, TAC
(ar) 3 16~18 CTG GOT, GCC, GCA, GCG 3 55~57 GCG GTT, GTC, GTA, GTG
3 376~378 TTC TAT, TAC 4 379~381 CTG TCT, TCC, TCA, TCG, AGU, AGC 4
478~480 CGG TGG 4 556~558 CTG CGT, CGC, CGA, CGG, AGA, AGG
TABLE-US-00011 TABLE V-2 (as) 3 16~18 CTG ACT, ACC, ACA, ACG 3
106~108 ACG ATG 3 376~378 TTC TAT, TAC 4 28~30 ACC GAT, GAC 4
352~354 TTC GTT, GTC, GTA, GTG 4 598~600 GCC GAA, GAG (at) 3 55~57
GCG GTT, GTC, GTA, GTG 3 211~213 CGT CAT, CAC 3 376~378 TTC TAT,
TAC 4 109~111 TTC TTA, TTG, CTT, CTC, CTA, CTG 4 322~324 GAG GAT,
GAC 4 598~600 GCC GAA, GAG (au) 3 55~57 GCG GTT, GTC, GTA, GTG 3
211~213 CGT CAT, CAC 3 376~378 TTC TAT, TAC 4 109~111 TTC GTT, GTC,
GTA, GTG 4 322~324 GAG GAT, GAC 4 598~600 GCC GAA, GAG (av) 3
106~108 ACG ATG 3 442~444 GGC GAT, GAC 3 610~612 GTC CGT, CGC, CGA,
CGG, AGA, AGG 4 121~123 TTC ATT, ATC, ATA 4 151~153 TTC GTT, GTC,
GTA, GTG 4 322~324 GAG GAT, GAC (aw) 3 442~444 GGC GAT, GAC 3
610~612 GTC CGT, CGC, CGA, CGG, AGA, AGG 4 322~324 GAG GAT, GAC 4
598~600 GCC GAA, GAG (ax) 3 106~108 ACG GGT, GGC, GGA, GGG 3
562~564 ACC GGT, GGC, GGA, GGG
TABLE-US-00012 TABLE V-3 (ay) 3 106~108 ACG GCT, GCC, GCA, GCG 3
142~144 AAC CAA, CAG (az) 3 142~144 AAC GAA, GAG 4 436~438 CGG GGT,
GGC, GGA, GGG (ba) 3 106~108 ACG TGG 4 526~528 TAC TGT, TGC (bb) 4
526~528 TAC ATG 4 649~651 GAC GGT, GGC, GGA, GGG (bc) 3 106~108 ACG
TCT, TCC, TCA, TCG, AGT, AGC 4 97~99 GCG GTT, GTC, GTA, GTG (bd) 4
526~528 TAC GCT, GCC, GCA, GCG 4 649~651 GAC GTT, GTC, GTA, GTG
(be) 4 118~120 ACG GTT, GTC, GTA, GTG 4 652~654 TGC ATG (bf) 4
97~99 GCG ATG 4 526~528 TAC ACT, ACC, ACA, ACG (bg) 4 118~120 ACG
TTA, TTG, CTT, CTC, CTA, CTG 4 649~651 GAC TTA, TTG, CTT, CTC, CTA,
CTG (bh) 4 118~120 ACG ATT, ATC, ATA 4 181~183 GCC GTT, GTC, GTA,
GTG (bi) 4 181~183 GCC ACT, ACC, ACA, ACG 4 652~654 TGC TCT, TCC,
TCA, TCG, AGT, AGC (bj) 4 334~336 AAG GTT, GTC, GTA, GTG 4 649~651
GAC ATG (bk) 4 181~183 GCC TGG 4 649~651 GAC CAT, CAC (bl) 4
181~183 GCC TTA, TTG, CTT, CTC, CTA, CTG 4 334~336 AAG ATT, ATC,
ATA
TABLE-US-00013 TABLE V-4 (bm) 4 436~438 CGG GGT, GGC, GGA, GGG 4
649~651 GAC TCT, TCC, TCA, TCG, AGT, AGC (bn) 4 511~513 AAG GCT,
GCC, GCA, GCG 4 649~651 GAC ACT, ACC, ACA, ACG (bo) 4 448~450 GCG
TCT, TCC, TCA, TCG, AGT, AGC 4 649~651 GAC TGT, TGC (bp) 4 181~183
GCC GGT, GGC, GGA, GGG 4 448~450 GCG AAT, AAC (bq) 4 181~183 GCC
TCT, TCC, TCA, TCG, AGT, AGC 4 478~480 CGG ATG (br) 4 478~480 CGG
TGT, TGC 4 502~504 ACG GAA, GAG
[0215] The plasmid can have, in addition to a gene encoding the
.alpha.-subunit of the nitrile hydratase variant, a gene encoding
the .beta.-subunit or a nitrile hydratase variant gene or nitryl
hydratase variant gene, a constitution which enables the production
of a nitrile hydratase by a transformant or a transformant strain
obtained by transforming an arbitrary host cell, such as the
regulatory region necessary for the expression of each gene, the
region necessary for autonomous replication or the like. The
arbitrary host cell as used herein may be exemplified by
Escherichia coli.
[0216] The regulatory region necessary for expression may include a
promoter sequence (including the transcription-regulating operator
sequence), a ribosome binding sequence (SD sequence), a
transcription-terminating sequence and the like. Specific examples
of the promoter sequence may include a trp promoter of tryptophan
operon and a lac promoter of lactose operon that are derived from
Escherichia coli, and a PL promoter and a PR promoter that are
derived from lambda phage. Further, artificially designed or
improved sequences such as a tac promoter or a trc promoter may
also be used.
[0217] The ribosome binding sequence is preferably a sequence
having TAAGGAGGT contained in SEQ ID No: 7. The sequence order of
these regulatory regions on a plasmid is preferably such that the
promoter sequence and the ribosome binding sequence are located
upstream to the 5'-terminal than the gene encoding the nitrile
hydratase variant, and the transcription-terminating sequence is
preferably located downstream to the 3'-terminal than the gene
encoding the nitrile hydratase variant. Also, the .alpha.-subunit
gene and the .beta.-subunit gene of the nitrile hydratase variant
may be expressed as individual independent cistrons by means of
such regulatory regions, or may be expressed as a polycistron by
means of a common regulatory region.
[0218] Examples of the plasmid vector satisfying the above
requirements may include pBR322, pUC18, pBluescript, pKK223-3 and
pSC101, which have a region capable of autonomous replication in
Escherichia coli.
[0219] For a method of constructing the plasmid of the present
invention by inserting the gene encoding the nitrile hydratase
variant of the present invention into such a plasmid vector,
together with those regions necessary for expression of the
activity of the nitrile hydratase variant, a method of transforming
the plasmid to a desired host cell and a method of producing
nitrile hydratase in the transformant, there may be used those
general methods and host cells known in the art of molecular
biology, biological engineering and genetic engineering as
described in, for example, "Molecular Cloning, 3rd Edition" (J.
Sambrook et al., Cold Spring Harbor Laboratory Press, 2001) or the
like.
[0220] The transformant obtained by transforming the above plasmid
to a desired host cell is cultivated in a culture medium, whereby
the nitrile hydratase variant can be produced based on the nitrile
hydratase gene carried by the plasmid. When the host cell is
Escherichia coli, LB medium, M9 medium or the like is generally
used as the culture medium for cultivating the transformant. More
preferably, these medium components may comprise Fe ions and Co
ions in an amount of 0.1 .mu.g/mL or more, or the transformant may
be inoculated and then cultivated at a suitable cultivating
temperature (in general, from 20 to 50 degrees centigrade).
[0221] When the nitrile hydratase variant having the desired enzyme
activity to express the gene encoding the nitrile hydratase variant
of the present invention is produced, a gene encoding a protein
involved in the activation of nitrile hydratase may be required in
some cases.
[0222] A protein involved in the activation of nitrile hydratase is
a protein having the property such that the presence or absence of
the expression of the protein directly controls the activation of
nitrile hydratase, and it can be exemplified by the protein
involved in the activation of Pseudonocardia thermophila-derived
nitrile hydratase (nitrile hydratase-activating protein) as
described in Japanese Patent Laid-open No. H11 (1999)-253168. The
sequence of the nitrile hydratase-activating protein is presented
in the Sequence Listing: 5 and 6.
[0223] The amide compound can be produced in the following manner
using the nitrile hydratase variant of the present invention.
First, the transformant or transformant strain to produce the
nitrile hydratase variant of the present invention is caltivated,
and a given cell or a given product obtained by processing the
cells is brought into contact with a nitrile compound in a solvent.
In this manner, a corresponding amide compound is produced.
[0224] The term "product obtained by processing the cells"
mentioned herein refers to an extract or a disruption product of
the transformant, a post-separation product such as a crude enzyme
preparation obtained by isolating the nitrile hydratase activated
fraction from such extract or disruption product, an enzyme
purification product obtained by further purification or the like,
and an immobilization product in which the transformant, or an
extract, a disruption product or a post-separation product of the
transformant is immobilized by using suitable means. The contact
temperature is not particularly limited, but it is preferably in
the range of not deactivating the nitrile hydratase variant, and
more preferably from 0 to 60 degrees centigrade. As the nitrile
compound, there is no particular limitation as long as it is a
compound which can act as the substrate for the nitrile hydratase
variant of the present invention, and it can be preferably
exemplified by nitrile compounds having 2 to 4 carbon atoms, such
as acetonitrile, propionitrile, acrylonitrile, methacrylonitrile,
n-butyronitrile, isobutyronitrile, crotononitrile,
.alpha.-hydroxyisobutyronitrile and the like. The concentration of
the nitrile compound in the aqueous medium is not particularly
limited. The reaction temperature is not particularly limited, but
it is preferably in the range of not deactivating the nitrile
hydratase, and more preferably from 0 to 60 degrees centigrade.
Furthermore, in order to produce an amide compound with a smaller
amount of the enzyme, it is preferable to use a nitrile hydratase
variant having a certain level of stability under conditions of
producing an amide compound.
[0225] Subsequently, the operational effect of the present
invention will be described in detail. The present inventors have
repeatedly conducted an extensive study and as a result, have found
a nitrile hydratase variant in which both physical properties
relating to the reaction itself and the enzyme stability are
improved as compared to the conventional nitrile hydratase,
comprising substitution of at least one amino acid with another
amino acid to improve two or more properties of nitrile hydratase
by substitution of one or more and three or less amino acids. In
particular, they have found that with respect to the nitrile
hydratase comprising the .alpha.-subunit defined in SEQ ID No: 1 in
the Sequence Listing and the .beta.-subunit defined in SEQ ID No: 2
in the Sequence Listing, at least one amino acid is substituted
with another amino acid, selected from substitution sites of the
amino acid consisting of the above (a) to (l), whereby enzyme
stability as well as the initial reaction rate of the nitrile
hydratase can be improved at the same time. In this way, both of
efficiency of the enzymatic reaction and handling of the enzyme can
be achieved. Also, by use of the nitrile hydratase in which both of
the initial reaction rate and enzyme stability are enhanced, the
activity value in a unit weight of the enzyme preparation can be
increased, and at the same time the risk of enzyme deactivation due
to temperature variation or the like for the industrial use can be
reduced. Accordingly, the amide compound can be stably produced
with a smaller amount of the enzyme so that the production costs
for producing the amide compound can be reduced.
[0226] As described above, embodiments of the present invention has
been described, but the embodiments described in the present
invention are illustrative only, and various other constructions
may also be adopted.
EXAMPLES
[0227] The present invention is now illustrated in detail below
with reference to the following Examples. However, the present
invention is not restricted to these Examples.
Example 1
Construction of Plasmid (1) Expressing Nitrile Hydratase with
Modified Ribosome Binding Sequence
[0228] A gene fragment of about 0.7 kbp was obtained by the PCR
reaction using a plasmid pPT-DB1 described in Example 3 of Patent
Document 1 as the template and the primers defined in SEQ ID Nos: 7
and 8 in the Sequence Listing. The above-mentioned PCR fragment was
cleaved by means of restriction endonucleases EcoRI and NotI, and
then this mixture treated with restriction endonucleases was
subjected to phenol/chloroform extraction and ethanol precipitation
to purify the DNA fragment. In the same manner, pPT-DB1 was cleaved
by means of EcoRI and NotI, and then subjected to agarose gel
electrophoresis, through which only the DNA fragment of about 3.9
kbp was cut out of the agarose gel. The thus obtained DNA fragments
of about 0.7 kbp and of about 3.9 kbp were subjected to DNA
ligation using a DNA ligation kit (manufactured by Takara Shuzo
Co., Ltd.) to prepare a plasmid (1) expressing the above-mentioned
nitrile hydratase with the modified ribosome binding sequence.
[0229] A competent cell of Escherichia coli HB101 (manufactured by
Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (1). Moreover, the plasmid was prepared from the above
microbial cells by the alkaline SDS extraction method, and the base
sequence of the nitrile hydratase gene was determined using a DNA
sequencer. Then, it was confirmed that the transformant (1) had the
modified ribosome binding sequence in pPT-DB1 as shown in Table
1.
[0230] In the production of an amide compound using the thus
obtained transformant (1) and a transformant MT-10822 containing
pPT-DB1 (deposited with the International Patent Organism
Depositary at the National Institute of Advanced Industrial Science
and Technology, 1-1-1 Higashi, Tsukuba-shi, Ibaraki-ken, Japan,
under the deposit number FERM BP-5785 from Feb. 7, 1996) to be its
base, the initial reaction rates were compared in the following
method.
[0231] Comparison of Initial Reaction Rate
[0232] 5 mL of a liquid LB medium containing 40 .mu.g/mL of ferric
sulfate heptahydrate and 10 .mu.g/mL of cobalt chloride dihydrate
was prepared in a test tube, and sterilized by autoclaving at 121
degrees centigrade for 20 minutes. Ampicillin was added to this
medium to have a final concentration of 100 .mu.g/mL. Then, on the
medium, one platinum loop of respective transformants was
inoculated and cultivated therein at 37 degrees centigrade for
about 20 hours with stirring at 200 rpm. 40 .mu.L of the resulting
culture was taken and suspended in 740 .mu.L of a 54 mM Tris-HCl
aqueous solution (pH 8.0). To this, 20 .mu.L of acrylonitrile was
added, and this mixture was gently stirred at 20 degrees centigrade
for 15 minutes to react, whereby acrylamide was produced. After
completion of the reaction, the content of acrylamide in the
reaction solution was analyzed through HPLC.
[0233] Comparison of Thermal Stability of Enzyme
[0234] Respective transformants were separated from the resulting
culture of the above-mentioned transformants by centrifugation
(5,000 G.times.15 minutes).
[0235] 0.1 g of the thus isolated transformants were respectively
suspended in 20 ml of a 50 mM Tris-HCl aqueous solution (pH 8.0),
and heated at 60 degrees centigrade for 2 hours. The temperature
was returned to 20 degrees centigrade after heating, and 0.5 ml of
acrylonitrile was added thereto as the substrate. The reaction was
carried out at 20 degrees centigrade for 15 minutes to measure the
initial reaction rate.
[0236] Analytical Conditions
[0237] Analytical Equipment: HPLC manufactured by JASCO
Corporation
[0238] Column: YMC Pack ODS-A (150.times.6.00 mm)
[0239] Analytical Temperature: 40 degrees centigrade
[0240] Mobile Phase: 3% acetonitrile, 10 mM phosphoric acid
[0241] Respective transformants were subjected to the reaction and
analysis three times or more to correct variations in the data by
means of a dispensing operation or the like.
[0242] As a result of comparison of the initial reaction rate and
thermal stability of the transformant (1) and MT-10822, that is,
the amount of produced acrylamide under the above reaction
conditions, improvement of the initial reaction rate by 1.15 times
was observed and thermal stability was maintained with the new
addition of the modified ribosome binding sequence shown in Table
1.
TABLE-US-00014 TABLE 14 Table 1 Effect on Effect on Improvement
Improvement of Reaction of Thermal Rate by Stability by Ribosome
Ribosome Change in Base Sequence Binding Binding Transformant
Mutated Before After Sequence Sequence No. Site Substitution
Substitution Substitution Substitution 1 Ribosome TGAGAGGAG
TAAGGAGGT 1.15 times 1.00 times Binding Sequence
Reference Example 1
Construction of a Transformant (2) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0243] In order to obtain a transformant (2) expressing the nitrile
hydratase variant obtained by mutating nitrile hydratase at (av)
amino acid substitution sites as shown in Table 2, the plasmid
described in Example 79 of Patent Document 2 was used as the
template, and the ribosome binding sequence was modified in the
method described in Example 1 to prepare a plasmid (2) encoding the
above-mentioned nitrile hydratase variant. A competent cell of
Escherichia coli HB101 (manufactured by Toyobo Co., Ltd.) was
transformed with the plasmid to obtain a transformant (2).
Reference Example 2
Construction of a Transformant (3) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0244] In order to obtain a transformant (3) expressing the nitrile
hydratase variant obtained by mutating nitrile hydratase at (bc)
amino acid substitution sites as shown in Table 2, introduction of
site-specific mutation was performed using a "LA PCR in vitro
mutagenesis Kit" manufactured by Takara Shuzo Co., Ltd.
(hereinafter referred to as the mutagenesis kit). The plasmid (1)
expressing nitrile hydratase with the modified ribosome binding
sequence described in Example 1 was used as the template to carry
out the PCR reaction.
[0245] For the PCR reaction No. 1, a reaction system of 50 .mu.L in
total containing 50 pmols of the primer having the sequence defined
in SEQ ID No: 9 in the Sequence Listing and 50 pmols of an M13
primer M4 (having the sequence defined in SEQ ID No: 10 in the
Sequence Listing) (for the composition of the system, the
instructions described in the mutagenesis kit were followed) was
used, and the reaction consisted of 25 PCR cycles, in which one PCR
cycle comprised thermal denaturation (98 degrees centigrade) for 15
seconds, annealing (55 degrees centigrade) for 30 seconds and chain
extension (72 degrees centigrade) for 120 seconds.
[0246] For the PCR reaction No. 2, a reaction system of 50 .mu.L in
total containing 50 pmols of an MUT4 primer (having the sequence
defined in SEQ ID No: 11 in the Sequence Listing) and an M13 primer
RV (having the sequence defined in SEQ ID No: 12 in the Sequence
Listing) (for the composition of the system, the instructions
described in the mutagenesis kit were followed) was used, and the
reaction was carried out following the same procedure as the PCR
reaction No. 1.
[0247] After completion of the PCR reaction Nos. 1 and 2, 5 .mu.L
of the reaction mixture was subjected to agarose gel
electrophoresis (where the agarose concentration was 1.0 weight %),
and an analysis of the DNA amplification product was carried out.
As a result, the presence of the amplified DNA product was
confirmed. From each of these PCR reaction mixtures, the excess
primers and dNTP were removed using Microcon 100 (manufactured by
Takara Shuzo Co., Ltd.), and then TE was added to each of the
mixtures to prepare 50 .mu.L each of TE solutions. An annealing
solution of 47.5 .mu.L in total containing 0.5 .mu.L of both of the
above TE solutions (for the composition of the system, the
instructions described in the mutagenesis kit were followed) was
prepared, and this solution was subjected to annealing by
performing thermal denaturation of the solution at 98 degrees
centigrade for 10 minutes, subsequently cooling the solution to 37
degrees centigrade at a constant cooling rate over a period of 60
minutes, and then maintaining it at 37 degrees centigrade for 15
minutes. To the thus annealed solution, 0.5 .mu.L of TaKaRa LA Taq
(manufactured by Takara Bio Inc.) was added, and the solution was
heated at 72 degrees centigrade for 3 minutes, thus completing the
formation of heterologous double-stranded DNA.
[0248] To this was added 50 pmols of an M13 primer M4 (having the
sequence defined in SEQ ID No: 10 in the Sequence Listing) and 50
pmols of an M13 primer RV (having the sequence defined in SEQ ID
No: 12 in the Sequence Listing) to give a reaction system of 50
.mu.L in total, and the reaction consisted of 25 PCR cycles, in
which one PCR cycle comprised thermal denaturation (98 degrees
centigrade) for 15 seconds, annealing (55 degrees centigrade) for
30 seconds and chain extension (72 degrees centigrade) for 120
seconds to carry out the PCR reaction No. 3. After completion of
the PCR reaction No. 3, 5 .mu.L of the reaction mixture was
subjected to agarose gel electrophoresis (using Type VII
low-melting-point agarose, a product by Sigma Corporation; agarose
concentration of 0.8 weight %), and an analysis of the DNA
amplification product was carried out. As a result, the presence of
the amplified DNA product of about 2 kb was confirmed.
[0249] Subsequently, an agarose fragment comprising only the DNA
fragment of about 2 kb was cut out of the agarose gel. The thus cut
agarose fragment (about 0.1 g) was finely pulverized, suspended in
1 ml of a TE solution, and then kept at 55 degrees centigrade for 1
hour, whereby the agarose fragment was completely melted. The
resulting agarose melt was then subjected to phenol/chloroform
extraction and ethanol precipitation to purify the DNA fragment.
The thus purified DNA fragment was finally dissolved in 10 .mu.L of
TE. The amplified DNA fragment of about 2 kb thus purified was
cleaved by means of restriction endonucleases EcoRI and HindIII,
and this mixture treated with restriction endonucleases was then
subjected to phenol/chloroform extraction and ethanol precipitation
to purify the DNA fragment. The thus purified DNA fragment was
finally dissolved in 10 .mu.L of TE.
[0250] Likewise, the plasmid (1) expressing nitrile hydratase with
the modified ribosome binding sequence described in Example 1 was
cleaved by means of EcoRI and HindIII, and then subjected to
agarose gel electrophoresis (using Type VII low-melting-point
agarose, a product by Sigma Corporation; agarose concentration of
0.7%). An agarose fragment comprising only the DNA fragment of
about 2.7 kb was cut out of the agarose gel. The thus cut agarose
fragment (about 0.1 g) was finely pulverized, suspended in 1 ml of
the TE solution, and then kept at 55 degrees centigrade for 1 hour,
whereby the agarose fragment was completely melted. The resulting
agarose melt was then subjected to phenol/chloroform extraction and
ethanol precipitation to purify the DNA fragment. The thus purified
DNA fragment was finally dissolved in 10 .mu.L of TE.
[0251] The thus obtained DNA fragments of about 2 kbp and of about
2.7 kbp were subjected to DNA ligation, using a DNA ligation kit
(manufactured by Takara Shuzo Co., Ltd.). Then, a competent cell of
Escherichia coli HB101 (manufactured by Toyobo Co., Ltd.) was
transformed. The above operation was carried out using the plasmid
extracted from the transformant as the template, and using the
primer having the sequence defined in SEQ ID No: 13 instead of the
primer defined in SEQ ID No: 9, whereby a plasmid (3) encoding the
above nitrile hydratase variant was prepared. A competent cell of
Escherichia coli HB101 (manufactured by Toyobo Co., Ltd.) was
transformed with the plasmid to obtain a transformant (3).
Reference Example 3
Construction of a Transformant (4) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0252] In order to obtain a transformant (4) expressing the nitrile
hydratase variant obtained by mutating nitrile hydratase at (bh)
amino acid substitution sites as shown in Table 2, introduction of
site-specific mutation was performed using the mutagenesis kit
described in the above Reference Example 2. The plasmid (1)
expressing nitrile hydratase with the modified ribosome binding
sequence described in Example 1 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 14 and 15 in the
Sequence Listing were used for repeatedly carrying out the method
described in Reference Example 2 per mutation point, whereby a
plasmid (4) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (4).
TABLE-US-00015 TABLE 2 Change in Amino Acid Change in Trans-
Sequence Base Sequence formant Mutated Before After Before After
No. Site Substitution Substitution Substitution Substitution 2
.alpha.-36th Thr Met ACG ATG .alpha.-148th Gly Asp GGC GAC
.alpha.-204th Val Arg GTC CGC .beta.-41st Phe Ile TTC ATC
.beta.-51st Phe Val TTC GTC .beta.-108th Glu Asp GAG GAT 3
.alpha.-36th Thr Ser ACG TCG .beta.-33rd Ala Val GCG GTG 4
.beta.-40th Thr Ile ACG ATT .beta.-61st Ala Val GCC GTC
Example 2
Construction of a Transformant (5) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0253] In order to obtain a transformant (5) expressing the nitrile
hydratase variant obtained by mutating nitrile hydratase at (a) and
(av) amino acid substitution sites as shown in Table 3, the plasmid
(2) recovered from the transformant (2) described in the above
Reference Example 1 was used as the template, and the primer having
the sequence defined in SEQ ID No: 16 in the Sequence Listing was
used for carrying out the method using the mutagenesis kit
described in Reference Example 2, whereby a plasmid (5) encoding
the above nitrile hydratase variant was prepared. A competent cell
of Escherichia coli HB101 (manufactured by Toyobo Co., Ltd.) was
transformed with the plasmid to obtain a transformant (5).
Moreover, the plasmid was prepared from the above-mentioned
microbial cells by the alkaline SDS extraction method, and the base
sequence of the nitrile hydratase gene was determined using a DNA
sequencer. Then, it was confirmed that the transformant (5) had
sequences according to the purpose in which mutation of 92nd Asp in
the .alpha.-subunit with Glu was newly added to the plasmid (2) of
Reference Example 1.
[0254] In the production of an amide compound using the thus
obtained transformant (5) and the transformant (2) to be its base,
the initial reaction rate and thermal stability were compared in
the same manner as in Example 1.
[0255] As a result, it was found that mutation of 92nd Asp in the
.alpha.-subunit with Glu was newly added to the transformant (5),
so that the initial reaction rate was improved by 1.65 times and
thermal stability was improved by 1.25 times, as compared to those
of the transformant (2).
Example 3
Construction of a Transformant (6) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0256] In order to obtain a transformant (6) expressing the nitrile
hydratase variant obtained by mutating nitrile hydratase at (a) and
(bc) amino acid substitution sites as shown in Table 3, the plasmid
(3) recovered from the transformant (3) described in the above
Reference Example 2 was used as the template, and the primer having
the sequence defined in SEQ ID No: 16 in the Sequence Listing was
used for carrying out the method using the mutagenesis kit
described in Reference Example 2, whereby a plasmid (6) encoding
the above nitrile hydratase variant was prepared. A competent cell
of Escherichia coli HB101 (manufactured by Toyobo Co., Ltd.) was
transformed with the plasmid to obtain a transformant (6).
Moreover, the plasmid was prepared from the above-mentioned
microbial cells by the alkaline SDS extraction method, and the base
sequence of the nitrile hydratase gene was determined using a DNA
sequencer. Then, it was confirmed that the transformant (6) had
sequences according to the purpose in which mutation of 92nd Asp in
the .alpha.-subunit with Glu was newly added to the plasmid (3) of
Reference Example 2. In the production of an amide compound using
the thus obtained transformant (6) and the transformant (3) to be
its base, the initial reaction rate and thermal stability were
compared in the same manner as in Example 1.
[0257] As a result, it was found that mutation of 92nd Asp in the
.alpha.-subunit with Glu was newly added to the transformant (6),
so that the initial reaction rate was improved by 1.63 times and
thermal stability was improved by 1.23 times, as compared to those
of the transformant (3).
Example 4
Construction of a Transformant (7) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0258] In order to obtain a transformant (7) expressing the nitrile
hydratase variant obtained by mutating nitrile hydratase at (a) and
(bh) amino acid substitution sites as shown in Table 3, the plasmid
(4) recovered from the transformant (4) described in the above
Reference Example 3 was used as the template, and the primer having
the sequence defined in SEQ ID No: 16 in the Sequence Listing was
used for carrying out the method using the mutagenesis kit
described in Reference Example 2, whereby a plasmid (7) encoding
the above nitrile hydratase variant was prepared. A competent cell
of Escherichia coli HB101 (manufactured by Toyobo Co., Ltd.) was
transformed with the plasmid to obtain a transformant (7).
Moreover, the plasmid was prepared from the above-mentioned
microbial cells by the alkaline SDS extraction method, and the base
sequence of the nitrile hydratase gene was determined using a DNA
sequencer. Then, it was confirmed that the transformant (7) had
sequences according to the purpose in which mutation of 92nd Asp in
the .alpha.-subunit with Glu was newly added to the plasmid (4) of
Reference Example 3. In the production of an amide compound using
the thus obtained transformant (7) and the transformant (4) to be
its base, the initial reaction rate and thermal stability were
compared in the same manner as in Example 1.
[0259] As a result, it was found that mutation of 92nd Asp in the
.alpha.-subunit with Glu was newly added to the transformant (7),
so that the initial reaction rate was improved by 1.58 times and
thermal stability was improved by 1.30 times, as compared to those
of the transformant (4).
TABLE-US-00016 TABLE 3 Change in Amino Acid Change in Base
Improvement of Sequence Sequence Improvement of Thermal Trans-
Before After Before After Reaction Rate by Stability by formant
Mutated Substitu- Substitu- Substitu- Substitu- .alpha.-92nd
.alpha.-92nd No. Site tion tion tion tion Substitution Substitution
5 .alpha.-36th Thr Met ACG ATG 1.65 times 1.25 times .alpha.-92nd
Asp Glu GAC GAG .alpha.-148th Gly Asp GGC GAC .alpha.-204th Val Arg
GTC CGC .beta.-41st Phe Ile TTC ATC .beta.-51st Phe Val TTC GTC
.beta.-108th Glu Asp GAG GAT 6 .alpha.-36th Thr Ser ACG TCG 1.63
times 1.23 times .alpha.-92nd Asp Glu GAC GAG .beta.-33rd Ala Val
GCG GTG 7 .alpha.-92nd Asp Glu GAC GAG 1.58 times 1.30 times
.beta.-40th Thr Ile ACG ATT .beta.-61st Ala Val GCC GTC
Reference Example 4
Construction of a Transformant (8) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0260] In order to obtain a transformant (8) expressing the nitrile
hydratase variant obtained by mutating nitrile hydratase at (ak)
amino acid substitution sites as shown in Table 4, the plasma
described in Example 68 of Patent Document 2 was used as the
template, and the ribosome binding sequence was modified according
to the method described in Example 1 to prepare a plasmid (8)
encoding the above nitrile hydratase variant. A competent cell of
Escherichia coli HB101 (manufactured by Toyobo Co., Ltd.) was
transformed with the plasmid to obtain a transformant (8).
Reference Example 5
Construction of a Transformant (9) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0261] In order to obtain a transformant (9) expressing the nitrile
hydratase variant obtained by mutating nitrile hydratase at (ap)
amino acid substitution sites as shown in Table 4, the plasma
described in Example 73 of Patent Document 2 was used as the
template, and the ribosome binding sequence was modified according
to the method described in Example 1 to prepare a plasmid (9)
encoding the above nitrile hydratase variant. A competent cell of
Escherichia coli HB101 (manufactured by Toyobo Co., Ltd.) was
transformed with the plasmid to obtain a transformant (9).
Reference Example 6
Construction of a Transformant (10) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0262] In order to obtain a transformant (10) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(bp) amino acid substitution sites as shown in Table 4,
introduction of site-specific mutation was performed using the
mutagenesis kit described in the above Reference Example 2. The
plasmid (1) expressing nitrile hydratase with the modified ribosome
binding sequence described in Example 1 was used as the template,
and the primers having the sequence defined in SEQ ID Nos: 17 and
18 in the Sequence Listing were used for repeatedly carrying out
the method described in Reference Example 2 per mutation point,
whereby a plasmid (10) encoding the above nitrile hydratase variant
was prepared. A competent cell of Escherichia coli HB101
(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid
to obtain a transformant (10).
TABLE-US-00017 TABLE 4 Change in Amino Acid Change in Trans-
Sequence Base Sequence formant Mutated Before After Before After
No. Site Substitution Substitution Substitution Substitution 8
.beta.-37th Phe Val TTC GTC .beta.-108th Glu Asp GAG GAT
.beta.-200th Ala Glu GCC GAG 9 .alpha.-6th Leu Thr CTG ACG
.alpha.-19th Ala Val GCG GTG .alpha.-126th Phe Tyr TTC TAC
.beta.-46th Met Lys ATG AAG .beta.-108th Glu Arg GAG CGG
.beta.-212th Ser Tyr TCC TAC 10 .beta.-61st Ala Gly GCC GGC
.beta.-150th Ala Asn GCG AAT
Example 5
Construction of a Transformant (11) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0263] In order to obtain a transformant (11) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(b) and (ak) amino acid substitution sites as shown in Table 5, the
plasmid (8) recovered from the transformant (8) described in the
above Reference Example 4 was used as the template, and the primer
having the sequence defined in SEQ ID No: 19 in the Sequence
Listing was used for carrying out the method using the mutagenesis
kit described in Reference Example 2, whereby a plasmid (11)
encoding the above nitrile hydratase variant was prepared. A
competent cell of Escherichia coli HB101 (manufactured by Toyobo
Co., Ltd.) was transformed with the plasmid to obtain a
transformant (11). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (11) had sequences according to the purpose in which
mutation of 94th Met in the .alpha.-subunit with Ile was newly
added to the plasmid (8) of Reference Example 4. In the production
of an amide compound using the thus obtained transformant (11) and
the transformant (8) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0264] As a result, it was found that mutation of 94th Met in the
.alpha.-subunit with Ile was newly added to the transformant (11),
so that the initial reaction rate was improved by 1.45 times and
thermal stability was improved by 1.38 times, as compared to those
of the transformant (8).
Example 6
Construction of a Transformant (12) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0265] In order to obtain a transformant (12) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(b) and (ap) amino acid substitution sites as shown in Table 5, the
plasmid (9) recovered from the transformant (9) described in the
above Reference Example 5 was used as the template, and the primer
having the sequence defined in SEQ ID No: 19 in the Sequence
Listing was used for carrying out the method using the mutagenesis
kit described in Reference Example 2, whereby a plasmid (12)
encoding the above nitrile hydratase variant was prepared. A
competent cell of Escherichia coli HB101 (manufactured by Toyobo
Co., Ltd.) was transformed with the plasmid to obtain a
transformant (12). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (12) had sequences according to the purpose in which
mutation of 94th Met in the .alpha.-subunit with Ile was newly
added to the plasmid (9) of Reference Example 5. In the production
of an amide compound using the thus obtained transformant (12) and
the transformant (9) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0266] As a result, it was found that mutation of 94th Met in the
.alpha.-subunit with Ile was newly added to the transformant (12),
so that the initial reaction rate was improved by 1.40 times and
thermal stability was improved by 1.25 times, as compared to those
of the transformant (9).
Example 7
Construction of a Transformant (13) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0267] In order to obtain a transformant (13) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(b) and (bp) amino acid substitution sites as shown in Table 5, the
plasmid (10) recovered from the transformant (10) described in the
above Reference Example 6 was used as the template, and the primer
having the sequence defined in SEQ ID No: 19 in the Sequence
Listing was used for carrying out the method using the mutagenesis
kit described in Reference Example 2, whereby a plasmid (13)
encoding the above nitrile hydratase variant was prepared. A
competent cell of Escherichia coli HB101 (manufactured by Toyobo
Co., Ltd.) was transformed with the plasmid to obtain a
transformant (13). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (13) had sequences according to the purpose in which
mutation of 94th Met in the .alpha.-subunit with Ile was newly
added to the plasmid (10) of Reference Example 6. In the production
of an amide compound using the thus obtained transformant (13) and
the transformant (10) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0268] As a result, it was found that mutation of 94th Met in the
.alpha.-subunit with Ile was newly added to the transformant (13),
so that the initial reaction rate was improved by 1.32 times and
thermal stability was improved by 1.35 times, as compared to those
of the transformant (10).
TABLE-US-00018 TABLE 5 Change in Amino Acid Change in Base
Improvement of Sequence Sequence Improvement of Thermal Trans-
Before After Before After Reaction Rate by Stability by formant
Mutated Substitu- Substitu- Substitu- Substitu- .alpha.-94th
.alpha.-94th No. Site tion tion tion tion Substitution Substitution
11 .alpha.-94th Met Ile ATG ATC 1.45 times 1.38 times .beta.-37th
Phe Val TTC GTC .beta.-108th Glu Asp GAG GAT .beta.-200th Ala Glu
GCC GAG 12 .alpha.-6th Leu Thr CTG ACG 1.40 times 1.25 times
.alpha.-19th Ala Val GCG GTG .alpha.-94th Met Ile ATG ATC
.alpha.-126th Phe Tyr TTC TAC .beta.-46th Met Lys ATG AAG
.beta.-108th Glu Arg GAG CGG .beta.-212th Ser Tyr TCC TAC 13
.alpha.-94th Met Ile ATG ATC 1.32 times 1.35 times .beta.-61st Ala
Gly GCC GGC .beta.-150th Ala Asn GCG AAT
Reference Example 7
Construction of a Transformant (14) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0269] In order to obtain a transformant (14) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(an) amino acid substitution sites as shown in Table 6, the plasma
described in Example 71 of Patent Document 2 was used as the
template, and the ribosome binding sequence was modified according
to the method described in Example 1 to prepare a plasmid (14)
encoding the above nitrile hydratase variant. A competent cell of
Escherichia coli HB101 (manufactured by Toyobo Co., Ltd.) was
transformed with the plasmid to obtain a transformant (14).
Reference Example 8
Construction of a Transformant (15) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0270] In order to obtain a transformant (15) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(be) amino acid substitution sites as shown in Table 6,
introduction of site-specific mutation was performed using the
mutagenesis kit described in the above Reference Example 2. The
plasmid (1) expressing nitrile hydratase with the modified ribosome
binding sequence described in Example 1 was used as the template,
and the primers having the sequence defined in SEQ ID Nos: 20 and
21 in the Sequence Listing were used for repeatedly carrying out
the method described in Reference Example 2 per mutation point,
whereby a plasmid (15) encoding the above nitrile hydratase variant
was prepared. A competent cell of Escherichia coli HB101
(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid
to obtain a transformant (15).
Reference Example 9
Construction of a Transformant (16) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0271] In order to obtain a transformant (16) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(br) amino acid substitution sites as shown in Table 6,
introduction of site-specific mutation was performed using the
mutagenesis kit described in the above Reference Example 2. The
plasmid (1) expressing nitrile hydratase with the modified ribosome
binding sequence described in Example 1 was used as the template,
and the primers having the sequence defined in SEQ ID Nos: 22 and
23 in the Sequence Listing were used for repeatedly carrying out
the method described in Reference Example 2 per mutation point,
whereby a plasmid (16) encoding the above nitrile hydratase variant
was prepared. A competent cell of Escherichia coli HB101
(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid
to obtain a transformant (16).
TABLE-US-00019 TABLE 6 Change in Amino Acid Change in Trans-
Sequence Base Sequence formant Mutated Before After Before After
No. Site Substitution Substitution Substitution Substitution 14
.beta.-48th Leu Val CTG GTG .beta.-108th Glu Arg GAG CGG
.beta.-212th Ser Tyr TCC TAC 15 .beta.-40th Thr Val ACG GTG
.beta.-218th Cys Met TGC ATG 16 .beta.-160th Arg Cys CGG TGT
.beta.-168th Thr Glu ACG GAG
Example 8
Construction of a Transformant (17) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0272] In order to obtain a transformant (17) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(c) and (an) amino acid substitution sites as shown in Table 7, the
plasmid (14) recovered from the transformant (14) described in the
above Reference Example 7 was used as the template, and the primer
having the sequence defined in SEQ ID No: 24 in the Sequence
Listing was used for carrying out the method using the mutagenesis
kit described in Reference Example 2, whereby a plasmid (17)
encoding the above nitrile hydratase variant was prepared. A
competent cell of Escherichia coli HB101 (manufactured by Toyobo
Co., Ltd.) was transformed with the plasmid to obtain a
transformant (17). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (17) had sequences according to the purpose in which
mutation of 197th Gly in the .alpha.-subunit with Cys was newly
added to the plasmid (14) of Reference Example 7. In the production
of an amide compound using the thus obtained transformant (17) and
the transformant (14) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0273] As a result, it was found that mutation of 197th Gly in the
.alpha.-subunit with Cys was newly added to the transformant (17),
so that the initial reaction rate was improved by 1.80 times and
thermal stability was improved by 1.25 times, as compared to those
of the transformant (14).
Example 9
Construction of a Transformant (18) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0274] In order to obtain a transformant (18) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(c) and (be) amino acid substitution sites as shown in Table 7, the
plasmid (15) recovered from the transformant (15) described in the
above Reference Example 8 was used as the template, and the primer
having the sequence defined in SEQ ID No: 24 in the Sequence
Listing was used for carrying out the method using the mutagenesis
kit described in Reference Example 2, whereby a plasmid (18)
encoding the above nitrile hydratase variant was prepared. A
competent cell of Escherichia coli HB101 (manufactured by Toyobo
Co., Ltd.) was transformed with the plasmid to obtain a
transformant (18). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (18) had sequences according to the purpose in which
mutation of 197th Gly in the .alpha.-subunit with Cys was newly
added to the plasmid (15) of Reference Example 8. In the production
of an amide compound using the thus obtained transformant (18) and
the transformant (15) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0275] As a result, it was found that mutation of 197th Gly in the
.alpha.-subunit with Cys was newly added to the transformant (18),
so that the initial reaction rate was improved by 1.86 times and
thermal stability was improved by 1.40 times, as compared to those
of the transformant (15).
Example 10
Construction of a Transformant (19) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0276] In order to obtain a transformant (19) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(c) and (br) amino acid substitution sites as shown in Table 7, the
plasmid (16) recovered from the transformant (16) described in the
above Reference Example 9 was used as the template, and the primer
having the sequence defined in SEQ ID No: 24 in the Sequence
Listing was used for carrying out the method using the mutagenesis
kit described in Reference Example 2, whereby a plasmid (19)
encoding the above nitrile hydratase variant was prepared. A
competent cell of Escherichia coli HB101 (manufactured by Toyobo
Co., Ltd.) was transformed with the plasmid to obtain a
transformant (19). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (19) had sequences according to the purpose in which
mutation of 197th Gly in the .alpha.-subunit with Cys was newly
added to the plasmid (16) of Reference Example 9. In the production
of an amide compound using the thus obtained transformant (19) and
the transformant (16) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0277] As a result, it was found that mutation of 197th Gly in the
.alpha.-subunit with Cys was newly added to the transformant (19),
so that the initial reaction rate was improved by 1.68 times and
thermal stability was improved by 1.20 times, as compared to those
of the transformant (16).
TABLE-US-00020 TABLE 7 Change in Amino Acid Change in Base
Improvement of Sequence Sequence Improvement of Thermal Trans-
Before After Before After Reaction Rate by Stability by formant
Mutated Substitu- Substitu- Substitu- Substitu- .alpha.-197th
.alpha.-197th No. Site tion tion tion tion Substitution
Substitution 17 .alpha.-197th Gly Cys GGC TGC 1.80 times 1.25 times
.beta.-48th Leu Val CTG GTG .beta.-108th Glu Arg GAG CGG
.beta.-212th Ser Tyr TCC TAC 18 .alpha.-197th Gly Cys GGC TGC 1.86
times 1.40 times .beta.-40th Thr Val ACG GTG .beta.-218th Cys Met
TGC ATG 19 .alpha.-197th Gly Cys GGC TGC 1.68 times 1.20 times
.beta.-160th Arg Cys CGG TGT .beta.-168th Thr Glu ACG GAG
Reference Example 10
Construction of a Transformant (20) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0278] In order to obtain a transformant (20) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(ar) amino acid substitution sites as shown in Table 8, the plasma
described in Example 75 of Patent Document 2 was used as the
template, and the ribosome binding sequence was modified according
to the method described in Example 1 to prepare a plasmid (20)
encoding the above nitrile hydratase variant. A competent cell of
Escherichia coli HB101 (manufactured by Toyobo Co., Ltd.) was
transformed with the plasmid to obtain a transformant (20).
Reference Example 11
Construction of a Transformant (21) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0279] In order to obtain a transformant (21) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(ax) amino acid substitution sites as shown in Table 8,
introduction of site-specific mutation was performed using the
mutagenesis kit described in the above Reference Example 2. The
plasmid (1) expressing nitrile hydratase with the modified ribosome
binding sequence described in Example 1 was used as the template,
and the primers having the sequence defined in SEQ ID Nos: 25 and
26 in the Sequence Listing were used for repeatedly carrying out
the method described in Reference Example 2 per mutation point,
whereby a plasmid (21) encoding the above nitrile hydratase variant
was prepared. A competent cell of Escherichia coli HB101
(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid
to obtain a transformant (21).
Reference Example 12
Construction of a Transformant (22) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0280] In order to obtain a transformant (22) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(bd) amino acid substitution sites as shown in Table 8,
introduction of site-specific mutation was performed using the
mutagenesis kit described in the above Reference Example 2. The
plasmid (1) expressing nitrile hydratase with the modified ribosome
binding sequence described in Example 1 was used as the template,
and the primers having the sequence defined in SEQ ID Nos: 27 and
28 in the Sequence Listing were used for repeatedly carrying out
the method described in Reference Example 2 per mutation point,
whereby a plasmid (22) encoding the above nitrile hydratase variant
was prepared. A competent cell of Escherichia coli HB101
(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid
to obtain a transformant (22).
TABLE-US-00021 TABLE 8 Change in Amino Acid Change in Trans-
Sequence Base Sequence formant Mutated Before After Before After
No. Site Substitution Substitution Substitution Substitution 20
.alpha.-6th Leu Ala CTG GCG .alpha.-19th Ala Val GCG GTG
.alpha.-126th Phe Tyr TTC TAC .beta.-127th Leu Ser CTG TCG
.beta.-160th Arg Trp CGG TGG .beta.-186th Leu Arg CTG CGG 21
.alpha.-36th Thr Gly ACG GGG .alpha.-188th Thr Gly ACC GGC 22
.beta.-176th Tyr Ala TAC GCC .beta.-217th Asp Val GAC GTC
Example 11
Construction of a Transformant (23) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0281] In order to obtain a transformant (23) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(d) and (ar) amino acid substitution sites as shown in Table 9, the
plasmid (20) recovered from the transformant (20) described in the
above Reference Example 10 was used as the template, and the primer
having the sequence defined in SEQ ID No: 29 in the Sequence
Listing was used for carrying out the method using the mutagenesis
kit described in Reference Example 2, whereby a plasmid (23)
encoding the above nitrile hydratase variant was prepared. A
competent cell of Escherichia coli HB101 (manufactured by Toyobo
Co., Ltd.) was transformed with the plasmid to obtain a
transformant (23). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (23) had sequences according to the purpose in which
mutation of 4th Val in the .beta.-subunit with Met was newly added
to the plasmid (20) of Reference Example 10. In the production of
an amide compound using the thus obtained transformant (23) and the
transformant (20) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0282] As a result, it was found that mutation of 4th Val in the
.beta.-subunit with Met was newly added to the transformant (23),
so that the initial reaction rate was improved by 1.25 times and
thermal stability was improved by 1.35 times, as compared to those
of the transformant (20).
Example 12
Construction of a Transformant (24) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0283] In order to obtain a transformant (24) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(d) and (ax) amino acid substitution sites as shown in Table 9, the
plasmid (21) recovered from the transformant (21) described in the
above Reference Example 11 was used as the template, and the primer
having the sequence defined in SEQ ID No: 29 in the Sequence
Listing was used for carrying out the method using the mutagenesis
kit described in Reference Example 2, whereby a plasmid (24)
encoding the above nitrile hydratase variant was prepared. A
competent cell of Escherichia coli HB101 (manufactured by Toyobo
Co., Ltd.) was transformed with the plasmid to obtain a
transformant (24). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (24) had sequences according to the purpose in which
mutation of 4th Val in the .beta.-subunit with Met was newly added
to the plasmid (21) of Reference Example 11. In the production of
an amide compound using the thus obtained transformant (24) and the
transformant (21) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0284] As a result, it was found that mutation of 4th Val in the
.beta.-subunit with Met was newly added to the transformant (24),
so that the initial reaction rate was improved by 1.32 times and
thermal stability was improved by 1.39 times, as compared to those
of the transformant (21).
Example 13
Construction of a Transformant (25) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0285] In order to obtain a transformant (25) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(d) and (bd) amino acid substitution sites as shown in Table 9, the
plasmid (22) recovered from the transformant (22) described in the
above Reference Example 12 was used as the template, and the primer
having the sequence defined in SEQ ID No: 29 in the Sequence
Listing was used for carrying out the method using the mutagenesis
kit described in Reference Example 2, whereby a plasmid (25)
encoding the above nitrile hydratase variant was prepared. A
competent cell of Escherichia coli HB101 (manufactured by Toyobo
Co., Ltd.) was transformed with the plasmid to obtain a
transformant (19).
[0286] Moreover, the plasmid was prepared from the above-mentioned
microbial cells by the alkaline SDS extraction method, and the base
sequence of the nitrile hydratase gene was determined using a DNA
sequencer. Then, it was confirmed that the transformant (25) had
sequences according to the purpose in which mutation of 4th Val in
the .beta.-subunit with Met was newly added to the plasmid (22) of
Reference Example 12. In the production of an amide compound using
the thus obtained transformant (25) and the transformant (22) to be
its base, the initial reaction rate and thermal stability were
compared in the same manner as in Example 1.
[0287] As a result of comparison of the initial reaction rate and
thermal stability between the transformant (25) and the
transformant (22), it was found that mutation of 4th Val in the
.beta.-subunit with Met was newly added to the transformant (25),
so that the initial reaction rate was improved by 1.25 times and
thermal stability was improved by 1.25 times, as compared to those
of the transformant (22).
TABLE-US-00022 TABLE 9 Change in Amino Acid Change in Base
Improvement of Sequence Sequence Improvement of Thermal Trans-
Before After Before After Reaction Rate by Stability by formant
Mutated Substitu- Substitu- Substitu- Substitu- .beta.-4th
.beta.-4th No. Site tion tion tion tion Substitution Substitution
23 .alpha.-6th Leu Ala CTG GCG 1.25 times 1.35 times .alpha.-19th
Ala Val GCG GTG .alpha.-126th Phe Tyr TTC TAC .beta.-4th Val Met
GTG ATG .beta.-127th Leu Ser CTG TCG .beta.-160th Arg Trp CGG TGG
.beta.-186th Leu Arg CTG CGG 24 .alpha.-36th Thr Gly ACG GGG 1.32
times 1.39 times .alpha.-188th Thr Gly ACC GGC .beta.-4th Val Met
GTG ATG 25 .beta.-4th Val Met GTG ATG 1.25 times 1.25 times
.beta.-176th Tyr Ala TAC GCC .beta.-217th Asp Val GAC GTC
Reference Example 13
Construction of a Transformant (26) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0288] In order to obtain a transformant (26) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(ao) amino acid substitution sites as shown in Table 10, the plasma
described in Example 72 of Patent Document 2 was used as the
template, and the ribosome binding sequence was modified according
to the method described in Example 1 to prepare a plasmid (26)
encoding the above nitrile hydratase variant. A competent cell of
Escherichia coli HB101 (manufactured by Toyobo Co., Ltd.) was
transformed with the plasmid to obtain a transformant (26).
Reference Example 14
Construction of a Transformant (27) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0289] In order to obtain a transformant (27) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(at) amino acid substitution sites as shown in Table 10, the plasma
described in Example 77 of Patent Document 2 was used as the
template, and the ribosome binding sequence was modified according
to the method described in Example 1 to prepare a plasmid (27)
encoding the above nitrile hydratase variant. A competent cell of
Escherichia coli HB101 (manufactured by Toyobo Co., Ltd.) was
transformed with the plasmid to obtain a transformant (27).
TABLE-US-00023 TABLE 10 Change in Amino Acid Change in Trans-
Sequence Base Sequence formant Mutated Before After Before After
No. Site Substitution Substitution Substitution Substitution 26
.beta.-127th Leu Ser CTG TCG .beta.-160th Arg Trp CGG TGG
.beta.-186th Leu Arg CTG CGG 27 .alpha.-19th Ala Val GCG GTG
.alpha.-71st Arg His CGT CAT .alpha.-126th Phe Tyr TTC TAC
.beta.-37th Phe Leu TTC CTC .beta.-108th Glu Asp GAG GAT
.beta.-200th Ala Glu GCC GAG
Comparative Example 1
Construction of a Transformant (28) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity
[0290] In order to obtain a transformant (28) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
8th of the .beta.-subunit and (ao) amino acid substitution sites as
shown in Table 11, the plasmid (26) recovered from the transformant
(26) described in the above Reference Example 13 was used as the
template, and the primer having the sequence defined in SEQ ID No:
30 in the Sequence Listing was used for carrying out the method
using the mutagenesis kit described in Reference Example 2, whereby
a plasmid (28) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (28). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (28) had sequences according to the purpose in which
mutation of 8th Gly in the .beta.-subunit with Ala was newly added
to the plasmid (26) of Reference Example 13. In the production of
an amide compound using the thus obtained transformant (28) and the
transformant (26) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0291] As a result of comparison, it was found that mutation of 8th
Gly in the .beta.-subunit with Ala was newly added to the
transformant (28), so that the initial reaction rate was improved
by 1.35 times and thermal stability was lowered by 0.65 times, as
compared to those of the transformant (26).
Comparative Example 2
Construction of a Transformant (29) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity
[0292] In order to obtain a transformant (29) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
8th of the .beta.-subunit and (at) amino acid substitution sites as
shown in Table 11, the plasmid (27) recovered from the transformant
(27) described in the above Reference Example 14 was used as the
template, and the primer having the sequence defined in SEQ ID No:
30 in the Sequence Listing was used for carrying out the method
using the mutagenesis kit described in Reference Example 2, whereby
a plasmid (29) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (29). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (29) had sequences according to the purpose in which
mutation of 8th Gly in the .beta.-subunit with Ala was newly added
to the plasmid (27) of Reference Example 14. In the production of
an amide compound using the thus obtained transformant (29) and the
transformant (27) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0293] As a result, it was found that mutation of 8th Gly in the
.beta.-subunit with Ala was newly added to the transformant (29),
so that the initial reaction rate was improved by 1.40 times and
thermal stability was lowered by 0.31 times, as compared to those
of the transformant (27).
Comparative Example 3
Construction of a Transformant (30) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity
[0294] In order to obtain a transformant (30) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
8th of the .beta.-subunit and (br) amino acid substitution sites as
shown in Table 11, the plasmid (16) recovered from the transformant
(16) described in the above Reference Example 9 was used as the
template, and the primer having the sequence defined in SEQ ID No:
30 in the Sequence Listing was used for carrying out the method
using the mutagenesis kit described in Reference Example 2, whereby
a plasmid (30) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (30). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (30) had sequences according to the purpose in which
mutation of 8th Gly in the .beta.-subunit with Ala was newly added
to the plasmid (16) of Reference Example 9. In the production of an
amide compound using the thus obtained transformant (30) and the
transformant (16) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0295] As a result, it was found that mutation of 8th Gly in the
.beta.-subunit with Ala was newly added to the transformant (30),
so that the initial reaction rate was improved by 1.32 times and
thermal stability was lowered by 0.52 times, as compared to those
of the transformant (16).
TABLE-US-00024 TABLE 11 Change in Amino Acid Change in Base
Improvement of Sequence Sequence Improvement of Thermal Trans-
Before After Before After Reaction Rate by Stability by formant
Mutated Substitu- Substitu- Substitu- Substitu- .beta.-8th
.beta.-8th No. Site tion tion tion tion Substitution Substitution
28 .alpha.-8th Gly Ala GGC GCC 1.35 times 0.65 times .beta.-127th
Leu Ser CTG TCG .beta.-160h Arg Trp CGG TGG .beta.-186th Leu Arg
CTG CGG 29 .alpha.-19th Ala Val GCG GTG 1.40 times 0.31 times
.alpha.-71st Arg His CGT CAT .alpha.-126th Phe Tyr TTC TAC
.beta.-8th Gly Ala GGC GCC .beta.-37th Phe Leu TTC CTC .beta.-108th
Glu Asp GAG GAT .beta.-200th Ala Glu GCC GAG 30 .beta.-8th Gly Ala
GGC GCC 1.32 times 0.52 times .beta.-160th Arg Cys CGG TGT
.beta.-168th Thr Glu ACG GAG
Reference Example 15
Construction of a Transformant (31) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0296] In order to obtain a transformant (31) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(au) amino acid substitution sites as shown in Table 12, the plasma
described in Example 78 of Patent Document 2 was used as the
template, and the ribosome binding sequence was modified according
to the method described in Example 1 to prepare a plasmid (31)
encoding the above nitrile hydratase variant. A competent cell of
Escherichia coli HB101 (manufactured by Toyobo Co., Ltd.) was
transformed with the plasmid to obtain a transformant (31).
Reference Example 16
Construction of a Transformant (32) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0297] In order to obtain a transformant (32) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(bf) amino acid substitution sites as shown in Table 12,
introduction of site-specific mutation was performed using the
mutagenesis kit described in the above Reference Example 2. The
plasmid (1) expressing nitrile hydratase with the modified ribosome
binding sequence described in Example 1 was used as the template,
and the primers having the sequence defined in SEQ ID Nos: 31 and
32 in the Sequence Listing were used for repeatedly carrying out
the method described in Reference Example 2 per mutation point,
whereby a plasmid (32) encoding the above nitrile hydratase variant
was prepared. A competent cell of Escherichia coli HB101
(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid
to obtain a transformant (32).
TABLE-US-00025 TABLE 12 Change in Amino Change in Trans- Acid
Sequence Base Sequence formant Mutated Before After Before After
No. Site Substitution Substitution Substitution Substitution 31
.alpha.-19th Ala Val GCG GTG .alpha.-71st Arg His CGT CAT
.alpha.-126th Phe Tyr TTC TAC .beta.-37th Phe Val TTC GTC
.beta.-108th Glu Asp GAG GAT .beta.-200th Ala Glu GCC GAG 32
.beta.-33rd Ala Met GCG ATG .beta.-176th Tyr Thr TAC ACC
Example 14
Construction of a Transformant (33) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0298] In order to obtain a transformant (33) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(f) and (au) amino acid substitution sites as shown in Table 13,
the plasmid (31) recovered from the transformant (31) described in
the above Reference Example 15 was used as the template, and the
primer having the sequence defined in SEQ ID No: 33 in the Sequence
Listing was used for carrying out the method using the mutagenesis
kit described in Reference Example 2, whereby a plasmid (33)
encoding the above nitrile hydratase variant was prepared. A
competent cell of Escherichia coli HB101 (manufactured by Toyobo
Co., Ltd.) was transformed with the plasmid to obtain a
transformant (33). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (33) had sequences according to the purpose in which
mutation of 79th His in the .beta.-subunit with Asn was newly added
to the plasmid (31) of Reference Example 15. In the production of
an amide compound using the thus obtained transformant (33) and the
transformant (31) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0299] As a result, it was found that mutation of 79th His in the
.beta.-subunit with Asn was newly added to the transformant (33),
so that the initial reaction rate was improved by 1.29 times and
thermal stability was improved by 1.82 times, as compared to those
of the transformant (31).
Example 15
Construction of a Transformant (34) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0300] In order to obtain a transformant (34) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(f) and (bf) amino acid substitution sites as shown in Table 13,
the plasmid (32) recovered from the transformant (32) described in
the above Reference Example 16 was used as the template, and the
primer having the sequence defined in SEQ ID No: 33 in the Sequence
Listing was used for carrying out the method using the mutagenesis
kit described in Reference Example 2, whereby a plasmid (34)
encoding the above nitrile hydratase variant was prepared. A
competent cell of Escherichia coli HB101 (manufactured by Toyobo
Co., Ltd.) was transformed with the plasmid to obtain a
transformant (34). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (34) had sequences according to the purpose in which
mutation of 79th His in the .beta.-subunit with Asn was newly added
to the plasmid (32) of Reference Example 16. In the production of
an amide compound using the thus obtained transformant (34) and the
transformant (32) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0301] As a result, it was found that mutation of 79th His in the
.beta.-subunit with Asn was newly added to the transformant (34),
so that the initial reaction rate was improved by 1.25 times and
thermal stability was improved by 1.76 times, as compared to those
of the transformant (32).
Example 16
Construction of a Transformant (35) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0302] In order to obtain a transformant (35) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(f) and (bp) amino acid substitution sites as shown in Table 13,
the plasmid (10) recovered from the transformant (10) described in
the above Reference Example 6 was used as the template, and the
primer having the sequence defined in SEQ ID No: 33 in the Sequence
Listing was used for carrying out the method using the mutagenesis
kit described in Reference Example 2, whereby a plasmid (35)
encoding the above nitrile hydratase variant was prepared. A
competent cell of Escherichia coli HB101 (manufactured by Toyobo
Co., Ltd.) was transformed with the plasmid to obtain a
transformant (35). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (35) had sequences according to the purpose in which
mutation of 79th His in the .beta.-subunit with Asn was newly added
to the plasmid (10) of Reference Example 6. In the production of an
amide compound using the thus obtained transformant (35) and the
transformant (10) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0303] As a result, it was found that mutation of 79th His in the
.beta.-subunit with Asn was newly added to the transformant (35),
so that the initial reaction rate was improved by 1.30 times and
thermal stability was improved by 1.72 times, as compared to those
of the transformant (10).
TABLE-US-00026 TABLE 13 Change in Amino Acid Change in Base
Improvement of Sequence Sequence Improvement of Thermal Trans-
Before After Before After Reaction Rate by Stability by formant
Mutated Substitu- Substitu- Substitu- Substitu- .beta.-79th
.beta.-79th No. Site tion tion tion tion Substitution Substitution
33 .alpha.-19th Ala Val GCG GTG 1.29 times 1.82 times .alpha.-71st
Arg His CGT CAT .alpha.-126th Phe Tyr TTC TAC .beta.-37th Phe Val
TTC GTC .beta.-79th His Asn CAC AAC .beta.-108h Glu Asp GAG GAT
.beta.-200th Ala Glu GCC GAG 34 .beta.-33rd Ala Met GCG ATG 1.25
times 1.76 times .beta.-79th His Asn CAC AAC .beta.-176th Tyr Thr
TAC ACC 35 .beta.-61st Ala Gly GCC GGC 1.30 times 1.72 times
.beta.-79th His Asn CAC AAC .beta.-150th Ala Asn GCG AAT
Reference Example 17
Construction of a Transformant (36) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0304] In order to obtain a transformant (36) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(aa) amino acid substitution sites as shown in Table 14, the plasma
described in Example 58 of Patent Document 2 was used as the
template, and the ribosome binding sequence was modified according
to the method described in Example 1 to prepare a plasmid (36)
encoding the above nitrile hydratase variant. A competent cell of
Escherichia coli HB101 (manufactured by Toyobo Co., Ltd.) was
transformed with the plasmid to obtain a transformant (36).
Reference Example 18
Construction of a Transformant (37) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0305] In order to obtain a transformant (37) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(ah) amino acid substitution sites as shown in Table 14, the plasma
described in Example 65 of Patent Document 2 was used as the
template, and the ribosome binding sequence was modified according
to the method described in Example 1 to prepare a plasmid (37)
encoding the above nitrile hydratase variant. A competent cell of
Escherichia coli HB101 (manufactured by Toyobo Co., Ltd.) was
transformed with the plasmid to obtain a transformant (37).
Reference Example 19
Construction of a Transformant (38) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0306] In order to obtain a transformant (38) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(aq) amino acid substitution sites as shown in Table 14, the plasma
described in Example 74 of Patent Document 2 was used as the
template, and the ribosome binding sequence was modified according
to the method described in Example 1 to prepare a plasmid (38)
encoding the above nitrile hydratase variant. A competent cell of
Escherichia coli HB101 (manufactured by Toyobo Co., Ltd.) was
transformed with the plasmid to obtain a transformant (38).
TABLE-US-00027 TABLE 14 Change in Amino Change in Trans- Acid
Sequence Base Sequence formant Mutated Before After Before After
No. Site Substitution Substitution Substitution Substitution 36
.alpha.-36th Thr Met ACG ATG .alpha.-126th Phe Tyr TTC TAC 37
.alpha.-36th Thr Met ACG ATG .alpha.-148th Gly Asp GGC GAC
.alpha.-204th Val Arg GTC CGC 38 .alpha.-6th Leu Thr CTG ACG
.alpha.-19th Ala Val GCG GTG .alpha.-126th Phe Tyr TTC TAC
.beta.-48th Leu Val CTG GTG .beta.-108th Glu Arg GAG CGG
.beta.-212th Ser Tyr TCC TAC
Example 17
Construction of a Transformant (39) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0307] In order to obtain a transformant (39) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(g) and (aa) amino acid substitution sites as shown in Table 15,
the plasmid (36) recovered from the transformant (36) described in
the above Reference Example 17 was used as the template, and the
primer having the sequence defined in SEQ ID No: 34 in the Sequence
Listing was used for carrying out the method using the mutagenesis
kit described in Reference Example 2, whereby a plasmid (39)
encoding the above nitrile hydratase variant was prepared. A
competent cell of Escherichia coli HB101 (manufactured by Toyobo
Co., Ltd.) was transformed with the plasmid to obtain a
transformant (39). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (39) had sequences according to the purpose in which
mutation of 96th Gln in the .beta.-subunit with Arg was newly added
to the plasmid (36) of Reference Example 17. In the production of
an amide compound using the thus obtained transformant (39) and the
transformant (36) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0308] As a result, it was found that mutation of 96th Gln in the
.beta.-subunit with Arg was newly added to the transformant (39),
so that the initial reaction rate was improved by 1.33 times and
thermal stability was improved by 1.25 times, as compared to those
of the transformant (36).
Example 18
Construction of a Transformant (40) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0309] In order to obtain a transformant (40) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(g) and (ah) amino acid substitution sites as shown in Table 15,
the plasmid (37) recovered from the transformant (37) described in
the above Reference Example 18 was used as the template, and the
primer having the sequence defined in SEQ ID No: 34 in the Sequence
Listing was used for carrying out the method using the mutagenesis
kit described in Reference Example 2, whereby a plasmid (40)
encoding the above nitrile hydratase variant was prepared. A
competent cell of Escherichia coli HB101 (manufactured by Toyobo
Co., Ltd.) was transformed with the plasmid to obtain a
transformant (40). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (40) had sequences according to the purpose in which
mutation of 96th Gln in the .beta.-subunit with Arg was newly added
to the plasmid (37) of Reference Example 18. In the production of
an amide compound using the thus obtained transformant (40) and the
transformant (37) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0310] As a result, it was found that mutation of 96th Gln in the
.beta.-subunit with Arg was newly added to the transformant (40),
so that the initial reaction rate was improved by 1.25 times and
thermal stability was improved by 1.36 times, as compared to those
of the transformant (37).
Example 19
Construction of a Transformant (41) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0311] In order to obtain a transformant (41) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(g) and (aq) amino acid substitution sites as shown in Table 15,
the plasmid (38) recovered from the transformant (38) described in
the above Reference Example 19 was used as the template, and the
primer having the sequence defined in SEQ ID No: 34 in the Sequence
Listing was used for carrying out the method using the mutagenesis
kit described in Reference Example 2, whereby a plasmid (41)
encoding the above nitrile hydratase variant was prepared. A
competent cell of Escherichia coli HB101 (manufactured by Toyobo
Co., Ltd.) was transformed with the plasmid to obtain a
transformant (41). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (41) had sequences according to the purpose in which
mutation of 96th Gln in the .beta.-subunit with Arg was newly added
to the plasmid (38) of Reference Example 19. In the production of
an amide compound using the thus obtained transformant (41) and the
transformant (38) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0312] As a result, it was found that mutation of 96th Gln in the
.beta.-subunit with Arg was newly added to the transformant (41),
so that the initial reaction rate was improved by 1.35 times and
thermal stability was improved by 1.42 times, as compared to those
of the transformant (38).
TABLE-US-00028 TABLE 15 Change in Amino Acid Change in Base
Improvement of Sequence Sequence Improvement of Thermal Trans-
Before After Before After Reaction Rate by Stability by formant
Mutated Substitu- Substitu- Substitu- Substitu- .beta.-96th
.beta.-96th No. Site tion tion tion tion Substitution Substitution
39 .alpha.-36th Thr Met ACG ATG 1.33 times 1.25 times .alpha.-126th
Phe Tyr TTC TAC .beta.-96th Gln Arg CAG CGT 40 .alpha.-36th Thr Met
ACG ATG 1.25 times 1.36 times .alpha.-148th Gly Asp GGC GAC
.alpha.-204th Val Arg GTC CGC .beta.-96th Gln Arg CAG CGT 41
.alpha.-6th Leu Thr CTG ACG 1.35 times 1.42 times .alpha.-19th Ala
Val GCG GTG .alpha.-126th Phe Tyr TTC TAC .beta.-48th Leu Val CTG
GTG .beta.-96th Gln Arg CAG CGT .beta.-108th Glu Arg GAG CGG
.beta.-212th Ser Tyr TCC TAC
Reference Example 20
Construction of a Transformant (42) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0313] In order to obtain a transformant (42) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(ae) amino acid substitution sites as shown in Table 16, the plasma
described in Example 62 of Patent Document 2 was used as the
template, and the ribosome binding sequence was modified according
to the method described in Example 1 to prepare a plasmid (42)
encoding the above nitrile hydratase variant. A competent cell of
Escherichia coli HB101 (manufactured by Toyobo Co., Ltd.) was
transformed with the plasmid to obtain a transformant (42).
Reference Example 21
Construction of a Transformant (43) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0314] In order to obtain a transformant (43) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(bk) amino acid substitution sites as shown in Table 16,
introduction of site-specific mutation was performed using the
mutagenesis kit described in the above Reference Example 2. The
plasmid (1) expressing nitrile hydratase with the modified ribosome
binding sequence described in Example 1 was used as the template,
and the primers having the sequence defined in SEQ ID Nos: 35 and
36 in the Sequence Listing were used for repeatedly carrying out
the method described in Reference Example 2 per mutation point,
whereby a plasmid (43) encoding the above nitrile hydratase variant
was prepared. A competent cell of Escherichia coli HB101
(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid
to obtain a transformant (43).
TABLE-US-00029 TABLE 16 Change in Amino Change in Trans- Acid
Sequence Base Sequence formant Mutated Before After Before After
No. Site Substitution Substitution Substitution Substitution 42
.beta.-160th Arg Trp CGG TGG .beta.-186th Leu Arg CTG CGG 43
.beta.-61st Ala Trp GCC TGG .beta.-217th Asp His GAC CAC
Example 20
Construction of a Transformant (44) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0315] In order to obtain a transformant (44) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(h) and (ae) amino acid substitution sites as shown in Table 17,
the plasmid (42) recovered from the transformant (42) described in
the above Reference Example 20 was used as the template, and the
primer having the sequence defined in SEQ ID No: 37 in the Sequence
Listing was used for carrying out the method using the mutagenesis
kit described in Reference Example 2, whereby a plasmid (44)
encoding the above nitrile hydratase variant was prepared. A
competent cell of Escherichia coli HB101 (manufactured by Toyobo
Co., Ltd.) was transformed with the plasmid to obtain a
transformant (44). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (44) had sequences according to the purpose in which
mutation of 107th Pro in the .beta.-subunit with Met was newly
added to the plasmid (42) of Reference Example 20. In the
production of an amide compound using the thus obtained
transformant (44) and the transformant (42) to be its base, the
initial reaction rate and thermal stability were compared in the
same manner as in Example 1.
[0316] As a result, it was found that mutation of 107th Pro in the
.beta.-subunit with Met was newly added to the transformant (44),
so that the initial reaction rate was improved by 1.34 times and
thermal stability was improved by 2.25 times, as compared to those
of the transformant (42).
Example 21
Construction (45) of a Transformant (45) Substituted Amino Acid
Having Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0317] In order to obtain a transformant (45) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(h) and (au) amino acid substitution sites as shown in Table 17,
the plasmid (31) recovered from the transformant (31) described in
the above Reference Example 15 was used as the template, and the
primer having the sequence defined in SEQ ID No: 68 in the Sequence
Listing was used for carrying out the method using the mutagenesis
kit described in Reference Example 2, whereby a plasmid (45)
encoding the above nitrile hydratase variant was prepared. A
competent cell of Escherichia coli HB101 (manufactured by Toyobo
Co., Ltd.) was transformed with the plasmid to obtain a
transformant (45). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (45) had sequences according to the purpose in which
mutation of 107th Pro in the .beta.-subunit with Met was newly
added to the plasmid (31) of Reference Example 15. In the
production of an amide compound using the thus obtained
transformant (45) and the transformant (31) to be its base, the
initial reaction rate and thermal stability were compared in the
same manner as in Example 1.
[0318] As a result, it was found that mutation of 107th Pro in the
.beta.-subunit with Met was newly added to the transformant (45),
so that the initial reaction rate was improved by 1.40 times and
thermal stability was improved by 2.12 times, as compared to those
of the transformant (31).
Example 22
Construction of a Transformant (46) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0319] In order to obtain a transformant (46) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(h) and (bk) amino acid substitution sites as shown in Table 17,
the plasmid (43) recovered from the transformant (43) described in
the above Reference Example 21 was used as the template, and the
primer having the sequence defined in SEQ ID No: 37 in the Sequence
Listing was used for carrying out the method using the mutagenesis
kit described in Reference Example 2, whereby a plasmid (46)
encoding the above nitrile hydratase variant was prepared. A
competent cell of Escherichia coli HB101 (manufactured by Toyobo
Co., Ltd.) was transformed with the plasmid to obtain a
transformant (46). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (46) had sequences according to the purpose in which
mutation of 107th Pro in the .beta.-subunit with Met was newly
added to the plasmid (43) of Reference Example 21. In the
production of an amide compound using the thus obtained
transformant (46) and the transformant (43) to be its base, the
initial reaction rate and thermal stability were compared in the
same manner as in Example 1.
[0320] As a result, it was found that mutation of 107th Pro in the
.beta.-subunit with Met was newly added to the transformant (46),
so that the initial reaction rate was improved by 1.32 times and
thermal stability was improved by 2.40 times, as compared to those
of the transformant (43).
TABLE-US-00030 TABLE 17 Change in Amino Acid Change in Base
Improvement of Sequence Sequence Improvement of Thermal Trans-
Before After Before After Reaction Rate by Stability by formant
Mutated Substitu- Substitu- Substitu- Substitu- .beta.-107th
.beta.-107th No. Site tion tion tion tion Substitution Substitution
44 .beta.-107th Pro Met CCC ATG 1.34 times 2.25 times .beta.-160th
Arg Trp CGG TGG .beta.-186th Leu Arg CTG CGG 45 .alpha.-19th Ala
Val GCG GTG 1.40 times 2.12 times .alpha.-71st Arg His CGT CAT
.alpha.-126th Phe Tyr TTC TAC .beta.-37th Phe Val TTC GTC
.beta.-107th Pro Met CCC ATG .beta.-108th Glu Asp GAG GAT
.beta.-200th Ala Glu GCC GAG 46 .beta.-61st Ala Trp GCC TGG 1.32
times 2.40 times .beta.-107th Pro Met CCC ATG .beta.-217th Asp His
GAC CAC
Example 23
Construction of a Transformant (47) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0321] In order to obtain a transformant (47) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(i) and (aa) amino acid substitution sites as shown in Table 18,
the plasmid (36) recovered from the transformant (36) described in
the above Reference Example 17 was used as the template, and the
primer having the sequence defined in SEQ ID No: 38 in the Sequence
Listing was used for carrying out the method using the mutagenesis
kit described in Reference Example 2, whereby a plasmid (47)
encoding the above nitrile hydratase variant was prepared. A
competent cell of Escherichia coli HB101 (manufactured by Toyobo
Co., Ltd.) was transformed with the plasmid to obtain a
transformant (47). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (47) had sequences according to the purpose in which
mutation of 226th Val in the .beta.-subunit with Ile was newly
added to the plasmid (36) of Reference Example 17. In the
production of an amide compound using the thus obtained
transformant (47) and the transformant (36) to be its base, the
initial reaction rate and thermal stability were compared in the
same manner as in Example 1.
[0322] As a result, it was found that mutation of 226th Val in the
.beta.-subunit with Ile was newly added to the transformant (47),
so that the initial reaction rate was improved by 1.26 times and
thermal stability was improved by 1.29 times, as compared to those
of the transformant (36).
Example 24
Construction (48) of a Transformant (48) Substituted Amino Acid
Having Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0323] In order to obtain a transformant (48) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(i) and (ak) amino acid substitution sites as shown in Table 18,
the plasmid (8) recovered from the transformant (8) described in
the above Reference Example 4 was used as the template, and the
primer having the sequence defined in SEQ ID No: 38 in the Sequence
Listing was used for carrying out the method using the mutagenesis
kit described in Reference Example 2, whereby a plasmid (48)
encoding the above nitrile hydratase variant was prepared. A
competent cell of Escherichia coli HB101 (manufactured by Toyobo
Co., Ltd.) was transformed with the plasmid to obtain a
transformant (48). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (48) had sequences according to the purpose in which
mutation of 226th Val in the .beta.-subunit with Ile was newly
added to the plasmid (8) of Reference Example 4. In the production
of an amide compound using the thus obtained transformant (48) and
the transformant (8) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0324] As a result, it was found that mutation of 226th Val in the
.beta.-subunit with Ile was newly added to the transformant (48),
so that the initial reaction rate was improved by 1.35 times and
thermal stability was improved by 1.27 times, as compared to those
of the transformant (8).
Example 25
Construction of a Transformant (49) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0325] In order to obtain a transformant (49) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(i) and (be) amino acid substitution sites as shown in Table 18,
the plasmid (15) recovered from the transformant (15) described in
the above Reference Example 8 was used as the template, and the
primer having the sequence defined in SEQ ID No: 38 in the Sequence
Listing was used for carrying out the method using the mutagenesis
kit described in Reference Example 2, whereby a plasmid (49)
encoding the above nitrile hydratase variant was prepared. A
competent cell of Escherichia coli HB101 (manufactured by Toyobo
Co., Ltd.) was transformed with the plasmid to obtain a
transformant (49). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (49) had sequences according to the purpose in which
mutation of 226th Val in the .beta.-subunit with Ile was newly
added to the plasmid (15) of Reference Example 8. In the production
of an amide compound using the thus obtained transformant (49) and
the transformant (15) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0326] As a result, it was found that mutation of 226th Val in the
.beta.-subunit with Ile was newly added to the transformant (49),
so that the initial reaction rate was improved by 1.25 times and
thermal stability was improved by 1.30 times, as compared to those
of the transformant (15).
TABLE-US-00031 TABLE 18 Change in Amino Acid Change in Base
Improvement of Sequence Sequence Improvement of Thermal Trans-
Before After Before After Reaction Rate by Stability by formant
Mutated Substitu- Substitu- Substitu- Substitu- .beta.-226th
.beta.-226th No. Site tion tion tion tion Substitution Substitution
47 .alpha.-36th Thr Met ACG ATG 1.26 times 1.29 times .alpha.-126th
Phe Tyr TTC TAC .beta.-226th Val Ile GTC ATC 48 .beta.-37th Phe Val
TTC GTC 1.35 times 1.27 times .beta.-108th Glu Asp GAG GAT
.beta.-200th Ala Glu GCC GAG .beta.-226th Val Ile GTC ATC 49
.beta.-40th Thr Val ACG GTG 1.25 times 1.30 times .beta.-218th Cys
Met TGC ATG .beta.-226th Val Ile GTC ATC
Reference Example 22
Construction of a Transformant (50) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0327] In order to obtain a transformant (50) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(af) amino acid substitution sites as shown in Table 19, the plasma
described in Example 63 of Patent Document 2 was used as the
template, and the ribosome binding sequence was modified according
to the method described in Example 1 to prepare a plasmid (50)
encoding the above nitrile hydratase variant. A competent cell of
Escherichia coli HB101 (manufactured by Toyobo Co., Ltd.) was
transformed with the plasmid to obtain a transformant (50).
Reference Example 23
Construction of a Transformant (51) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0328] In order to obtain a plasmid encoding the nitrile hydratase
variant obtained by mutating nitrile hydratase at (bq) amino acid
substitution sites as shown in Table 19, introduction of
site-specific mutation was performed using the mutagenesis kit
described in the above Reference Example 2. The plasmid (1)
expressing nitrile hydratase with the modified ribosome binding
sequence described in Example 1 was used as the template, the
primers having the sequence defined in SEQ ID Nos: 39 and 40 in the
Sequence Listing were used for repeatedly carrying out the method
described in Reference Example 2 per mutation point, whereby a
plasmid (51) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (51).
Reference Example 24
Construction of a Transformant (52) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0329] In order to obtain a transformant (52) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(bj) amino acid substitution sites as shown in Table 19,
introduction of site-specific mutation was performed using the
mutagenesis kit described in the above Reference Example 2. The
plasmid (1) expressing nitrile hydratase with the modified ribosome
binding sequence described in Example 1 was used as the template,
and the primers having the sequence defined in SEQ ID Nos: 41 and
42 in the Sequence Listing were used for repeatedly carrying out
the method described in Reference Example 2 per mutation point,
whereby a plasmid (52) encoding the above nitrile hydratase variant
was prepared. A competent cell of Escherichia coli HB101
(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid
to obtain a transformant (52).
TABLE-US-00032 TABLE 19 Change in Amino Change in Trans- Acid
Sequence Base Sequence formant Mutated Before After Before After
No. Site Substitution Substitution Substitution Substitution 50
.alpha.-6th Leu Thr CTG ACG .alpha.-36th Thr Met ACG ATG
.alpha.-126th Phe Tyr TTC TAC 51 .beta.-61st Ala Ser GCC TCG
.beta.-160th Arg Met CGG ATG 52 .beta.-112th Lys Val AAG GTG
.beta.-217th Asp Met GAC ATG
Example 26
Construction of a Transformant (53) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0330] In order to obtain a transformant (53) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(m-1) and (af) amino acid substitution sites as shown in Table 20,
the plasmid (50) recovered from the transformant (50) described in
the above Reference Example 22 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 43 and 19 in the
Sequence Listing were used for repeatedly carrying out the method
described in Reference Example 2 per mutation point, whereby a
plasmid (53) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (53). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (53) had sequences according to the purpose in which
mutation of 13th Ile in the .alpha.-subunit with Leu and mutation
of 94th Met in the .alpha.-subunit with Ile were newly added to the
plasmid (50) of Reference Example 22. In the production of an amide
compound using the thus obtained transformant (53) and the
transformant (50) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0331] As a result, it was found that mutation of 13th Ile in the
.alpha.-subunit with Leu and mutation of 94th Met in the
.alpha.-subunit with Ile were newly added to the transformant (53),
so that the initial reaction rate was improved by 1.67 times and
thermal stability was improved by 1.45 times, as compared to those
of the transformant (50).
Example 27
Construction of a Transformant (54) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0332] In order to obtain a transformant (54) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(m-1) and (bq) amino acid substitution sites as shown in Table 20,
the plasmid (51) recovered from the transformant (51) described in
the above Reference Example 23 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 43 and 19 in the
Sequence Listing were used for repeatedly carrying out the method
described in Reference Example 2 per mutation point, whereby a
plasmid (54) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (54). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (54) had sequences according to the purpose in which
mutation of 13th Ile in the .alpha.-subunit with Leu and mutation
of 94th Met in the .alpha.-subunit with Ile were newly added to the
plasmid (51) of Reference Example 23. In the production of an amide
compound using the thus obtained transformant (54) and the
transformant (51) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0333] As a result, it was found that mutation of 13th Ile in the
.alpha.-subunit with Leu and mutation of 94th Met in the
.alpha.-subunit with Ile were newly added to the transformant (54),
so that the initial reaction rate was improved by 1.59 times and
thermal stability was improved by 1.32 times, as compared to those
of the transformant (51).
Example 28
Construction of a Transformant (55) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0334] In order to obtain a transformant (55) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(m-1) and (bj) amino acid substitution sites as shown in Table 20,
the plasmid (52) recovered from the transformant (52) described in
the above Reference Example 24 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 43 and 19 in the
Sequence Listing were used for repeatedly carrying out the method
described in Reference Example 2 per mutation point, whereby a
plasmid (55) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (55). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (55) had sequences according to the purpose in which
mutation of 13th Ile in the .alpha.-subunit with Leu and mutation
of 94th Met in the .alpha.-subunit with Ile were newly added to the
plasmid (52) of Reference Example 24. In the production of an amide
compound using the thus obtained transformant (55) and the
transformant (52) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0335] As a result, it was found that mutation of 13th Ile in the
.alpha.-subunit with Leu and mutation of 94th Met in the
.alpha.-subunit with Ile were newly added to the transformant (55),
so that the initial reaction rate was improved by 1.62 times and
thermal stability was improved by 1.26 times, as compared to those
of the transformant (52).
TABLE-US-00033 TABLE 20 Improvement of Change in Amino Acid Change
in Base Improvement of Thermal Sequence Sequence Reaction Rate by
Stability by Trans- Before After Before After .alpha.-13th and
.alpha.-13th and formant Mutated Substitu- Substitu- Substitu-
Substitu- .alpha.-94th .alpha.-94th No. Site tion tion tion tion
Substitution Substitution 53 .alpha.-6th Leu Thr CTG ACG 1.67 times
1.45 times .alpha.-13th Ile Leu ATC CTC .alpha.-36th Thr Met ACG
ATG .alpha.-94th Met Ile ATG ATC .alpha.-126th Phe Tyr TTC TAC 54
.alpha.-13th Ile Leu ATC CTC 1.59 times 1.32 times .alpha.-94th Met
Ile ATG ATC .beta.-61st Ala Ser GCC TCG .beta.-160th Arg Met CGG
ATG 55 .alpha.-13th Ile Leu ATC CTC 1.62 times 1.26 times
.alpha.-94th Met Ile ATG ATC .beta.-112th Lys Val AAG GTG
.beta.-217th Asp Met GAC ATG
Reference Example 25
Construction of a Transformant (56) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0336] In order to obtain a transformant (56) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(am) amino acid substitution sites as shown in Table 21, the plasma
described in Example 70 of Patent Document 2 was used as the
template, and the ribosome binding sequence was modified according
to the method described in Example 1 to prepare a plasmid (56)
encoding the above nitrile hydratase variant. A competent cell of
Escherichia coli HB101 (manufactured by Toyobo Co., Ltd.) was
transformed with the plasmid to obtain a transformant (56).
Reference Example 26
Construction of a Transformant (57) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0337] In order to obtain a transformant (57) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(ay) amino acid substitution sites as shown in Table 21,
introduction of site-specific mutation was performed using the
mutagenesis kit described in the above Reference Example 2. The
plasmid (1) expressing nitrile hydratase with the modified ribosome
binding sequence described in Example 1 was used as the template,
and the primers having the sequence defined in SEQ ID Nos: 44 and
45 in the Sequence Listing were used for repeatedly carrying out
the method described in Reference Example 2 per mutation point,
whereby a plasmid (57) encoding the above nitrile hydratase variant
was prepared. A competent cell of Escherichia coli HB101
(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid
to obtain a transformant (57).
TABLE-US-00034 TABLE 21 Change in Amino Change in Trans- Acid
Sequence Base Sequence formant Mutated Before After Before After
No. Site Substitution Substitution Substitution Substitution 56
.beta.-46th Met Lys ATG AAG .beta.-108th Glu Arg GAG CGG
.beta.-212th Ser Tyr TCC TAC 57 .alpha.-36th Thr Ala ACG GCG
.alpha.-48th Asn Gln AAC CAA
Example 29
Construction of a Transformant (58) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0338] In order to obtain a transformant (58) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(m-2) and (am) amino acid substitution sites as shown in Table 22,
the plasmid (56) recovered from the transformant (56) described in
the above Reference Example 25 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 43 and 34 in the
Sequence Listing were used for repeatedly carrying out the method
described in Reference Example 2 per mutation point, whereby a
plasmid (58) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (58). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (56) had sequences according to the purpose in which
mutation of 13th Ile in the .alpha.-subunit with Leu and mutation
of 96th Gln in the .beta.-subunit with Arg were newly added to the
plasmid (56) of Reference Example 25. In the production of an amide
compound using the thus obtained transformant (58) and the
transformant (56) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0339] As a result, it was found that mutation of 13th Ile in the
.alpha.-subunit with Leu and mutation of 96th Gln in the
.beta.-subunit with Arg were newly added to the transformant (58),
so that the initial reaction rate was improved by 1.53 times and
thermal stability was improved by 1.32 times, as compared to those
of the transformant (56).
Example 30
Construction of a Transformant (59) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0340] In order to obtain a transformant (59) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(m-2) and (at) amino acid substitution sites as shown in Table 22,
the plasmid (27) recovered from the transformant (27) described in
the above Reference Example 14 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 43 and 34 in the
Sequence Listing were used for repeatedly carrying out the method
described in Reference Example 2 per mutation point, whereby a
plasmid (59) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (59). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (59) had sequences according to the purpose in which
mutation of 13th Ile in the .alpha.-subunit with Leu and mutation
of 96th Gln in the .beta.-subunit with Arg were newly added to the
plasmid (27) of Reference Example 14. In the production of an amide
compound using the thus obtained transformant (59) and the
transformant (27) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0341] As a result, it was found that mutation of 13th Ile in the
.alpha.-subunit with Leu and mutation of 96th Gln in the
.beta.-subunit with Arg were newly added to the transformant (59),
so that the initial reaction rate was improved by 1.49 times and
thermal stability was improved by 1.28 times, as compared to those
of the transformant (27).
Example 31
Construction of a Transformant (60) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0342] In order to obtain a transformant (60) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(m-2) and (ay) amino acid substitution sites as shown in Table 22,
the plasmid (57) recovered from the transformant (57) described in
the above Reference Example 26 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 43 and 34 in the
Sequence Listing were used for repeatedly carrying out the method
described in Reference Example 2 per mutation point, whereby a
plasmid (60) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (60). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (60) had sequences according to the purpose in which
mutation of 13th Ile in the .alpha.-subunit with Leu and mutation
of 96th Gln in the .beta.-subunit with Arg were newly added to the
plasmid (57) of Reference Example 26. In the production of an amide
compound using the thus obtained transformant (60) and the
transformant (57) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0343] As a result, it was found that mutation of 13th Ile in the
.alpha.-subunit with Leu and mutation of 96th Gln in the
.beta.-subunit with Arg were newly added to the transformant (60),
so that the initial reaction rate was improved by 1.39 times and
thermal stability was improved by 1.45 times, as compared to those
of the transformant (57).
TABLE-US-00035 TABLE 22 Improvement of Change in Amino Acid Change
in Base Improvement of Thermal Sequence Sequence Reaction Rate by
Stability by Trans- Before After Before After .alpha.-13th and
.alpha.-13th and formant Mutated Substitu- Substitu- Substitu-
Substitu- .beta.-96th .beta.-96th No. Site tion tion tion tion
Substitutions Substitutions 58 .alpha.-13th Ile Leu ATC CTC 1.53
times 1.32 times .beta.-46th Met Lys ATG AAG .beta.-96th Gln Arg
CAG CGT .beta.-108th Glu Arg GAG CGG .beta.-212th Ser Tyr TCC TAC
59 .alpha.-13th Ile Leu ATC CTC 1.49 times 1.28 times .alpha.-19th
Ala Val GCG GTG .alpha.-71st Arg His CGT CAT .alpha.-126th Phe Tyr
TTC TAC .beta.-37th Phe Leu TTC CTC .beta.-96th Gln Arg CAG CGT
.beta.-108th Glu Asp GAG GAT .beta.-200th Ala Glu GCC GAG 60
.alpha.-13th Ile Leu ATC CTC 1.39 times 1.45 times .alpha.-36th Thr
Ala ACG GCG .alpha.-48th Asn Gln AAC CAA .beta.-96th Gln Arg CAG
CGT
Example 32
Construction of a Transformant (61) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0344] In order to obtain a transformant (61) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(n-1) and (af) amino acid substitution sites as shown in Table 23,
the plasmid (50) recovered from the transformant (50) described in
the above Reference Example 22 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 46 and 19 in the
Sequence Listing were used for repeatedly carrying out the method
described in Reference Example 2 per mutation point, whereby a
plasmid (61) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (61). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (61) had sequences according to the purpose in which
mutation of 27th Met in the .alpha.-subunit with Ile and mutation
of 94th Met in the .alpha.-subunit with Ile were newly added to the
plasmid (50) of Reference Example 22. In the production of an amide
compound using the thus obtained transformant (61) and the
transformant (50) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0345] As a result, it was found that mutation of 27th Met in the
.alpha.-subunit with Ile and mutation of 94th Met in the
.alpha.-subunit with Ile were newly added to the transformant (61),
so that the initial reaction rate was improved by 1.65 times and
thermal stability was improved by 1.36 times, as compared to those
of the transformant (50).
Example 33
Construction of a Transformant (62) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0346] In order to obtain a transformant (62) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(n-1) and (ao) amino acid substitution sites as shown in Table 23,
the plasmid (26) recovered from the transformant (26) described in
the above Reference Example 13 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 46 and 19 in the
Sequence Listing were used for repeatedly carrying out the method
described in Reference Example 2 per mutation point, whereby a
plasmid (62) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (62). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (62) had sequences according to the purpose in which
mutation of 27th Met in the .alpha.-subunit with Ile and mutation
of 94th Met in the .alpha.-subunit with Ile were newly added to the
plasmid (26) of Reference Example 13. In the production of an amide
compound using the thus obtained transformant (62) and the
transformant (26) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0347] As a result, it was found that mutation of 27th Met in the
.alpha.-subunit with Ile and mutation of 94th Met in the
.alpha.-subunit with Ile were newly added to the transformant (62),
so that the initial reaction rate was improved by 1.72 times and
thermal stability was improved by 1.47 times, as compared to those
of the transformant (26).
Example 34
Construction of a Transformant (63) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0348] In order to obtain a transformant (63) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(n-1) and (ax) amino acid substitution sites as shown in Table 23,
the plasmid (21) recovered from the transformant (21) described in
the above Reference Example 11 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 46 and 19 in the
Sequence Listing were used for repeatedly carrying out the method
described in Reference Example 2 per mutation point, whereby a
plasmid (63) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (63). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (63) had sequences according to the purpose in which
mutation of 27th Met in the .alpha.-subunit with Ile and mutation
of 94th Met in the .alpha.-subunit with Ile were newly added to the
plasmid (21) of Reference Example 11. In the production of an amide
compound using the thus obtained transformant (63) and the
transformant (21) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0349] As a result, it was found that mutation of 27th Met in the
.alpha.-subunit with Ile and mutation of 94th Met in the
.alpha.-subunit with Ile were newly added to the transformant (63),
so that the initial reaction rate was improved by 1.55 times and
thermal stability was improved by 1.27 times, as compared to those
of the transformant (21).
TABLE-US-00036 TABLE 23 Improvement of Change in Amino Acid Change
in Base Improvement of Thermal Sequence Sequence Reaction Rate by
Stability by Trans- Before After Before After .alpha.-27th and
.alpha.-27th and formant Mutated Substitu- Substitu- Substitu-
Substitu- .alpha.-94th .alpha.-94th No. Site tion tion tion tion
Substitutions Substitutions 61 .alpha.-6th Leu Thr CTG ACG 1.65
times 1.36 times .alpha.-27th Met Ile ATG ATC .alpha.-36th Thr Met
ACG ATG .alpha.-94th Met Ile ATG ATC .alpha.-126th Phe Tyr TTC TAC
62 .alpha.-27th Met Ile ATG ATC 1.72 times 1.47 times .alpha.-94th
Met Ile ATG ATC .beta.-127th Leu Ser CTG TCG .beta.-160th Arg Trp
CGG TGG .beta.-186th Leu Arg CTG CGG 63 .alpha.-27th Met Ile ATG
ATC 1.55 times 1.27 times .alpha.-36th Thr Gly ACG GGG .alpha.-94th
Met Ile ATG ATC .alpha.-188th Thr Gly ACC GGC
Reference Example 27
Construction of a Transformant (64) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0350] In order to obtain a transformant (64) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(aj) amino acid substitution sites as shown in Table 24, the plasma
described in Example 67 of Patent Document 2 was used as the
template, and the ribosome binding sequence was modified according
to the method described in Example 1 to prepare a plasmid (64)
encoding the above nitrile hydratase variant. A competent cell of
Escherichia coli HB101 (manufactured by Toyobo Co., Ltd.) was
transformed with the plasmid to obtain a transformant (64).
Reference Example 28
Construction of a Transformant (65) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0351] In order to obtain a transformant (65) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(as) amino acid substitution sites as shown in Table 24, the plasma
described in Example 76 of Patent Document 2 was used as the
template, and the ribosome binding sequence was modified according
to the method described in Example 1 to prepare a plasmid (65)
encoding the above nitrile hydratase variant. A competent cell of
Escherichia coli HB101 (manufactured by Toyobo Co., Ltd.) was
transformed with the plasmid to obtain a transformant (65).
Reference Example 29
Construction of a Transformant (66) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0352] In order to obtain a transformant (66) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(bb) amino acid substitution sites as shown in Table 24,
introduction of site-specific mutation was performed using the
mutagenesis kit described in the above Reference Example 2. The
plasmid (1) expressing nitrile hydratase with the modified ribosome
binding sequence described in Example 1 was used as the template,
and the primers having the sequence defined in SEQ ID Nos: 47 and
48 in the Sequence Listing were used for repeatedly carrying out
the method described in Reference Example 2 per mutation point,
whereby a plasmid (66) encoding the above nitrile hydratase variant
was prepared. A competent cell of Escherichia coli HB101
(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid
to obtain a transformant (66).
TABLE-US-00037 TABLE 24 Change in Amino Change in Trans- Acid
Sequence Base Sequence formant Mutated Before After Before After
No. Site Substitution Substitution Substitution Substitution 64
.beta.-37th Phe Leu TTC CTC .beta.-108th Glu Asp GAG GAT
.beta.-200th Ala Glu GCC GAG 65 .alpha.-6th Leu Thr CTG ACG
.alpha.-36th Thr Met ACG ATG .alpha.-126th Phe Tyr TTC TAC
.beta.-10th Thr Asp ACC GAC .beta.-118th Phe Val TTC GTC
.beta.-200th Ala Glu GCC GAG 66 .beta.-176th Tyr Met TAC ATG
.beta.-217th Asp Gly GAC GGC
Example 35
Construction of a Transformant (67) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0353] In order to obtain a transformant (67) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(n-2) and (aj) amino acid substitution sites as shown in Table 25,
the plasmid (64) recovered from the transformant (64) described in
the above Reference Example 27 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 46 and 68 in the
Sequence Listing were used for repeatedly carrying out the method
described in Reference Example 2 per mutation point, whereby a
plasmid (67) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (67). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (67) had sequences according to the purpose in which
mutation of 27th Met in the .alpha.-subunit with Ile and mutation
of 107th Pro in the .beta.-subunit with Met were newly added to the
plasmid (64) of Reference Example 27. In the production of an amide
compound using the thus obtained transformant (67) and the
transformant (64) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0354] As a result, it was found that mutation of 27th Met in the
.alpha.-subunit with Ile and mutation of 107th Pro in the
.beta.-subunit with Met were newly added to the transformant (67),
so that the initial reaction rate was improved by 1.53 times and
thermal stability was improved by 2.23 times, as compared to those
of the transformant (64).
Example 36
Construction of a Transformant (68) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0355] In order to obtain a transformant (68) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(n-2) and (as) amino acid substitution sites as shown in Table 25,
the plasmid (65) recovered from the transformant (65) described in
the above Reference Example 28 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 46 and 37 in the
Sequence Listing were used for repeatedly carrying out the method
described in Reference Example 2 per mutation point, whereby a
plasmid (68) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (68). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (68) had sequences according to the purpose in which
mutation of 27th Met in the .alpha.-subunit with Ile and mutation
of 107th Pro in the .beta.-subunit with Met were newly added to the
plasmid (65) of Reference Example 28. In the production of an amide
compound using the thus obtained transformant (68) and the
transformant (65) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0356] As a result, it was found that mutation of 27th Met in the
.alpha.-subunit with Ile and mutation of 107th Pro in the
.beta.-subunit with Met were newly added to the transformant (68),
so that the initial reaction rate was improved by 1.55 times and
thermal stability was improved by 2.15 times, as compared to those
of the transformant (65).
Example 37
Construction of a Transformant (69) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0357] In order to obtain a transformant (69) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(n-2) and (bb) amino acid substitution sites as shown in Table 25,
the plasmid (66) recovered from the transformant (66) described in
the above Reference Example 29 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 46 and 37 in the
Sequence Listing were used for repeatedly carrying out the method
described in Reference Example 2 per mutation point, whereby a
plasmid (69) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (69). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (69) had sequences according to the purpose in which
mutation of 27th Met in the .alpha.-subunit with Ile and mutation
of 107th Pro in the .beta.-subunit with Met were newly added to the
plasmid (66) of Reference Example 29. In the production of an amide
compound using the thus obtained transformant (69) and the
transformant (66) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0358] As a result, it was found that mutation of 27th Met in the
.alpha.-subunit with Ile and mutation of 107th Pro in the
.beta.-subunit with Met were newly added to the transformant (69),
so that the initial reaction rate was improved by 1.46 times and
thermal stability was improved by 1.92 times, as compared to those
of the transformant (66).
TABLE-US-00038 TABLE 25 Improvement of Change in Amino Acid Change
in Base Improvement of Thermal Sequence Sequence Reaction Rate by
Stability by Trans- Before After Before After .alpha.-27th and
.alpha.-27th and formant Mutated Substitu- Substitu- Substitu-
Substitu- .beta.-107th .beta.-107th No. Site tion tion tion tion
Substitutions Substitutions 67 .alpha.-27th Met Ile ATG ATC 1.53
times 2.23 times .beta.-37th Phe Leu TTC CTC .beta.-107th Pro Met
CCC ATG .beta.-108th Glu Asp GAG GAT .beta.-200th Ala Glu GCC GAG
68 .alpha.-6th Leu Thr CTG ACG 1.55 times 2.15 times .alpha.-27th
Met Ile ATG ATC .alpha.-36th Thr Met ACG ATG .alpha.-126th Phe Tyr
TTC TAC .beta.-10th Thr Asp ACC GAC .beta.-107th Pro Met CCC ATG
.beta.-118th Phe Val TTC GTC .beta.-200th Ala Glu GCC GAG 69
.alpha.-27th Met Ile ATG ATC 1.46 times 1.92 times .beta.-107th Pro
Met CCC ATG .beta.-176th Tyr Met TAC ATG .beta.-217th Asp Gly GAC
GGC
Reference Example 30
Construction of a Transformant (70) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0359] In order to obtain a transformant (70) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(al) amino acid substitution sites as shown in Table 26, the plasma
described in Example 69 of Patent Document 2 was used as the
template, and the ribosome binding sequence was modified according
to the method described in Example 1 to prepare a plasmid (70)
encoding the above nitrile hydratase variant. A competent cell of
Escherichia coli HB101 (manufactured by Toyobo Co., Ltd.) was
transformed with the plasmid to obtain a transformant (70).
Reference Example 31
Construction of a Transformant (71) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0360] In order to obtain a transformant (71) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(aw) amino acid substitution sites as shown in Table 26, the plasma
described in Example 80 of Patent Document 2 was used as the
template, and the ribosome binding sequence was modified according
to the method described in Example 1 to prepare a plasmid (71)
encoding the above nitrile hydratase variant. A competent cell of
Escherichia coli HB101 (manufactured by Toyobo Co., Ltd.) was
transformed with the plasmid to obtain a transformant (71).
Reference Example 32
Construction of a Transformant (72) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0361] In order to obtain a transformant (72) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(bl) amino acid substitution sites as shown in Table 26,
introduction of site-specific mutation was performed using the
mutagenesis kit described in the above Reference Example 2. The
plasmid (1) expressing nitrile hydratase with the modified ribosome
binding sequence described in Example 1 was used as the template,
and the primers having the sequence defined in SEQ ID Nos: 49 and
50 in the Sequence Listing were used for repeatedly carrying out
the method described in Reference Example 2 per mutation point,
whereby a plasmid (72) encoding the above nitrile hydratase variant
was prepared. A competent cell of Escherichia coli HB101
(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid
to obtain a transformant (72).
TABLE-US-00039 TABLE 26 Change in Amino Change in Trans- Acid
Sequence Base Sequence formant Mutated Before After Before After
No. Site Substitution Substitution Substitution Substitution 70
.beta.-41st Phe Ile TTC ATC .beta.-51st Phe Val TTC GTC
.beta.-108th Glu Asp GAG GAT 71 .alpha.-148th Gly Asp GGC GAC
.alpha.-204th Val Arg GTC CGC .beta.-108th Glu Asp GAG GAT
.beta.-200th Ala Glu GCC GAG 72 .beta.-61st Ala Leu GCC CTC
.beta.-112th Lys Ile AAG ATT
Example 38
Construction of a Transformant (73) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0362] In order to obtain a transformant (73) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(q) and (al) amino acid substitution sites as shown in Table 27,
the plasmid (70) recovered from the transformant (70) described in
the above Reference Example 30 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 16 and 38 in the
Sequence Listing were used for repeatedly carrying out the method
described in Reference Example 2 per mutation point, whereby a
plasmid (73) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (73). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (73) had sequences according to the purpose in which
mutation of 92nd Asp in the .alpha.-subunit with Glu and mutation
of 226th Val in the .beta.-subunit with Ile were newly added to the
plasmid (70) of Reference Example 30. In the production of an amide
compound using the thus obtained transformant (73) and the
transformant (70) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0363] As a result, it was found that mutation of 92nd Asp in the
.alpha.-subunit with Glu and mutation of 226th Val in the
.beta.-subunit with Ile were newly added to the transformant (73),
so that the initial reaction rate was improved by 2.00 times and
thermal stability was improved by 1.52 times, as compared to those
of the transformant (70).
Example 39
Construction of a Transformant (74) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0364] In order to obtain a transformant (74) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(q) and (aw) amino acid substitution sites as shown in Table 27,
the plasmid (71) recovered from the transformant (71) described in
the above Reference Example 31 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 16 and 38 in the
Sequence Listing were used for repeatedly carrying out the method
described in Reference Example 2 per mutation point, whereby a
plasmid (74) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (74). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (74) had sequences according to the purpose in which
mutation of 92nd Asp in the .alpha.-subunit with Glu and mutation
of 226th Val in the .beta.-subunit with Ile were newly added to the
plasmid (71) of Reference Example 31. In the production of an amide
compound using the thus obtained transformant (74) and the
transformant (71) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0365] As a result, it was found that mutation of 92nd Asp in the
.alpha.-subunit with Glu and mutation of 226th Val in the
.beta.-subunit with Ile were newly added to the transformant (74),
so that the initial reaction rate was improved by 1.78 times and
thermal stability was improved by 1.44 times, as compared to those
of the transformant (71).
Example 40
Construction of a Transformant (75) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0366] In order to obtain a transformant (75) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(q) and (bl) amino acid substitution sites as shown in Table 27,
the plasmid (72) recovered from the transformant (72) described in
the above Reference Example 32 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 16 and 38 in the
Sequence Listing were used for repeatedly carrying out the method
described in Reference Example 2 per mutation point, whereby a
plasmid (75) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (75). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (75) had sequences according to the purpose in which
mutation of 92nd Asp in the .alpha.-subunit with Glu and mutation
of 226th Val in the .beta.-subunit with Ile were newly added to the
plasmid (72) of Reference Example 32. In the production of an amide
compound using the thus obtained transformant (75) and the
transformant (72) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0367] As a result, it was found that mutation of 92nd Asp in the
.alpha.-subunit with Glu and mutation of 226th Val in the
.beta.-subunit with Ile were newly added to the transformant (75),
so that the initial reaction rate was improved by 1.85 times and
thermal stability was improved by 1.38 times, as compared to those
of the transformant (72).
TABLE-US-00040 TABLE 27 Improvement of Change in Amino Acid Change
in Base Improvement of Thermal Sequence Sequence Reaction Rate by
Stability by Trans- Before After Before After .alpha.-92nd and
.alpha.-92nd and formant Mutated Substitu- Substitu- Substitu-
Substitu- .beta.-226th .beta.-226th No. Site tion tion tion tion
Substitution Substitution 73 .alpha.-92nd Asp Glu GAC GAG 2.00
times 1.52 times .beta.-41st Phe Ile TTC ATC .beta.-51st Phe Val
TTC GTC .beta.-108th Glu Asp GAG GAT .beta.-226th Val Ile GTC ATC
74 .alpha.-92nd Asp Glu GAC GAG 1.78 times 1.44 times .alpha.-148th
Gly Asp GGC GAC .alpha.-204th Val Arg GTC CGC .beta.-108th Glu Asp
GAG GAT .beta.-200th Ala Glu GCC GAG .beta.-226th Val Ile GTC ATC
75 .alpha.-92nd Asp Glu GAC GAG 1.85 times 1.38 times .beta.-61st
Ala Leu GCC CTC .beta.-112th Lys Ile AAG ATT .beta.-226th Val Ile
GTC ATC
Reference Example 33
Construction of a Transformant (76) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0368] In order to obtain a transformant (76) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(bi) amino acid substitution sites as shown in Table 28,
introduction of site-specific mutation was performed using the
mutagenesis kit described in the above Reference Example 2. The
plasmid (1) expressing nitrile hydratase with the modified ribosome
binding sequence described in Example 1 was used as the template,
and the primers having the sequence defined in SEQ ID Nos: 51 and
52 in the Sequence Listing were used for repeatedly carrying out
the method described in Reference Example 2 per mutation point,
whereby a plasmid (76) encoding the above nitrile hydratase variant
was prepared. A competent cell of Escherichia coli HB101
(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid
to obtain a transformant (76).
Reference Example 34
Construction of a Transformant (77) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0369] In order to obtain a transformant (77) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(bm) amino acid substitution sites as shown in Table 28,
introduction of site-specific mutation was performed using the
mutagenesis kit described in the above Reference Example 2. The
plasmid (1) expressing nitrile hydratase with the modified ribosome
binding sequence described in Example 1 was used as the template,
and the primers having the sequence defined in SEQ ID Nos: 53 and
54 in the Sequence Listing were used for repeatedly carrying out
the method described in Reference Example 2 per mutation point,
whereby a plasmid (77) encoding the above nitrile hydratase variant
was prepared. A competent cell of Escherichia coli HB101
(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid
to obtain a transformant (77).
TABLE-US-00041 TABLE 28 Change in Amino Change in Trans- Acid
Sequence Base Sequence formant Mutated Before After Before After
No. Site Substitution Substitution Substitution Substitution 76
.beta.-61st Ala Thr GCC ACG .beta.-218th Cys Ser TGC TCC 77
.beta.-146th Arg Gly CGG GGG .beta.-217th Asp Ser GAC AGC
Example 41
Construction of a Transformant (78) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0370] In order to obtain a transformant (78) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(o) and (an) amino acid substitution sites as shown in Table 29,
the plasmid (14) recovered from the transformant (14) described in
the above Reference Example 7 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 29 and 33 in the
Sequence Listing were used for repeatedly carrying out the method
described in Reference Example 2 per mutation point, whereby a
plasmid (78) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (78). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (78) had sequences according to the purpose in which
mutation of 4th Val in the .beta.-subunit with Met and mutation of
79th His in the .beta.-subunit with Asn were newly added to the
plasmid (14) of Reference Example 7. In the production of an amide
compound using the thus obtained transformant (78) and the
transformant (14) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0371] As a result, it was found that mutation of 4th Val in the
.beta.-subunit with Met and mutation of 79th His in the
.beta.-subunit with Asn were newly added to the transformant (78),
so that the initial reaction rate was improved by 1.60 times and
thermal stability was improved by 1.46 times, as compared to those
of the transformant (14).
Example 42
Construction of a Transformant (79) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0372] In order to obtain a transformant (79) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(o) and (bi) amino acid substitution sites as shown in Table 29,
the plasmid (76) recovered from the transformant (76) described in
the above Reference Example 33 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 29 and 33 in the
Sequence Listing were used for repeatedly carrying out the method
described in Reference Example 2 per mutation point, whereby a
plasmid (79) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (79). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (79) had sequences according to the purpose in which
mutation of 4th Val in the .beta.-subunit with Met and mutation of
79th His in the .beta.-subunit with Asn were newly added to the
plasmid (76) of Reference Example 33. In the production of an amide
compound using the thus obtained transformant (79) and the
transformant (76) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0373] As a result, it was found that mutation of 4th Val in the
.beta.-subunit with Met and mutation of 79th His in the
.beta.-subunit with Asn were newly added to the transformant (79),
so that the initial reaction rate was improved by 1.38 times and
thermal stability was improved by 1.35 times, as compared to those
of the transformant (76).
Example 43
Construction of a Transformant (80) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0374] In order to obtain a transformant (80) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(o) and (bm) amino acid substitution sites as shown in Table 29,
the plasmid (77) recovered from the transformant (77) described in
the above Reference Example 34 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 29 and 33 in the
Sequence Listing were used for repeatedly carrying out the method
described in Reference Example 2 per mutation point, whereby a
plasmid (80) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (80). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (80) had sequences according to the purpose in which
mutation of 4th Val in the .beta.-subunit with Met and mutation of
79th His in the .beta.-subunit with Asn were newly added to the
plasmid (77) of Reference Example 34. In the production of an amide
compound using the thus obtained transformant (80) and the
transformant (77) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0375] As a result, it was found that mutation of 4th Val in the
.beta.-subunit with Met and mutation of 79th His in the
.beta.-subunit with Asn were newly added to the transformant (80),
so that the initial reaction rate was improved by 1.52 times and
thermal stability was improved by 1.28 times, as compared to those
of the transformant (77).
TABLE-US-00042 TABLE 29 Improvement of Change in Amino Acid Change
in Base Improvement of Thermal Sequence Sequence Reaction Rate by
Stability by Trans- Before After Before After .beta.-4th and
.beta.-4th and formant Mutated Substitu- Substitu- Substitu-
Substitu- .beta.-79th .beta.-79th No. Site tion tion tion tion
Substitution Substitution 78 .beta.-4th Val Met GTG ATG 1.60 times
1.46 times .beta.-48th Leu Val CTG GTG .beta.-79th His Asn CAC AAC
.beta.-108th Glu Arg GAG CGG .beta.-212th Ser Tyr TCC TAC 79
.beta.-4th Val Met GTG ATG 1.38 times 1.35 times .beta.-61st Ala
Thr GCC ACG .beta.-79th His Asn CAC AAC .beta.-218th Cys Ser TGC
TCC 80 .beta.-4th Val Met GTG ATG 1.52 times 1.28 times .beta.-79th
His Asn CAC AAC .beta.-146th Arg Gly CGG GGG .beta.-217th Asp Ser
GAC AGC
Reference Example 35
Construction of a Transformant (81) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0376] In order to obtain a transformant (81) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(ag) amino acid substitution sites as shown in Table 30, the plasma
described in Example 64 of Patent Document 2 was used as the
template, and the ribosome binding sequence was modified according
to the method described in Example 1 to prepare a plasmid (81)
encoding the above nitrile hydratase variant. A competent cell of
Escherichia coli HB101 (manufactured by Toyobo Co., Ltd.) was
transformed with the plasmid to obtain a transformant (81).
Reference Example 36
Construction of a Transformant (82) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0377] In order to obtain a transformant (82) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(ai) amino acid substitution sites as shown in Table 30, the plasma
described in Example 66 of Patent Document 2 was used as the
template, and the ribosome binding sequence was modified according
to the method described in Example 1 to prepare a plasmid (82)
encoding the above nitrile hydratase variant. A competent cell of
Escherichia coli HB101 (manufactured by Toyobo Co., Ltd.) was
transformed with the plasmid to obtain a transformant (82).
TABLE-US-00043 TABLE 30 Change in Amino Change in Trans- Acid
Sequence Base Sequence formant Mutated Before After Before After
No. Site Substitution Substitution Substitution Substitution 81
.alpha.-19th Ala Val GCG GTG .alpha.-71st Arg His CGT CAT
.alpha.-126th Phe Tyr TTC TAC 82 .beta.-10th Thr Asp ACC GAC
.beta.-118th Phe Val TTC GTC .beta.-200th Ala Glu GCC GAG
Example 44
Construction of a Transformant (83) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0378] In order to obtain a transformant (83) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(p) and (ag) amino acid substitution sites as shown in Table 31,
the plasmid (81) recovered from the transformant (81) described in
the above Reference Example 35 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 33 and 60 in the
Sequence Listing were used for repeatedly carrying out the method
described in Reference Example 2 per mutation point, whereby a
plasmid (83) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (83). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (83) had sequences according to the purpose in which
mutation of 79th His in the .beta.-subunit with Asn and mutation of
230th Ala in the .beta.-subunit with Glu were newly added to the
plasmid (81) of Reference Example 35. In the production of an amide
compound using the thus obtained transformant (83) and the
transformant (81) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0379] As a result, it was found that mutation of 79th His in the
.beta.-subunit with Asn and mutation of 230th Ala in the
.beta.-subunit with Glu were newly added to the transformant (83),
so that the initial reaction rate was improved by 1.25 times and
thermal stability was improved by 2.16 times, as compared to those
of the transformant (81).
Example 45
Construction of a Transformant (84) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0380] In order to obtain a transformant (84) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(p) and (ai) amino acid substitution sites as shown in Table 31,
the plasmid (82) recovered from the transformant (82) described in
the above Reference Example 36 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 33 and 60 in the
Sequence Listing were used for repeatedly carrying out the method
described in Reference Example 2 per mutation point, whereby a
plasmid (84) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (84). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (84) had sequences according to the purpose in which
mutation of 79th His in the .beta.-subunit with Asn and mutation of
230th Ala in the .beta.-subunit with Glu were newly added to the
plasmid (82) of Reference Example 36. In the production of an amide
compound using the thus obtained transformant (84) and the
transformant (82) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0381] As a result, it was found that mutation of 79th His in the
.beta.-subunit with Asn and mutation of 230th Ala in the
.beta.-subunit with Glu were newly added to the transformant (84),
so that the initial reaction rate was improved by 1.27 times and
thermal stability was improved by 2.10 times, as compared to those
of the transformant (82).
Example 46
Construction (85) of a Transformant (85) Substituted Amino Acid
Having Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0382] In order to obtain a transformant (85) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(p) and (aq) amino acid substitution sites as shown in Table 31,
the plasmid (38) recovered from the transformant (38) described in
the above Reference Example 19 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 33 and 60 in the
Sequence Listing were used for repeatedly carrying out the method
described in Reference Example 2 per mutation point, whereby a
plasmid (85) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (85). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (85) had sequences according to the purpose in which
mutation of 79th His in the .beta.-subunit with Asn and mutation of
230th Ala in the .beta.-subunit with Glu were newly added to the
plasmid (38) of Reference Example 31. In the production of an amide
compound using the thus obtained transformant (85) and the
transformant (38) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0383] As a result, it was found that mutation of 79th His in the
.beta.-subunit with Asn and mutation of 230th Ala in the
.beta.-subunit with Glu were newly added to the transformant (85),
so that the initial reaction rate was improved by 1.33 times and
thermal stability was improved by 2.52 times, as compared to those
of the transformant (38).
TABLE-US-00044 TABLE 31 Improvement of Change in Amino Acid Change
in Base Improvement of Thermal Sequence Sequence Reaction Rate by
Stability by Trans- Before After Before After .beta.-79th and
.beta.-79th and formant Mutated Substitu- Substitu- Substitu-
Substitu- .beta.-230th .beta.-230th No. Site tion tion tion tion
Substitution Substitution 83 .alpha.-19th Ala Val GCG GTG 1.25
times 2.16 times .alpha.-71st Arg His CGT CAT .alpha.-126th Phe Tyr
TTC TAC .beta.-79th His Asn CAC AAC .beta.-230th Ala Glu GCG GAG 84
.beta.-10th Thr Asp ACC GAC 1.27 times 2.10 times .beta.-79th His
Asn CAC AAC .beta.-118th Phe Val TTC GTC .beta.-200th Ala Glu GCC
GAG .beta.-230th Ala Glu GCG GAG 85 .alpha.-6th Leu Thr CTG ACG
1.33 times 2.52 times .alpha.-19th Ala Val GCG GTG .alpha.-126th
Phe Tyr TTC TAC .beta.-48th Leu Val CTG GTG .beta.-79th His Asn CAC
AAC .beta.-108th Glu Arg GAG CGG .beta.-212th Ser Tyr TCC TAC
.beta.-230th Ala Glu GCG GAG
Reference Example 37
Construction of a Transformant (86) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0384] In order to obtain a transformant (86) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(ad) amino acid substitution sites as shown in Table 32, the plasma
described in Example 61 of Patent Document 2 was used as the
template, and the ribosome binding sequence was modified according
to the method described in Example 1 to prepare a plasmid (86)
encoding the above nitrile hydratase variant. A competent cell of
Escherichia coli HB101 (manufactured by Toyobo Co., Ltd.) was
transformed with the plasmid to obtain a transformant (86).
Reference Example 38
Construction of a Transformant (87) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0385] In order to obtain a transformant (87) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(bg) amino acid substitution sites as shown in Table 32,
introduction of site-specific mutation was performed using the
mutagenesis kit described in the above Reference Example 2. The
plasmid (1) expressing nitrile hydratase with the modified ribosome
binding sequence described in Example 1 was used as the template,
and the primers having the sequence defined in SEQ ID Nos: 55 and
56 in the Sequence Listing were used for repeatedly carrying out
the method described in Reference Example 2 per mutation point,
whereby a plasmid (87) encoding the above nitrile hydratase variant
was prepared. A competent cell of Escherichia coli HB101
(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid
to obtain a transformant (87).
TABLE-US-00045 TABLE 32 Change in Amino Change in Trans- Acid
Sequence Base Sequence formant Mutated Before After Before After
No. Site Substitution Substitution Substitution Substitution 86
.beta.-118th Phe Val TTC GTC .beta.-200th Ala Glu GCC GAG 87
.beta.-40th Thr Leu ACG CTG .beta.-217th Asp Leu GAC CTC
Example 47
Construction of a Transformant (88) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0386] In order to obtain a transformant (88) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(j) and (ad) amino acid substitution sites as shown in Table 33,
the plasmid (86) recovered from the transformant (86) described in
the above Reference Example 37 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 57 and 58 in the
Sequence Listing were used for repeatedly carrying out the method
described in Reference Example 2 per mutation point, whereby a
plasmid (88) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (88). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (88) had sequences according to the purpose in which
mutation of 110th Glu in the .beta.-subunit with Asn and mutation
of 231st Ala in the .beta.-subunit with Val were newly added to the
plasmid (86) of Reference Example 37. In the production of an amide
compound using the thus obtained transformant (88) and the
transformant (86) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0387] As a result, it was found that mutation of 110th Glu in the
.beta.-subunit with Asn and mutation of 231st Ala in the
.beta.-subunit with Val were newly added to the transformant (88),
so that the initial reaction rate was improved by 1.29 times and
thermal stability was improved by 1.62 times, as compared to those
of the transformant (86).
Example 48
Construction of a Transformant (89) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0388] In order to obtain a transformant (89) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(j) and (aj) amino acid substitution sites as shown in Table 33,
the plasmid (64) recovered from the transformant (64) described in
the above Reference Example 27 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 57 and 58 in the
Sequence Listing were used for repeatedly carrying out the method
described in Reference Example 2 per mutation point, whereby a
plasmid (89) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (89). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (89) had sequences according to the purpose in which
mutation of 110th Glu in the .beta.-subunit with Asn and mutation
of 231st Ala in the .beta.-subunit with Val were newly added to the
plasmid (64) of Reference Example 27. In the production of an amide
compound using the thus obtained transformant (89) and the
transformant (64) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0389] As a result, it was found that mutation of 110th Glu in the
.beta.-subunit with Asn and mutation of 231st Ala in the
.beta.-subunit with Val were newly added to the transformant (89),
so that the initial reaction rate was improved by 1.34 times and
thermal stability was improved by 1.83 times, as compared to those
of the transformant (64).
Example 49
Construction of a Transformant (90) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0390] In order to obtain a transformant (90) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(j) and (bg) amino acid substitution sites as shown in Table 33,
the plasmid (87) recovered from the transformant (87) described in
the above Reference Example 38 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 57 and 58 in the
Sequence Listing were used for repeatedly carrying out the method
described in Reference Example 2 per mutation point, whereby a
plasmid (90) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (90). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (90) had sequences according to the purpose in which
mutation of 110th Glu in the .beta.-subunit with Asn and mutation
of 231st Ala in the .beta.-subunit with Val were newly added to the
plasmid (87) of Reference Example 38. In the production of an amide
compound using the thus obtained transformant (90) and the
transformant (87) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0391] As a result, it was found that mutation of 110th Glu in the
.beta.-subunit with Asn and mutation of 231st Ala in the
.beta.-subunit with Val were newly added to the transformant (90),
so that the initial reaction rate was improved by 1.25 times and
thermal stability was improved by 1.46 times, as compared to those
of the transformant (87).
TABLE-US-00046 TABLE 33 Improvement of Change in Amino Acid Change
in Base Improvement of Thermal Sequence Sequence Reaction Rate by
Stability by Trans- Before After Before After .beta.-110th and
.beta.-110th and formant Mutated Substitu- Substitu- Substitu-
Substitu- .beta.-231st .beta.-231st No. Site tion tion tion tion
Substitution Substitution 88 .beta.-110th Glu Asn GAG AAC 1.29
times 1.62 times .beta.-118th Phe Val TTC GTC .beta.-200th Ala Glu
GCC GAG .beta.-231st Ala Val GCC GTC 89 .beta.-37th Phe Leu TTC CTC
1.34 times 1.83 times .beta.-108th Glu Asp GAG GAT .beta.-110th Glu
Asn GAG AAC .beta.-200th Ala Glu GCC GAG .beta.-231st Ala Val GCC
GTC 90 .beta.-40th Thr Leu ACG CTG 1.25 times 1.46 times
.beta.-110th Glu Asn GAG AAC .beta.-217th Asp Leu GAC CTC
.beta.-231st Ala Val GCC GTC
Example 50
Construction of a Transformant (91) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0392] In order to obtain a transformant (91) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(k) and (ad) amino acid substitution sites as shown in Table 34,
the plasmid (86) recovered from the transformant (86) described in
the above Reference Example 37 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 59 and 60 in the
Sequence Listing were used for repeatedly carrying out the method
described in Reference Example 2 per mutation point, whereby a
plasmid (91) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (91). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (91) had sequences according to the purpose in which
mutation of 206th Pro in the .beta.-subunit with Leu and mutation
of 230th Ala in the .beta.-subunit with Glu were newly added to the
plasmid (86) of Reference Example 37. In the production of an amide
compound using the thus obtained transformant (91) and the
transformant (86) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0393] As a result, it was found that mutation of 206th Pro in the
.beta.-subunit with Leu and mutation of 230th Ala in the
.beta.-subunit with Glu were newly added to the transformant (91),
so that the initial reaction rate was improved by 1.44 times and
thermal stability was improved by 1.42 times, as compared to those
of the transformant (86).
Example 51
Construction of a Transformant (92) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0394] In order to obtain a transformant (92) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(k) and (as) amino acid substitution sites as shown in Table 34,
the plasmid (65) recovered from the transformant (65) described in
the above Reference Example 28 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 59 and 60 in the
Sequence Listing were used for repeatedly carrying out the method
described in Reference Example 2 per mutation point, whereby a
plasmid (92) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (92). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (92) had sequences according to the purpose in which
mutation of 206th Pro in the .beta.-subunit with Leu and mutation
of 230th Ala in the .beta.-subunit with Glu were newly added to the
plasmid (65) of Reference Example 28. In the production of an amide
compound using the thus obtained transformant (92) and the
transformant (65) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0395] As a result, it was found that mutation of 206th Pro in the
.beta.-subunit with Leu and mutation of 230th Ala in the
.beta.-subunit with Glu were newly added to the transformant (92),
so that the initial reaction rate was improved by 1.48 times and
thermal stability was improved by 1.39 times, as compared to those
of the transformant (65).
Example 52
Construction of a Transformant (93) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0396] In order to obtain a transformant (93) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(k) and (av) amino acid substitution sites as shown in Table 34,
the plasmid (2) recovered from the transformant (2) described in
the above Reference Example 1 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 59 and 60 in the
Sequence Listing were used for repeatedly carrying out the method
described in Reference Example 2 per mutation point, whereby a
plasmid (93) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (93). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (93) had sequences according to the purpose in which
mutation of 206th Pro in the .beta.-subunit with Leu and mutation
of 230th Ala in the .beta.-subunit with Glu were newly added to the
plasmid (2) of Reference Example 1. In the production of an amide
compound using the thus obtained transformant (93) and the
transformant (2) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0397] As a result, it was found that mutation of 206th Pro in the
.beta.-subunit with Leu and mutation of 230th Ala in the
.beta.-subunit with Glu were newly added to the transformant (93),
so that the initial reaction rate was improved by 1.36 times and
thermal stability was improved by 1.52 times, as compared to those
of the transformant (2).
TABLE-US-00047 TABLE 34 Improvement of Change in Amino Acid Change
in Base Improvement of Thermal Sequence Sequence Reaction Rate by
Stability by Trans- Before After Before After .beta.-206th and
.beta.-206th and formant Mutated Substitu- Substitu- Substitu-
Substitu- .beta.-230th .beta.-230th No. Site tion tion tion tion
Substitution Substitution 91 .beta.-118th Phe Val TCC GTC 1.44
times 1.42 times .beta.-200th Ala Glu GCC GAG .beta.-206th Pro Leu
CCG CTG .beta.-230th Ala Glu GCG GAG 92 .alpha.-6th Leu Thr CTG ACG
1.48 times 1.39 times .alpha.-36th Thr Met ACG ATG .alpha.-126th
Phe Tyr TTC TAC .beta.-10th Thr Asp ACC GAC .beta.-118th Phe Val
TTC GTC .beta.-200th Ala Glu GCC GAG .beta.-206th Pro Leu CCG CTG
.beta.-230th Ala Glu GCG GAG 93 .alpha.-36th Thr Met ACG ATG 1.36
times 1.52 times .alpha.-148th Gly Asp GGC GAC .alpha.-204th Val
Arg GTC CGC .beta.-41st Phe Ile TTC ATC .beta.-51st Phe Val TTC GTC
.beta.-108th Glu Asp GAG GAT .beta.-206th Pro Leu CCG CTG
.beta.-230th Ala Glu GCG GAG
Reference Example 39
Construction of a Transformant (94) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0398] In order to obtain a transformant (94) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(bo) amino acid substitution sites as shown in Table 35,
introduction of site-specific mutation was performed using the
mutagenesis kit described in the above Reference Example 2. The
plasmid (1) expressing nitrile hydratase with the modified ribosome
binding sequence described in Example 1 was used as the template,
and the primers having the sequence defined in SEQ ID Nos: 61 and
62 in the Sequence Listing were used for repeatedly carrying out
the method described in Reference Example 2 per mutation point,
whereby a plasmid (94) encoding the above nitrile hydratase variant
was prepared. A competent cell of Escherichia coli HB101
(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid
to obtain a transformant (94).
TABLE-US-00048 TABLE 35 Change in Amino Change in Trans- Acid
Sequence Base Sequence formant Mutated Before After Before After
No. Site Substitution Substitution Substitution Substitution 94
.beta.-150th Ala Ser GCG TCG .beta.-217th Asp Cys GAC TGT
Example 53
Construction of a Transformant (95) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0399] In order to obtain a transformant (95) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(1) and (ag) amino acid substitution sites as shown in Table 36,
the plasmid (81) recovered from the transformant (81) described in
the above Reference Example 35 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 43, 46 and 57 in
the Sequence Listing were used for repeatedly carrying out the
method described in Reference Example 2 per mutation point, whereby
a plasmid (95) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (95). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (95) had sequences according to the purpose in which
mutation of 13th Ile in the .alpha.-subunit with Leu, mutation of
27th Met in the .alpha.-subunit with Ile and mutation of 110th Glu
in the .beta.-subunit with Asn were newly added to the plasmid (81)
of Reference Example 35. In the production of an amide compound
using the thus obtained transformant (95) and the transformant (81)
to be its base, the initial reaction rate and thermal stability
were compared in the same manner as in Example 1.
[0400] As a result, it was found that mutation of 13th Ile in the
.alpha.-subunit with Leu, mutation of 27th Met in the
.alpha.-subunit with Ile and mutation of 110th Glu in the
.beta.-subunit with Asn were newly added to the transformant (95),
so that the initial reaction rate was improved by 1.53 times and
thermal stability was improved by 1.76 times, as compared to those
of the transformant (81).
Example 54
Construction of a Transformant (96) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0401] In order to obtain a transformant (96) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(1) and (am) amino acid substitution sites as shown in Table 36,
the plasmid (56) recovered from the transformant (56) described in
the above Reference Example 25 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 43, 46 and 57 in
the Sequence Listing were used for repeatedly carrying out the
method described in Reference Example 2 per mutation point, whereby
a plasmid (96) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (96). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (96) had sequences according to the purpose in which
mutation of 13th Ile in the .alpha.-subunit with Leu, mutation of
27th Met in the .alpha.-subunit with Ile and mutation of 110th Glu
in the .beta.-subunit with Asn were newly added to the plasmid (56)
of Reference Example 25. In the production of an amide compound
using the thus obtained transformant (96) and the transformant (56)
to be its base, the initial reaction rate and thermal stability
were compared in the same manner as in Example 1.
[0402] As a result, it was found that mutation of 13th Ile in the
.alpha.-subunit with Leu, mutation of 27th Met in the
.alpha.-subunit with Ile and mutation of 110th Glu in the
.beta.-subunit with Asn were newly added to the transformant (96),
so that the initial reaction rate was improved by 1.49 times and
thermal stability was improved by 1.69 times, as compared to those
of the transformant (56).
Example 55
Construction of a Transformant (97) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0403] In order to obtain a transformant (97) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(1) and (bo) amino acid substitution sites as shown in Table 36,
the plasmid (94) recovered from the transformant (94) described in
the above Reference Example 39 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 43, 46 and 57 in
the Sequence Listing were used for repeatedly carrying out the
method described in Reference Example 2 per mutation point, whereby
a plasmid (97) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (97). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (97) had sequences according to the purpose in which
mutation of 13th Ile in the .alpha.-subunit with Leu, mutation of
27th Met in the .alpha.-subunit with Ile and mutation of 110th Glu
in the .beta.-subunit with Asn were newly added to the plasmid (94)
of Reference Example 39. In the production of an amide compound
using the thus obtained transformant (97) and the transformant (94)
to be its base, the initial reaction rate and thermal stability
were compared in the same manner as in Example 1.
[0404] As a result, it was found that mutation of 13th Ile in the
.alpha.-subunit with Leu, mutation of 27th Met in the
.alpha.-subunit with Ile and mutation of 110th Glu in the
.beta.-subunit with Asn were newly added to the transformant (97),
so that the initial reaction rate was improved by 1.37 times and
thermal stability was improved by 1.83 times, as compared to those
of the transformant (94).
TABLE-US-00049 TABLE 36 Improvement of Change in Amino Acid Change
in Base Improvement of Thermal Sequence Sequence Reaction Rate by
Stability by Trans- Before After Before After .alpha.-13th,
.alpha.-27th .alpha.-13th, .alpha.-27th formant Mutated Substitu-
Substitu- Substitu- Substitu- and .beta.-110th and .beta.-110th No.
Site tion tion tion tion Substitution Substitution 95 .alpha.-13th
Ile Leu ATC CTC 1.53 times 1.76 times .alpha.-19th Ala Val GCG GTG
.alpha.-27th Met Ile ATG ATC .alpha.-71st Arg His CGT CAT
.alpha.-126th Phe Tyr TTC TAC .beta.-110th Glu Asn GAG AAC 96
.alpha.-13th Ile Leu ATC CTC 1.49 times 1.69 times .alpha.-27th Met
Ile ATG ATC .beta.-46th Met Lys ATG AAG .beta.-108th Glu Arg GAG
CGG .beta.-110th Glu Asn GAG AAC .beta.-212th Ser Tyr TCC TAC 97
.alpha.-13th Ile Leu ATC CTC 1.37 times 1.83 times .alpha.-27th Met
Ile ATG ATC .beta.-110th Glu Asn GAG AAC .beta.-150th Ala Ser GCG
TCG .beta.-217th Asp Cys GAC TGT
Reference Example 40
Construction of a Transformant (98) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0405] In order to obtain a transformant (98) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(ab) amino acid substitution sites as shown in Table 37, the plasma
described in Example 59 of Patent Document 2 was used as the
template, and the ribosome binding sequence was modified according
to the method described in Example 1 to prepare a plasmid (98)
encoding the above nitrile hydratase variant. A competent cell of
Escherichia coli HB101 (manufactured by Toyobo Co., Ltd.) was
transformed with the plasmid to obtain a transformant (98).
TABLE-US-00050 TABLE 37 Change in Amino Change in Trans- Acid
Sequence Base Sequence formant Mutated Before After Before After
No. Site Substitution Substitution Substitution Substitution 98
.alpha.-148th Gly Asp GGC GAC .alpha.-204th Val Arg GTC CGC
Example 56
Construction of a Transformant (99) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0406] In order to obtain a transformant (99) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(r) and (ab) amino acid substitution sites as shown in Table 38,
the plasmid (98) recovered from the transformant (98) described in
the above Reference Example 40 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 43, 59 and 38 in
the Sequence Listing were used for repeatedly carrying out the
method described in Reference Example 2 per mutation point, whereby
a plasmid (99) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (99). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (99) had sequences according to the purpose in which
mutation of 13th Ile in the .alpha.-subunit with Leu, mutation of
206th Pro in the .beta.-subunit with Leu and mutation of 226th Val
in the .beta.-subunit with Ile were newly added to the plasmid (98)
of Reference Example 40. In the production of an amide compound
using the thus obtained transformant (99) and the transformant (98)
to be its base, the initial reaction rate and thermal stability
were compared in the same manner as in Example 1.
[0407] As a result, it was found that mutation of 13th Ile in the
.alpha.-subunit with Leu, mutation of 206th Pro in the
.beta.-subunit with Leu and mutation of 226th Val in the
.beta.-subunit with Ile were newly added to the transformant (99),
so that the initial reaction rate was improved by 1.85 times and
thermal stability was improved by 1.36 times, as compared to those
of the transformant (98).
Example 57
Construction of a Transformant (100) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0408] In order to obtain a transformant (100) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(r) and (ai) amino acid substitution sites as shown in Table 38,
the plasmid (82) recovered from the transformant (82) described in
the above Reference Example 36 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 43, 59 and 38 in
the Sequence Listing were used for repeatedly carrying out the
method described in Reference Example 2 per mutation point, whereby
a plasmid (100) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (100). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (100) had sequences according to the purpose in which
mutation of 13th Ile in the .alpha.-subunit with Leu, mutation of
206th Pro in the .beta.-subunit with Leu and mutation of 226th Val
in the .beta.-subunit with Ile were newly added to the plasmid (82)
of Reference Example 36. In the production of an amide compound
using the thus obtained transformant (100) and the transformant
(82) to be its base, the initial reaction rate and thermal
stability were compared in the same manner as in Example 1.
[0409] As a result, it was found that mutation of 13th Ile in the
.alpha.-subunit with Leu, mutation of 206th Pro in the
.beta.-subunit with Leu and mutation of 226th Val in the
.beta.-subunit with Ile were newly added to the transformant (100),
so that the initial reaction rate was improved by 1.72 times and
thermal stability was improved by 1.42 times, as compared to those
of the transformant (82).
Example 58
Construction of a Transformant (101) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0410] In order to obtain a transformant (101) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(r) and (bh) amino acid substitution sites as shown in Table 38,
the plasmid (4) recovered from the transformant (4) described in
the above Reference Example 3 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 43, 59 and 38 in
the Sequence Listing were used for repeatedly carrying out the
method described in Reference Example 2 per mutation point, whereby
a plasmid (101) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (101). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (101) had sequences according to the purpose in which
mutation of 13th Ile in the .alpha.-subunit with Leu, mutation of
206th Pro in the .beta.-subunit with Leu and mutation of 226th Val
in the .beta.-subunit with Ile were newly added to the plasmid (4)
of Reference Example 3. In the production of an amide compound
using the thus obtained transformant (101) and the transformant (4)
to be its base, the initial reaction rate and thermal stability
were compared in the same manner as in Example 1.
[0411] As a result, it was found that mutation of 13th Ile in the
.alpha.-subunit with Leu, mutation of 206th Pro in the
.beta.-subunit with Leu and mutation of 226th Val in the
.beta.-subunit with Ile were newly added to the transformant (101),
so that the initial reaction rate was improved by 1.65 times and
thermal stability was improved by 1.29 times, as compared to those
of the transformant (4).
TABLE-US-00051 TABLE 38 Improvement of Change in Amino Acid Change
in Base Improvement of Thermal Sequence Sequence Reaction Rate by
Stability by Trans- Before After Before After .alpha.-13th,
.beta.-206th .alpha.-13th, .beta.-206th formant Mutated Substitu-
Substitu- Substitu- Substitu- and .beta.-226th and .beta.-226th No.
Site tion tion tion tion Substitution Substitution 99 .alpha.-13th
Ile Leu ATC CTC 1.85 times 1.36 times .alpha.-148th Gly Asp GGC GAC
.alpha.-204th Val Arg GTC CGC .beta.-206th Pro Leu CCG CTG
.beta.-226th Val Ile GTC ATC 100 .alpha.-13th Ile Leu ATC CTC 1.72
times 1.42 times .beta.-10th Thr Asp ACC GAC .beta.-118th Phe Val
TTC GTC .beta.-200th Ala Glu GCC GAG .beta.-206th Pro Leu CCG CTG
.beta.-226th Val Ile GTC ATC 101 .alpha.-13th Ile Leu ATC CTC 1.65
times 1.29 times .beta.-40th Thr Ile ACG ATT .beta.-61st Ala Val
GCC GTC .beta.-206th Pro Leu CCG CTG .beta.-226th Val Ile GTC
ATC
Reference Example 41
Construction of a Transformant (102) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0412] In order to obtain a transformant (102) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(ac) amino acid substitution sites as shown in Table 39, the plasma
described in Example 60 of Patent Document 2 was used as the
template, and the ribosome binding sequence was modified according
to the method described in Example 1 to prepare a plasmid (102)
encoding the above nitrile hydratase variant. A competent cell of
Escherichia coli HB101 (manufactured by Toyobo Co., Ltd.) was
transformed with the plasmid to obtain a transformant (102).
TABLE-US-00052 TABLE 39 Change in Amino Change in Trans- Acid
Sequence Base Sequence formant Mutated Before After Before After
No. Site Substitution Substitution Substitution Substitution 102
.beta.-51st Phe Val TTC GTC .beta.-108th Glu Asp GAG GAT
Example 59
Construction of a Transformant (103) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0413] In order to obtain a transformant (103) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(s) and (ab) amino acid substitution sites as shown in Table 40,
the plasmid (98) recovered from the transformant (98) described in
the above Reference Example 40 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 16, 29 and 59 in
the Sequence Listing were used for repeatedly carrying out the
method described in Reference Example 2 per mutation point, whereby
a plasmid (103) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (103). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (103) had sequences according to the purpose in which
mutation of 92nd Asp in the .alpha.-subunit with Glu, mutation of
4th Val in the .beta.-subunit with Met and mutation of 206th Pro in
the .beta.-subunit with Leu were newly added to the plasmid (98) of
Reference Example 40. In the production of an amide compound using
the thus obtained transformant (103) and the transformant (98) to
be its base, the initial reaction rate and thermal stability were
compared in the same manner as in Example 1.
[0414] As a result, it was found that mutation of 92nd Asp in the
.alpha.-subunit with Glu, mutation of 4th Val in the .beta.-subunit
with Met and mutation of 206th Pro in the .beta.-subunit with Leu
were newly added to the transformant (103), so that the initial
reaction rate was improved by 2.50 times and thermal stability was
improved by 1.57 times, as compared to those of the transformant
(98).
Example 60
Construction of a Transformant (104) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0415] In order to obtain a transformant (104) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(s) and (ah) amino acid substitution sites as shown in Table 40,
the plasmid (37) recovered from the transformant (37) described in
the above Reference Example 18 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 16, 29 and 59 in
the Sequence Listing were used for repeatedly carrying out the
method described in Reference Example 2 per mutation point, whereby
a plasmid (104) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (104). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (104) had sequences according to the purpose in which
mutation of 92nd Asp in the .alpha.-subunit with Glu, mutation of
4th Val in the .beta.-subunit with Met and mutation of 206th Pro in
the .beta.-subunit with Leu were newly added to the plasmid (37) of
Reference Example 18. In the production of an amide compound using
the thus obtained transformant (104) and the transformant (37) to
be its base, the initial reaction rate and thermal stability were
compared in the same manner as in Example 1.
[0416] As a result, it was found that mutation of 92nd Asp in the
.alpha.-subunit with Glu, mutation of 4th Val in the .beta.-subunit
with Met and mutation of 206th Pro in the .beta.-subunit with Leu
were newly added to the transformant (104), so that the initial
reaction rate was improved by 1.82 times and thermal stability was
improved by 1.41 times, as compared to those of the transformant
(37).
Example 61
Construction of a Transformant (105) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0417] In order to obtain a transformant (105) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(s) and (ac) amino acid substitution sites as shown in Table 40,
the plasmid (102) recovered from the transformant (102) described
in the above Reference Example 41 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 16, 29 and 59 in
the Sequence Listing were used for repeatedly carrying out the
method described in Reference Example 2 per mutation point, whereby
a plasmid (105) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (105). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (105) had sequences according to the purpose in which
mutation of 92nd Asp in the .alpha.-subunit with Glu, mutation of
4th Val in the .beta.-subunit with Met and mutation of 206th Pro in
the .beta.-subunit with Leu were newly added to the plasmid (102)
of Reference Example 41. In the production of an amide compound
using the thus obtained transformant (105) and the transformant
(102) to be its base, the initial reaction rate and thermal
stability were compared in the same manner as in Example 1.
[0418] As a result, it was found that mutation of 92nd Asp in the
.alpha.-subunit with Glu, mutation of 4th Val in the .beta.-subunit
with Met and mutation of 206th Pro in the .beta.-subunit with Leu
were newly added to the transformant (105), so that the initial
reaction rate was improved by 1.67 times and thermal stability was
improved by 1.61 times, as compared to those of the transformant
(102).
TABLE-US-00053 TABLE 40 Improvement of Change in Amino Acid Change
in Base Improvement of Thermal Sequence Sequence Reaction Rate by
Stability by Trans- Before After Before After .alpha.-92nd,
.beta.-4th .alpha.-92nd, .beta.-4th formant Mutated Substitu-
Substitu- Substitu- Substitu- and .beta.-206th and .beta.-206th No.
Site tion tion tion tion Substitution Substitution 103 .alpha.-92nd
Asp Glu GAC GAG 2.50 times 1.57 times .alpha.-148th Gly Asp GGC GAC
.alpha.-204th Val Arg GTC CGC .beta.-4th Val Met GTG ATG
.beta.-206th Pro Leu CCG CTG 104 .alpha.-36th Thr Met ACG ATG 1.82
times 1.41 times .alpha.-92nd Asp Glu GAC GAG .alpha.-148th Gly Asp
GGC GAC .alpha.-204th Val Arg GTC CGC .beta.-4th Val Met GTG ATG
.beta.-206th Pro Leu CCG CTG 105 .alpha.-92nd Asp Glu GAC GAG 1.67
times 1.61 times .beta.-4th Val Met GTG ATG .beta.-51st Phe Val TTC
GTC .beta.-108th Glu Asp GAG GAT .beta.-206th Pro Leu CCG CTG
Reference Example 42
Construction of a Transformant (106) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0419] In order to obtain a transformant (106) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(az) amino acid substitution sites as shown in Table 41,
introduction of site-specific mutation was performed using the
mutagenesis kit described in the above Reference Example 2. The
plasmid (1) expressing nitrile hydratase with the modified ribosome
binding sequence described in Example 1 was used as the template,
and the primers having the sequence defined in SEQ ID Nos: 65 and
53 in the Sequence Listing were used for repeatedly carrying out
the method described in Reference Example 2 per mutation point,
whereby a plasmid (106) encoding the above nitrile hydratase
variant was prepared. A competent cell of Escherichia coli HB101
(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid
to obtain a transformant (106).
TABLE-US-00054 TABLE 41 Change in Amino Change in Trans- Acid
Sequence Base Sequence formant Mutated Before After Before After
No. Site Substitution Substitution Substitution Substitution 106
.alpha.-48th Asn Glu AAC GAA .alpha.-146th Arg Gly CGG GGG
Example 62
Construction of a Transformant (107) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0420] In order to obtain a transformant (107) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(t) and (ac) amino acid substitution sites as shown in Table 42,
the plasmid (102) recovered from the transformant (102) described
in the above Reference Example 41 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 24, 37 and 60 in
the Sequence Listing were used for repeatedly carrying out the
method described in Reference Example 2 per mutation point, whereby
a plasmid (107) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (107). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (107) had sequences according to the purpose in which
mutation of 197th Gly in the .alpha.-subunit with Cys, mutation of
107th Pro in the .beta.-subunit with Met and mutation of 230th Ala
in the .beta.-subunit with Glu were newly added to the plasmid
(102) of Reference Example 41. In the production of an amide
compound using the thus obtained transformant (107) and the
transformant (102) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0421] As a result, it was found that mutation of 197th Gly in the
.alpha.-subunit with Cys, mutation of 107th Pro in the
.beta.-subunit with Met and mutation of 230th Ala in the
.beta.-subunit with Glu were newly added to the transformant (107),
so that the initial reaction rate was improved by 2.11 times and
thermal stability was improved by 1.88 times, as compared to those
of the transformant (102).
Example 63
Construction of a Transformant (108) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0422] In order to obtain a transformant (108) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(t) and (al) amino acid substitution sites as shown in Table 42,
the plasmid (70) recovered from the transformant (70) described in
the above Reference Example 30 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 24, 37 and 60 in
the Sequence Listing were used for repeatedly carrying out the
method described in Reference Example 2 per mutation point, whereby
a plasmid (108) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (108). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (108) had sequences according to the purpose in which
mutation of 197th Gly in the .alpha.-subunit with Cys, mutation of
107th Pro in the .beta.-subunit with Met and mutation of 230th Ala
in the .beta.-subunit with Glu were newly added to the plasmid (70)
of Reference Example 30. In the production of an amide compound
using the thus obtained transformant (108) and the transformant
(70) to be its base, the initial reaction rate and thermal
stability were compared in the same manner as in Example 1.
[0423] As a result, it was found that mutation of 197th Gly in the
.alpha.-subunit with Cys, mutation of 107th Pro in the
.beta.-subunit with Met and mutation of 230th Ala in the
.beta.-subunit with Glu were newly added to the transformant (108),
so that the initial reaction rate was improved by 1.98 times and
thermal stability was improved by 2.34 times, as compared to those
of the transformant (70).
Example 64
Construction (109) of a Transformant (109) Substituted Amino Acid
Having Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0424] In order to obtain a transformant (109) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(t) and (az) amino acid substitution sites as shown in Table 42,
the plasmid (106) recovered from the transformant (106) described
in the above Reference Example 42 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 24, 37 and 60 in
the Sequence Listing were used for repeatedly carrying out the
method described in Reference Example 2 per mutation point, whereby
a plasmid (109) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (109). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (109) had sequences according to the purpose in which
mutation of 197th Gly in the .alpha.-subunit with Cys, mutation of
107th Pro in the .beta.-subunit with Met and mutation of 230th Ala
in the .beta.-subunit with Glu were newly added to the plasmid
(106) of Reference Example 43. In the production of an amide
compound using the thus obtained transformant (109) and the
transformant (106) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0425] As a result, it was found that mutation of 197th Gly in the
.alpha.-subunit with Cys, mutation of 107th Pro in the
.beta.-subunit with Met and mutation of 230th Ala in the
.beta.-subunit with Glu were newly added to the transformant (109),
so that the initial reaction rate was improved by 2.05 times and
thermal stability was improved by 1.62 times, as compared to those
of the transformant (106).
TABLE-US-00055 TABLE 42 Improvement of Change in Amino Acid Change
in Base Improvement of Thermal Sequence Sequence Reaction Rate by
Stability by Trans- Before After Before After .alpha.-197th,
.beta.-107th .alpha.-197th, .beta.-107th formant Mutated Substitu-
Substitu- Substitu- Substitu- and .beta.-230th and .beta.-230th No.
Site tion tion tion tion Substitution Substitution 107
.alpha.-197th Gly Cys GGC TGC 2.11 times 1.88 times .beta.-51st Phe
Val TTC GTC .beta.-107th Pro Met CCC ATG .beta.-108th Glu Asp GAG
GAT .beta.-230th Ala Glu GCG GAG 108 .alpha.-197th Gly Cys GGC TGC
1.98 times 2.34 times .beta.-41st Phe Ile TTC ATC .beta.-51st Phe
Val TTC GTC .beta.-107th Pro Met CCC ATG .beta.-108th Glu Asp GAG
GAT .beta.-230th Ala Glu GCG GAG 109 .alpha.-48th Asn Glu AAC GAA
2.05 times 1.62 times .alpha.-197th Gly Cys GGC TGC .beta.-107th
Pro Met CCC ATG .beta.-146th Arg Gly CGG GGG .beta.-230th Ala Glu
GCG GAG
Reference Example 43
Construction of a Transformant (110) Substituted Amino Acid Having
Nitrile Hydratase Activity
[0426] In order to obtain a transformant (110) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(ba) amino acid substitution sites as shown in Table 43,
introduction of site-specific mutation was performed using the
mutagenesis kit described in the above Reference Example 2. The
plasmid (1) expressing nitrile hydratase with the modified ribosome
binding sequence described in Example 1 was used as the template,
and the primers having the sequence defined in SEQ ID Nos: 66 and
67 in the Sequence Listing were used for repeatedly carrying out
the method described in Reference Example 2 per mutation point,
whereby a plasmid (110) encoding the above nitrile hydratase
variant was prepared. A competent cell of Escherichia coli HB101
(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid
to obtain a transformant (110).
TABLE-US-00056 TABLE 43 Change in Amino Change in Trans- Acid
Sequence Base Sequence formant Mutated Before After Before After
No. Site Substitution Substitution Substitution Substitution 110
.alpha.-36th Thr Trp ACG TGG .beta.-176th Tyr Cys TAC TGC
Example 65
Construction of a Transformant (111) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0427] In order to obtain a transformant (111) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(u) and (aq) amino acid substitution sites as shown in Table 44,
the plasmid (38) recovered from the transformant (38) described in
the above Reference Example 19 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 33 and 69 in the
Sequence Listing were used for repeatedly carrying out the method
described in Reference Example 2 per mutation point, whereby a
plasmid (111) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (111). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (111) had sequences according to the purpose in which
mutation of 79th His in the .beta.-subunit with Asn, mutation of
230th Ala in the .beta.-subunit with Glu and mutation of 231st Ala
in the .beta.-subunit with Val were newly added to the plasmid (38)
of Reference Example 19. In the production of an amide compound
using the thus obtained transformant (111) and the transformant
(38) to be its base, the initial reaction rate and thermal
stability were compared in the same manner as in Example 1.
[0428] As a result, it was found that mutation of 79th His in the
.beta.-subunit with Asn, mutation of 230th Ala in the
.beta.-subunit with Glu and mutation of 231st Ala in the
.beta.-subunit with Val were newly added to the transformant (111),
so that the initial reaction rate was improved by 1.46 times and
thermal stability was improved by 1.43 times, as compared to those
of the transformant (38).
Example 66
Construction of a Transformant (112) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0429] In order to obtain a transformant (112) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(u) and (ap) amino acid substitution sites as shown in Table 44,
the plasmid (9) recovered from the transformant (9) described in
the above Reference Example 5 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 33 and 69 in the
Sequence Listing were used for repeatedly carrying out the method
described in Reference Example 2 per mutation point, whereby a
plasmid (112) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (112). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (112) had sequences according to the purpose in which
mutation of 79th His in the .beta.-subunit with Asn, mutation of
230th
[0430] Ala in the .beta.-subunit with Glu and mutation of 231st Ala
in the .beta.-subunit with Val were newly added to the plasmid (9)
of Reference Example 5. In the production of an amide compound
using the thus obtained transformant (112) and the transformant (9)
to be its base, the initial reaction rate and thermal stability
were compared in the same manner as in Example 1.
[0431] As a result, it was found that mutation of 79th His in the
.beta.-subunit with Asn, mutation of 230th Ala in the
.beta.-subunit with Glu and mutation of 231st Ala in the
.beta.-subunit with Val were newly added to the transformant (112),
so that the initial reaction rate was improved by 1.42 times and
thermal stability was improved by 1.66 times, as compared to those
of the transformant (9).
Example 67
Construction of a Transformant (113) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0432] In order to obtain a transformant (113) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(u) and (ba) amino acid substitution sites as shown in Table 44,
the plasmid (110) recovered from the transformant (110) described
in the above Reference Example 43 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 33 and 69 in the
Sequence Listing were used for repeatedly carrying out the method
described in Reference Example 2 per mutation point, whereby a
plasmid (113) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (113). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (113) had sequences according to the purpose in which
mutation of 79th His in the .beta.-subunit with Asn, mutation of
230th Ala in the .beta.-subunit with Glu and mutation of 231st Ala
in the .beta.-subunit with Val were newly added to the plasmid
(110) of Reference Example 43. In the production of an amide
compound using the thus obtained transformant (113) and the
transformant (110) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0433] As a result, it was found that mutation of 79th His in the
.beta.-subunit with Asn, mutation of 230th Ala in the
.beta.-subunit with Glu and mutation of 231st Ala in the
.beta.-subunit with Val were newly added to the transformant (113),
so that the initial reaction rate was improved by 1.39 times and
thermal stability was improved by 1.38 times, as compared to those
of the transformant (110).
TABLE-US-00057 TABLE 44 Improvement of Change in Amino Acid Change
in Base Improvement of Thermal Sequence Sequence Reaction Rate by
Stability by Trans- Before After Before After .beta.-79th,
.beta.-230th .beta.-79th, .beta.-230th formant Mutated Substitu-
Substitu- Substitu- Substitu- and .beta.-231st and .beta.-231st No.
Site tion tion tion tion Substitution Substitution 111 .alpha.-6th
Leu Thr CTG ACG 1.46 times 1.43 times .alpha.-19th Ala Val GCG GTG
.alpha.-126th Phe Tyr TTC TAC .beta.-48th Leu Val CTG GTG
.beta.-79th His Asn CAC AAC .beta.-108th Glu Arg GAG CGG
.beta.-212th Ser Tyr TCC TAC .beta.-230th Ala Glu GCG GAG
.beta.-231st Ala Val GCC GTC 112 .alpha.-6th Leu Thr CTG ACG 1.42
times 1.66 times .alpha.-19th Ala Val GCG GTG .alpha.-126th Phe Tyr
TTC TAC .beta.-46th Met Lys ATG AAG .beta.-79th His Asn CAC AAC
.beta.-108th Glu Arg GAG CGG .beta.-212th Ser Tyr TCC TAC
.beta.-230th Ala Glu GCG GAG .beta.-231st Ala Val GCC GTC 113
.alpha.-36th Thr Trp ACG TGG 1.39 times 1.38 times .beta.-79th His
Asn CAC AAC .beta.-176th Tyr Cys TAC TGC .beta.-230th Ala Glu GCG
GAG .beta.-231st Ala Val GCC GTC
Example 68
Construction of a Transformant (114) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0434] In order to obtain a transformant (114) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(v) and (al) amino acid substitution sites as shown in Table 45,
the plasmid (70) recovered from the transformant (70) described in
the above Reference Example 30 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 16, 38 and 70 in
the Sequence Listing were used for repeatedly carrying out the
method described in Reference Example 2 per mutation point, whereby
a plasmid (114) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (114). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (114) had sequences according to the purpose in which
mutation of 92nd Asp in the .alpha.-subunit with Glu, mutation of
24th Val in the .beta.-subunit with Ile and mutation of 226th Val
in the .beta.-subunit with Ile were newly added to the plasmid (70)
of Reference Example 30. In the production of an amide compound
using the thus obtained transformant (114) and the transformant
(70) to be its base, the initial reaction rate and thermal
stability were compared in the same manner as in Example 1.
[0435] As a result, it was found that mutation of 92nd Asp in the
.alpha.-subunit with Glu, mutation of 24th Val in the
.beta.-subunit with Ile and mutation of 226th Val in the
.beta.-subunit with Ile were newly added to the transformant (114),
so that the initial reaction rate was improved by 2.43 times and
thermal stability was improved by 1.63 times, as compared to those
of the transformant (70).
TABLE-US-00058 TABLE 45 Improvement of Change in Amino Acid Change
in Base Improvement of Thermal Sequence Sequence Reaction Rate by
Stability by Trans- Before After Before After .alpha.-92nd,
.beta.-24th .alpha.-92nd, .beta.-24th formant Mutated Substitu-
Substitu- Substitu- Substitu- and .beta.-226th and .beta.-226th No.
Site tion tion tion tion Substitution Substitution 114 .alpha.-92nd
Asp Glu GAC GAG 2.43 times 1.63 times .beta.-24th Val Ile GTC ATC
.beta.-41st Phe Ile TTC ATC .beta.-51st Phe Val TTC GTC
.beta.-108th Glu Asp GAG GAT .beta.-226th Val Ile GTC ATC
Example 69
Construction of a Transformant (115) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0436] In order to obtain a transformant (115) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(w) and (al) amino acid substitution sites as shown in Table 46,
the plasmid (70) recovered from the transformant (70) described in
the above Reference Example 30 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 24, 37, 60 and
70 in the Sequence Listing were used for repeatedly carrying out
the method described in Reference Example 2 per mutation point,
whereby a plasmid (115) encoding the above nitrile hydratase
variant was prepared. A competent cell of Escherichia coli HB101
(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid
to obtain a transformant (115). Moreover, the plasmid was prepared
from the above-mentioned microbial cells by the alkaline SDS
extraction method, and the base sequence of the nitrile hydratase
gene was determined using a DNA sequencer. Then, it was confirmed
that the transformant (115) had sequences according to the purpose
in which mutation of 197th Gly in the .alpha.-subunit with Cys,
mutation of 24th Val in the .beta.-subunit with Ile, mutation of
107th Pro in the .beta.-subunit with Met, and mutation of 230th Ala
in the .beta.-subunit with Glu were newly added to the plasmid (70)
of Reference Example 30. In the production of an amide compound
using the thus obtained transformant (115) and the transformant
(70) to be its base, the initial reaction rate and thermal
stability were compared in the same manner as in Example 1.
[0437] As a result, it was found that mutation of 197th Gly in the
.alpha.-subunit with Cys, mutation of 24th Val in the
.beta.-subunit with Ile, mutation of 107th Pro in the
.beta.-subunit with Met, and mutation of 230th Ala in the
.beta.-subunit with Glu were newly added to the transformant (115),
so that the initial reaction rate was improved by 2.23 times and
thermal stability was improved by 2.51 times, as compared to those
of the transformant (70).
Example 70
Construction of a Transformant (116) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0438] In order to obtain a transformant (116) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(w) and (az) amino acid substitution sites as shown in Table 46,
the plasmid (106) recovered from the transformant (106) described
in the above Reference Example 42 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 24, 37, 60 and
70 in the Sequence Listing were used for repeatedly carrying out
the method described in Reference Example 2 per mutation point,
whereby a plasmid (116) encoding the above nitrile hydratase
variant was prepared. A competent cell of Escherichia coli HB101
(manufactured by Toyobo Co., Ltd.) was transformed with the plasmid
to obtain a transformant (116). Moreover, the plasmid was prepared
from the above-mentioned microbial cells by the alkaline SDS
extraction method, and the base sequence of the nitrile hydratase
gene was determined using a DNA sequencer. Then, it was confirmed
that the transformant (116) had sequences according to the purpose
in which mutation of 197th Gly in the .alpha.-subunit with Cys,
mutation of 24th Val in the .beta.-subunit with Ile, mutation of
107th Pro in the .beta.-subunit with Met, and mutation of 230th Ala
in the .beta.-subunit with Glu were newly added to the plasmid
(106) of Reference Example 42. In the production of an amide
compound using the thus obtained transformant (116) and the
transformant (106) to be its base, the initial reaction rate and
thermal stability were compared in the same manner as in Example
1.
[0439] As a result, it was found that mutation of 197th Gly in the
.alpha.-subunit with Cys, mutation of 24th Val in the
.beta.-subunit with Ile, mutation of 107th Pro in the
.beta.-subunit with Met, and mutation of 230th Ala in the
.beta.-subunit with Glu were newly added to the transformant (116),
so that the initial reaction rate was improved by 2.35 times and
thermal stability was improved by 1.87 times, as compared to those
of the transformant (106).
TABLE-US-00059 TABLE 46 Improvement of Improvement of Thermal
Change in Amino Acid Change in Base Reaction Rate by Stability by
Sequence Sequence .alpha.-197th, .beta.-24th, .alpha.-197th,
.beta.-24th, Trans- Before After Before After .beta.-107th and
.beta.-107th and formant Mutated Substitu- Substitu- Substitu-
Substitu- .beta.-230th .beta.-230th No. Site tion tion tion tion
Substitution Substitution 115 .alpha.-197th Gly Cys GGC TGC 2.23
times 2.51 times .beta.-24th Val Ile GTC ATC .beta.-41st Phe Ile
TTC ATC .beta.-51st Phe Val TTC GTC .beta.-107th Pro Met CCC ATG
.beta.-108th Glu Asp GAG GAT .beta.-230th Ala Glu GCG GAG 116
.alpha.-48th Asn Glu AAC GAA 2.35 times 1.87 times .alpha.-197th
Gly Cys GGC TGC .beta.-24th Val Ile GTC ATC .beta.-107th Pro Met
CCC ATG .beta.-146th Arg Gly CGG GGG .beta.-230th Ala Glu GCG
GAG
Example 71
Construction of a Transformant (117) Substituted Amino Acid Having
Improved Nitrile Hydratase Activity and Improved Thermal
Stability
[0440] In order to obtain a transformant (117) expressing the
nitrile hydratase variant obtained by mutating nitrile hydratase at
(x) and (aq) amino acid substitution sites as shown in Table 47,
the plasmid (38) recovered from the transformant (38) described in
the above Reference Example 19 was used as the template, and the
primers having the sequence defined in SEQ ID Nos: 33, 69 and 70 in
the Sequence Listing were used for repeatedly carrying out the
method described in Reference Example 2 per mutation point, whereby
a plasmid (117) encoding the above nitrile hydratase variant was
prepared. A competent cell of Escherichia coli HB101 (manufactured
by Toyobo Co., Ltd.) was transformed with the plasmid to obtain a
transformant (117). Moreover, the plasmid was prepared from the
above-mentioned microbial cells by the alkaline SDS extraction
method, and the base sequence of the nitrile hydratase gene was
determined using a DNA sequencer. Then, it was confirmed that the
transformant (117) had sequences according to the purpose in which
mutation of 24th Val in the .beta.-subunit with Ile, mutation of
79th His in the .beta.-subunit with Asn, mutation of 230th Ala in
the .beta.-subunit with Glu, and mutation of 231st Ala in the
.beta.-subunit with Val were newly added to the plasmid (38) of
Reference Example 19. In the production of an amide compound using
the thus obtained transformant (117) and the transformant (38) to
be its base, the initial reaction rate and thermal stability were
compared in the same manner as in Example 1.
[0441] As a result, it was found that mutation of 24th Val in the
.beta.-subunit with Ile, mutation of 79th His in the .beta.-subunit
with Asn, mutation of 230th Ala in the .beta.-subunit with Glu, and
mutation of 231st Ala in the .beta.-subunit with Val were newly
added to the transformant (117), so that the initial reaction rate
was improved by 1.73 times and thermal stability was improved by
1.50 times, as compared to those of the transformant (38).
TABLE-US-00060 TABLE 47 Improvement of Improvement of Thermal
Change in Amino Acid Change in Base Reaction Rate by Stability by
Sequence Sequence .beta.-24th, .beta.-79th, .beta.-24th,
.beta.-79th, Trans- Before After Before After .beta.-230th and
.beta.-230th and formant Mutated Substitu- Substitu- Substitu-
Substitu- .beta.-231st .beta.-231st No. Site tion tion tion tion
Substitution Substitution 117 .alpha.-6th Leu Thr CTG ACG 1.73
times 1.50 times .alpha.-19th Ala Val GCG GTG .alpha.-126th Phe Tyr
TTC TAC .beta.-24th Val Ile GTC ATC .beta.-48th Leu Val CTG GTG
.beta.-79th His Asn CAC AAC .beta.-108th Glu Arg GAG CGG
.beta.-212th Ser Tyr TCC TAC .beta.-230th Ala Glu GCG GAG
.beta.-231st Ala Val GCC GTC
Sequence CWU 1
1
701205PRTPseudonocardia thermophila 1Met Thr Glu Asn Ile Leu Arg
Lys Ser Asp Glu Glu Ile Gln Lys Glu 1 5 10 15 Ile Thr Ala Arg Val
Lys Ala Leu Glu Ser Met Leu Ile Glu Gln Gly 20 25 30 Ile Leu Thr
Thr Ser Met Ile Asp Arg Met Ala Glu Ile Tyr Glu Asn 35 40 45 Glu
Val Gly Pro His Leu Gly Ala Lys Val Val Val Lys Ala Trp Thr 50 55
60 Asp Pro Glu Phe Lys Lys Arg Leu Leu Ala Asp Gly Thr Glu Ala Cys
65 70 75 80 Lys Glu Leu Gly Ile Gly Gly Leu Gln Gly Glu Asp Met Met
Trp Val 85 90 95 Glu Asn Thr Asp Glu Val His His Val Val Val Cys
Thr Leu Cys Ser 100 105 110 Cys Tyr Pro Trp Pro Val Leu Gly Leu Pro
Pro Asn Trp Phe Lys Glu 115 120 125 Pro Gln Tyr Arg Ser Arg Val Val
Arg Glu Pro Arg Gln Leu Leu Lys 130 135 140 Glu Glu Phe Gly Phe Glu
Val Pro Pro Ser Lys Glu Ile Lys Val Trp 145 150 155 160 Asp Ser Ser
Ser Glu Met Arg Phe Val Val Leu Pro Gln Arg Pro Ala 165 170 175 Gly
Thr Asp Gly Trp Ser Glu Glu Glu Leu Ala Thr Leu Val Thr Arg 180 185
190 Glu Ser Met Ile Gly Val Glu Pro Ala Lys Ala Val Ala 195 200 205
2233PRTPseudonocardia thermophila 2Met Asn Gly Val Tyr Asp Val Gly
Gly Thr Asp Gly Leu Gly Pro Ile 1 5 10 15 Asn Arg Pro Ala Asp Glu
Pro Val Phe Arg Ala Glu Trp Glu Lys Val 20 25 30 Ala Phe Ala Met
Phe Pro Ala Thr Phe Arg Ala Gly Phe Met Gly Leu 35 40 45 Asp Glu
Phe Arg Phe Gly Ile Glu Gln Met Asn Pro Ala Glu Tyr Leu 50 55 60
Glu Ser Pro Tyr Tyr Trp His Trp Ile Arg Thr Tyr Ile His His Gly 65
70 75 80 Val Arg Thr Gly Lys Ile Asp Leu Glu Glu Leu Glu Arg Arg
Thr Gln 85 90 95 Tyr Tyr Arg Glu Asn Pro Asp Ala Pro Leu Pro Glu
His Glu Gln Lys 100 105 110 Pro Glu Leu Ile Glu Phe Val Asn Gln Ala
Val Tyr Gly Gly Leu Pro 115 120 125 Ala Ser Arg Glu Val Asp Arg Pro
Pro Lys Phe Lys Glu Gly Asp Val 130 135 140 Val Arg Phe Ser Thr Ala
Ser Pro Lys Gly His Ala Arg Arg Ala Arg 145 150 155 160 Tyr Val Arg
Gly Lys Thr Gly Thr Val Val Lys His His Gly Ala Tyr 165 170 175 Ile
Tyr Pro Asp Thr Ala Gly Asn Gly Leu Gly Glu Cys Pro Glu His 180 185
190 Leu Tyr Thr Val Arg Phe Thr Ala Gln Glu Leu Trp Gly Pro Glu Gly
195 200 205 Asp Pro Asn Ser Ser Val Tyr Tyr Asp Cys Trp Glu Pro Tyr
Ile Glu 210 215 220 Leu Val Asp Thr Lys Ala Ala Ala Ala 225 230
3618DNAPseudonocardia thermophila 3atgaccgaga acatcctgcg caagtcggac
gaggagatcc agaaggagat cacggcgcgg 60gtcaaggccc tggagtcgat gctcatcgaa
cagggcatcc tcaccacgtc gatgatcgac 120cggatggccg agatctacga
gaacgaggtc ggcccgcacc tcggcgcgaa ggtcgtcgtg 180aaggcctgga
ccgacccgga gttcaagaag cgtctgctcg ccgacggcac cgaggcctgc
240aaggagctcg gcatcggcgg cctgcagggc gaggacatga tgtgggtgga
gaacaccgac 300gaggtccacc acgtcgtcgt gtgcacgctc tgctcctgct
acccgtggcc ggtgctgggg 360ctgccgccga actggttcaa ggagccgcag
taccgctccc gcgtggtgcg tgagccccgg 420cagctgctca aggaggagtt
cggcttcgag gtcccgccga gcaaggagat caaggtctgg 480gactccagct
ccgagatgcg cttcgtcgtc ctcccgcagc gccccgcggg caccgacggg
540tggagcgagg aggagctcgc caccctcgtc acccgcgagt cgatgatcgg
cgtcgaaccg 600gcgaaggcgg tcgcgtga 6184702DNAPseudonocardia
thermophila 4atgaacggcg tgtacgacgt cggcggcacc gatgggctgg gcccgatcaa
ccggcccgcg 60gacgaaccgg tcttccgcgc cgagtgggag aaggtcgcgt tcgcgatgtt
cccggcgacg 120ttccgggccg gcttcatggg cctggacgag ttccggttcg
gcatcgagca gatgaacccg 180gccgagtacc tcgagtcgcc gtactactgg
cactggatcc gcacctacat ccaccacggc 240gtccgcaccg gcaagatcga
tctcgaggag ctggagcgcc gcacgcagta ctaccgggag 300aaccccgacg
ccccgctgcc cgagcacgag cagaagccgg agttgatcga gttcgtcaac
360caggccgtct acggcgggct gcccgcaagc cgggaggtcg accgaccgcc
caagttcaag 420gagggcgacg tggtgcggtt ctccaccgcg agcccgaagg
gccacgcccg gcgcgcgcgg 480tacgtgcgcg gcaagaccgg gacggtggtc
aagcaccacg gcgcgtacat ctacccggac 540accgccggca acggcctggg
cgagtgcccc gagcacctct acaccgtccg cttcacggcc 600caggagctgt
gggggccgga aggggacccg aactccagcg tctactacga ctgctgggag
660ccctacatcg agctcgtcga cacgaaggcg gccgcggcat ga
7025144PRTPseudonocardia thermophila 5Met Ser Ala Glu Ala Lys Val
Arg Leu Lys His Cys Pro Thr Ala Glu 1 5 10 15 Asp Arg Ala Ala Ala
Asp Ala Leu Leu Ala Gln Leu Pro Gly Gly Asp 20 25 30 Arg Ala Leu
Asp Arg Gly Phe Asp Glu Pro Trp Gln Leu Arg Ala Phe 35 40 45 Ala
Leu Ala Val Ala Ala Cys Arg Ala Gly Arg Phe Glu Trp Lys Gln 50 55
60 Leu Gln Gln Ala Leu Ile Ser Ser Ile Gly Glu Trp Glu Arg Thr His
65 70 75 80 Asp Leu Asp Asp Pro Ser Trp Ser Tyr Tyr Glu His Phe Val
Ala Ala 85 90 95 Leu Glu Ser Val Leu Gly Glu Glu Gly Ile Val Glu
Pro Glu Ala Leu 100 105 110 Asp Glu Arg Thr Ala Glu Val Leu Ala Asn
Pro Pro Asn Lys Asp His 115 120 125 His Gly Pro His Leu Glu Pro Val
Ala Val His Pro Ala Val Arg Ser 130 135 140 6435DNAPseudonocardia
thermophila 6gtgagcgccg aggcgaaggt ccgcctgaag cactgcccca cggccgagga
ccgggcggcg 60gccgacgcgc tgctcgcgca gctgcccggc ggcgaccgcg cgctcgaccg
cggcttcgac 120gagccgtggc agctgcgggc gttcgcgctg gcggtcgcgg
cgtgcagggc gggccggttc 180gagtggaagc agctgcagca ggcgctgatc
tcctcgatcg gggagtggga gcgcacccac 240gatctcgacg atccgagctg
gtcctactac gagcacttcg tcgccgcgct ggaatccgtg 300ctcggcgagg
aagggatcgt cgagccggag gcgctggacg agcgcaccgc ggaggtcttg
360gccaacccgc cgaacaagga tcaccatgga ccgcatctgg agcccgtcgc
ggtccacccg 420gccgtgcggt cctga 435733DNAArtificial
SequenceOligonucleotide to act as a PCR primer 7tacgaattct
aaggaggtct cagcatgaac ggc 33821DNAArtificial
SequenceOligonucleotide to act as a PCR primer 8ctcggtcatg
ccgcggccgc c 21924DNAArtificial SequenceOligonucleotide to act as a
PCR primer 9atcctcacct cgtcgatgat cgac 241017DNAArtificial
SequenceOligonucleotide to act as a PCR primer 10caggaaacag ctatgac
171120DNAArtificial SequenceOligonucleotide to act as a PCR primer
11ggccagtgcc tagcttacat 201217DNAArtificial SequenceOligonucleotide
to act as a PCR primer 12gttttcccag tcacgac 171324DNAArtificial
SequenceOligonucleotide to act as a PCR primer 13gagaaggtcg
tgttcgcgat gttc 241424DNAArtificial SequenceOligonucleotide to act
as a PCR primer 14ttcccggcga ttttccgggc cggc 241524DNAArtificial
SequenceOligonucleotide to act as a PCR primer 15atgaacccgg
tcgagtacct cgag 241624DNAArtificial SequenceOligonucleotide to act
as a PCR primer 16cagggcgagg agatgatgtg ggtg 241724DNAArtificial
SequenceOligonucleotide to act as a PCR primer 17atgaacccgg
gcgagtacct cgag 241824DNAArtificial SequenceOligonucleotide to act
as a PCR primer 18ttctccacca atagcccgaa gggc 241924DNAArtificial
SequenceOligonucleotide to act as a PCR primer 19gaggacatga
tgtgggtgga gaac 242024DNAArtificial SequenceOligonucleotide to act
as a PCR primer 20ttcccggcgg tgttccgggc cggc 242124DNAArtificial
SequenceOligonucleotide to act as a PCR primer 21tactacgaca
tgtgggagcc ctac 242224DNAArtificial SequenceOligonucleotide to act
as a PCR primer 22cggcgcgcgt gttacgtgcg cggc 242324DNAArtificial
SequenceOligonucleotide to act as a PCR primer 23aagaccgggg
aggtggtcaa gcac 242424DNAArtificial SequenceOligonucleotide to act
as a PCR primer 24tcgatgatct gcgtcgaacc ggcg 242524DNAArtificial
SequenceOligonucleotide to act as a PCR primer 25atcctcaccg
ggtcgatgat cgac 242624DNAArtificial SequenceOligonucleotide to act
as a PCR primer 26gagctcgccg gcctcgtcac ccgc 242724DNAArtificial
SequenceOligonucleotide to act as a PCR primer 27cacggcgcgg
ccatctaccc ggac 242824DNAArtificial SequenceOligonucleotide to act
as a PCR primer 28gtctactacg tctgctggga gccc 242924DNAArtificial
SequenceOligonucleotide to act as a PCR primer 29atgaacggca
tgtacgacgt cggc 243024DNAArtificial SequenceOligonucleotide to act
as a PCR primer 30tacgacgtcg ccggcaccga tggg 243124DNAArtificial
SequenceOligonucleotide to act as a PCR primer 31gagaaggtca
tgttcgcgat gttc 243224DNAArtificial SequenceOligonucleotide to act
as a PCR primer 32cacggcgcga ccatctaccc ggac 243324DNAArtificial
SequenceOligonucleotide to act as a PCR primer 33tacatccaca
acggcgtccg cacc 243424DNAArtificial SequenceOligonucleotide to act
as a PCR primer 34cgccgcacgc gttactaccg ggag 243524DNAArtificial
SequenceOligonucleotide to act as a PCR primer 35atgaacccgt
gggagtacct cgag 243624DNAArtificial SequenceOligonucleotide to act
as a PCR primer 36gtctactacc actgctggga gccc 243724DNAArtificial
SequenceOligonucleotide to act as a PCR primer 37gccccgctga
tggagcacga gcag 243824DNAArtificial SequenceOligonucleotide to act
as a PCR primer 38atcgagctca tcgacacgaa ggcg 243924DNAArtificial
SequenceOligonucleotide to act as a PCR primer 39atgaacccgt
cggagtacct cgag 244024DNAArtificial SequenceOligonucleotide to act
as a PCR primer 40cggcgcgcga tgtacgtgcg cggc 244124DNAArtificial
SequenceOligonucleotide to act as a PCR primer 41cacgagcagg
tgccggagtt gatc 244224DNAArtificial SequenceOligonucleotide to act
as a PCR primer 42gtctactaca tgtgctggga gccc 244324DNAArtificial
SequenceOligonucleotide to act as a PCR primer 43gacgaggagc
tccagaagga gatc 244424DNAArtificial SequenceOligonucleotide to act
as a PCR primer 44atcctcaccg cgtcgatgat cgac 244524DNAArtificial
SequenceOligonucleotide to act as a PCR primer 45atctacgagc
aagaggtcgg cccg 244624DNAArtificial SequenceOligonucleotide to act
as a PCR primer 46ctggagtcga tcctcatcga acag 244724DNAArtificial
SequenceOligonucleotide to act as a PCR primer 47cacggcgcga
tgatctaccc ggac 244824DNAArtificial SequenceOligonucleotide to act
as a PCR primer 48gtctactacg gctgctggga gccc 244924DNAArtificial
SequenceOligonucleotide to act as a PCR primer 49atgaacccgc
tggagtacct cgag 245024DNAArtificial SequenceOligonucleotide to act
as a PCR primer 50cacgagcaga ttccggagtt gatc 245124DNAArtificial
SequenceOligonucleotide to act as a PCR primer 51atgaacccga
cggagtacct cgag 245224DNAArtificial SequenceOligonucleotide to act
as a PCR primer 52tactacgact cctgggagcc ctac 245324DNAArtificial
SequenceOligonucleotide to act as a PCR primer 53gacgtggtgg
ggttctccac cgcg 245424DNAArtificial SequenceOligonucleotide to act
as a PCR primer 54gtctactaca gctgctggga gccc 245524DNAArtificial
SequenceOligonucleotide to act as a PCR primer 55ttcccggcgc
tgttccgggc cggc 245624DNAArtificial SequenceOligonucleotide to act
as a PCR primer 56gtctactacc tctgctggga gccc 245724DNAArtificial
SequenceOligonucleotide to act as a PCR primer 57cccgagcaca
accagaagcc ggag 245824DNAArtificial SequenceOligonucleotide to act
as a PCR primer 58acgaaggcgg tcgcggcatg accg 245924DNAArtificial
SequenceOligonucleotide to act as a PCR primer 59ctgtgggggc
tggaagggga cccg 246024DNAArtificial SequenceOligonucleotide to act
as a PCR primer 60gacacgaagg aggccgcggc atga 246124DNAArtificial
SequenceOligonucleotide to act as a PCR primer 61ttctccacct
cgagcccgaa gggc 246224DNAArtificial SequenceOligonucleotide to act
as a PCR primer 62gtctactact gttgctggga gccc 246324DNAArtificial
SequenceOligonucleotide to act as a PCR primer 63acggtggtcg
cgcaccacgg cgcg 246424DNAArtificial SequenceOligonucleotide to act
as a PCR primer 64gtctactaca cctgctggga gccc 246524DNAArtificial
SequenceOligonucleotide to act as a PCR primer 65atctacgagg
aagaggtcgg cccg 246624DNAArtificial SequenceOligonucleotide to act
as a PCR primer 66atcctcacct ggtcgatgat cgac 246724DNAArtificial
SequenceOligonucleotide to act as a PCR primer 67cacggcgcgt
gcatctaccc ggac 246824DNAArtificial SequenceOligonucleotide to act
as a PCR primer 68gccccgctga tggatcacga gcag 246924DNAArtificial
SequenceOligonucleotide to act as a PCR primer 69gacacgaagg
aggcgcggca tgac 247024DNAArtificial SequenceOligonucleotide to act
as a PCR primer 70gacgaaccga tcttccgcgc cgag 24
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