U.S. patent application number 13/471757 was filed with the patent office on 2012-11-15 for cellulase variants.
This patent application is currently assigned to NOVOZYMES A/S. Invention is credited to Kim Vilbour Andersen, Lars Christiansen, Bo Damgaard, Martin Schulein, Claus Von der Osten.
Application Number | 20120289450 13/471757 |
Document ID | / |
Family ID | 8100020 |
Filed Date | 2012-11-15 |
United States Patent
Application |
20120289450 |
Kind Code |
A1 |
Andersen; Kim Vilbour ; et
al. |
November 15, 2012 |
Cellulase Variants
Abstract
The present invention relates to cellulase variants, i.e.,
endo-beta-1,4-glucanase variants, derived from a parental
cellulase, i.e., endo-beta-1,4-glucanase, by substitution,
insertion and/or deletion, which variant has a catalytic core
domain, in which the variant at position 5 holds an alanine residue
(A), a serine residue (S), or a threonine residue (T); at position
8 holds a phenylalanine residue (F), or a tyrosine residue (Y); at
position 9 holds a phenylalanine residue (F), a tryptophan residue
(W), or a tyrosine residue (Y); at position 10 holds an aspartic
acid residue (D); and at position 121 holds an aspartic acid
residue (D).
Inventors: |
Andersen; Kim Vilbour;
(Copenhagen, DK) ; Schulein; Martin; (Copenhagen,
DK) ; Christiansen; Lars; (Virum, DK) ;
Damgaard; Bo; (Lausanne, CH) ; Von der Osten;
Claus; (Lyngby, DK) |
Assignee: |
NOVOZYMES A/S
Bagsvaerd
DK
|
Family ID: |
8100020 |
Appl. No.: |
13/471757 |
Filed: |
May 15, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13162636 |
Jun 17, 2011 |
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13471757 |
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12394202 |
Feb 27, 2009 |
7993898 |
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13162636 |
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11830063 |
Jul 30, 2007 |
8017372 |
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12394202 |
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10919195 |
Aug 16, 2004 |
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11830063 |
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09261329 |
Mar 3, 1999 |
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10919195 |
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PCT/DK97/00393 |
Sep 17, 1997 |
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09261329 |
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Current U.S.
Class: |
510/392 ;
435/209 |
Current CPC
Class: |
C11D 3/38645 20130101;
C12N 9/2437 20130101; C12Y 302/01004 20130101 |
Class at
Publication: |
510/392 ;
435/209 |
International
Class: |
C12N 9/42 20060101
C12N009/42; C11D 3/386 20060101 C11D003/386 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 19, 1996 |
DK |
1013/96 |
Claims
1-88. (canceled)
89. A modified cellulase having endoglucanase activity, comprising
a mutation in an amino acid sequence of a cellulase, wherein the
mutation comprises a substitution at one or more positions selected
from the group consisting of: 21a, 22, 24, 25, 26, 29, 32, 33, 34,
35, 36, 38, 40, 42, 42a, 43, 49, 49a, 49b, 50, 53, 54, 57, 64, 68,
69, 70, 71, 76, 79, 80, 82, 83, 84, 85, 86, 88, 91, 92, 93, 95d,
95h, 95i, 95j, 106, 127, 132a, 137, 138, 139, 140, 140a, 141, 143a,
149, 150b, 150e, 150j, 151, 152, 153, 154, 155, 156, 157, 159,
160c, 160e, 160k, 161, 164, 165, 166, 168, 169, 170, 171, 172, 173,
174, 188, 191, 192, 193, 195, 197, 200, and 201, wherein each
mutation is independently a substitution, insertion or deletion and
each position is numbered according to the amino acid sequence of
the cellulase of SEQ ID NO: 1.
90. A detergent composition comprising a cellulase variant of claim
89 and a surfactant.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a division of U.S. application Ser. No.
13/162,636 filed on Jun. 17, 2011, now pending, which is a division
of U.S. application Ser. No. 12/394,202 filed on Feb. 27, 2009, now
U.S. Pat. No. 7,993,898, which is a division of U.S. application
Ser. No. 11/830,063 filed on Jul. 30, 2007, now U.S. Pat. No.
8,017,372, which is a continuation of U.S. application Ser. No.
10/919,195 filed on Aug. 16, 2004, now abandoned, which is a
continuation of U.S. application Ser. No. 09/261,329 filed on Mar.
3, 1999, now abandoned, which is a continuation of PCT/DK97/00393
filed on Sep. 17, 1997, which claims priority under 35 U.S.C. 119
of Danish application no. 1013/96 filed on Sep. 17, 1996, the
contents of which are fully incorporated herein by reference.
SEQUENCE LISTING
[0002] The present application contains information in the form of
a sequence listing, which is submitted on a data carrier
accompanying this application. The data carrier is fully
incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] 1. Field of the Invention
[0004] The present invention relates to cellulase variants, i.e.,
endo-beta-1,4-glucanase variants, derived from a parental
cellulase, i.e., endo-beta-1,4-glucanase, by substitution,
insertion and/or deletion, which variant has a catalytic core
domain, in which the variant at position 5 holds an alanine residue
(A), a serine residue (S), or a threonine residue (T); at position
8 holds a phenylalanine residue (F), or a tyrosine residue (Y); at
position 9 holds a phenylalanine residue (F), a tryptophan residue
(W), or a tyrosine residue (Y); at position 10 holds an aspartic
acid residue (D); and at position 121 holds an aspartic acid
residue (D).
[0005] 2. Description of Related Art
[0006] Cellulases or cellulolytic enzymes are enzymes involved in
hydrolysis of cellulose. In the hydrolysis of native cellulose, it
is known that there are three major types of cellulase enzymes
involved, namely cellobiohydrolase (1,4-beta-D-glucan
cellobiohydrolase, EC 3.2.1.91), endo-beta-1,4-glucanase
(endo-1,4-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.4) and
beta-glucosidase (EC 3.2.1.21).
[0007] Especially the endo-beta-1,4-glucanases (EC No. 3.2.1.4)
constitute an interesting group of hydrolases for the mentioned
industrial uses. Endoglucanases catalyses endo hydrolysis of
1,4-beta-D-glycosidic linkages in cellulose, cellulose derivatives
(such as carboxy methyl cellulose and hydroxy ethyl cellulose),
lichenin, beta-1,4 bonds in mixed beta-1,3 glucans such as cereal
beta-D-glucans or xyloglucans and other plant material containing
cellulosic parts. The authorized name is endo-1,4-beta-D-glucan
4-glucano hydrolase, but the abbreviated term endoglucanase is used
in the present specification. Reference can be made to T.-M.
Enveri, "Microbial Cellulases" in W. M. Fogarty, Microbial Enzymes
and Biotechnology, Applied Science Publishers, p. 183-224 (1983);
Methods in Enzymology, 1988, Vol. 160, pp. 200-391 (edited by Wood,
W. A. and Kellogg, S. T.); Beguin, P., "Molecular Biology of
Cellulose Degradation", Annu. Rev. Microbiol., 1990, Vol. 44, pp.
219-248; Beguin, P. and Aubert, J-P., "The biological degradation
of cellulose", FEMS Microbiology Reviews, 1994, Vol. 13, pp. 25-58;
Henrissat, B., "Cellulases and their interaction with cellulose",
Cellulose, 1994, Vol. 1, pp. 169-196.
[0008] Cellulases are synthesized by a large number of
microorganisms which include fungi, actinomycetes, myxobacteria and
true bacteria but also by plants. Especially endoglucanases of a
wide variety of specificities have been identified.
[0009] A very important industrial use of cellulolytic enzymes is
the use for treatment of cellulosic textile or fabric, e.g., as
ingredients in detergent compositions or fabric softener
compositions, for bio-polishing of new fabric (garment finishing),
and for obtaining a "stone-washed" look of cellulose-containing
fabric, especially denim, and several methods for such treatment
have been suggested, e.g., in GB-A-1 368 599, EP-A-0 307 564 and
EP-A-0 435 876, WO 91/17243, WO 91/10732, WO 91/17244,
PCT/DK95/000108 and PCT/DK95/00132. Another important industrial
use of cellulolytic enzymes is the use for treatment of paper pulp,
e.g., for improving the drainage or for deinking of recycled
paper.
[0010] It is also known that cellulases may or may not have a
cellulose binding domain (a CBD). The CBD enhances the binding of
the enzyme to a cellulose-containing fiber and increases the
efficacy of the catalytic active part of the enzyme
[0011] Fungi and bacteria produces a spectrum of cellulolytic
enzymes (cellulases) which, on the basis of sequence similarities
(hydrophobic cluster analysis), can be classified into different
families of glycosyl hydrolases [Henrissat B & Bairoch A;
Biochem. J., 1993, 293: 781-788]. At present are known cellulases
belonging to the families 5, 6, 7, 8, 9, 10, 12, 26, 44, 45, 48,
60, and 61 of glycosyl hydrolases.
[0012] Industrially well-performing endo-beta-1,4-glucanases are
described in, e.g., WO 91/17243, WO 91/17244 and WO 91/10732, and
specific cellulase variants are described in WO 94/07998.
[0013] It is an object of the present invention to provide novel
variants of cellulolytic enzymes, which variants, when compared to
the parental enzyme, show improved performance.
SUMMARY OF THE INVENTION
[0014] In a cellulolytic enzyme useful in industrial processes,
i.e., an endo-1,4-glucanase, a number of amino acid residue
positions important for the properties of the enzyme and thereby
for the performance thereof in these processes has been
identified.
[0015] Accordingly, in a first aspect the present invention
provides a method for improving the properties of a cellulolytic
enzyme by amino acid substitution, deletion or insertion, the
method comprising the steps of:
[0016] a. constructing a multiple alignment of at least two amino
acid sequences known to have three-dimensional structures similar
to endoglucanase V (EGV) from Humicola insolens known from Protein
Data Bank entry 4ENG;
[0017] b. constructing a homology-built three-dimensional structure
of the cellulolytic enzyme based on the structure of the EGV;
[0018] c. identifying amino acid residue positions present in a
distance from the substrate binding cleft of not more than 5
.ANG.;
[0019] d. identifying surface-exposed amino acid residues of the
enzyme;
[0020] e. identifying all charged or potentially charged amino acid
residue positions of the enzyme;
[0021] f. choosing one or more positions wherein the amino acid
residue is to be substituted, deleted or where an insertion is to
be provided;
[0022] g. carrying out the substitution, deletion or insertion by
using conventional protein engineering techniques.
[0023] By using the method of the invention, it is now possible
effectively to transfer desirable properties from one cellulase to
another by protein engineering methods which are known per se.
[0024] More particular the invention provides cellulase variants
improved with respect to altered (increased or decreased) catalytic
activity; and/or altered sensitivity to anionic tensides; and/or
altered pH optimum and pH profile activity-wise as well as
stability-wise.
[0025] Accordingly, in a further aspect, the invention provides a
cellulase variant derived from a parental cellulase by
substitution, insertion and/or deletion, which variant has a
catalytic core domain, in which the variant
[0026] at position 5 holds an alanine residue (A), a serine residue
(S), or a threonine residue (T);
[0027] at position 8 holds a phenylalanine residue (F), or a
tyrosine residue (Y);
[0028] at position 9 holds a phenylalanine residue (F), a
tryptophan residue (W), or a tyrosine residue (Y);
[0029] at position 10 holds an aspartic acid residue (D); and
[0030] at position 121 holds an aspartic acid residue (D)
(cellulase numbering).
DETAILED DISCLOSURE OF THE INVENTION
Cellulase Variants
[0031] The present invention provides new cellulase variants
derived from a parental cellulase by substitution, insertion and/or
deletion. A cellulase variant of this invention is a cellulase
variant or mutated cellulase, having an amino acid sequence not
found in nature. The cellulase variants of the invention show
improved performance, in particular with respect to increased
catalytic activity; and/or altered sensitivity to anionic tensides;
and/or altered pH optimum; and/or altered thermostability.
[0032] Formally the cellulase variant or mutated cellulase of this
invention may be regarded a functional derivative of a parental
cellulase (i.e., the native or wild-type enzyme), and may be
obtained by alteration of a DNA nucleotide sequence of the parental
gene or its derivatives, encoding the parental enzyme. The
cellulase variant or mutated cellulase may be expressed and
produced when the DNA nucleotide sequence encoding the cellulase
variant is inserted into a suitable vector in a suitable host
organism. The host organism is not necessarily identical to the
organism from which the parental gene originated.
[0033] In the literature, enzyme variants have also been referred
to as mutants or muteins.
Amino Acids
[0034] In the context of this invention the following symbols and
abbreviations for amino acids and amino acid residues are used:
[0035] A=Ala=Alanine
[0036] C=Cys=Cysteine
[0037] D=Asp=Aspartic acid
[0038] E=Glu=Glutamic acid
[0039] F=Phe=Phenylalanine
[0040] G=Gly=Glycine
[0041] H=His=Histidine
[0042] I=Ile=Isoleucine
[0043] K=Lys=Lysine
[0044] L=Leu=Leucine
[0045] M=Met=Methionine
[0046] N=Asn=Asparagine
[0047] P=Pro=Proline
[0048] Q=Gln=Glutamine
[0049] R=Arg=Arginine
[0050] S=Ser=Serine
[0051] T=Thr=Threonine
[0052] V=Val=Valine
[0053] W=Trp=Tryptophan
[0054] Y=Tyr=Tyrosine
[0055] B=Asx=Asp or Asn
[0056] Z=Glx=Glu or Gln
[0057] X=Xaa=Any amino acid
[0058] *=Deletion or absent amino acid
Cellulase Numbering
[0059] In the context of this invention a specific numbering of
amino acid residue positions in cellulolytic enzymes is employed.
By aligning the amino acid sequences of known cellulases, as in
Table 1 below, it is possible to unambiguously allot an amino acid
position number to any amino acid residue in any cellulolytic
enzyme, if its amino acid sequence is known.
[0060] In Table 1, below, 11 selected amino acid sequences of
cellulases of different microbial origin are aligned. These are (a)
Humicola insolens; (b) Acremonium sp.; (c) Volutella
collectotrichoides; (d) Sordaria fimicola; (e) Thielavia
terrestris; (f) Fusarium oxysporum; (g) Myceliophthora thermophila;
(h) Crinipellis scabella; (i) Macrophomina phaseolina; (j)
Pseudomonas fluorescens; (k) Ustilago maydis. The cellulases (a-i)
are described in WO 96/29397, (j) is described in GeneBank under
the accession number G45498, and (k) is described in GeneBank under
the accession number S81598 and in Biol. Chem. Hoppe-Seyler, 1995,
376 (10): 617-625.
[0061] Using the numbering system originating from the amino acid
sequence of the cellulase (endo-beta-1,4-glucanase) obtained from
the strain of Humicola insolens DSM 1800, disclosed in, e.g., WO
91/17243, which sequence is shown in the first column of Table 1,
aligned with the amino acid sequence of a number of other
cellulases, it is possible to indicate the position of an amino
acid residue in a cellulolytic enzyme unambiguously.
[0062] In describing the various cellulase variants produced or
contemplated according to the invention, the following
nomenclatures are adapted for ease of reference: [0063] [Original
amino acid; Position; Substituted amino acid]
[0064] Accordingly, the substitution of glutamine with histidine in
position 119 is designated as Q119H.
[0065] Amino acid residues which represent insertions in relation
to the amino acid sequence of the cellulase from Humicola insolens,
are numbered by the addition of letters in alphabetical order to
the preceding cellulase number, such as, e.g., position *21aV for
the "inserted" valine (V), where no amino acid residue is present,
between lysine at position 21 and alanine at position 22 of the
amino acid sequence of the cellulase from Humicola insolens, cf.
Table 1.
[0066] Deletion of a proline (P) at position 49 in the amino acid
sequence of the cellulase from Humicola insolens is indicated as
P49*.
[0067] Multiple mutations are separated by slash marks ("/"), e.g.,
Q119H/Q146R, representing mutations in positions 119 and 146
substituting glutamine (Q) with histidine (H), and glutamine (Q)
acid with arginine (R), respectively.
[0068] If a substitution is made by mutation in, e.g., a cellulase
derived from a strain of Humicola insolens, the product is
designated, e.g., "Humicola insolens/*49P".
[0069] All positions referred to in this application by cellulase
numbering refer to the cellulase numbers described above, and are
determined relative to the amino acid sequence of the cellulase
derived from Humicola insolens, cf. Table 1, (a).
TABLE-US-00001 TABLE 1 Amino Acid Sequence Alignment Cellulase
Numbering of Selected Cellulases of Different Microbial Origin (a)
Humicola insolens (SEQ ID NO: 1); (b) Acremonium sp. (SEQ ID NO:
2); (c) Volutella collectotrichoides (SEQ ID NO: 3); (d) Sordaria
fimicola (SEQ ID NO: 4); (e) Thielavia terrestris (SEQ ID NO: 5);
(f) Fusarium oxysporum (SEQ ID NO: 6); (g) Myceliophthora
thermophila (SEQ ID NO: 7); (h) Crinipellis scabella (SEQ ID NO:
8); (i) Macrophomina phaseolina (SEQ ID NO: 9); (j) Pseudomonas
fluorescens (SEQ ID NO: 10); (k) Ustilago maydis (SEQ ID NO: 11). a
b c d e f g h i j k 1 A G G G G G G T T C * 2 D S T S S S I A S N *
3 G G G G G G G G G G G 4 R H R K Q H Q V V Y M 5 S T T S S S T T T
A A 6 T T T T T T T T T T T 7 R R R R R R R R R R R 8 Y Y Y Y Y Y Y
Y Y Y Y 9 W W W W W W W W W W W 10 D D D D D D D D D D D 11 C C C C
C C C C C C C 12 C C C C C C C C C C C 13 K K K K K K K K K K L 14
P P P P P P P P P P A 15 S S S S S S S S S H S 16 C C C C C C C C C
C A 17 G A G A A S A G A G S 18 W W W W W W W W W W W 19 A D D S P
S P S T S E 20 K E E G G G G G G A G 21 K K K K K K K K K N K 21a *
* * * * * * * * V * 22 A A A A A A G A A P A 23 P A S S A A P S S S
P 24 V V V V V V * V V L V 25 N S S N S N S S S V Y 26 Q R Q R Q A
S A K S A 27 P P P P P P P P P P P 28 V V V V V A V V V L V 29 F T
K L Y L Q R G Q D 30 S T T A A T A T T S A 31 C C C C C C C C C C C
32 N D D D D D D D D S K 33 A R R A A K K R I A A 34 N N N N N N N
N N N D 35 F N N N F D D G D N G 36 Q S N N Q N N N N T V 37 R P P
P R P P T A R T 38 I L L L L I F L Q L L 39 T S A N S S N G T S I
40 D P S D D N D P P D D 41 F * * A F T G * S V S 42 D G T N N N G
* D S K 42a * * * * * * S D L * K 43 A A A V V A T V L V D 44 K V R
K Q V R K K G P 45 S S S S S N S S S S S 46 G G G G G G G G S S G
47 C C C C C C C C C C Q 48 E D D D N E D D D D S 49 P P S * * G A
S * * G 49a * * * * * * * * * * C 49b * * * * * * * * * * N 50 G N
N G G G G G G G G 51 G G G G G G G G G G G 52 V V V S S S S T S G N
53 A A A A A A A S A G K 54 Y F Y Y Y Y Y F Y Y F 55 S T T T S A M
T Y M M 56 C C C C C C C C C C C 57 A N N A A T S A S W S 58 D D D
N D N S N N D C 59 Q N N N Q Y Q N Q K M 60 T Q Q S T S S G G I Q
61 P P P P P P P P P P P 62 W W W W W W W F W F F 63 A A A A A A A
A A A D 64 V V V V V V V I V V D 65 N N N N N N S D N S E 66 D N D
D D D D N D P T 67 D N N N N E E N S T D 68 F V L L L L L T L L P
69 A A A A A A S A S A T 70 L Y Y Y Y Y Y Y Y Y L 71 G G G G G G G
G G G A 72 F F F F F F W F F Y F 73 A A A A A A A A A A G 74 A A A
A A A A A A A F 75 T T T T T T V A A T G 76 S A A K S K K H K S A
77 I F F L I I L L L S F 78 A P S S A S A A S G T 79 G G G G G G G
G G D T 80 S G G G G G S S K V G 81 N N S T S S S S Q * Q 82 E E E
E E E E E E * E 83 A A A S S A S A T * S 84 G S S S S S Q A D * D
85 W W W W W W W W W * T 86 C C C C C C C C C C D 87 C C C C C C C
C C G C 88 A A A A A A A Q G R A 89 C C C C C C C C C C C 90 Y Y Y
Y Y Y Y Y Y Y F 91 E A A A A A E E K Q Y 92 L L L L L L L L L L A
93 T Q Q T T T T T T Q E 94 F F F F F F F F F F F 95 T T T T T T T
T T T E 95a * * * * * * * * * G * 95b * * * * * * * * * S * 95c * *
* * * * * * * S * 95d * * * * * * * * * Y * 95e * * * * * * * * * N
* 95f * * * * * * * * * A * 95g * * * * * * * * * P * 95h * * * * *
* * * * G H 95i * * * * * * * * * D D 95j * * * * * * * * * P A 95k
* * * * * * * * * G Q 96 S S S S S T S S S S G 97 G G G G G G G G T
A K 98 P P P P P P P P A A A 99 V V V V V V V V V L M 100 A A A S A
K A V S A K 101 G G G G G G G G G G R
102 K K K K K K K K K K N 103 K T T T T K K K Q T K 104 M M M L M M
M L M M L 105 V V V V V I I T I I I 106 V V V V V V V V V V F 107 Q
Q Q Q Q Q Q Q Q Q Q 108 S S S S S S A V I A V 109 T T T T T T T T T
T T 110 S N N S S N N N N N N 111 T T T T T T T T T I V 112 G G G G
G G G G G G G 113 G G G G G G G G G Y G 114 D D D D D D D D D D D
115 L L L L L L L L L V V 116 G S S G G G G G G S Q 117 S G G S S D
D N N G S 118 N T N N N N N N N G Q 119 H H H H Q H H H H Q N 120 F
F F F F F F F F F F 121 D D D D D D D D D D D 122 L I I L I L L L I
I F 123 N Q L N A M A M A L Q 124 I M M M M M I I M V I 125 P P P P
P P P P P P P 126 G G G G G G G G G G G 127 G G G G G G G G G G G
128 G G G G G G G G G G G 129 V L L V V V V V V V L 130 G G G G G G
G G G G G 131 I I I L I I I L I A A 132 F F F F F F F F F F F 132a
* * * * * * * T * * P 133 D D D D N D N Q N N K 134 G G G G G G A G
G A G 135 C C C C C C C C C C C 136 T T T K S T T P S S P 137 P P P
R S S D A K A A 138 Q Q Q E Q E Q Q Q Q Q 139 F F W F F F Y F W W W
140 G G G G G G G G N G G 140a * F V * * K A S G V V 141 G T S G G
A P W I S E 142 L F F L L L P N * N A 143 P P P P P G N G * A S
143a * * * * * * G * N E L 143b * * * * * * W * L L W 144 G G G G G
G G G G G G 145 Q N N A A A D A N A D 146 R R R Q Q Q R Q Q Q Q 147
Y Y Y Y Y Y Y Y Y Y Y 148 G G G G G G G G G G G 149 G G G G G G G G
G G G 150 I T T I I I I V F F V 150a * * * * * * * * * L * 150b * *
* * * * * * * A * 150c * * * * * * * * * A * 150d * * * * * * * * *
C * 150e * * * * * * * * * K * 150f * * * * * * * * * Q * 150g * *
* * * * * * * Q * 150h * * * * * * * * * L * 150i * * * * * * * * *
G * 150j * * * * * * * * * Y * 150k * * * * * * * * * N * 151 S T T
S S S H S T A K 152 S S S S S S S S D S S 153 R R R R R R K R R L A
154 N S S S D S E D S S T 155 E Q Q E Q E E Q Q Q E 156 C C C C C C
C C C Y C 157 D A S D D D E S A K S 158 R E Q S S S S Q T S K 159 F
L I F F Y F L L C L 160 P P P P P P P P P V P 160a * * * * * * * *
* L * 160b * * * * * * * * * N * 160c * * * * * * * * * R * 160d *
* * * * * * * * C * 160e * * * * * * * * * D * 160f * * * * * * * *
* S * 160g * * * * * * * * * V * 160h * * * * * * * * * F * 160i *
* * * * * * * * G * 160j * * * * * * * * * S * 160k * * * * * * * *
* R * 160l * * * * * * * * * G * 160m * * * * * * * * * L * 161 D S
S A A E E A S T K 162 A V A A P L A A K Q P 163 L L L L L L L V W L
L 164 K R Q K K K K Q Q Q Q 165 P D P P P D P A A Q E 166 G G G G G
G G G S G G 167 C C C C C C C C C C C 168 Y H N Q Q H N Q N T K 169
W W W W W W W F W W W 170 R R R R R R R R R F R 171 F Y Y F F F F F
F A F 172 D D D D D D D D D E S 173 W W W W W W W W W W E 174 F F F
F F F F M F F W 175 K N N K Q E Q G E E G 176 N D D N N N N G N A D
177 A A A A A A A A A A N 178 D D D D D D D D D D P 179 N N N N N N
N N N N V 180 P P P P P P P P P P L 181 S N D E T D S N T S K 182 F
V V F F F V V V L G 183 S N S T T T T T D K S 184 F W W F F F F F W
Y P 185 R R R K Q E Q R E K K 186 Q R R Q Q Q E P P E R 187 V V V V
V V V V V V V 188 Q R Q Q Q Q A T T P K 189 C C C C C C C C C C C
190 P P P P P P P P P P P 191 A A A S A K S A Q A K 192 E A A E E A
E Q E E S 193 L L L L I L L L L L L 194 V T T T V L T T V T I 195 A
N D S A D S N A T D 196 R R R R R I K I R R R 197 T S T T S S S S T
S S 198 G G G G G G G G G G G
199 C C C C C C C C C M C 200 R V R K K K S V S N Q 201 R R R R R R
R R R R R * Amino acid residue absent in this position
The Enzyme (Endo-Beta-1,4-Glucanase) Variants of the Invention
[0070] The present invention relates to cellulase variants. More
specifically the present invention provides cellulase variant
derived from a parental cellulase by substitution, insertion and/or
deletion, which variant has a catalytic core domain, in which the
variant [0071] at position 5 holds an alanine residue (A), a serine
residue (S), or a threonine residue (T); [0072] at position 8 holds
a phenylalanine residue (F) or a tyrosine residue (Y); [0073] at
position 9 holds a phenylalanine residue (F), a tryptophan residue
(W), or a tyrosine residue (Y); [0074] at position 10 holds an
aspartic acid residue (D); and [0075] at position 121 holds an
aspartic acid residue (D) (cellulase numbering).
[0076] The endoglucanase of the invention may comprise a cellulose
binding domain (CBD) existing as an integral part of the enzyme, or
a CBD from another origin may be introduced into the endoglucanase
thus creating an enzyme hybride. In this context, the term
"cellulose-binding domain" is intended to be understood as defined
by Peter Tomme et al. "Cellulose-Binding Domains: Classification
and Properties" in "Enzymatic Degradation of Insoluble
Carbohydrates", John N. Saddler and Michael H. Penner (Eds.), ACS
Symposium Series, No. 618, 1996. This definition classifies more
than 120 cellulose-binding domains into 10 families (I-X), and
demonstrates that CBDs are found in various enzymes such as
cellulases, xylanases, mannanases, arabinofuranosidases, acetyl
esterases and chitinases. CBDs have also been found in algae, e.g.,
the red alga Porphyra purpurea as a non-hydrolytic
polysaccharide-binding protein, see Tomme et al., op. cit. However,
most of the CBDs are from cellulases and xylanases, CBDs are found
at the N and C termini of proteins or are internal. Enzyme hybrids
are known in the art, see, e.g., WO 90/00609 and WO 95/16782, and
may be prepared by transforming into a host cell a DNA construct
comprising at least a fragment of DNA encoding the
cellulose-binding domain ligated, with or without a linker, to a
DNA sequence encoding the endoglucanase and growing the host cell
to express the fused gene.
[0077] Enzyme hybrids may be described by the following
formula:
CBD-MR-X or X-MR-CBD
wherein CBD is the N-terminal or the C-terminal region of an amino
acid sequence corresponding to at least the cellulose-binding
domain; MR is the middle region (the linker), and may be a bond, or
a short linking group preferably of from about 2 to about 100
carbon atoms, more preferably of from 2 to 40 carbon atoms; or is
preferably from about 2 to about 100 amino acids, more preferably
of from 2 to 40 amino acids; and X is an N-terminal or C-terminal
region of the enzyme according to the invention.
THE METHOD OF THE INVENTION
[0078] In another aspect, the present invention relates to a method
for improving the properties of a cellulolytic enzyme by amino acid
substitution, deletion or insertion, the method comprising the
steps of:
[0079] a. constructing a multiple alignment of at least two amino
acid sequences known to have three-dimensional structures similar
to endoglucanase V (EGV) from Humicola insolens known from Protein
Data Bank entry 4ENG;
[0080] b. constructing a homology-built three-dimensional structure
of the cellulolytic enzyme based on the structure of the EGV;
[0081] c. identifying amino acid residue positions present in a
distance from the substrate binding cleft of not more than 5
.ANG.;
[0082] d. identifying surface-exposed amino acid residues of the
enzyme;
[0083] e. identifying all charged or potentially charged amino acid
residue positions of the enzyme;
[0084] f. choosing one or more positions wherein the amino acid
residue is to be substituted, deleted or where an insertion is to
be provided;
[0085] g. carrying out the substitution, deletion or insertion by
using conventional protein engineering techniques.
[0086] Step f. of the method is preferably carried out by choosing
positions which, as a result of the alignment of step a., carry the
same amino acid residue in a majority of the aligned sequences;
more preferably in at least 63% of the aligned sequences; even more
preferably positions which, in the aligned sequences, carries
different amino acid residues, cf. below.
[0087] In a preferred embodiment, the specific activity of the
cellulase can be improved, preferably by carrying out a
substitution, deletion or insertion at amino acid residue positions
present in a distance from the substrate binding cleft of not more
than 5 .ANG., more preferably not more than 3 .ANG., even more
preferably not more than 2.5 .ANG.. It is believed that residues
present in a distance of not more than 2.5 .ANG. are capable of
being in direct contact with the substrate.
[0088] In another preferred embodiment, the pH activity profile,
the pH activity optimum, the pH stability profile, or the pH
stability optimum of the cellulase can be altered, preferably by
carrying out a substitution, deletion or insertion at amino acid
residue positions present either in a distance from the substrate
binding cleft of not more than 5 .ANG., more preferably not more
than 3 .ANG., even more preferably not more than 2.5 .ANG.; or at
surface-exposed amino acid residue positions of the enzyme, thereby
altering the electrostatic environment either locally or globally.
It is preferred to perform a substitution involving a charged or
potentially charged residue, this residue either being the original
residue or the replacement residue. In the present context, charged
or potentially charged residues are meant to include: Arg, Lys,
His, Cys (if not part of a disulfide bridge), Tyr, Glu, and
Asp.
[0089] In yet another preferred embodiment, the stability of the
cellulase in the presence of an anionic tenside or anionic
detergent component can be altered, preferably by carrying out a
substitution, deletion or insertion at surface-exposed amino acid
residue positions of the enzyme, thereby altering the electrostatic
environment either locally or globally. It is preferred to perform
a substitution involving a charged or potentially charged residue,
this residue either being the original residue or the replacement
residue. In the present context, charged or potentially charged
residues are meant to include: Arg, Lys, His, Cys (if not part of a
disulfide bridge), Tyr, Glu, and Asp. Mutations towards a more
negatively charged amino acid residue result in improved stability
of the cellulase in the presence of an anionic tenside, whereas
mutations towards a more positively charged aa residue decreases
the stability of the cellulase towards anionic tensides.
[0090] Further, cellulase variants comprising any combination of
two or more of the amino acid substitutions, deletions or
insertions disclosed herein are also within the scope of the
present invention, cf. the exemplified variants.
Multiple Sequence Alignment of Cellulases
[0091] The multiple sequence alignment is performed using the
Pileup algorithm as implemented in the Wisconsin Sequence Analysis
Package version 8.1-UNIX (GCG, Genetics Computer Group, Inc.). The
method used is similar to the method described by Higgens and Sharp
(CARBIOS, 1989, 5, 151-153). A gap creation penalty of 3.0 and a
gap extension penalty of 0.1 are used together with a scoring
matrix as described in Nucl. Acids Res., 1986, 14 (16), 6745-6763
(Dayhoff table (Schwartz, R. M. and Dayhoff, M. O.; Atlas of
Protein Sequence and Structure (Dayhoff, M. O. Ed.); National
Biomedical Research Foundation, Washington D.C., 1979, 353-358)
rescaled by dividing each value by the sum of its row and column,
and normalizing to a mean of 0 and standard deviation of 1.0. The
value for FY (Phe-Tyr)=RW=1.425. Perfect matches are set to 1.5 and
no matches on any row are better than perfect matches).
Pair-Wise Sequence Alignment of Cellulases
[0092] A pair-wise sequence alignment is performed using the
algorithm described by Needleman & Wunsch (J. Mol. Biol., 1970,
48, 443-453), as implemented in the GAP routine in the Wisconsin
Sequence Analysis Package (GCG). The parameters used for the GAP
routine are the same as mentioned for the Pileup routine
earlier.
Pair-Wise Sequence Alignment of Cellulases with Forced Pairing
[0093] A pair-wise sequence alignment with forced pairing of
residues is performed using the algorithm described by Needleman
& Wunsch (J. Mol. Biol., 1970, 48, 443-453), as implemented in
the GAP routine in the Wisconsin Sequence Analysis Package (GCG).
The parameters used for the GAP routine are the same as mentioned
for the Pileup routine earlier, where the scoring matrix is
modified to incorporate a residue named X which symbolizes the
residues to be paired. The diagonal value for X paired with X is
set to 9.0 and all off diagonal values involving X is set to 0.
Complex Between Humicola insolens Endoglucanase and
Celloheptaose
[0094] Based on the X-ray structure of the core domain of the
Humicola insolens EGV endoglucanase inactive variant (D10N) in
complex with cellohexaose (Davies et. al.; Biochemistry, 1995, 34,
16210-12220, PDB entry 4ENG) a model of the structure of the native
Humicola insolens EGV endoglucanase core domain in complex with
celloheptaose is build using the following steps:
1. Using the Biopolymer module of the Insight II 95.0 (Insight II
95.0 User Guide, October 1995. San Diego: Biosym/MSI, 1995) replace
N10 with a aspartic acid. 2. Make a copy of the sugar unit
occupying subsite -3 by copying all the molecule and delete the
extra atoms. Manually move the new sugar unit to best fit the
unoccupied -1 binding site. Create the bonds to bind the new sugar
unit to the two existing cellotriose units. 3. Delete overlapping
crystal water molecules. These are identified by using the Subset
Interface By_Atom 2.5 command. 4. Build hydrogens at a pH of 8.0
and applying charged terminals 5. Protonate D121 using the Residue
Replace <D121 residue name> ASP L command. 6. Apply the CVFF
forcefield template through the command Potentials Fix. 7. Fix all
atoms except the new sugar unit. 8. Relax the atomic position of
the new sugar unit using 300 cycles of simple energy minimization
followed by 5000 steps of 1 fs simple molecular dynamics ending by
300 cycles of simple energy minimization all using the molecular
mechanics program Discover 95.0/3.0.1 (Discover 95.0/3.0.0 User
Guide, October 1995. San Diego: Biosym/MSI, 1995.).
Homology Building of Cellulases
[0095] The construction of a structural model of a cellulase with
known amino acid sequence based on a known X-ray structure of the
Humicola insolens EGV cellulase consists of the following
steps:
1. Define the approximate extend of the core region of the
structure to be modeled and the alignment of the cysteine based on
multiple sequence alignment between many known industrially useful
cellulase sequences. 2. Pair-wise sequence alignment between the
new sequence and the sequence of the known X-ray structure. 3.
Define Structurally Conserved Regions (SCRs) based on the sequence
alignment. 4. Assign coordinates for the model structure within the
SCRs. 5. Find structures for the loops or Variable Regions (VRs)
between the SCRs by a search in a loop structure database. 6.
Assign coordinates for the VRs in the model structure from the
database search result. 7. Create disulfide bonds and set
protonation state. 8. Refine the build structure using molecular
mechanics.
[0096] The known X-ray structure of the Humicola insolens EGV
cellulase will in the following be termed the reference structure.
The structure to be modeled will be termed the model structure.
[0097] Ad 1: The approximate extent of the core part of the enzyme
is determined by a multiple sequence alignment including many known
cellulase sequences. Since the reference structure contains only
atomic coordinates for the core part of the enzyme only the
residues in the sequence to be modeled which align with the core
part of the reference structure can be included in the model
building. This alignment also determines the alignment of the
cysteine. The multiple sequence alignment is performed using the
Pileup algorithm as described earlier.
[0098] Ad 2: A pair-wise sequence alignment is performed as
described earlier. If the cysteine in the conserved disulfide
bridges and/or the active site residues (D10 and D121) does not
align, a pair-wise sequence alignment using forced pairing of the
cysteines in the conserved disulfide bridges and/or the active site
residues is performed as described earlier. The main purpose of the
sequence alignment is to define SCRs (see later) to be used for a
model structure generation.
[0099] Ad 3: Based on the sequence alignment Structurally Conserved
Regions (SCRs) are defined as continuous regions of overlapping
sequence with no insertions or deletions.
[0100] Ad 4: Using the computer program Homology 95.0 (Homology
User Guide, October 1995. San Diego: Biosym/MSI, 1995.) atomic
coordinates in the model structure can be generated from the atomic
coordinates of the reference structure using the command
AssignCoords Sequences.
[0101] Ad 5: Using the computer program Homology 95.0 possible
conformations for the remaining regions, named Variable Regions
(VRs) are found by a search in the loop structure database included
in Homology 95.0. This procedure is performed for each VR.
[0102] Ad 6: If the VR length is smaller than six residues the
first loop structure in the database search result is selected for
coordinate generation. In cases where longer loops are generated
the first solution in the list which does not have severe atomic
overlap are selected. The degree of atomic overlap can be analyzed
using the Bump Monitor Add Intra command in the computer program
Insight II 95.0 (Insight II 95.0 User Guide, October 1995. San
Diego: Biosym/MSI, 1995.) a parameter of 0.25 for the Bump command
will show the severe overlap. If more than ten bumps exists between
the inserted loop region and the remaining part of the protein the
next solution is tested. If no solution is found with these
parameters, the solution with the fewest bumps is selected. The
coordinates for the VR regions are generated using the command
AssignCoords Loops in the program Homology 95.0.
[0103] Ad 7: The disulfide bonds are created using the Bond Create
command in the Biopolymer module of Insight II 95.0 and the
protonation state is set to match pH 8.0 with charged caps using
the Hydrogens command. Finally the active proton donor (the residue
equivalent to D121 in the reference structure) is protonated using
the residue replace <D121 residue name> ASP L command. To
finalize the data of the model the appropriate forcefield template
is applied using the CVFF forcefield through the command Potentials
Fix.
[0104] Ad 8: Finally the modeled structure is subjected to 500
cycles energy minimization using the molecular mechanics program
Discover 95.0/3.0.1 (Discover 95.0/3.0.0 User Guide, October 1995.
San Diego: Biosym/MSI, 1995.). The output from the above described
procedure is atomic coordinates describing a structural model for
the core domain of a new cellulase based on sequence homology to
the Humicola insolens EGV cellulase.
Superpositioning of Cellulase Structures
[0105] To overlay two cellulase structures a superposition of the
structures are performed using the Structure Alignment command of
the Homology 95.0 (Homology User Guide, October 1995. San Diego:
Biosym/MSI, 1995.). All parameters for the command are chosen as
the default values.
Determination of Residues within 3 .ANG. and 5 .ANG. from the
Substrate
[0106] In order to determine the amino acid residues within a
specified distance from the substrate, a given cellulase structure
is superimposed on the cellulase part of the model structure of the
complex between Humicola insolens EGV endoglucanase and
celloheptaose as described above. The residues within a specified
distance of the substrate are then found using the Interface Subset
command of the Insight II 95.0 (Insight II 95.0 User Guide, October
1995, San Diego Biosym/MSI). The specified distance is supplied as
parameter to the program.
[0107] The results of this determination are presented in Tables 2
and 3 below.
Determination of Surface Accessibility
[0108] To determine the solvent accessibility the Access_Surf
command in Homology 95.0 (Homology User Guide, October 1995; San
Diego: Biosym/MSI, 1995) was used. The program uses the definition
proposed by Lee and Richards (Lee, B. & Richards, F. M. "The
interpretation of protein structures: Estimation of static
accessibility", J. Mol. Biol., 1971, 55, 379-400). A solvent probe
radius of 1.4 .ANG. was used and only heavy atoms (i.e.,
non-hydrogen atoms) were included in the calculation. Residues with
zero accessibility are defined as being buried, all other residues
are defined as being solvent exposed and on the surface of the
enzyme structure.
Transferring Level of Specific Activity Between Cellulases
[0109] In order to transfer the level of catalytic activity between
two cellulases, the following protocol is applied using the methods
described above. This method will pinpoint amino acid residues
responsible for the difference in specific activity, and one or
more of those amino acid residues must be replaced in one sequence
in order to transfer the level of specific activity from the
comparison cellulase:
1) Perform multiple sequence alignment of all known industrially
useful cellulases (excluding the Trichoderma reesei cellulases).
From this identify conserved disulfide bridges amongst the two
involved sequences and the sequence of the Humicola insolens EGV
cellulase are identified and the active site residues (D10 and
D121) are located; 2) Perform pair-wise sequence alignment of each
sequence with the Humicola insolens EGV cellulase core domain
(residues 1-201). If the cysteines in the conserved disulfide
bridges do not align at the same positions and/or if the two active
site residues (D10 and D121) do not align at the same positions
then use the pair-wise sequence alignment of cellulases with forced
pairing method. Include only residues in the sequences overlapping
with the core domain (residues 1-201) of the Humicola insolens EGV
cellulase; 3) Create a homology build structure of each sequence;
4) Determination of residues within 3 .ANG. from the substrate in
each of the homology build structures. Differences between the
sequences in these positions will most probably be the residues
responsible for the difference in specific activity. In the case
where residues in inserts are found in any of the sequences within
the above mentioned distance, the complete insert can be
responsible for the difference in specific activity, and the
complete insert must be transferred to the sequence without the
insert or the complete insert must be deleted in the sequence with
the insert; 5) If not all specific activity was restored by
substitution of residues within 3 .ANG. of the substrate,
determination of residues within 5 .ANG. from the substrate in each
of the homology build structures will reveal the most probable
residues responsible for the remaining difference in specific
activity. In the case where residues in inserts are found in any of
the sequences within the above mentioned distance, the complete
insert can be responsible for the difference in specific activity,
and the complete insert must be transferred to the sequence without
the insert or the complete insert must be deleted in the sequence
with the insert.
Transferring the Level of Stability Towards Anionic Tensides
Between Cellulases
[0110] In order to transfer level of stability towards anionic
tensides between two cellulases, the following protocol is applied
using the methods described above. This method will pinpoint amino
acid residues responsible for the difference in level of stability
towards anionic tensides, and one or more of those amino acid
residues must be replaced in one sequence in order to transfer the
level of specific activity from the comparison cellulase:
1) Perform multiple sequence alignment of all known industrially
useful cellulases (excluding Trichoderma reesei cellulases). From
this identify conserved disulfide bridges amongst the two involved
sequences and the sequence of the Humicola insolens EGV cellulase
are identified and the active site residues (D10 and D121) are
located; 2) Perform pair-wise sequence alignment of each sequence
with the Humicola insolens EGV cellulase core domain (residues
1-201). If the cysteines in the conserved disulfide bridges do not
align at the same positions and/or if the two active site residues
(D10 and D121) do not align at the same positions then use the
pair-wise sequence alignment of cellulases with forced pairing
method. Include only residues in the sequences overlapping with the
core domain (residues 1-201) of the Humicola insolens EGV
cellulase; 3) Create a homology build structure of each sequence;
4) Determination of residues located at the surface of the enzyme.
This is done by calculation the surface accessibility. Residues
with a surface accessibility greater than 0.0 .ANG..sup.2 are
exposed to the surface; 5) Any residue exposed to the surface
belonging to the following group of amino acids: D, E, H, K, R and
C if not involved in a disulfide bridge which differs between the
two sequences will most probably be responsible for the difference
in level of stability towards anionic tensides. In the case where
residues in inserts are found in any of the sequences within the
above mentioned group of amino acid types, the complete insert can
be responsible for the difference in level of stability towards
anionic tensides, and the complete insert must be transferred to
the sequence without the insert or the complete insert must be
deleted in the sequence with the insert.
Disulfide Bridges
[0111] Disulfide bridges (i.e., Cys-Cys bridges) stabilize the
structure of the enzyme. It is believed that a certain number of
stabilizing disulfide bridges is necessary to maintain a proper
stability of the enzyme. However, it is also contemplated that
disulfide bridges can be removed from the protein structure
resulting in an enzyme variant which is less stable, especially
less thermostable, but which still has significant activity.
[0112] Therefore, in another aspect, the invention provides a
cellulase variant which variant holds 4 or more of the following
disulfide bridges: C11-C135; C12-C47; C16-C86; C31-C56; C87-C199;
C89-C189; and C156-C167 (cellulase numbering). In a more specific
embodiment the variant of the invention holds 5 or more of the
following disulfide bridges: C11-C135; C12-C47; C16-C86; C31-C56;
C87-C199; C89-C189; and C156-C167 (cellulase numbering). In its
most specific embodiment, the variant of the invention holds 6 or
more of the following disulfide bridges: C11-C135; C12-C47;
C16-C86; C31-C56; C87-C199; C89-C189; and C156-C167 (cellulase
numbering).
[0113] In another embodiment the invention provides a cellulase
variant in which cysteine has been replaced by another natural
amino acid at one or more of the positions 16, 86, 87, 89, 189,
and/or 199 (cellulase numbering).
Binding Cleft Substitutions
[0114] In a further aspect, the invention provides a cellulase
variant derived from a parental cellulase by substitution,
insertion and/or deletion at one or more amino acid residues
located in the substrate binding cleft. Mutations introduced at
positions close to the substrate affect the enzyme-substrate
interactive bindings.
[0115] An appropriate way of determining the residues interacting
with a potential substrate in a structure is to partitionate the
structure in "shells". The shells are defined as: 1st shell are
residues directly interacting with the substrate, i.e., closest
inter atomic distance between substrate and residue both including
hydrogen atoms are smaller than 2.5 .ANG. which will include all
direct interaction via hydrogen bonds and other non bonded
interactions. The subsequent (2nd, 3rd e.t.c.) shells are defined
in the same way, as the residues with inter atomic distances
smaller than 2.5 .ANG. to the substrate or all previously
determined shells. In this way the structure will be partitioned in
shells. The routine "subset zone" in the program Insight II 95.0
(Insight II 95.0 User Guide, October 1995. San Diego: Biosym/MSI,
1995.) can be used to determine the shells.
[0116] In a preferred embodiment, the amino acid residue
contemplated according to this invention is located in the
substrate binding cleft at a distance of up to 5 .ANG. from the
substrate.
[0117] When subjecting the aligned cellulases to the computer
modeling method disclosed above, the following positions within a
distance of up to 5 .ANG. from the substrate are revealed: 4, 5, 6,
7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 19, 20, 21, 21a, 42, 44,
45, 47, 48, 49, 49a, 49b, 74, 82, 95j, 110, 111, 112, 113, 114,
115, 116, 119, 121, 123, 127, 128, 129, 130, 131, 132, 132a, 133,
145, 146, 147, 148, 149, 150b, 178, and/or 179 (cellulase
numbering), cf. Table 2.
[0118] Accordingly, in a more specific embodiment, the invention
provides a cellulase variant which has been derived from a parental
cellulase by substitution, insertion and/or deletion at one or more
of these acid residues. In a particular embodiment, the cellulase
variant is derived from one of the cellulases identified in Table 2
((a) Humicola insolens; (b) Acremonium sp.; (c) Volutella
collectotrichoides; (d) Sordaria fimicola; (e) Thielavia
terrestris; (f) Fusarium oxysporum; (g) Myceliophthora thermophila;
(h) Crinipellis scabella; (i) Macrophomina phaseolina; (j)
Pseudomonas fluorescens; (k) Ustilago maydis), by substitution,
insertion and/or deletion at one or more of the positions
identified in Table 2 for these cellulases.
TABLE-US-00002 TABLE 2 Amino Acid Residues less than 5 A from the
Substrate Positions Identified by Cellulase Numbering (a) Humicola
insolens; (b) Acremonium sp.; (c) Volutella collectotrichoides; (d)
Sordaria fimicola; (e) Thielavia terrestris; (f) Fusarium
oxysporum; (g) Myceliophthora thermophila; (h) Crinipellis
scabella; (i) Macrophomina phaseolina; (j) Pseudomonas fluorescens;
(k) Ustilago maydis. a b c d e f g h i j k 4 Y 5 S T T S S S T T T
A A 6 T T T T T T T T T T T 7 R R R R R R R R R R R 8 Y Y Y Y Y Y Y
Y Y Y Y 9 W W W W W W W W W W W 10 D D D D D D D D D D D 11 C 12 C
C C C C C C C C C C 13 K K K K K K K K K K L 14 P P P P P P P P P P
A 15 S S S S S S S S S H S 16 C 18 W W W W W W W W W W W 19 A D D S
P S P S T E 20 K E E G G G A 21 K K K K K K K K K K 21a V 42 G T D
44 V R K Q V R G 45 S S S S S N S S S S 47 C C C C C C C C C C 48 E
D D D N E D D D D S 49 P 49a C 49b N 74 A A A A A A A A A A F 82 E
E E E E E E E E 95j P 110 S N N S S N N N N N N 111 T T T T T T T T
T I V 112 G G G G G G G G G G G 113 G G G G G G G G G Y G 114 D D D
D D D D D D D D 115 L L L L L L L L L V V 116 G 119 H H H H Q H H H
H Q N 121 D D D D D D D D D D D 123 Q 127 G G G G G G G G G G G 128
G G G G G G G G G G G 129 V L L V V V V V V V L 130 G G G G G G G G
G G G 131 I I I L I I I L I A A 132 F F F F F F F F F F F 132a T P
133 N N 145 A D 146 R R R Q Q Q R Q Q Q Q 147 Y Y Y Y Y Y Y Y Y Y Y
148 G G G G G G G G G G G 149 G G G G G 150b A 178 D D D D D D D D
D D P 179 N N N N N N N N N N V
[0119] In another preferred embodiment, the amino acid residue
contemplated according to this invention is located in the
substrate binding cleft at a distance of up to 3 .ANG. from the
substrate.
[0120] When subjecting the aligned cellulases to the computer
modeling method disclosed above, the following positions within a
distance of up to 3 .ANG. from the substrate are revealed: 6, 7, 8,
10, 12, 13, 14, 15, 18, 20, 21, 45, 48, 74, 110, 111, 112, 113,
114, 115, 119, 121, 127, 128, 129, 130, 131, 132, 132a, 146, 147,
148, 150b, 178, and/or 179 (cellulase numbering). cf. Table 3.
[0121] Accordingly, in a more specific embodiment, the invention
provides a cellulase variant which has been derived from a parental
cellulase by substitution, insertion and/or deletion at one or more
of these acid residues. In a particular embodiment, the cellulase
variant is derived from one of the cellulases identified in Table 3
((a) Humicola insolens; (b) Acremonium sp.; (c) Volutella
collectotrichoides; (d) Sordaria fimicola; (e) Thielavia
terrestris; (f) Fusarium oxysporum; (g) Myceliophthora thermophila;
(h) Crinipellis scabella; (i) Macrophomina phaseolina; (j)
Pseudomonas fluorescens; (k) Ustilago maydis), by substitution,
insertion and/or deletion at one or more of the positions
identified in Table 3 for these cellulases.
TABLE-US-00003 TABLE 3 Amino Acid Residues less than 3 A from the
Substrate Positions Identified by Cellulase Numbering (a) Humicola
insolens; (b) Acremonium sp.; (c) Volutella collectotrichoides; (d)
Sordaria fimicola; (e) Thielavia terrestris; (f) Fusarium
oxysporum; (g) Myceliophthora thermophila; (h) Crinipellis
scabella; (i) Macrophomina phaseolina; (j) Pseudomonas fluorescens;
(k) Ustilago maydis. a b c D e f g h i j K 6 T T T T T T T T T T T
7 R R R R R R R R R R R 8 Y Y Y Y Y Y Y Y Y Y Y 10 D D D D D D D D
D D 12 C C C C C C C C C C C 13 K K K K K K L 14 P P P P P P A 15 S
S S S S S S S S H S 18 W W W W W W W W W W W 20 E E 21 K K 45 S S S
S S N S S S S S 48 D N E D D 74 A A A A A A A F 110 N N S N N N N N
N 111 T T T T T T T T T 112 G G G G G G G G G G G 113 G G G G G G Y
G 114 D D D D D D D D D D D 115 L L L L L L L L L V V 119 H H H Q H
H Q 121 D D D D D D D D D D D 127 G G G G G G 128 G G G G G G G G
129 V L L V V V V V V V L 130 G G G G G G G G G G G 131 I I I L I I
I L I A A 132 F F F F F F F F F F F 132a T P 146 Q Q Q Q 147 Y Y Y
Y Y Y Y Y Y Y Y 148 G G G G G G G G G G G 150b A 178 D D D D D D P
179 N N N N N N N N N N
Partly Conserved Amino Acid Residues
[0122] As defined herein a "partly conserved amino acid residue" is
an amino acid residue identified according to Table 1, at a
position at which position between 7 to 10 amino acid residues of
the 11 residues (i.e., more than 63%) indicated in Table 1 for that
position, are identical.
[0123] Accordingly, the invention further provides a cellulase
variant, in which variant an amino acid residue has been changed
into a conserved amino acid residue at one or more positions
according to Table 1, at which position(s) between 7 and 10 amino
acid residues of the 11 residues identified in Table 1, are
identical.
[0124] In a preferred embodiment the invention provides a cellulase
variant, which has been derived from a parental cellulase by
substitution, insertion and/or deletion at one or more of the
following positions: 13, 14, 15, 20, 21, 22, 24, 28, 32, 34, 45,
48, 50, 53, 54, 62, 63, 64, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75,
79, 85, 88, 90, 92, 93, 95, 96, 97, 98, 99, 104, 106, 110, 111,
113, 115, 116, 118, 119, 131, 134, 138, 140, 146, 152, 153, 163,
166, 169, 170, 171, 172, 173, 174, 174, 177, 178, 179, 180, 193,
196, and/or 197 (cellulase numbering).
[0125] In a more specific embodiment the invention provides a
cellulase variant that has been subjected to substitutions,
insertions and/or deletions, so as to comprise one or more of the
amino acid residues at the positions identified in Table 4, below.
The positions in Table 4 reflects the "partly conserved amino acid
residue positions" as well as the non-conserved positions present
within 5 .ANG. of the substrate in binding cleft all of which
indeed are present in the aligned sequences in Table 1.
TABLE-US-00004 TABLE 4 Selected Substitutions, Insertions and/or
Deletions Positions Identified by Cellulase Numbering Position
Amino Acid Residue 4 R, H, K, Q, V, Y, M 5 S, T, A 13 K, L 14 P, A
15 H, S 16 C, A 19 A, D, S, P, T, E 20 A, E, G, K 21 K, N 21a V, *
22 A, G, P 24 *, L, V 28 A, L, V 32 D, K, N, S 34 D, N 38 F, I, L,
Q 42 D, G, T, N, S, K, * 44 K, V, R, Q, G, P 45 N, S 46 G, S 47 C,
Q 48 D, E, N, S 49 P, S, A, G, * 49a C, * 49b N, * 50 G, N 53 A, G,
K, S 54 F, Y 62 F, W 63 A, D 64 D, I V 65 D, E, N, S 66 D, N, P, T
68 F, L, P, T, V 69 A, S, T 70 L, Y 71 A, G 72 F, W, Y 73 A, G 74
A, F 75 A, G, T, V 79 G, T 82 E, * 88 A, G, Q, R 90 F, Y 92 A, L 93
E, Q, T 95 E, T 95j P, * 96 S, T 97 A, G, T 98 A, P 99 L, V 104 L,
M 106 F, V 110 N, S 111 I, T, V 113 G, Y 115 L, V 116 G, Q, S 118
G, N, Q, T 119 H, N, Q 129 L, V 131 A, I, L 132 A, P, T, * 133 D,
K, N, Q 134 A, G 138 E, Q 145 A, D, N, Q 146 Q, R 150b A, * 152 D,
S 153 A, K, L, R 163 L, V, W 166 G, S 169 F, W 170 F, R 171 A, F, Y
172 D, E, S 173 E, W 174 F, M, W 177 A, N 178 D, P 179 N, V 180 L,
P 193 I, L 196 I, K, R 197 S, T
[0126] In a yet more preferred embodiment, the invention provides a
cellulase variant derived from a parental cellulase by
substitution, insertion and/or deletion at one or more amino acid
residues as indicated in Tables 5-6, below. The cellulase variant
may be derived from any parental cellulase holding the amino acid
residue stated at the position indicated. In particular the
parental cellulase may be a Humicola insolens cellulase; an
Acremonium sp. Cellulase; a Volutella collectotrichoides cellulase;
a Sordaria fimicola cellulase; a Thielavia terrestris cellulase; a
Fusarium oxysporum cellulase; a Myceliophthora thermophila
cellulase; a Crinipellis scabella cellulase; a Macrophomina
phaseolina cellulase; a Pseudomonas fluorescens cellulase; or a
Ustilago maydis cellulase.
[0127] Moreover, the cellulase variant may be characterized by
having improved performance, in particular with respect to
[0128] 1. improved performance defined as increased catalytic
activity;
[0129] 2. altered sensitivity to anionic tenside; and/or
[0130] 3. altered pH optimum;
as also indicated in Tables 5-6. The positions listed in Table 5
reflect transfer of properties between the different cellulases
aligned in Table 1. The positions listed in Table 6 reflect
transfer of properties from Humicola insolens EGV to the other
cellulases aligned in Table 1.
TABLE-US-00005 TABLE 5 Preferred Cellulase Variants Positions
Identified by Cellulase Numbering K13L, L13K (1, 2, 3); P14A, A14P
(1); S15H, H15S (1, 3); K20E, K20G, K20A, E20K, G20K, A20K, E20G,
E20A, G20E, A20E, G20A, A20G (1, 2, 3); K21N, N21K (1, 2, 3); A22G,
A22P, G22A, P22A, G22P, P22G (1); V24*, V24L, *24V, L24V, *24L,
L24* (1); V28A, V28L, A28V, L28V, A28L, L28A (1); N32D, N32S, N32K,
D32N, S32N, K32N, D32S, D32K, S32D, K32D, S32K, K32S (2, 3); N34D,
D34N (2); I38L, I38F, I38Q, L38I, F38I, Q38I, L38F, L38Q, F38L,
Q38L, F38Q, Q38F (1) S45N, N45S (1); G46S, S46G (1); E48D, E48N,
D48E, N48E, D48N, N48D (1, 2, 3); G50N, N50G (1); A53S, A53G, A53K,
S53A, G53A, K53A, S53G, S53K, G53S, K53S, G53K, K53G (1); Y54F,
F54Y (1, 3); W62F, F62W (1, 2); A63D, D63A (2, 3); V64I, V64D,
I64V, D64V, I64D, D64I (2); N65S, N65D, N65E, S65N, D65N, E65N,
S65D, S65E, D65S, E65S, D65E, E65D (2); D66N, D66P, D66T, N66D,
P66D, T66D, N66P, N66T, P66N, T66N, P66T, T66P (2, 3); F68V, F68L,
F68T, F68P, V68F, L68F, T68F, P68F, V68L, V68T, V68P, L68V, T68V,
P68V, L68T, L68P, T68L, P68L, T68P, P68T (1, 2); A69S, A69T, S69A,
T69A, S69T, T69S (1); L70Y, Y70L (1); G71A, A71G (1); F72W, F72Y,
W72F, Y72F, W72Y, Y72W (1); A73G, G73A (1); A74F, F74A (1); T75V,
T75A, T75G, V75T, A75T, G75T, V75A, V75G, A75V, G75V, A75G, G75A
(1); G79T, T79G (1); W85T, T85W (1); A88Q, A88G, A88R, Q88A, G88A,
R88A, Q88G, Q88R, G88Q, R88Q, G88R, R88G (1, 2, 3); Y90F, F90Y (1);
L92A, A92L (1); T93Q, T93E, Q93T, E93T, Q93E, E93Q (2); T95E, E95T
(2); S96T, T96S (1); G97T, G97A, T97G, A97G, T97A, A97T (1); P98A,
A98P (1); V99L, L99V (1); M104L, L104M (1); V106F, F106V (1, 3);
S110N, N110S (1); T111I, T111V, I111T, V111T, I111V, V111I (1);
G113Y, Y113G (1, 3); L115V, V115L (1); G116S, G116Q, S116G, Q116G,
S116Q, Q116S (1); N118T, N118G, N118Q, T118N, G118N, Q118N, T118G,
T118Q, G118T, Q118T, G118Q, Q118G (1); H119Q, H119N, Q119H, N119H
(1, 2); V129L, L129V (1); I131L, I131A, L131I, A131I, L131A, A131L
(1); G134A, A134G (1); Q138E, E138Q (1, 2, 3); G140N, N140G (1);
R146Q, Q146R (1, 2, 3); S152D, D152S (2); R153K, R153L, R153A,
K153R, L153R, A153R, K153L, K153A, L153K, A153K, L153A, A153L (2);
L163V, L163W, V163L, W163L, V163W, W163V (1); G166S, S166G (1);
W169F, F169W (1); R170F, F170R (1, 2, 3); F171Y, F171A, Y171F,
A171F, Y171A, A171Y (1); D172E, D172S, E172D, S172D, E172S, S172E
(2); W173E, E173W (1, 2, 3); F174M, F174W, M174F, W174F, M174W,
W174M (1); A177N, N177A (1); D178P, P178D (1, 2, 3); N179V, V179N
(1); P180L, L180P (1); L193I, I193L (1); R196I, R196K, I196R,
K196R, I196K, K196I (2, 3); T197S, S197T (1)
TABLE-US-00006 TABLE 6 Preferred Cellulase Variants Positions
Identified by Cellulase Numbering L13K (1, 2, 3); A14P (1); H15S
(1, 3); E20K, G20K, A20K (1, 2, 3); N21K (1, 2, 3); G22A, P22A (1);
*24V, L24V (1); A28V, L28V (1); D32N, S32N, K32N (2, 3); D34N (2);
L38I, F38I, Q38I (1); N45S (1); S46G (1); D48E, N48E (1, 2, 3);
N50G (1); S53A, G53A, K53A (1); F54Y (1, 3); F62W (1, 2); D63A (2,
3); I64V, D64V (2); S65N, D65N, E65N (2) N66D, P66D, T66D (2, 3);
V68F, L68F, T68F, P68F (1, 2); S69A, T69A (1) Y70L (1) A71G (1)
W72F, Y72F (1) G73A (1) F74A (1) V75T, A75T, G75T (1) T79G (1);
T85W (1); Q88A, G88A, R88A (1, 2, 3) F90Y (1) A92L (1) Q93T, E93T
(2); E95T (2); T96S (1); T97G, A97G (1); A98P (1); L99V (1); L104M
(1); F106V (1, 3); N110S (1); I111T, V111T (1); Y113G (1, 3); V115L
(1); S116G, Q116G (1); T118N, G118N, Q118N (1); Q119H, N119H (1,
2); L129V (1); L131I, A131I (1); A134G (1); E138Q (1, 2, 3); N140G
(1); Q146R (1, 2, 3); D152S (2); K153R, L153R, A153R (2); V163L,
W163L (1); S166G (1); F169W (1); F170R (1, 2, 3); Y171F, A171F (1);
E172D, S172D (2); E173W (1, 2, 3); M174F, W174F (1); N177A (1);
P178D (1, 2, 3); V179N (1); L180P (1); I193L (1); I196R, K196R (2,
3); S197T (1)
Altered Sensibility Towards Anionic Tensides
[0131] As mentioned above, anionic tensides are products frequently
incorporated into detergent compositions. Sometimes cellulolytic
enzymes having an increased stability towards anionic tensides are
desired, and sometimes cellulolytic enzymes having an increased
sensitivity are preferred. In a further aspect the invention
provides cellulase variants having an altered anionic tenside
sensitivity.
[0132] Accordingly, a cellulase variant of the invention of altered
anionic tenside sensitivity is a cellulase variant which has been
derived from a parental cellulase by substitution, insertion and/or
deletion at one or more of the following positions: 2, 4, 7, 8, 10,
13, 15, 19, 20, 21, 25, 26, 29, 32, 33, 34, 35, 37, 40, 42, 42a,
43, 44, 48, 53, 54, 55, 58, 59, 63, 64, 65, 66, 67, 70, 72, 76, 79,
80, 82, 84, 86, 88, 90, 91, 93, 95, 95d, 95h, 95j, 97, 100, 101,
102, 103, 113, 114, 117, 119, 121, 133, 136, 137, 138, 139, 140a,
141, 143a, 145, 146, 147, 150e, 150j, 151, 152, 153, 154, 155, 156,
157, 158, 159, 160c, 160e, 160k, 161, 162, 164, 165, 168, 170, 171,
172, 173, 175, 176, 178, 181, 183, 184, 185, 186, 188, 191, 192,
195, 196, 200, and/or 201 (cellulase numbering). These positions
contain, in at least one of the cellulase sequences aligned in
Table 1, a charged or potentially charged aa residue.
[0133] In a particular embodiment, the cellulase variant is derived
from one of the cellulases identified in Table 7, below, ((a)
Humicola insolens; (b) Acremonium sp.; (c) Volutella
collectotrichoides; (d) Sordaria fimicola; (e) Thielavia
terrestris; (f) Fusarium oxysporum; (g) Myceliophthora thermophila;
(h) Crinipellis scabella; (i) Macrophomina phaseolina; (j)
Pseudomonas fluorescens; (k) Ustilago maydis), by substitution,
insertion and/or deletion at one or more of the positions
identified in Table 7 for these cellulases.
TABLE-US-00007 TABLE 7 Altered Sensitivity towards Anionic Tensides
Positions identified by Cellulase Numbering (a) Humicola insolens;
(b) Acremonium sp.; (c) Volutella collectotrichoides; (d) Sordaria
fimicola; (e) Thielavia terrestris; (f) Fusarium oxysporum; (g)
Myceliophthora thermophila; (h) Crinipellis scabella; (i)
Macrophomina phaseolina; (j) Pseudomonas fluorescens; (k) Ustilago
maydis. a b c d e f g h i j k 2 D 4 R H R K H Y 7 R R R R R R R R R
R R 8 Y Y Y Y Y Y Y Y Y Y Y 10 D D D D D D D D D D D 13 K K K K K K
K K K K 15 H 19 D D E 20 K E E 21 K K K K K K K K K K 25 Y 26 R R K
29 K Y R D 32 D D D D D D D D K 33 R R K K R 34 D 35 D D D 37 R R R
40 D D D D D D 42 D D K 42a D K 43 D 44 K R K R K K 48 E D D D E D
D D D 53 K 54 Y Y Y Y Y Y Y Y 55 Y 58 D D D D D 59 Y K 63 D 64 D 65
D E 66 D D D D D D D 67 D E E D 70 Y Y Y Y Y Y Y Y Y 72 Y 76 K K K
H K 79 D 80 K 82 E E E E E E E E E E 84 D D 86 D 88 R 90 Y Y Y Y Y
Y Y Y Y Y 91 E E E K Y 93 E 95 E 95d Y 95h H 95i D D 97 K 100 K K
101 R 102 K K K K K K K K K K 103 K K K K K 113 Y 114 D D D D D D D
D D D D 117 D D 119 H H H H H H H H 121 D D D D D D D D D D D 133 D
D D D D K 136 K 137 R D K 138 E E 139 Y 140a K 141 E 143a E L 145 D
D 146 R R R R 147 Y Y Y Y Y Y Y Y Y Y Y 150e K 150j Y 151 H K 152 D
153 R R R R R R K R R 154 D E D 155 E E E E E 156 Y 157 D D D D E K
158 R E K 159 Y 160c R 160e D 160k R 161 D E E K 162 K 164 K R K K
K K 165 D D E 168 Y H H K 170 R R R R R R R R R R 171 Y Y 172 D D D
D D D D D D E 173 E 175 K K E E E 176 D D D 178 D D D D D D D D D D
181 D E D K 183 D K 184 Y 185 R R R K E R E K K 186 R R E E R 188 R
K 191 K K 192 E E E E E E 195 D D D 196 R R R R R K R R R 200 R R K
K K 201 R R R R R R R R R R R
Enzyme Compositions
[0134] In a still further aspect, the present invention relates to
an enzyme composition comprising an enzyme exhibiting cellulolytic
activity as described above.
[0135] The enzyme composition of the invention may, in addition to
the cellulase of the invention, comprise one or more other enzyme
types, for instance hemi-cellulase such as xylanase and mannanase,
other cellulase components, chitinase, lipase, esterase, pectinase,
cutinase, phytase, oxidoreductase, peroxidase, laccase, oxidase,
pactinmethylesterase, polygalacturonase, protease, or amylase.
[0136] The enzyme composition may be prepared in accordance with
methods known in the art and may be in the form of a liquid or a
dry composition. For instance, the enzyme composition may be in the
form of a granulate or a microgranulate. The enzyme to be included
in the composition may be stabilized in accordance with methods
known in the art.
[0137] Examples are given below of preferred uses of the enzyme
composition of the invention. The dosage of the enzyme composition
of the invention and other conditions under which the composition
is used may be determined on the basis of methods known in the
art.
[0138] The enzyme composition according to the invention may be
useful for at least one of the following purposes.
Uses
[0139] During washing and wearing, dyestuff from dyed fabrics or
garment will conventionally bleed from the fabric which then looks
faded and worn. Removal of surface fibers from the fabric will
partly restore the original colors and looks of the fabric. By the
term "color clarification", as used herein, is meant the partly
restoration of the initial colors of fabric or garment throughout
multiple washing cycles.
[0140] The term "de-pilling" denotes removing of pills from the
fabric surface.
[0141] The term "soaking liquor" denotes an aqueous liquor in which
laundry may be immersed prior to being subjected to a conventional
washing process. The soaking liquor may contain one or more
ingredients conventionally used in a washing or laundering
process.
[0142] The term "washing liquor" denotes an aqueous liquor in which
laundry is subjected to a washing process, i.e., usually a combined
chemical and mechanical action either manually or in a washing
machine. Conventionally, the washing liquor is an aqueous solution
of a powder or liquid detergent composition.
[0143] The term "rinsing liquor" denotes an aqueous liquor in which
laundry is immersed and treated, conventionally immediately after
being subjected to a washing process, in order to rinse the
laundry, i.e., essentially remove the detergent solution from the
laundry. The rinsing liquor may contain a fabric conditioning or
softening composition.
[0144] The laundry subjected to the method of the present invention
may be conventional washable laundry. Preferably, the major part of
the laundry is sewn or un-sewn fabrics, including knits, wovens,
denims, yarns, and toweling, made from cotton, cotton blends or
natural or manmade cellulosics (e.g., originating from
xylan-containing cellulose fibers such as from wood pulp) or blends
thereof. Examples of blends are blends of cotton or rayon/viscose
with one or more companion material such as wool, synthetic fibers
(e.g., polyamide fibers, acrylic fibers, polyester fibers,
polyvinyl alcohol fibers, polyvinyl chloride fibers, polyvinylidene
chloride fibers, polyurethane fibers, polyurea fibers, aramid
fibers), and cellulose-containing fibers (e.g., rayon/viscose,
ramie, flax/linen, jute, cellulose acetate fibers, lyocell).
Detergent Disclosure and Examples
Surfactant System
[0145] The detergent compositions according to the present
invention comprise a surfactant system, wherein the surfactant can
be selected from nonionic and/or anionic and/or cationic and/or
ampholytic and/or zwitterionic and/or semi-polar surfactants.
[0146] The surfactant is typically present at a level from 0.1% to
60% by weight.
[0147] The surfactant is preferably formulated to be compatible
with enzyme components present in the composition. In liquid or gel
compositions the surfactant is most preferably formulated in such a
way that it promotes, or at least does not degrade, the stability
of any enzyme in these compositions.
[0148] Preferred systems to be used according to the present
invention comprise as a surfactant one or more of the nonionic
and/or anionic surfactants described herein.
[0149] Polyethylene, polypropylene, and polybutylene oxide
condensates of alkyl phenols are suitable for use as the nonionic
surfactant of the surfactant systems of the present invention, with
the polyethylene oxide condensates being preferred. These compounds
include the condensation products of alkyl phenols having an alkyl
group containing from about 6 to about 14 carbon atoms, preferably
from about 8 to about 14 carbon atoms, in either a straight chain
or branched-chain configuration with the alkylene oxide. In a
preferred embodiment, the ethylene oxide is present in an amount
equal to from about 2 to about 25 moles, more preferably from about
3 to about 15 moles, of ethylene oxide per mole of alkyl phenol.
Commercially available nonionic surfactants of this type include
Igepal.TM. CO-630, marketed by the GAF Corporation; and Triton.TM.
X-45, X-114, X-100 and X-102, all marketed by the Rohm & Haas
Company. These surfactants are commonly referred to as alkylphenol
alkoxylates (e.g., alkyl phenol ethoxylates).
[0150] The condensation products of primary and secondary aliphatic
alcohols with about 1 to about 25 moles of ethylene oxide are
suitable for use as the nonionic surfactant of the nonionic
surfactant systems of the present invention. The alkyl chain of the
aliphatic alcohol can either be straight or branched, primary or
secondary, and generally contains from about 8 to about 22 carbon
atoms. Preferred are the condensation products of alcohols having
an alkyl group containing from about 8 to about 20 carbon atoms,
more preferably from about 10 to about 18 carbon atoms, with from
about 2 to about 10 moles of ethylene oxide per mole of alcohol.
About 2 to about 7 moles of ethylene oxide and most preferably from
2 to 5 moles of ethylene oxide per mole of alcohol are present in
said condensation products. Examples of commercially available
nonionic surfactants of this type include Tergitol.TM. 15-S-9 (The
condensation product of C11-C15 linear alcohol with 9 moles
ethylene oxide), Tergitol.TM. 24-L-6 NMW (the condensation product
of C12-C14 primary alcohol with 6 moles ethylene oxide with a
narrow molecular weight distribution), both marketed by Union
Carbide Corporation; Neodol.TM. 45-9 (the condensation product of
C14-C15 linear alcohol with 9 moles of ethylene oxide), Neodol.TM.
23-3 (the condensation product of C12-C13 linear alcohol with 3
moles of ethylene oxide), Neodol.TM. 45-7 (the condensation product
of C14-C15 linear alcohol with 7 moles of ethylene oxide),
Neodol.TM. 45-5 (the condensation product of C14-C15 linear alcohol
with 5 moles of ethylene oxide) marketed by Shell Chemical Company,
Kyro.TM. EOB (the condensation product of C13-C15 alcohol with 9
moles ethylene oxide), marketed by The Procter & Gamble
Company, and Genapol LA 050 (the condensation product of C12-C14
alcohol with 5 moles of ethylene oxide) marketed by Hoechst.
Preferred range of HLB in these products is from 8-11 and most
preferred from 8-10.
[0151] Also useful as the nonionic surfactant of the surfactant
systems of the present invention are alkylpolysaccharides disclosed
in U.S. Pat. No. 4,565,647, having a hydrophobic group containing
from about 6 to about 30 carbon atoms, preferably from about 10 to
about 16 carbon atoms and a polysaccharide, e.g., a polyglycoside,
hydrophilic group containing from about 1.3 to about 10, preferably
from about 1.3 to about 3, most preferably from about 1.3 to about
2.7 saccharide units. Any reducing saccharide containing 5 or 6
carbon atoms can be used, e.g., glucose, galactose and galactosyl
moieties can be substituted for the glucosyl moieties (optionally
the hydrophobic group is attached at the 2-, 3-, 4-, etc. positions
thus giving a glucose or galactose as opposed to a glucoside or
galactoside). The intersaccharide bonds can be, e.g., between the
one position of the additional saccharide units and the 2-, 3-, 4-,
and/or 6-positions on the preceding saccharide units.
[0152] The preferred alkylpolyglycosides have the formula
R2O(CnH.sub.2nO)t(glycosyl)x
wherein R2 is selected from the group consisting of alkyl,
alkylphenyl, hydroxyalkyl, hydroxyalkylphenyl, and mixtures thereof
in which the alkyl groups contain from about 10 to about 18,
preferably from about 12 to about 14, carbon atoms; n is 2 or 3,
preferably 2; t is from 0 to about 10, preferably 0; and x is from
about 1.3 to about 10, preferably from about 1.3 to about 3, most
preferably from about 1.3 to about 2.7. The glycosyl is preferably
derived from glucose. To prepare these compounds, the alcohol or
alkylpolyethoxy alcohol is formed first and then reacted with
glucose, or a source of glucose, to form the glucoside (attachment
at the 1-position). The additional glycosyl units can then be
attached between their 1-position and the preceding glycosyl units
2-, 3-, 4-, and/or 6-position, preferably predominantly the
2-position.
[0153] The condensation products of ethylene oxide with a
hydrophobic base formed by the condensation of propylene oxide with
propylene glycol are also suitable for use as the additional
nonionic surfactant systems of the present invention. The
hydrophobic portion of these compounds will preferably have a
molecular weight from about 1500 to about 1800 and will exhibit
water insolubility. The addition of polyoxyethylene moieties to
this hydrophobic portion tends to increase the water solubility of
the molecule as a whole, and the liquid character of the product is
retained up to the point where the polyoxyethylene content is about
50% of the total weight of the condensation product, which
corresponds to condensation with up to about 40 moles of ethylene
oxide. Examples of compounds of this type include certain of the
commercially available Pluronic.TM. surfactants, marketed by
BASF.
[0154] Also suitable for use as the nonionic surfactant of the
nonionic surfactant system of the present invention, are the
condensation products of ethylene oxide with the product resulting
from the reaction of propylene oxide and ethylenediamine. The
hydrophobic moiety of these products consists of the reaction
product of ethylenediamine and excess propylene oxide, and
generally has a molecular weight of from about 2500 to about 3000.
This hydrophobic moiety is condensed with ethylene oxide to the
extent that the condensation product contains from about 40% to
about 80% by weight of polyoxyethylene and has a molecular weight
of from about 5,000 to about 11,000. Examples of this type of
nonionic surfactant include certain of the commercially available
Tetronic.TM. compounds, marketed by BASF.
[0155] Preferred for use as the nonionic surfactant of the
surfactant systems of the present invention are polyethylene oxide
condensates of alkyl phenols, condensation products of primary and
secondary aliphatic alcohols with from about 1 to about 25 moles of
ethyleneoxide, alkylpolysaccharides, and mixtures hereof. Most
preferred are C8-C14 alkyl phenol ethoxylates having from 3 to 15
ethoxy groups and C8-C18 alcohol ethoxylates (preferably C10 avg.)
having from 2 to 10 ethoxy groups, and mixtures thereof.
[0156] Highly preferred nonionic surfactants are polyhydroxy fatty
acid amide surfactants of the formula
##STR00001##
wherein R1 is H, or R1 is C1-4 hydrocarbyl, 2-hydroxyethyl,
2-hydroxypropyl or a mixture thereof, R2 is C5-31 hydrocarbyl, and
Z is a polyhydroxyhydrocarbyl having a linear hydrocarbyl chain
with at least 3 hydroxyls directly connected to the chain, or an
alkoxylated derivative thereof. Preferably, R1 is methyl, R2 is
straight C11-15 alkyl or C16-18 alkyl or alkenyl chain such as
coconut alkyl or mixtures thereof, and Z is derived from a reducing
sugar such as glucose, fructose, maltose or lactose, in a reductive
amination reaction.
[0157] Highly preferred anionic surfactants include alkyl
alkoxylated sulfate surfactants. Examples hereof are water soluble
salts or acids of the formula RO(A)mSO3M wherein R is an
unsubstituted C10-C-24 alkyl or hydroxyalkyl group having a C10-C24
alkyl component, preferably a C12-C20 alkyl or hydro-xyalkyl, more
preferably C12-C18 alkyl or hydroxyalkyl, A is an ethoxy or propoxy
unit, m is greater than zero, typically between about 0.5 and about
6, more preferably between about 0.5 and about 3, and M is H or a
cation which can be, for example, a metal cation (e.g., sodium,
potassium, lithium, calcium, magnesium, etc.), ammonium or
substituted-ammonium cation. Alkyl ethoxylated sulfates as well as
alkyl propoxylated sulfates are contemplated herein. Specific
examples of substituted ammonium cations include methyl-, dimethyl,
trimethyl-ammonium cations and quaternary ammonium cations such as
tetramethyl-ammonium and dimethyl piperidinium cations and those
derived from alkylamines such as ethylamine, diethylamine,
triethylamine, mixtures thereof, and the like. Exemplary
surfactants are C12-C18 alkyl polyethoxylate (1.0) sulfate
(C12-C18E(1.0)M), C12-C18 alkyl polyethoxylate (2.25) sulfate
(C12-C18(2.25)M, and C12-C18 alkyl polyethoxylate (3.0) sulfate
(C12-C18E(3.0)M), and C12-C18 alkyl polyethoxylate (4.0) sulfate
(C12-C18E(4.0)M), wherein M is conveniently selected from sodium
and potassium.
[0158] Suitable anionic surfactants to be used are alkyl ester
sulfonate surfactants including linear esters of C8-C20 carboxylic
acids (i.e., fatty acids) which are sulfonated with gaseous
SO.sub.3 according to "The Journal of the American Oil Chemists
Society", 52, 1975, pp. 323-329. Suitable starting materials would
include natural fatty substances as derived from tallow, palm oil,
etc.
[0159] The preferred alkyl ester sulfonate surfactant, especially
for laundry applications, comprises alkyl ester sulfonate
surfactant of the structural formula:
##STR00002##
wherein R3 is a C8-C20 hydrocarbyl, preferably an alkyl, or
combination thereof, R4 is a C1-C6 hydrocarbyl, preferably an
alkyl, or combination thereof, and M is a cation which forms a
water soluble salt with the alkyl ester sulfonate. Suitable
salt-forming cations include metals such as sodium, potassium, and
lithium, and substituted or unsubstituted ammonium cations, such as
monoethanolamine, diethonolamine, and triethanolamine. Preferably,
R3 is C10-C16 alkyl, and R4 is methyl, ethyl or isopropyl.
Especially preferred are the methyl ester sulfonates wherein R3 is
C10-C16 alkyl.
[0160] Other suitable anionic surfactants include the alkyl sulfate
surfactants which are water soluble salts or acids of the formula
ROSO.sub.3M wherein R preferably is a C10-C24 hydrocarbyl,
preferably an alkyl or hydroxyalkyl having a C10-C20 alkyl
component, more preferably a C12-C18 alkyl or hydroxyalkyl, and M
is H or a cation, e.g., an alkali metal cation (e.g., sodium,
potassium, lithium), or ammonium or substituted ammonium (e.g.,
methyl-, dimethyl-, and trimethyl ammonium cations and quaternary
ammonium cations such as tetramethyl-ammonium and dimethyl
piperidinium cations and quaternary ammonium cations derived from
alkylamines such as ethylamine, diethylamine, triethylamine, and
mixtures thereof, and the like). Typically, alkyl chains of C12-C16
are preferred for lower wash temperatures (e.g., below about
50.degree. C.) and C16-C18 alkyl chains are preferred for higher
wash temperatures (e.g., above about 50.degree. C.).
[0161] Other anionic surfactants useful for detersive purposes can
also be included in the laundry detergent compositions of the
present invention. Theses can include salts (including, for
example, sodium, potassium, ammonium, and substituted ammonium
salts such as mono- di- and triethanolamine salts) of soap, C8-C22
primary or secondary alkanesulfonates, C8-C24 olefinsulfonates,
sulfonated polycarboxylic acids prepared by sulfonation of the
pyrolyzed product of alkaline earth metal citrates, e.g., as
described in British patent specification No. 1,082,179, C8-C24
alkylpolyglycolethersulfates (containing up to 10 moles of ethylene
oxide); alkyl glycerol sulfonates, fatty acyl glycerol sulfonates,
fatty oleyl glycerol sulfates, alkyl phenol ethylene oxide ether
sulfates, paraffin sulfonates, alkyl phosphates, isethionates such
as the acyl isethionates, N-acyl taurates, alkyl succinamates and
sulfosuccinates, monoesters of sulfosuccinates (especially
saturated and unsaturated C12-C18 monoesters) and diesters of
sulfosuccinates (especially saturated and unsaturated C6-C12
diesters), acyl sarcosinates, sulfates of alkylpolysaccharides such
as the sulfates of alkylpolyglucoside (the nonionic nonsulfated
compounds being described below), branched primary alkyl sulfates,
and alkyl polyethoxy carboxylates such as those of the formula
RO(CH.sub.2CH.sub.2O)k-CH.sub.2C00-M+ wherein R is a C8-C22 alkyl,
k is an integer from 1 to 10, and M is a soluble salt forming
cation. Resin acids and hydrogenated resin acids are also suitable,
such as rosin, hydrogenated rosin, and resin acids and hydrogenated
resin acids present in or derived from tall oil.
[0162] Alkylbenzene sulfonates are highly preferred. Especially
preferred are linear (straight-chain) alkyl benzene sulfonates
(LAS) wherein the alkyl group preferably contains from 10 to 18
carbon atoms.
[0163] Further examples are described in "Surface Active Agents and
Detergents" (Vols. I and II by Schwartz, Perrry and Berch). A
variety of such surfactants are also generally disclosed in U.S.
Pat. No. 3,929,678 (column 23, line 58 through column 29, line 23,
herein incorporated by reference).
[0164] When included therein, the laundry detergent compositions of
the present invention typically comprise from about 1% to about
40%, preferably from about 3% to about 20% by weight of such
anionic surfactants.
[0165] The laundry detergent compositions of the present invention
may also contain cationic, ampholytic, zwitterionic, and semi-polar
surfactants, as well as the nonionic and/or anionic surfactants
other than those already described herein.
[0166] Cationic detersive surfactants suitable for use in the
laundry detergent compositions of the present invention are those
having one long-chain hydrocarbyl group. Examples of such cationic
surfactants include the ammonium surfactants such as
alkyltrimethylammonium halogenides, and those surfactants having
the formula:
[R2(OR3)y][R4(OR3)y]2R5N+X--
wherein R2 is an alkyl or alkyl benzyl group having from about 8 to
about 18 carbon atoms in the alkyl chain, each R3 is selected form
the group consisting of --CH.sub.2CH.sub.2--,
--CH.sub.2CH(CH.sub.3)--, --CH.sub.2CH(CH.sub.2OH)--,
--CH.sub.2CH.sub.2CH.sub.2--, and mixtures thereof; each R4 is
selected from the group consisting of C1-C4 alkyl, C1-C4
hydroxyalkyl, benzyl ring structures formed by joining the two R4
groups, --CH.sub.2CHOHCHOHCOR6CHOHCH.sub.2OH, wherein R6 is any
hexose or hexose polymer having a molecular weight less than about
1000, and hydrogen when y is not 0; R5 is the same as R4 or is an
alkyl chain, wherein the total number of carbon atoms or R2 plus R5
is not more than about 18; each y is from 0 to about 10, and the
sum of the y values is from 0 to about 15; and X is any compatible
anion.
[0167] Highly preferred cationic surfactants are the water soluble
quaternary ammonium compounds useful in the present composition
having the formula:
R1R2R3R4N+X-- (i)
wherein R1 is C8-C16 alkyl, each of R2, R3 and R4 is independently
C1-C4 alkyl, C1-C4 hydroxy alkyl, benzyl, and --(C2H40)xH where x
has a value from 2 to 5, and X is an anion. Not more than one of
R2, R3 or R4 should be benzyl.
[0168] The preferred alkyl chain length for R1 is C12-C15,
particularly where the alkyl group is a mixture of chain lengths
derived from coconut or palm kernel fat or is derived synthetically
by olefin build up or OXO alcohols synthesis.
[0169] Preferred groups for R2, R3 and R4 are methyl and
hydroxyethyl groups and the anion
[0170] X may be selected from halide, methosulphate, acetate and
phosphate ions.
[0171] Examples of suitable quaternary ammonium compounds of
formulae (i) for use herein are:
[0172] coconut trimethyl ammonium chloride or bromide;
[0173] coconut methyl dihydroxyethyl ammonium chloride or
bromide;
[0174] decyl triethyl ammonium chloride;
[0175] decyl dimethyl hydroxyethyl ammonium chloride or
bromide;
[0176] C12-15 dimethyl hydroxyethyl ammonium chloride or
bromide;
[0177] coconut dimethyl hydroxyethyl ammonium chloride or
bromide;
[0178] myristyl trimethyl ammonium methyl sulphate;
[0179] lauryl dimethyl benzyl ammonium chloride or bromide;
[0180] lauryl dimethyl (ethenoxy)4 ammonium chloride or
bromide;
[0181] choline esters (compounds of formula (i) wherein R1 is
##STR00003##
[0182] di-alkyl imidazolines [compounds of formula (i)].
[0183] Other cationic surfactants useful herein are also described
in U.S. Pat. No. 4,228,044 and in EP 000 224.
[0184] When included therein, the laundry detergent compositions of
the present invention typically comprise from 0.2% to about 25%,
preferably from about 1% to about 8% by weight of such cationic
surfactants.
[0185] Ampholytic surfactants are also suitable for use in the
laundry detergent compositions of the present invention. These
surfactants can be broadly described as aliphatic derivatives of
secondary or tertiary amines, or aliphatic derivatives of
heterocyclic secondary and tertiary amines in which the aliphatic
radical can be straight or branched-chain. One of the aliphatic
substituents contains at least about 8 carbon atoms, typically from
about 8 to about 18 carbon atoms, and at least one contains an
anionic water-solubilizing group, e.g., carboxy, sulfonate,
sulfate. See U.S. Pat. No. 3,929,678 (column 19, lines 18-35) for
examples of ampholytic surfactants.
[0186] When included therein, the laundry detergent compositions of
the present invention typically comprise from 0.2% to about 15%,
preferably from about 1% to about 10% by weight of such ampholytic
surfactants.
[0187] Zwitterionic surfactants are also suitable for use in
laundry detergent compositions. These surfactants can be broadly
described as derivatives of secondary and tertiary amines,
derivatives of heterocyclic secondary and tertiary amines, or
derivatives of quaternary ammonium, quaternary phosphonium or
tertiary sulfonium compounds. See U.S. Pat. No. 3,929,678 (column
19, line 38 through column 22, line 48) for examples of
zwitterionic surfactants.
[0188] When included therein, the laundry detergent compositions of
the present invention typically comprise from 0.2% to about 15%,
preferably from about 1% to about 10% by weight of such
zwitterionic surfactants.
[0189] Semi-polar nonionic surfactants are a special category of
nonionic surfactants which include water-soluble amine oxides
containing one alkyl moiety of from about 10 to about 18 carbon
atoms and 2 moieties selected from the group consisting of alkyl
groups and hydroxyalkyl groups containing from about 1 to about 3
carbon atoms; water soluble phosphine oxides containing one alkyl
moiety of from about 10 to about 18 carbon atoms and 2 moieties
selected from the group consisting of alkyl groups and hydroxyalkyl
groups containing from about 1 to about 3 carbon atoms; and
water-soluble sulfoxides containing one alkyl moiety from about 10
to about 18 carbon atoms and a moiety selected from the group
consisting of alkyl and hydroxyalkyl moieties of from about 1 to
about 3 carbon atoms.
[0190] Semi-polar nonionic detergent surfactants include the amine
oxide surfactants having the formula:
##STR00004##
wherein R3 is an alkyl, hydroxyalkyl, or alkyl phenyl group or
mixtures thereof containing from about 8 to about 22 carbon atoms;
R4 is an alkylene or hydroxyalkylene group containing from about 2
to about 3 carbon atoms or mixtures thereof; x is from 0 to about
3: and each R5 is an alkyl or hydroxyalkyl group containing from
about 1 to about 3 carbon atoms or a polyethylene oxide group
containing from about 1 to about 3 ethylene oxide groups. The R5
groups can be attached to each other, e.g., through an oxygen or
nitrogen atom, to form a ring structure.
[0191] These amine oxide surfactants in particular include C10-C18
alkyl dimethyl amine oxides and C8-C12 alkoxy ethyl dihydroxy ethyl
amine oxides.
[0192] When included therein, the laundry detergent compositions of
the present invention typically comprise from 0.2% to about 15%,
preferably from about 1% to about 10% by weight of such semi-polar
nonionic surfactants.
Builder System
[0193] The compositions according to the present invention may
further comprise a builder system. Any conventional builder system
is suitable for use herein including aluminosilicate materials,
silicates, polycarboxylates and fatty acids, materials such as
ethylenediamine tetraacetate, metal ion sequestrants such as
aminopolyphosphonates, particularly ethylenediamine tetramethylene
phosphonic acid and diethylene triamine pentamethylenephosphonic
acid. Though less preferred for obvious environmental reasons,
phosphate builders can also be used herein.
[0194] Suitable builders can be an inorganic ion exchange material,
commonly an inorganic hydrated aluminosilicate material, more
particularly a hydrated synthetic zeolite such as hydrated zeolite
A, X, B, HS or MAP.
[0195] Another suitable inorganic builder material is layered
silicate, e.g., SKS-6 (Hoechst). SKS-6 is a crystalline layered
silicate consisting of sodium silicate
(Na.sub.2Si.sub.2O.sub.5).
[0196] Suitable polycarboxylates containing one carboxy group
include lactic acid, glycolic acid and ether derivatives thereof as
disclosed in Belgian Patent Nos. 831,368, 821,369 and 821,370.
Polycarboxylates containing two carboxy groups include the
water-soluble salts of succinic acid, malonic acid,
(ethylenedioxy)diacetic acid, maleic acid, diglycollic acid,
tartaric acid, tartronic acid and fumaric acid, as well as the
ether carboxylates described in German Offenle-enschrift 2,446,686,
and 2,446,487, U.S. Pat. No. 3,935,257 and the sulfinyl
carboxylates described in Belgian Patent No. 840,623.
Polycarboxylates containing three carboxy groups include, in
particular, water-soluble citrates, aconitrates and citraconates as
well as succinate derivatives such as the
carboxymethyloxysuccinates described in British Patent No.
1,379,241, lactoxysuccinates described in Netherlands Application
7205873, and the oxypolycarboxylate materials such as
2-oxa-1,1,3-propane tricarboxylates described in British Patent No.
1,387,447.
[0197] Polycarboxylates containing four carboxy groups include
oxydisuccinates disclosed in British Patent No. 1,261,829,
1,1,2,2,-ethane tetracarboxylates, 1,1,3,3-propane
tetracarboxylates containing sulfo substituents include the
sulfosuccinate derivatives disclosed in British Patent Nos.
1,398,421 and 1,398,422 and in U.S. Pat. No. 3,936,448, and the
sulfonated pyrolysed citrates described in British Patent No.
1,082,179, while polycarboxylates containing phosphone substituents
are disclosed in British Patent No. 1,439,000.
[0198] Alicyclic and heterocyclic polycarboxylates include
cyclopentane-cis,cis-cis-tetracarboxylates, cyclopentadienide
pentacarboxylates, 2,3,4,5-tetrahydro-furan-cis, cis,
cis-tetracarboxylates, 2,5-tetrahydro-furan-cis, discarboxylates,
2,2,5,5,-tetrahydrofuran-tetracarboxylates,
1,2,3,4,5,6-hexane-hexacarboxylates and carboxymethyl derivatives
of polyhydric alcohols such as sorbitol, mannitol and xylitol.
Aromatic polycarboxylates include mellitic acid, pyromellitic acid
and the phthalic acid derivatives disclosed in British Patent No.
1,425,343.
[0199] Of the above, the preferred polycarboxylates are
hydroxy-carboxylates containing up to three carboxy groups per
molecule, more particularly citrates.
[0200] Preferred builder systems for use in the present
compositions include a mixture of a water-insoluble aluminosilicate
builder such as zeolite A or of a layered silicate (SKS-6), and a
water-soluble carboxylate chelating agent such as citric acid.
[0201] A suitable chelant for inclusion in the detergent
compositions in accordance with the invention is
ethylenediamine-N,N'-disuccinic acid (EDDS) or the alkali metal,
alkaline earth metal, ammonium, or substituted ammonium salts
thereof, or mixtures thereof. Preferred EDDS compounds are the free
acid form and the sodium or magnesium salt thereof. Examples of
such preferred sodium salts of EDDS include Na.sub.2EDDS and
Na.sub.4EDDS. Examples of such preferred magnesium salts of EDDS
include MgEDDS and Mg.sub.2EDDS. The magnesium salts are the most
preferred for inclusion in compositions in accordance with the
invention.
[0202] Preferred builder systems include a mixture of a
water-insoluble aluminosilicate builder such as zeolite A, and a
water soluble carboxylate chelating agent such as citric acid.
[0203] Other builder materials that can form part of the builder
system for use in granular compositions include inorganic materials
such as alkali metal carbonates, bicarbonates, silicates, and
organic materials such as the organic phosphonates, amino
polyalkylene phosphonates and amino polycarboxylates.
[0204] Other suitable water-soluble organic salts are the homo- or
co-polymeric acids or their salts, in which the polycarboxylic acid
comprises at least two carboxyl radicals separated form each other
by not more than two carbon atoms.
[0205] Polymers of this type are disclosed in GB-A-1,596,756.
Examples of such salts are polyacrylates of MW 2000-5000 and their
copolymers with maleic anhydride, such copolymers having a
molecular weight of from 20,000 to 70,000, especially about
40,000.
[0206] Detergency builder salts are normally included in amounts of
from 5% to 80% by weight of the composition. Preferred levels of
builder for liquid detergents are from 5% to 30%.
Enzymes
[0207] Preferred detergent compositions, in addition to the enzyme
preparation of the invention, comprise other enzyme(s) which
provides cleaning performance and/or fabric care benefits.
[0208] Such enzymes include proteases, lipases, cutinases,
amylases, cellulases, peroxidases, oxidases (e.g., laccases).
[0209] Proteases: Any protease suitable for use in alkaline
solutions can be used. Suitable proteases include those of animal,
vegetable or microbial origin. Microbial origin is preferred.
Chemically or genetically modified mutants are included. The
protease may be a serine protease, preferably an alkaline microbial
protease or a trypsin-like protease. Examples of alkaline proteases
are subtilisins, especially those derived from Bacillus, e.g.,
subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin
147 and subtilisin 168 (described in WO 89/06279). Examples of
trypsin-like proteases are trypsin (e.g., of porcine or bovine
origin) and the Fusarium protease described in WO 89/06270.
[0210] Preferred commercially available protease enzymes include
those sold under the trade names Alcalase, Savinase, Primase,
Durazym, and Esperase by Novo Nordisk A/S (Denmark), those sold
under the tradename Maxatase, Maxacal, Maxapem, Properase, Purafect
and Purafect OXP by Genencor International, and those sold under
the tradename Opticlean and Optimase by Solvay Enzymes. Protease
enzymes may be incorporated into the compositions in accordance
with the invention at a level of from 0.00001% to 2% of enzyme
protein by weight of the composition, preferably at a level of from
0.0001% to 1% of enzyme protein by weight of the composition, more
preferably at a level of from 0.001% to 0.5% of enzyme protein by
weight of the composition, even more preferably at a level of from
0.01% to 0.2% of enzyme protein by weight of the composition.
[0211] Lipases: Any lipase suitable for use in alkaline solutions
can be used. Suitable lipases include those of bacterial or fungal
origin. Chemically or genetically modified mutants are
included.
[0212] Examples of useful lipases include a Humicola lanuginosa
lipase, e.g., as described in EP 258 068 and EP 305 216, a
Rhizomucor miehei lipase, e.g., as described in EP 238 023, a
Candida lipase, such as a C. antarctica lipase, e.g., the C.
antarctica lipase A or B described in EP 214 761, a Pseudomonas
lipase l such as a P. alcaligenes and P. pseudoalcaligenes lipase,
e.g., as described in EP 218 272, a P. cepacia lipase, e.g., as
described in EP 331 376, a P. stutzeri lipase, e.g., as disclosed
in GB 1,372,034, a P. fluorescens lipase, a Bacillus lipase, e.g.,
a B. subtilis lipase (Dartois et al., 1993, Biochemica et
Biophysica acta 1131, 253-260), a B. stearothermophilus lipase (JP
64/744992) and a B. pumilus lipase (WO 91/16422).
[0213] Furthermore, a number of cloned lipases may be useful,
including the Penicillium camembertii lipase described by Yamaguchi
et al., 1991, Gene 103, 61-67), the Geotricum candidum lipase
(Schimada, Y. et al., 1989, J. Biochem., 106, 383-388), and various
Rhizopus lipases such as a R. delemar lipase (Hass, M. J et al.,
1991, Gene 109, 117-113), a R. niveus lipase (Kugimiya et al.,
1992, Biosci. Biotech. Biochem. 56, 716-719) and an R. oryzae
lipase.
[0214] Other types of lipolytic enzymes such as cutinases may also
be useful, e.g., a cutinase derived from Pseudomonas mendocina as
described in WO 88/09367, or a cutinase derived from Fusarium
solani pisi (e.g., described in WO 90/09446).
[0215] Especially suitable lipases are lipases such as M1
Lipase.TM., Luma Fast.TM. and Lipomax.TM. (Genencor), Lipolase.TM.
and Lipolase Ultra.TM. (Novo Nordisk A/S), and Lipase P "Amano"
(Amano Pharmaceutical Co. Ltd.).
[0216] The lipases are normally incorporated in the detergent
composition at a level of from 0.00001% to 2% of enzyme protein by
weight of the composition, preferably at a level of from 0.0001% to
1% of enzyme protein by weight of the composition, more preferably
at a level of from 0.001% to 0.5% of enzyme protein by weight of
the composition, even more preferably at a level of from 0.01% to
0.2% of enzyme protein by weight of the composition.
[0217] Amylases: Any amylase (alpha and/or beta) suitable for use
in alkaline solutions can be used. Suitable amylases include those
of bacterial or fungal origin. Chemically or genetically modified
mutants are included. Amylases include, for example, a-amylases
obtained from a special strain of B. licheniformis, described in
more detail in GB 1,296,839. Commercially available amylases are
Duramyl.TM., Termamyl.TM., Fungamyl.TM. and BAN.TM. (available from
Novo Nordisk A/S) and Rapidase.TM. and Maxamyl P.TM. (available
from Genencor).
[0218] The amylases are normally incorporated in the detergent
composition at a level of from 0.00001% to 2% of enzyme protein by
weight of the composition, preferably at a level of from 0.0001% to
1% of enzyme protein by weight of the composition, more preferably
at a level of from 0.001% to 0.5% of enzyme protein by weight of
the composition, even more preferably at a level of from 0.01% to
0.2% of enzyme protein by weight of the composition.
[0219] Cellulases: Any cellulase suitable for use in alkaline
solutions can be used. Suitable cellulases include those of
bacterial or fungal origin. Chemically or genetically modified
mutants are included. Suitable cellulases are disclosed in U.S.
Pat. No. 4,435,307, which discloses fungal cellulases produced from
Humicola insolens. Especially suitable cellulases are the
cellulases having color care benefits. Examples of such cellulases
are cellulases described in European patent application No. 0 495
257 and the endoglucanase of the present invention.
[0220] Commercially available cellulases include Celluzyme.TM.
produced by a strain of Humicola insolens (Novo Nordisk A/S), and
KAC-500(B).TM. (Kao Corporation).
[0221] Cellulases are normally incorporated in the detergent
composition at a level of from 0.00001% to 2% of enzyme protein by
weight of the composition, preferably at a level of from 0.0001% to
1% of enzyme protein by weight of the composition, more preferably
at a level of from 0.001% to 0.5% of enzyme protein by weight of
the composition, even more preferably at a level of from 0.01% to
0.2% of enzyme protein by weight of the composition.
[0222] Peroxidases/Oxidases: Peroxidase enzymes are used in
combination with hydrogen peroxide or a source thereof (e.g., a
percarbonate, perborate or persulfate). Oxidase enzymes are used in
combination with oxygen. Both types of enzymes are used for
"solution bleaching", i.e., to prevent transfer of a textile dye
from a dyed fabric to another fabric when said fabrics are washed
together in a wash liquor, preferably together with an enhancing
agent as described in, e.g., WO 94/12621 and WO 95/01426. Suitable
peroxidases/oxidases include those of plant, bacterial or fungal
origin. Chemically or genetically modified mutants are
included.
[0223] Peroxidase and/or oxidase enzymes are normally incorporated
in the detergent composition at a level of from 0.00001% to 2% of
enzyme protein by weight of the composition, preferably at a level
of from 0.0001% to 1% of enzyme protein by weight of the
composition, more preferably at a level of from 0.001% to 0.5% of
enzyme protein by weight of the composition, even more preferably
at a level of from 0.01% to 0.2% of enzyme protein by weight of the
composition.
[0224] Mixtures of the above mentioned enzymes are encompassed
herein, in particular a mixture of a protease, an amylase, a lipase
and/or a cellulase.
[0225] The enzyme of the invention, or any other enzyme
incorporated in the detergent composition, is normally incorporated
in the detergent composition at a level from 0.00001% to 2% of
enzyme protein by weight of the composition, preferably at a level
from 0.0001% to 1% of enzyme protein by weight of the composition,
more preferably at a level from 0.001% to 0.5% of enzyme protein by
weight of the composition, even more preferably at a level from
0.01% to 0.2% of enzyme protein by weight of the composition.
Bleaching Agents
[0226] Additional optional detergent ingredients that can be
included in the detergent compositions of the present invention
include bleaching agents such as PB1, PB4 and percarbonate with a
particle size of 400-800 microns. These bleaching agent components
can include one or more oxygen bleaching agents and, depending upon
the bleaching agent chosen, one or more bleach activators. When
present oxygen bleaching compounds will typically be present at
levels of from about 1% to about 25%. In general, bleaching
compounds are optional added components in non-liquid formulations,
e.g., granular detergents.
[0227] The bleaching agent component for use herein can be any of
the bleaching agents useful for detergent compositions including
oxygen bleaches as well as others known in the art.
[0228] The bleaching agent suitable for the present invention can
be an activated or non-activated bleaching agent.
[0229] One category of oxygen bleaching agent that can be used
encompasses percarboxylic acid bleaching agents and salts thereof.
Suitable examples of this class of agents include magnesium
monoperoxyphthalate hexahydrate, the magnesium salt of meta-chloro
perbenzoic acid, 4-nonylamino-4-oxoperoxybutyric acid and
diperoxydodecanedioic acid. Such bleaching agents are disclosed in
U.S. Pat. No. 4,483,781, EP 0 133 354 and U.S. Pat. No. 4,412,934.
Highly preferred bleaching agents also include
6-nonylamino-6-oxoperoxycaproic acid as described in U.S. Pat. No.
4,634,551.
[0230] Another category of bleaching agents that can be used
encompasses the halogen bleaching agents. Examples of hypohalite
bleaching agents, for example, include trichloro isocyanuric acid
and the sodium and potassium dichloroisocyanurates and N-chloro and
N-bromo alkane sulphonamides. Such materials are normally added at
0.5-10% by weight of the finished product, preferably 1-5% by
weight.
[0231] The hydrogen peroxide releasing agents can be used in
combination with bleach activators such as
tetra-acetylethylenediamine (TAED), nonanoyloxybenzenesulfonate
(NOBS, described in U.S. Pat. No. 4,412,934),
3,5-trimethyl-hexsanoloxybenzenesulfonate (ISONOBS, described in EP
120 591) or pentaacetylglucose (PAG), which are perhydrolyzed to
form a peracid as the active bleaching species, leading to improved
bleaching effect. In addition, very suitable are the bleach
activators C8 (6-octanamido-caproyl)oxybenzene-sulfonate, C9
(6-nonanamido caproyl)oxybenzenesulfonate and C10 (6-decanamido
caproyl) oxybenzenesulfonate or mixtures thereof. Also suitable
activators are acylated citrate esters such as disclosed in
European Patent Application No. 91870207.7.
[0232] Useful bleaching agents, including peroxyacids and bleaching
systems comprising bleach activators and peroxygen bleaching
compounds for use in cleaning compositions according to the
invention are described in application U.S. Ser. No.
08/136,626.
[0233] The hydrogen peroxide may also be present by adding an
enzymatic system (i.e., an enzyme and a substrate therefore) which
is capable of generation of hydrogen peroxide at the beginning or
during the washing and/or rinsing process. Such enzymatic systems
are disclosed in European Patent Application EP 0 537 381.
[0234] Bleaching agents other than oxygen bleaching agents are also
known in the art and can be utilized herein. One type of non-oxygen
bleaching agent of particular interest includes photoactivated
bleaching agents such as the sulfonated zinc and/or aluminium
phthalocyanines. These materials can be deposited upon the
substrate during the washing process. Upon irradiation with light,
in the presence of oxygen, such as by hanging clothes out to dry in
the daylight, the sulfonated zinc phthalocyanine is activated and,
consequently, the substrate is bleached. Preferred zinc
phthalocyanine and a photoactivated bleaching process are described
in U.S. Pat. No. 4,033,718. Typically, detergent composition will
contain about 0.025% to about 1.25%, by weight, of sulfonated zinc
phthalocyanine.
[0235] Bleaching agents may also comprise a manganese catalyst. The
manganese catalyst may, e.g., be one of the compounds described in
"Efficient manganese catalysts for low-temperature bleaching",
Nature, 1994, Vol. 369, pp. 637-639.
Suds Suppressors
[0236] Another optional ingredient is a suds suppressor,
exemplified by silicones, and silica-silicone mixtures. Silicones
can generally be represented by alkylated polysiloxane materials,
while silica is normally used in finely divided forms exemplified
by silica aerogels and xerogels and hydrophobic silicas of various
types. Theses materials can be incorporated as particulates, in
which the suds suppressor is advantageously releasably incorporated
in a water-soluble or water-dispersible, substantially non
surface-active detergent impermeable carrier. Alternatively the
suds suppressor can be dissolved or dispersed in a liquid carrier
and applied by spraying on to one or more of the other
components.
[0237] A preferred silicone suds controlling agent is disclosed in
U.S. Pat. No. 3,933,672. Other particularly useful suds suppressors
are the self-emulsifying silicone suds suppressors, described in
German Patent Application DTOS 2,646,126. An example of such a
compound is DC-544, commercially available form Dow Corning, which
is a siloxane-glycol copolymer. Especially preferred suds
controlling agent are the suds suppressor system comprising a
mixture of silicone oils and 2-alkyl-alkanols. Suitable
2-alkyl-alkanols are 2-butyl-octanol which are commercially
available under the trade name Isofol 12 R.
[0238] Such suds suppressor system are described in European Patent
Application EP 0 593 841.
[0239] Especially preferred silicone suds controlling agents are
described in European Patent Application No. 92201649.8. Said
compositions can comprise a silicone/silica mixture in combination
with fumed nonporous silica such as AerosilR.
[0240] The suds suppressors described above are normally employed
at levels of from 0.001% to 2% by weight of the composition,
preferably from 0.01% to 1% by weight.
Other Components
[0241] Other components used in detergent compositions may be
employed such as soil-suspending agents, soil-releasing agents,
optical brighteners, abrasives, bactericides, tarnish inhibitors,
coloring agents, and/or encapsulated or nonencapsulated
perfumes.
[0242] Especially suitable encapsulating materials are water
soluble capsules which consist of a matrix of polysaccharide and
polyhydroxy compounds such as described in GB 1,464,616.
[0243] Other suitable water soluble encapsulating materials
comprise dextrins derived from ungelatinized starch acid esters of
substituted dicarboxylic acids such as described in U.S. Pat. No.
3,455,838. These acid-ester dextrins are, preferably, prepared from
such starches as waxy maize, waxy sorghum, sago, tapioca and
potato. Suitable examples of said encapsulation materials include
N-Lok manufactured by National Starch. The N-Lok encapsulating
material consists of a modified maize starch and glucose. The
starch is modified by adding monofunctional substituted groups such
as octenyl succinic acid anhydride.
[0244] Antiredeposition and soil suspension agents suitable herein
include cellulose derivatives such as methylcellulose,
carboxymethylcellulose and hydroxyethylcellulose, and homo- or
co-polymeric polycarboxylic acids or their salts. Polymers of this
type include the polyacrylates and maleic anhydride-acrylic acid
copolymers previously mentioned as builders, as well as copolymers
of maleic anhydride with ethylene, methylvinyl ether or methacrylic
acid, the maleic anhydride constituting at least 20 mole percent of
the copolymer. These materials are normally used at levels of from
0.5% to 10% by weight, more preferably form 0.75% to 8%, most
preferably from 1% to 6% by weight of the composition.
[0245] Preferred optical brighteners are anionic in character,
examples of which are disodium
4,4'-bis-(2-diethanolamino-4-anilino-s-triazin-6-ylamino)stilbene-2:2'
disulphonate, disodium
4,4'-bis-(2-morpholino-4-anilino-s-triazin-6-ylamino-stilbene-2:2'-disulp-
honate, disodium
4,4'-bis-(2,4-dianilino-s-triazin-6-ylamino)stilbene-2:2'-disulphonate,
monosodium 4',4''-bis-(2,4-dianilino-s-triazin-6
ylamino)stilbene-2-sulphonate, disodium
4,4'-bis-(2-anilino-4-(N-methyl-N-2-hydroxyethylamino)-s-triazin-6-ylamin-
o)stilbene-2,2'-disulphonate, disodium
4,4'-bis-(4-phenyl-2,1,3-triazol-2-yl)-stilbene-2,2'-disulphonate,
disodium
4,4'-bis(2-anilino-4-(1-methyl-2-hydroxyethylamino)-s-triazin-6--
ylamino)stilbene-2,2'-disulphonate, sodium
2(stilbyl-4''-(naphtho-1',2':4,5)-1,2,3-triazole-2''-sulphonate and
4,4'-bis(2-sulphostyryl)biphenyl.
[0246] Other useful polymeric materials are the polyethylene
glycols, particularly those of molecular weight 1000-10000, more
particularly 2000 to 8000 and most preferably about 4000. These are
used at levels of from 0.20% to 5% more preferably from 0.25% to
2.5% by weight. These polymers and the previously mentioned homo-
or co-polymeric poly-carboxylate salts are valuable for improving
whiteness maintenance, fabric ash deposition, and cleaning
performance on clay, proteinaceous and oxidizable soils in the
presence of transition metal impurities.
[0247] Soil release agents useful in compositions of the present
invention are conventionally copolymers or terpolymers of
terephthalic acid with ethylene glycol and/or propylene glycol
units in various arrangements. Examples of such polymers are
disclosed in U.S. Pat. Nos. 4,116,885 and 4,711,730 and EP 0 272
033. A particular preferred polymer in accordance with EP 0 272 033
has the formula:
(CH.sub.3(PEG)43)0.75(POH)0.25[T-PO)2.8(T-PEG)0.4]T(POH)0.25((PEG)43CH.s-
ub.3)0.75
where PEG is --(OC.sub.2H.sub.4)0-, PO is (OC.sub.3H.sub.6O) and T
is (pOOC.sub.6H.sub.4CO).
[0248] Also very useful are modified polyesters as random
copolymers of dimethyl terephthalate, dimethyl sulfoisophthalate,
ethylene glycol and 1,2-propanediol, the end groups consisting
primarily of sulphobenzoate and secondarily of mono esters of
ethylene glycol and/or 1,2-propanediol. The target is to obtain a
polymer capped at both end by sulphobenzoate groups, "primarily",
in the present context most of said copolymers herein will be
endcapped by sulphobenzoate groups. However, some copolymers will
be less than fully capped, and therefore their end groups may
consist of monoester of ethylene glycol and/or 1,2-propanediol,
thereof consist "secondarily" of such species.
[0249] The selected polyesters herein contain about 46% by weight
of dimethyl terephthalic acid, about 16% by weight of
1,2-propanediol, about 10% by weight ethylene glycol, about 13% by
weight of dimethyl sulfobenzoic acid and about 15% by weight of
sulfoisophthalic acid, and have a molecular weight of about 3.000.
The polyesters and their method of preparation are described in
detail in EP 311 342.
Softening Agents
[0250] Fabric softening agents can also be incorporated into
laundry detergent compositions in accordance with the present
invention. These agents may be inorganic or organic in type.
Inorganic softening agents are exemplified by the smectite clays
disclosed in GB-A-1 400 898 and in U.S. Pat. No. 5,019,292. Organic
fabric softening agents include the water insoluble tertiary amines
as disclosed in GB-A-1 514 276 and EP 0 011 340 and their
combination with mono C12-C14 quaternary ammonium salts are
disclosed in EP-B-0 026 528 and di-long-chain amides as disclosed
in EP 0 242 919. Other useful organic ingredients of fabric
softening systems include high molecular weight polyethylene oxide
materials as disclosed in EP 0 299 575 and 0 313 146.
[0251] Levels of smectite clay are normally in the range from 5% to
15%, more preferably from 8% to 12% by weight, with the material
being added as a dry mixed component to the remainder of the
formulation. Organic fabric softening agents such as the
water-insoluble tertiary amines or dilong chain amide materials are
incorporated at levels of from 0.5% to 5% by weight, normally from
1% to 3% by weight whilst the high molecular weight polyethylene
oxide materials and the water soluble cationic materials are added
at levels of from 0.1% to 2%, normally from 0.15% to 1.5% by
weight. These materials are normally added to the spray dried
portion of the composition, although in some instances it may be
more convenient to add them as a dry mixed particulate, or spray
them as molten liquid on to other solid components of the
composition.
Polymeric Dye-Transfer Inhibiting Agents
[0252] The detergent compositions according to the present
invention may also comprise from 0.001% to 10%, preferably from
0.01% to 2%, more preferably form 0.05% to 1% by weight of
polymeric dye-transfer inhibiting agents. Said polymeric
dye-transfer inhibiting agents are normally incorporated into
detergent compositions in order to inhibit the transfer of dyes
from colored fabrics onto fabrics washed therewith. These polymers
have the ability of complexing or adsorbing the fugitive dyes
washed out of dyed fabrics before the dyes have the opportunity to
become attached to other articles in the wash.
[0253] Especially suitable polymeric dye-transfer inhibiting agents
are polyamine N-oxide polymers, copolymers of N-vinyl-pyrrolidone
and N-vinylimidazole, polyvinylpyrrolidone polymers,
polyvinyloxazolidones and polyvinylimidazoles or mixtures
thereof.
[0254] Addition of such polymers also enhances the performance of
the enzymes according the invention.
[0255] The detergent composition according to the invention can be
in liquid, paste, gels, bars or granular forms.
[0256] Non-dusting granulates may be produced, e.g., as disclosed
in U.S. Pat. Nos. 4,106,991 and 4,661,452 (both to Novo Industri
A/S) and may optionally be coated by methods known in the art.
Examples of waxy coating materials are poly(ethylene oxide)
products (polyethyleneglycol, PEG) with mean molecular weights of
1000 to 20000; ethoxylated nonylphenols having from 16 to 50
ethylene oxide units; ethoxylated fatty alcohols in which the
alcohol contains from 12 to 20 carbon atoms and in which there are
15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and
mono- and di- and triglycerides of fatty acids. Examples of
film-forming coating materials suitable for application by fluid
bed techniques are given in GB 1483591.
[0257] Granular compositions according to the present invention can
also be in "compact form", i.e., they may have a relatively higher
density than conventional granular detergents, i.e., form 550 to
950 g/l; in such case, the granular detergent compositions
according to the present invention will contain a lower amount of
"Inorganic filler salt", compared to conventional granular
detergents; typical filler salts are alkaline earth metal salts of
sulphates and chlorides, typically sodium sulphate; "Compact"
detergent typically comprise not more than 10% filler salt. The
liquid compositions according to the present invention can also be
in "concentrated form", in such case, the liquid detergent
compositions according to the present invention will contain a
lower amount of water, compared to conventional liquid detergents.
Typically, the water content of the concentrated liquid detergent
is less than 30%, more preferably less than 20%, most preferably
less than 10% by weight of the detergent compositions.
[0258] The compositions of the invention may for example, be
formulated as hand and machine laundry detergent compositions
including laundry additive compositions and compositions suitable
for use in the pretreatment of stained fabrics, rinse added fabric
softener compositions, and compositions for use in general
household hard surface cleaning operations and dishwashing
operations.
[0259] The following examples are meant to exemplify compositions
for the present invention, but are not necessarily meant to limit
or otherwise define the scope of the invention.
[0260] In the detergent compositions, the abbreviated component
identifications have the following meanings:
LAS: Sodium linear C12 alkyl benzene sulphonate TAS: Sodium tallow
alkyl sulphate XYAS: Sodium C1X--C1Y alkyl sulfate SS: Secondary
soap surfactant of formula 2-butyl octanoic acid 25EY: A C12-C15
predominantly linear primary alcohol condensed with an average of Y
moles of ethylene oxide 45EY: A C14-C15 predominantly linear
primary alcohol condensed with an average of Y moles of ethylene
oxide XYEZS: C1X--C1Y sodium alkyl sulfate condensed with an
average of Z moles of ethylene oxide per mole Nonionic: C13-C15
mixed ethoxylated/propoxylated fatty alcohol with an average degree
of ethoxylation of 3.8 and an average degree of propoxylation of
4.5 sold under the tradename Plurafax LF404 by BASF Gmbh CFAA:
C12-C14 alkyl N-methyl glucamide TFAA: C16-C18 alkyl N-methyl
glucamide Silicate: Amorphous Sodium Silicate (SiO.sub.2:Na.sub.2O
ratio=2.0) NaSKS-6: Crystalline layered silicate of formula
d-Na.sub.2Si.sub.2O.sub.5 Carbonate: Anhydrous sodium carbonate
Phosphate: Sodium tripolyphosphate MA/AA: Copolymer of 1:4
maleic/acrylic acid, average molecular weight about 80,000
Polyacrylate: Polyacrylate homopolymer with an average molecular
weight of 8,000 sold under the tradename PA30 by BASF Gmbh Zeolite
A: Hydrated Sodium Aluminosilicate of formula
Na.sub.12(AlO.sub.2SiO.sub.2).sub.12.27H.sub.2O having a primary
particle size in the range from 1 to 10 micrometers Citrate:
Tri-sodium citrate dihydrate
Citric: Citric Acid
[0261] Perborate: Anhydrous sodium perborate monohydrate bleach,
empirical formula NaBO.sub.2.H.sub.2O.sub.2 PB4: Anhydrous sodium
perborate tetrahydrate Percarbonate: Anhydrous sodium percarbonate
bleach of empirical formula 2Na.sub.2CO.sub.3.3H.sub.2O.sub.2 TAED:
Tetraacetyl ethylene diamine CMC: Sodium carboxymethyl cellulose
DETPMP: Diethylene triamine penta(methylene phosphonic acid),
marketed by Monsanto under the Tradename Dequest 2060 PVP:
Polyvinylpyrrolidone polymer EDDS: Ethylenediamine-N,N'-disuccinic
acid, [S,S] isomer in the form of the sodium salt Suds Suppressor:
25% paraffin wax Mpt 50.degree. C., 17% hydrophobic silica, 58%
paraffin oil Granular Suds suppressor: 12% Silicone/silica, 18%
stearyl alcohol, 70% starch in granular form Sulphate: Anhydrous
sodium sulphate HMWPEO: High molecular weight polyethylene oxide
TAE 25: Tallow alcohol ethoxylate (25)
Detergent Example I
[0262] A granular fabric cleaning composition in accordance with
the invention may be prepared as follows:
TABLE-US-00008 Sodium linear C12 alkyl 6.5 benzene sulfonate Sodium
sulfate 15.0 Zeolite A 26.0 Sodium nitrilotriacetate 5.0 Enzyme of
the invention 0.1 PVP 0.5 TAED 3.0 Boric acid 4.0 Perborate 18.0
Phenol sulphonate 0.1 Minors Up to 100
Detergent Example II
[0263] A compact granular fabric cleaning composition (density 800
g/l) in accord with the invention may be prepared as follows:
TABLE-US-00009 45AS 8.0 25E3S 2.0 25E5 3.0 25E3 3.0 TFAA 2.5
Zeolite A 17.0 NaSKS-6 12.0 Citric acid 3.0 Carbonate 7.0 MA/AA 5.0
CMC 0.4 Enzyme of the invention 0.1 TAED 6.0 Percarbonate 22.0 EDDS
0.3 Granular suds suppressor 3.5 water/minors Up to 100%
Detergent Example III
[0264] Granular fabric cleaning compositions in accordance with the
invention which are especially useful in the laundering of coloured
fabrics were prepared as follows:
TABLE-US-00010 LAS 10.7 -- TAS 2.4 -- TFAA -- 4.0 45AS 3.1 10.0
45E7 4.0 -- 25E3S -- 3.0 68E11 1.8 -- 25E5 -- 8.0 Citrate 15.0 7.0
Carbonate -- 10 Citric acid 2.5 3.0 Zeolite A 32.1 25.0 Na-SKS-6 --
9.0 MA/AA 5.0 5.0 DETPMP 0.2 0.8 Enzyme of the invention 0.10 0.05
Silicate 2.5 -- Sulphate 5.2 3.0 PVP 0.5 -- Poly
(4-vinylpyridine)-N- -- 0.2 Oxide/copolymer of vinyl- imidazole and
vinyl- pyrrolidone Perborate 1.0 -- Phenol sulfonate 0.2 --
Water/Minors Up to 100%
Detergent Example IV
[0265] Granular fabric cleaning compositions in accordance with the
invention which provide "Softening through the wash" capability may
be prepared as follows:
TABLE-US-00011 45AS -- 10.0 LAS 7.6 -- 68AS 1.3 -- 45E7 4.0 -- 25E3
-- 5.0 Coco-alkyl-dimethyl hydroxy- 1.4 1.0 ethyl ammonium chloride
Citrate 5.0 3.0 Na-SKS-6 -- 11.0 Zeolite A 15.0 15.0 MA/AA 4.0 4.0
DETPMP 0.4 0.4 Perborate 15.0 -- Percarbonate -- 15.0 TAED 5.0 5.0
Smectite clay 10.0 10.0 HMWPEO -- 0.1 Enzyme of the invention 0.10
0.05 Silicate 3.0 5.0 Carbonate 10.0 10.0 Granular suds suppressor
1.0 4.0 CMC 0.2 0.1 Water/Minors Up to 100%
Detergent Example V
[0266] Heavy duty liquid fabric cleaning compositions in accordance
with the invention may be prepared as follows:
TABLE-US-00012 I II LAS acid form -- 25.0 Citric acid 5.0 2.0 25AS
acid form 8.0 -- 25AE2S acid form 3.0 -- 25AE7 8.0 -- CFAA 5.0 --
DETPMP 1.0 1.0 Fatty acid 8.0 -- Oleic acid -- 1.0 Ethanol 4.0 6.0
Propanediol 2.0 6.0 Enzyme of the invention 0.10 0.05 Coco-alkyl
dimethyl -- 3.0 hydroxy ethyl ammonium chloride Smectite clay --
5.0 PVP 2.0 -- Water/Minors Up to 100%
Textile Applications
[0267] In another embodiment, the present invention relates to use
of the endoglucanase of the invention in the bio-polishing process.
Bio-Polishing is a specific treatment of the yarn surface which
improves fabric quality with respect to handle and appearance
without loss of fabric wettability. The most important effects of
Bio-Polishing can be characterized by less fuzz and pilling,
increased gloss/luster, improved fabric handle, increased durable
softness and altered water absorbency. Bio-Polishing usually takes
place in the wet processing of the manufacture of knitted and woven
fabrics. Wet processing comprises such steps as, e.g., desizing,
scouring, bleaching, washing, dying/printing and finishing. During
each of these steps, the fabric is more or less subjected to
mechanical action. In general, after the textiles have been knitted
or woven, the fabric proceeds to a desizing stage, followed by a
scouring stage, etc. Desizing is the act of removing size from
textiles. Prior to weaving on mechanical looms, warp yarns are
often coated with size starch or starch derivatives in order to
increase their tensile strength. After weaving, the size coating
must be removed before further processing the fabric in order to
ensure a homogeneous and wash-proof result. It is known that in
order to achieve the effects of Bio-Polishing, a combination of
cellulytic and mechanical action is required. It is also known that
"super-softness" is achievable when the treatment with a cellulase
is combined with a conventional treatment with softening agents. It
is contemplated that use of the endoglucanase of the invention for
bio-polishing of cellulosic fabrics is advantageous, e.g., a more
thorough polishing can be achieved. Bio-polishing may be obtained
by applying the method described, e.g., in WO 93/20278.
Stone-Washing
[0268] It is known to provide a "stone-washed" look (localized
abrasion of the color) in dyed fabric, especially in denim fabric
or jeans, either by washing the denim or jeans made from such
fabric in the presence of pumice stones to provide the desired
localized lightening of the color of the fabric or by treating the
fabric enzymatically, in particular with cellulytic enzymes. The
treatment with an endoglucanase of the present invention may be
carried out either alone such as disclosed in U.S. Pat. No.
4,832,864, together with a smaller amount of pumice than required
in the traditional process, or together with perlite such as
disclosed in WO 95/09225.
Pulp and Paper Applications
[0269] In the papermaking pulp industry, the endoglucanase of the
present invention may be applied advantageously, e.g., as follows:
[0270] For debarking: pretreatment with the endoglucanase may
degrade the cambium layer prior to debarking in mechanical drums
resulting in advantageous energy savings. [0271] For defibration:
treatment of a material containing cellulosic fibers with the
endoglucanase prior to refining or beating may result in reduction
of the energy consumption due to the hydrolyzing effect of the
cellulase on the interfiber surfaces. Use of the endoglucanase may
result in improved energy savings as compared to the use of known
enzymes, since it is believed that the enzyme composition of the
invention may possess a higher ability to penetrate fiber walls.
[0272] For fiber modification, i.e., improvement of fiber
properties where partial hydrolysis across the fiber wall is needed
which requires deeper penetrating enzymes (e.g., in order to make
coarse fibers more flexible). Deep treatment of fibers has so far
not been possible for high yield pulps, e.g., mechanical pulps or
mixtures of recycled pulps. This has been ascribed to the nature of
the fiber wall structure that prevents the passage of enzyme
molecules due to physical restriction of the pore matrix of the
fiber wall. It is contemplated that the present endoglucanase is
capable of penetrating into the fiber wall. [0273] For drainage
improvement. The drainability of papermaking pulps may be improved
by treatment of the pulp with hydrolysing enzymes, e.g.,
cellulases. Use of the present endoglucanase may be more effective,
e.g., result in a higher degree of loosening bundles of strongly
hydrated micro-fibrils in the fines fraction (consisting of fiber
debris) that limits the rate of drainage by blocking hollow spaces
between fibers and in the wire mesh of the paper machine. The
Canadian standard freeness (CSF) increases and the Schopper-Riegler
drainage index decreases when pulp in subjected to cellulase
treatment, see, e.g., U.S. Pat. No. 4,923,565; TAPPI T227, SCAN
C19:65.ence. [0274] For inter fiber bonding. Hydrolytic enzymes are
applied in the manufacture of papermaking pulps for improving the
inter fiber bonding. The enzymes rinse the fiber surfaces for
impurities, e.g., cellulosic debris, thus enhancing the area of
exposed cellulose with attachment to the fiber wall, thus improving
the fiber-to-fiber hydrogen binding capacity. This process is also
referred to as dehornification. Paper and board produced with a
cellulase containing enzyme preparation may have an improved
strength or a reduced grammage, a smoother surface and an improved
printability. [0275] For enzymatic deinking. Partial hydrolysis of
recycled paper during or upon pulping by use of hydrolysing enzymes
such as cellulases are known to facilitate the removal and
agglomeration of ink particles. Use of the present endoglucanse may
give a more effective loosening of ink from the surface structure
due to a better penetration of the enzyme molecules into the
fibrillar matrix of the fiber wall, thus softening the surface
whereby ink particles are effectively loosened. The agglomeration
of loosened ink particles are also improved, due to a more
efficient hydrolysis of cellulosic fragments found attached to ink
particles originating from the fibers.
[0276] The treatment of lignocellulosic pulp may, e.g., be
performed as described in WO 91/14819, WO 91/14822, WO 92/17573 and
WO 92/18688.
Degradation of Plant Material
[0277] In yet another embodiment, the present invention relates to
use of the endoglucanase and/or enzyme preparation according to the
invention for degradation of plant material, e.g., cell walls.
[0278] It is contemplated that the novel endoglucanase and/or
enzyme preparation of the invention is useful in the preparation of
wine, fruit or vegetable juice in order to increase yield.
Endoglucanases according to the invention may also be applied for
enzymatic hydrolysis of various plant cell-wall derived materials
or waste materials, e.g., agricultural residues such as
wheat-straw, corn cobs, whole corn plants, nut shells, grass,
vegetable hulls, bean hulls, spent grains, sugar beet pulp, and the
like. The plant material may be degraded in order to improve
different kinds of processing, facilitate purification or
extraction of other components like purification of beta-glucan or
beta-glucan oligomers from cereals, improve the feed value,
decrease the water binding capacity, improve the degradability in
waste water plants, improve the conversion of, e.g., grass and corn
to ensilage, etc.
EXAMPLES
[0279] The invention is further illustrated in the following
examples which are not intended to be in any way limiting to the
scope of the invention as claimed.
Materials and Methods
Cellulolytic Activity
[0280] The cellulase variants of the invention show improved
performance. Some of the variants may show improved performance
with respect to increased catalytic activity.
[0281] In the context of this invention, cellulase activity can be
expressed in S-CEVU. Cellulolytic enzymes hydrolyse CMC, thereby
increasing the viscosity of the incubation mixture. The resulting
reduction in viscosity may be determined by a vibration
viscosimeter (e.g., MIVI 3000 from Sofraser, France).
[0282] Determination of the cellulolytic activity, measured in
terms of S-CEVU, may be determined according to the following
analysis method (assay): The S-CEVU assay quantifies the amount of
catalytic activity present in the sample by measuring the ability
of the sample to reduce the viscosity of a solution of
carboxy-methylcellulose (CMC). The assay is carried out at
40.degree. C.; pH 7.5; 0.1 M phosphate buffer; time 30 min; using a
relative enzyme standard for reducing the viscosity of the CMC
(carboxymethylcellulose Hercules 7 LFD) substrate; enzyme
concentration approx. 0.15 S-CEVU/ml. The arch standard is defined
to 8200 S-CEVU/g.
Example 1
Preparation of Cellulase Variants
[0283] Based on the disclosed sequence alignment (Table 1) and
computer modeling method, position 119 was identified as a
particular point of interest for making cellulase variants.
Position 119 (cellulase numbering) is located within 3 .ANG. from
the substrate. In position 119 the wild-type Humicola insolens
cellulase holds a histidine residue (H), whereas the wild-type
Thielavia terrestris cellulase holds a glutamine residue (Q).
[0284] In this experiment, histidine was substituted for glutamine
in the Thielavia terrestris cellulase (thereby obtaining the
cellulase variant Thielavia terrestris/Q119H). The variant obtained
was tested for specific activity.
[0285] All Humicola insolens variants are, unless otherwise stated,
constructed by application of the Chameleon.TM. Double-stranded,
site-directed Mutagenesis kit, from Stratagene. The following
synthetic oligo-nucleotides were used as selection primers:
TABLE-US-00013 (SEQ ID NO: 12) S/M GAATGACTTGGTTGACGCGTCACCAGTCAC,
or (SEQ ID NO: 13) M/S GAATGACTTGGTTGAGTACTCACCAGTCAC.
[0286] S/M replaces the ScaI site in the beta-lactamase gene of the
plasmid with a MluI site and M/S does the reverse. The latter is
used to introduce secondary mutations in variants generated by the
first selection primer.
[0287] For construction of Thielavia terrestis cellulase variants,
the Thielavia terrestis EG V cellulase cDNA obtainable from the
plasmid deposited as DSM 10811 was used. DSM 10811 was deposited at
the Deutsche Sammlung von Mikroorganismen and Zellkulturen on 30
Jun. 1995 according to the Budapest Treaty. The plasmid was
digested with the restriction endonucleases BamHI and NotI The 4153
bp vector part and the 1211 bp BamHI-NotI fragment were isolated.
Equal portions of the 1211 bp fragment were digested with
respectively HgiAI and EcoRV and the 487 bp BamHI-HgiAI and 690 bp
EcoRV-NotI fragments were isolated.
[0288] These fragments and the vector part were ligated in the
presence of 5 fold molar excess of a synthetic DNA fragment,
resulting from the annealing of two single stranded DNA
oligomers:
TABLE-US-00014 (SEQ ID NO: 14) 18802:
CACTGGCGGCGACCTGGGATCTAACCACTTCGAT (SEQ ID NO: 15) 18803:
ATCGAAGTGGTTAGATCCCAGGTCGCCGCCTGTGCTC
[0289] The ligation mixture was transformed into E. coli strain
XL1, and from the resulting transformants Thielavia
terrestris/Q119H was isolated and verified by DNA sequencing.
[0290] All the cellulase variants ware produced by cloning the gene
and transforming the gene into Aspergillus oryzae using a plasmid
with the gene inserted between the fungal amylase promoter and the
AMG terminator from A. niger [Christensen, T. Woldike, H. Boel, E.,
Mortensen, S. B., Hjortshoj, K., Thim, L. and Hansen, M. T. (1988)
Biotechnology 6: 1419-1422].
[0291] The cellulases with a cellulose binding domain CBD were
purified by exploiting their binding to Avicel. The cloned product
was recovered after fermentation by separation of the extracellular
fluid from the production organism. The cellulase was then highly
purified by affinity chromatography using 150 gram of Avicel in a
slurry with 20 mM sodiumphosphate pH 7.5. The Avicel slurry was
mixed with the crude fermentation broth which in total contains
about 1 gram of protein. After mixing at 4.degree. C. for 20 min,
the Avicel-bound enzyme is packed into a column with a dimension of
50 times 200 mm about 400 ml total.
[0292] The column is washed with the 200 ml buffer, then washed
with 0.5 M NaCl in the same buffer until no more protein elutes,
and washed with 500 ml buffer (20 mM Tris pH 8.5). Finally the pure
full length enzyme is eluted with 1% Triethylamine pH 11.8. The
eluted enzyme solution is adjusted to pH 8 and concentrated using
an Amicon cell unit with a membrane DOW GR61PP (polypropylene with
a cut off of 20 KD) to 5 mg protein per ml. The enzymes have all
been purified yielding a single band on SDS-PAGE.
[0293] Cellulases which natural lack CBD or the linker has been
proteolytic cleaved or in which the CBD has been removed by
introducing a stop codon after the catalytic domain, can not be
purified using Avicel. The extracellular proteins are recovered
free from the production organism. The core cellulases were
purified free of Aspergillus proteins by cation exchange
chromatography. The fermentation broth was adjusted to pH 3.5 and
filtered to remove the precipitating proteins. Then the proteins
were ultra filtrated (concentrated and washed with water) on a DOW
GR81PP membrane with a cut off 6 KD until the conductivity of the
eluate is below 1000 mS/cm. The sample was finally applied to an
S-Sepharose column equilibrated with a 20 mM citrate buffer pH
3.5.
[0294] The enzyme will bind to the S-Sepharose at this low pH and
it is eluted as a single peak using a NaCl gradient from 0 to 500
mM. The eluted pure enzyme was concentrated on a Amicon cell with
the DOW GR81PP membrane. All purified cellulases gave a single band
in SDS-PAGE.
[0295] The specific activity data are summarized in the following
table:
TABLE-US-00015 Enzyme/variant Specific activity [%] Humicola
insolens 100 Thielavia terrestris 35 Thielavia terrestris/Q119H
92
[0296] From this experiment it is seen that by introducing the
mutation Q119H into the Thielavia terrestris cellulase, the
specific activity if the resulting cellulase variants was increased
to the level of that of the homologous Humicola insolens
cellulase.
Example 2
Thielavia terrestris Variant with Improved Alkaline Performance
Profile
[0297] In this experiment the Thielavia terrestris/Q119D was
constructed as described in example 1 but using the following
construction: For easy cassette swap and standard primer
utilization, the CT1 encoding DNA was furnished with a C-terminal
Xba1 site and subcloned into the pCaHj418 vector as described
below. PCT1 was used as template in a Pwo polymerase PCR,
94.degree. C., 2'-3x(94.degree. C., 30''-72.degree. C.,
1')-25x(94.degree. C., 30''-55.degree. C., 30''-72.degree. C.,
1')-72.degree. C., 5' applying the two primers
TABLE-US-00016 (SEQ ID NO: 16) 8939: CGACTTCAATGTCCAGTCGG (SEQ ID
NO: 17) 25335: GCGCTCTAGAGGATTAAAGGCACTGC
[0298] The resulting 718 bp PCR product was digested with Sal1 and
Xba1 and the 165 bp fragment was isolated. This fragment was
ligated together with the 833 bp BamH1-Sal1 fragment from pCT1-2
into the 4.1 kb Xba1-BamH1 vector fragment of pCaHj418.
[0299] From this ligation pCT1418 was isolated from E. coli
transformants.
[0300] PCT2 was constructed by the Chameleon.TM. Double-stranded,
site-directed Mutagenesis kit (from Stratagene) as described above
with pCT1418 as template, the S/M primer as selection primer and
the following mutagenic primer:
TABLE-US-00017 (SEQ ID NO: 18) 109330:
CGACCTGGGATCGAACGACTTCGATATCGCCATGC
[0301] A successfully mutated plasmid pCT2 was isolated, verified
by DNA sequencing and transformed into Aspergillus oryzae strain
JaL228.
[0302] The Thielavia terrestris cellulase and the Thielavia
terrestris/Q119D variant was tested for activity towards PASC as
described in example 9 at pH 7.0 and pH 10.0.
[0303] The results are presented in the table below which shows the
activity at pH10 compared to the activity at pH 7. This
demonstrates that the Thielavia terrestris/Q119D variant has
relatively more alkaline activity as compared to the parent
Thielavia terrestris.
Relative activity
[0304] pH 10/pH 7 [%]Thielavia terrestris 27 Thielavia
terrestris/Q119D 62
Example 3
Construction of a Cellulase Hybrid Variant
[0305] The plasmid pCT3 embodies DNA encoding the Thielavia
terrestris endoglucanase core enzyme and followed by the linker CBD
of Humicola grisea.
[0306] pCT3 was constructed by means of sequence overlap extension
PCR, applying PWO polymerase.
[0307] From a cDNA clone of Humicola grisea a 415 bp fragment was
generated by the following primers:
TABLE-US-00018 (SEQ ID NO: 19) 109452:
CGACTCCAGCTTCCCCGTCTTCACGCCCCC (SEQ ID NO: 20) 107819:
CGAGCTTCTAGATCTCGACTAGAGGCACTGGGAG
[0308] From pCT1418 (disclosed in example 2) an 876 bp PCR fragment
was generated by the following primers:
TABLE-US-00019 (SEQ ID NO: 21) 101621 :GGATGCCATGCTTGGAGGATAGCAACC
(SEQ ID NO: 22) 107823 :GGGGGCGTGAAGACGGGAAGCTGGAGTCG
[0309] For both reactions the following set up was used: 96.degree.
C., 1'-3x(94.degree. C., 30''-50.degree. C., 1'-72.degree. C.,
1')-25x(94.degree. C., 30''-61.degree. C., 30''-72.degree. C.,
1')-72.degree. C., 7'.
[0310] The isolated PCR fragments were applied as template in an
assembly PCR reaction with primers 101621 and 107819: 94.degree.
C., 1'-3x(94.degree. C., 30''-70.degree. C., 1.degree.-72.degree.
C., 2')-20x(94.degree. C., 30''-61.degree. C., 30''-72.degree. C.,
1.5.degree.)-72.degree. C., 7'. The resulting 1261 bp PCR product
was isolated cut by restriction enzymes BamH1 and Xba1 and the
resulting 1172 bp DNA fragment was isolated and ligated into the
4.1 kb vector fragment of BamH1-Xba1 digested pCaHj418.
[0311] Correct clones were isolated and verified by DNA sequencing
of plasmids isolated from E. coli XL1 transformants resulting above
ligation reaction.
cDNA sequence of Humicola grisea (SEQ ID NO: 23):
TABLE-US-00020
CAAGAACCTCACACTCATTTTATTCACGCTCATTTATTCTAAAACTTCAATATGCGCTCTGCTCCTA
TTTTCCGCACGGCCCTGGCGGCTGCGCTCCCCCTTGCCGCACTCGCCGCCGATGGCAAGTCGACCAG
ATACTGGGACTGCTGCAAGCCATCGTGCTCTTGGCCCGGAAAGGCACTCGTGAACCAGCCTGTCTTC
ACTTGCGACGCCAAATTCCAGCGCATCACCGACCCCAATACCAAGTCGGGCTGCGATGGCGGCTCGG
CCTTTTCGTGTGCTGACCAGACCCCCTGGGCTCTGAACGACGATGTCGCCTATGGCTTCGCTGCCAC
GGCTATTTCGGGTGGATCGGAAGCCTCGTGGTGCTGCGCATGCTACGCTCTTACTTTCACCTCGGGC
CCTGTGGCCGGCAAGACCATGGTCGTCCAGTCGACCAACACCGGCGGCGATCTCGGCAGCAACCATT
TCGACCTCCAGATTCCAGGCGGCGGTGTCGGCATCTTTGATGGGTGCACCCCCCAGTTCGGAGGTCT
CGCTGGCGAACGCTACGGTGGCATCTCAGACCGCAGCTCCTGCGACTCGTTCCCTGCGGCGCTCAAG
CCCGGCTGCCTCTGGCGCTTCGATTGGTTCAAGAACGCCGACAACCCGACCTTTACCTTCAAGCAGG
TGCAGTGCCCCGCCGAGCTTGTTGCCAGGACCGGCTGCAAGCGCGAGGATGACGGCAACTTCCCCGT
CTTCACGCCCCCCGCGGGTAGCAACACCGGCGGTAGCCAGTCGAGCTCCACTATCGCTTCCAGCTCG
ACCTCCAAGGCTCAGACTTCGGCCGCCAGCTCCACCTCCAAGGCTGTCGTGACTCCCGTCTCCAGCT
CCACCTCGAAGGCCGCTGAGGTCCCCAAATCCAGCTCGACCTCCAAGGCTGCCGAGGTCGCCAAGCC
CAGCTCAACTTCGACCTCGACCTCGACCTCGACCAAGGTCAGCTGCTCTGCGACCGGTGGCTCCTGC
GTCGCTCAGAAGTGGGCGCAGTGCGGCGGCAATGGCTTCACCGGCTGCACGTCGTGCGTCAGCGGCA
CCACCTGCCAGAAGCAAAATGACTGGTACTCCCAGTGCCTCTAAGTCGTTTGTAGTAGCAGTTTGAA
GGATGTCAGGGATGAGGGAGGGAGGAGTGGGGGAAAAGTACGCCGCAGTTTTTTGGTAGACTTACTG
TATTGTTGAGTAATTACCCATTCGCTTCTTGTACGAAAAAAAAAAAAAAAAAAAA
Example 4
Construction of Variants of a Hybrid Cellulase
[0312] The plasmid pPsF45 embodies DNA encoding the Pseudomonas
cellolytica endoglucanase core enzyme headed by the H. insolens EGV
endoglucanase signal peptide and followed by the linker CBD of same
enzyme.
[0313] Two variants of this hybrid enzyme were constructed by means
of the above-mentioned Stratagene Chameleon.RTM. kit:
[0314] PsF45/H15S and PsF45/Q119H (cellulase numbering) by
application of the following mutagenic primers
TABLE-US-00021 PsF45/H15S: (SEQ ID NO: 24)
GCTGCAAGCCGTCCTGTGGCTGGAGCGCTAACGTGCCCGCG PsF45/Q119H: (SEQ ID NO:
25) CGATGTTTCCGGAGGCCACTTTGACATTCTGGTTCC
[0315] Deviations from template sequence are indicated in bold
type.
[0316] The selection primer was converting the unike Sca1 site in
the lactamase gene of the plasmid to a Mlu1 site:
TABLE-US-00022 (SEQ ID NO: 26) GAATGACTTGGTTGACGCGTCACCAGTCAC
[0317] The two variants were verified by DNA sequencing and one
correct version of each variant was identified.
[0318] The two plasmids emharboring the variant sequences
pPsF45H15S and pPsF45Q119H were used to transform A. oryzae strain
JaL142 together with the AMDS selection plasmid pToC202. From the
resulting transformants LaC2829 and LaC 2830 were isolated after 3
reisolation steps via spores.
Example 5
Removal of Disulfide Bridges
[0319] Disulfide bridges are known to stabilize protein structures.
The removal of disulfide bridges in a cellulase will destabilizes
the enzyme (thermostability) while retaining significant activity.
This can be useful in applications where a fast inactivation of the
enzyme is preferred, e.g., in denim or textile applications or for
low temperature processes.
[0320] In this example Humicola insolens EGV cellulase and five
variants of Humicola insolens cellulase were constructed mutating
either one or both residues involved in a disulfide bridge. The
specific activity was measured as disclosed under Materials and
Methods. The melting temperature of the enzymes was measured using
Differential Scanning Calometry, DSC. DSC was done at neutral pH
(7.0) using a MicroCalc Inc. MC calorimeter with a constant scan
rate and raising the temperature from 20.degree. C. to 90.degree.
C. at a rate of 90.degree. C. per hour.
[0321] The results are presented in the table below which shows
that removal of a disulfide bridge leads to a variant with a
significantly lower melting temperature but retaining significant
activity.
TABLE-US-00023 Specific activity Melting temp. [%] [.degree. C.]
Humicola insolens 100 81 Humicola insolens/C12G, C47M 15 63.7
Humicola insolens/C12M, C47G 53 64.3 Humicola insolens/C47G 48 57.3
Humicola insolens/C87M, C199G 75 63.4 Humicola insolens/C16M, C86G
103 59.2
Example 6
Mutation of Conserved Residues in the Binding Cleft <5 .ANG.
from Substrate
[0322] When comparing the positions within a distance of 5 .ANG.
from the substrate to the sequence alignment in Table 1 the type of
amino acid residue at these positions are conserved in the aligned
cellulases for the following positions: 6, 7, 8, 9, 10, 11, 12, 18,
45, 112, 114, 121, 127, 128, 130, 132, 147, 148, and 149. Conserved
residues are normally thought to be extremely important for the
activity, but the inventors have found that a certain variability
is allowed while maintaining significant activity. Only the two
residues D10 and D121 (cellulase numbering) are necessary to
maintain reasonable activity.
[0323] Variants of the Humicola insolens EGV cellulase were
prepared and the specific activity was measured as disclosed in
Materials and Methods.
[0324] The type of mutations and the variants specific activity are
summarized in the following table:
TABLE-US-00024 Variant Specific activity [%] Humicola insolens 100
Humicola insolens/T6S 34 Humicola insolens/R7I 33 Humicola
insolens/R7W 29 Humicola insolens/Y8F 67 Humicola insolens/W9F 83
Humicola insolens/C12M, C47G 53 Humicola insolens/W18Y 49 Humicola
insolens/W18F 53 Humicola insolens/S45T 85 Humicola insolens/S45N
85 Humicola insolens/D114N 6 Humicola insolens/F132D 11 Humicola
insolens/Y147D 34 Humicola insolens/Y147C 30 Humicola
insolens/Y147W 74 Humicola insolens/Y147V 33 Humicola
insolens/Y147R 45 Humicola insolens/Y147G 34 Humicola
insolens/Y147Q 41 Humicola insolens/Y147N 53 Humicola
insolens/Y147K 45 Humicola insolens/Y147H 75 Humicola
insolens/Y147F 57 Humicola insolens/Y147S 55
[0325] From this experiment it is seen that mutating conserved
residues in the binding cleft can be performed while retaining
significant activity of the cellulase variant.
Example 7
Mutation of Non-Conserved Residues in the Binding Cleft <5 .ANG.
from the Substrate
[0326] Based on the sequence alignment in Table 1 and the disclosed
computer modeling method the following residues located within a
distance of 5 .ANG. from the substrate and not being conserved
amongst the aligned sequences in were identified as points of
interest for making cellulase variants.
[0327] In this experiment non-conserved residues located no more
than 5 .ANG. from the substrate were modified in the Humicola
insolens EGV cellulase and the specific activity was measured as
described under Materials and Methods.
[0328] The type of mutations and the variants specific activity are
summarized in the following table:
TABLE-US-00025 Specific activity [%] Humicola insolens 100 Humicola
insolens/R4H 73 Humicola insolens/R4Q 70 Humicola insolens/K13L 37
Humicola insolens/K13R 100 Humicola insolens/K13Q 38 Humicola
insolens/P14A 99 Humicola insolens/P14T 71 Humicola insolens/S15T
18 Humicola insolens/S15H 10 Humicola insolens/C16M, C86G 103
Humicola insolens/A19P 51 Humicola insolens/A19T 84 Humicola
insolens/A19G 78 Humicola insolens/A19S 89 Humicola insolens/K20G
91 Humicola insolens/D42Y 102 Humicola insolens/D42W 103 Humicola
insolens/C47G 48 Humicola insolens/E48D 93 Humicola insolens/E48Q
71 Humicola insolens/E48D, P49* 88 Humicola insolens/E48N, P49* 79
Humicola insolens/S110N 94 Humicola insolens/L115I 18 Humicola
insolens/G116D 71 Humicola insolens/H119R 15 Humicola
insolens/H119Q 39 Humicola insolens/H119F 11 Humicola
insolens/N123A 61 Humicola insolens/N123M 80 Humicola
insolens/N123Q 76 Humicola insolens/N123Y 8 Humicola insolens/N123D
86 Humicola insolens/V129L 72 Humicola insolens/D133N 102 Humicola
insolens/D178N 81
[0329] From this experiment it is seen that most of the
non-conserved residues in the binding cleft can be mutated while
retaining all or most of the activity of the cellulase.
Example 8
Resistance to Anionic Surfactants in Detergent
[0330] A. Variants of the present invention may show improved
performance with respect to an altered sensitivity towards anionic
tensides. Anionic tensides are products frequently incorporated
into detergent compositions. Unfolding of cellulases tested so far,
is accompanied by a decay in the intrinsic fluorescence of the
proteins. The intrinsic fluorescence derives from Trp side chains
(and to a smaller extent Tyr side chains) and is sensitive to the
hydrophobicity of the side chain environment. Unfolding leads to a
more hydrophilic environment as the side-chains become more exposed
to solvent, and this quenches fluorescence.
[0331] Fluorescence is followed on a Perkin/Elmer.TM. LS50
luminescence spectrometer. In practice, the greatest change in
fluorescence on unfolding is obtained by excitation at 280 nm and
emission at 345 nm. Slit widths (which regulate the magnitude of
the signal) are usually 5 nm for both emission and excitation at a
protein concentration of 5 micrograms/ml. Fluorescence is measured
in 2-ml quartz cuvettes thermostatted with a circulating water bath
and stirred with a small magnet. The magnet-stirrer is built into
the spectrometer.
[0332] Unfolding can be followed in real time using the available
software. Rapid unfolding (going to completion within less than
5-10 minutes) is monitored in the TimeDrive option, in which the
fluorescence is measured every few (2-5) seconds. For slower
unfolding, four cuvettes can be measured at a time in the
cuvette-holder using the Wavelength Program option, in which the
fluorescence of each cuvette is measured every 30 seconds. In all
cases, unfolding is initiated by adding a small volume (typically
50 microliters) of concentrated enzyme solution to the
thermostatted cuvette solution where mixing is complete within a
few seconds due to the rapid rotation of the magnet.
[0333] Data are measured in the software program GraphPad Prism.
Unfolding fits in all cases to a single-exponential function from
which a single half-time of unfolding (or unfolding rate constant)
can be obtained.
[0334] Typical unfolding conditions are:
a. 10 mM CAPS pH 10, 1000 ppm LAS, 40.degree. C.
b. 10 mM HEPES pH 10, 200 ppm LAS, 25.degree. C.
[0335] In both cases, the protein concentration is 5-10
micrograms/ml (the protein concentration is not crucial, as LAS is
in excess). Under these conditions, the unfolding of Humicola
insolens cellulase can be compared with other enzymes (Table 1).
This enables us to draw up the following ranking order for
stability against anionic tenside:
[0336] Thielavia terrestris/Q119H.apprxeq.Thielavia
terrestris>>Humicola insolens.apprxeq.Humicola
insolens/H119Q.
TABLE-US-00026 t1/2 pH 7 (s) t1/2 pH 10 (s) (200 ppm LAS, Cellulase
(1000 ppm LAS, 40.degree. C.) 25.degree. C.) Humicola insolens 48
28 Humicola insolens/H119Q 63 9a Thielavia terrestris 970 690
Thielavia terrestris/Q119H 1100 550 a Unfolding is
double-exponential. The t1/2 of the slower phase is approx. 120
sec.
[0337] B. The alteration of the surface electrostatics of an enzyme
will influence the sensibility towards anionic tensides such as LAS
(linear alkylbenzenesulfonate). Especially variants where positive
charged residues have been removed and/or negatively charged
residues have been introduced will increase the resistance towards
LAS, whereas the opposite, i.e., the introduction of positively
charged residues and/or the removal of negatively charged residues
will lower the resistance towards LAS. The residues Arg (R), Lys
(K) and His (H) are viewed as positively or potentially positively
charged residue and the residues Asp (D), Glu (E) and Cys (C) if
not included in a disulphide bridge are viewed as negatively or
potentially negatively charged residues. Positions already
containing one of these residues are the primary target for
mutagenesis, secondary targets are positions which have one of
these residues on an equivalent position in another cellulase, and
third target are any surface exposed residue. In this experiment
wild type Humicola insolens cellulase are being compared to
Humicola insolens cellulase variants belonging to all three of the
above groups, comparing the stability towards LAS in detergent.
[0338] Cellulase resistance to anionic surfactants was measured as
activity on PASC (phosphoric acid swollen cellulose) in the
presence of anionic surfactant vs. activity on PASC in the absence
of anionic surfactant.
[0339] The reaction medium contained 5.0 g/l of a commercial
regular powder detergent from the detergent manufacturer NOPA
Denmark. The detergent was formulated without surfactants for this
experiment and pH adjusted to pH 7.0. Further the reaction medium
included 0.5 g/l PASC and was with or without 1 g/l LAS (linear
alkylbenzenesulphonate), which is an anionic surfactant, and the
reaction proceeded at the temperature 30.degree. C. for 30 minutes.
Cellulase was dosed at 0.20 S-CEVU/I. After the 30 minutes of
incubation the reaction was stopped with 2 N NaOH and the amount of
reducing sugar ends determined through reduction of
p-hydroxybenzoic acid hydrazide. The decrease in absorption of
reduced p-hydroxybenzoic acid hydrazide relates to the cellulase
activity.
[0340] The type of mutation and the resistance towards LAS for
variants with increased LAS resistance is summarized in the
following table:
TABLE-US-00027 Relative LAS Variant resistance [%] Humicola
insolens 100 Humicola insolens/R158E 341 Humicola insolens/Y8F,
W62E, A162P, 179 Humicola insolens/R158E, A162P 347 Humicola
insolens/R158G 322 Humicola insolens/S152D 161 Humicola
insolens/R158E/R196E 319 Humicola insolens/R158E, D161P, A162P 351
Humicola insolens/R4H, R158E, D161P, A162P 344 Humicola
insolens/H119Q 148 Humicola insolens/Y8F, W62E, R252L, Y280F 131
Humicola insolens/R252L, Y280F 133 Humicola insolens/W62E, A162P
130 Humicola insolens/W62E, A162P 129 Humicola insolens/S117D 143
Humicola insolens/A57C, A162C 134 Humicola insolens/N154D 149
Humicola insolens/R4H, D161P, A162P, R196E 134
[0341] From this table it is seen that mutations of residues
resulting in the removal of positively charged residue and/or the
introduction of a negatively charged residue increase the
resistance towards LAS.
[0342] As described above the type of mutation and the resistance
towards LAS for variants with decreased LAS resistance is
summarized in the following table:
TABLE-US-00028 Relative LAS Variant resistance [%] Humicola
insolens 100 Humicola insolens/Y147H 71 Humicola insolens/E192P 52
Humicola insolens/D161P, A162P 64 Humicola insolens/D67T 44
Humicola insolens/Q36T, D67T 67 Humicola insolens/D66N 47 Humicola
insolens/D67N 71 Humicola insolens/V64R 58 Humicola insolens/N65R
48 Humicola insolens/T93R 60 Humicola insolens/Q36T, D67T, A83T 64
Humicola insolens/E91Q 71 Humicola insolens/A191K 63 Humicola
insolens/D42W 67 Humicola insolens/S117K 62 Humicola insolens/R4H,
A63R, N65R, D67R 54 Humicola insolens/D133N 0 Humicola
insolens/D58A 15 Humicola insolens/D67R 39 Humicola insolens/A63R
38 Humicola insolens/R37N, D58A 6 Humicola insolens/K175R 32
Humicola insolens/D2N 43 Humicola insolens/N65R, D67R 40 Humicola
insolens/T136D, G141R 5 Humicola insolens/Y147K 17 Humicola
insolens/Y147R 1 Humicola insolens/D161P 35 Humicola insolens/D66P
40 Humicola insolens/D66A, D67T 39 Humicola insolens/D67T, *143NGT
7 Humicola insolens/Q36T, D67T, *143NGT 0 Humicola insolens/N65R,
D67R, S76K 22 Humicola insolens/W62R 25 Humicola insolens/S117R,
F120S 31 Humicola insolens/K13R 16 Humicola insolens/D10E 0
[0343] From this table it is seen that mutations of residues
resulting in the introduction of positively charged residue and/or
the removal of a negatively charged residue decrease the resistance
towards LAS.
Example 9
[0344] Alteration of pH Activity Profile
[0345] The pH activity profile of a cellulase is governed by the pH
dependent behavior of specific titratable groups, typically the
acidic residues in the active site. The pH profile can be altered
by changing the electrostatic environment of these residues, either
by substitution of residues involving charged or potentially
charged groups such as Arg (R), Lys (K), Tyr (Y), His (H), Glu (E),
Asp (D) or Cys (C) if not involved in a disulphide bridge or by
changes in the surface accessibility of these specific titratable
groups by mutations in the biding cleft within 5 .ANG. of the
substrate.
[0346] In this example Humicola insolens cellulase and variants of
Humicola insolens cellulase involving substitution of charged or
potentially charged residues have been tested for activity towards
PASC at pH 7 and pH 10, respectively.
[0347] In order to determine the pH optimum for cellulases we have
selected organic buffers because it is common known that, e.g.,
borate forms covalent complexes with mono- and oligo-saccharides
and phosphate can precipitate with Ca-ions. In DATA FOR BIOCHEMICAL
RESEARCH Third Edition OXFORD SCIENCE PUBLICATIONS page 223 to 241,
suitable organic buffers have been found. In respect of their pKa
values we decided to use Na-acetate in the range 4-5.5, MES at 6.0,
MOPS in the range 6.5-7.5, Na-barbiturate 8.0-8.5 and glycine in
the range 9.0-10.5.
Method:
[0348] The method is enzymatic degradation of
carboxy-methyl-cellulose, at different pH's. Buffers are prepared
in the range 4.0 to 10.5 with intervals of 0.5 pH unit. The
analysis is based on formation of new reducing ends in
carboxy-methyl-cellulose, these are visualized by reaction with
PHBAH in strong alkaline environment, were they forms a yellow
compound with absorption maximum at 410 nm.
Experimental Protocol:
[0349] Buffer preparation: 0.2 mol of each buffer substance is
weighed out and dissolved in 1 liter of Milli Q water. 250 ml 0.2 M
buffer solution and 200 ml Milli Q water is mixed. The pH is
measured using Radiometer PHM92 labmeter calibrated using standard
buffer solutions from Radiometer. The pH of the buffers are
adjusted to actual pH using 4 M NaOH or 4 M HCl and adjusted to
total 500 ml with water. When adjusting Na-barbiturate to pH 8.0
there might be some precipitation, this can be re-dissolved by
heating to 50.degree. C.
Acetic acid 100% 0.2 mol=12.01 g. MES 0.2 mol=39.04 g. MOPS 0.2
mol=41.86 g. Na-barbiturate 0.2 mol=41.24 g. Glycine 0.2 mol=15.01
g.
Buffers:
pH: 4.0, 4.5, 5.0 & 5.5 Na-acetate 0.1 M
pH: 6.0 Na-MES 0.1 M
pH: 6.5, 7.0 & 7.5 Na-MOPS 0.1 M
pH: 8.0 & 8.5 Na-barbiturate 0.1 M
[0350] pH: 9.0, 9.5, 10.0 & 10.5 Na.glycine 0.1 M The actual pH
is measured in a series treated as the main values, but without
stop reagent, pH is measured after 20 min. incubation at 40.degree.
C.
Substrate Preparation:
[0351] 2.0 g CMC, in 250 ml conic glass flask with a magnet rod, is
moistened with 2.5 ml. 96% ethanol, 100 ml. Milli Q water is added
and then boiled to transparency on a heating magnetic stirrer.
Approximately 2 min. boiling. Cooled to room temperature on
magnetic stirrer.
Stop Reagent:
[0352] 1.5 g PHBAH and 5 g K--Na-tartrate dissolved in 2% NaOH.
Procedure:
[0353] There are made 3 main values and 2 blank value using 5 ml
glass test tubes. (1 main value for pH determination)
TABLE-US-00029 Main values Blank value Buffer 1.0 ml. 1.0 ml.
Substrate CMC 0.75 ml. 0.75 ml. Mix 5 sec. 5 sec. Preheat 10
min./40.degree. C. -- Enzyme 0.25 ml. -- Mix 5 sec. -- Incubation
20 min./40.degree. C. room temp. PHBAH-reagent 1 ml. 1 ml. Mix 5
sec. -- Enzyme -- 0.25 ml. Mix -- 5 sec.
[0354] Mixing on a Heidolph REAX 2000 mixer with permanent mix and
maximum speed (9). No stirring during incubation on water bath with
temperature control. Immediately after adding PHBAH-reagent and
mixing the samples are boiled 10 min. Cooled in cold tap water for
5 min. Absorbance read at 410 nm.
Determination of Activity
[0355] The absorbance at 410 nm from the 2 Main values are added
and divided by 2 and the 2 Blank values are added and divided by 2,
the 2 mean values are subtracted. The percentages are calculated by
using the highest value as 100%.
[0356] The measured pH is plotted against the relative
activity.
Buffer Reagents:
[0357] Acetic acid 100% from MERCK cat. no. 1.00063, batch no.
K20928263 422, pKa 4.76, MW 60.05; MES
(2[N-Morpholino]ethanesulfonic acid) from SIGMA cat. no. M-8250,
batch no. 68F-5625, pKa 6.09, MW 195.2; MOPS
(3-[N-Morpholino]propanesulfonic acid) from SIGMA cat. no. M-1254,
batch no. 115F-5629, pKa 7.15, MW 209.3; Na-barbiturate
(5,5-Diethylbarbituric acid sodium salt) from MERCK cat. no. 6318,
batch no. K20238018 404, pKa 7.98, MW 206.2; Glycine from MERCK
cat. no. 4201, batch no. K205535601 405, pKa 9.78, MW 75.07; PHBAH
(p-hydroxy benzoic acid hydrazide) from SIGMA cat. no. H-9882,
batch no. 53H7704; K--Na-tartrate (Potassium sodium tartrate
tetrahydrate) from MERCK cat. no. 8087, batch no. A653387 304; NaOH
(Sodium hydroxide) from MERCK cat. no. 1.06498, batch no. C294798
404; CMC (Carboxy Methyl Cellulose) supplied by Hercules (FMC)7LF
(November 1989).
[0358] Cellulase resistance to anionic surfactants was measured as
activity on PASC (phosphoric acid swollen cellulose) at neutral pH
(pH 7.0) vs. activity on PASC at alkaline pH (pH 10.0).
[0359] The reaction medium contained 5.0 g/l of a commercial
regular powder detergent from the detergent manufacturer NOPA
Denmark. The pH was adjusted to pH 7.0 and pH 10.0, respectively.
Further the reaction medium included 0.5 g/l PASC, and the reaction
proceeded at the temperature 30.degree. C. for 30 minutes.
Cellulase was dosed at 0.20 S-CEVU/I. After the 30 minutes of
incubation the reaction was stopped with 2 N NaOH and the amount of
reducing sugar ends determined through reduction of
p-hydroxybenzoic acid hydrazide. The decrease in absorption of
reduced p-hydroxybenzoic acid hydrazide relates to the cellulase
activity.
Results:
[0360] The results are presented in the table below, the activity
at pH 10 relative to pH 7 is compared to that of wild type Humicola
insolens cellulase.
TABLE-US-00030 PASC activity pH 10/pH 7 Variant relative to wild
type [%] Humicola insolens 100 Humicola insolens/S76K, S117D 120
Humicola insolens/V129L 133 Humicola insolens/R4H, A63R, N65R, 120
D67R Humicola insolens/R252L, Y280F 115 Humicola insolens/D161P,
A162P 117 Humicola insolens/A57C, A162C 110 Humicola insolens/S76K
117 Humicola insolens/D161P, A162P, R196E 113 Humicola
insolens/Q36T, D67T, A83T 111 Humicola insolens/W62R 112 Humicola
insolens/D42Y 110 Humicola insolens/S76K, A78K 114 Humicola
insolens/S76K, A78R 118
[0361] From the above table it is seen that the relative alkaline
activity can be increased by creating variants involving
potentially charged residues and/or by altering residues in the
binding cleft less that 5 .ANG. from the substrate.
[0362] Similarly the following table shows that the relative acidic
activity can be increased by other mutations involving potentially
charged residues and/or by altering residues in the binding cleft
less than 5 .ANG. from the substrate.
TABLE-US-00031 PASC activity pH 10/pH 7 Variant relative to wild
type [%] Humicola insolens 100 Humicola insolens/D58A 83 Humicola
insolens/Y280W 90 Humicola insolens/D67R 89 Humicola insolens/A63R
85 Humicola insolens/Y8F 82 Humicola insolens/W62E 82 Humicola
insolens/R37N, D58A 84 Humicola insolens/K175G 81 Humicola
insolens/K175R 82 Humicola insolens/Y8F, M104Q 83 Humicola
insolens/Y8F, W62E, R252L, 83 Y280F Humicola insolens/W62E, A162P
87 Humicola insolens/Y8F, W62E, A162P, 88 Humicola insolens/Y147H
90 Humicola insolens/Y147N 90 Humicola insolens/Y147Q 85 Humicola
insolens/Y147W 85 Humicola insolens/E192P 89 Humicola
insolens/R158G 83 Humicola insolens/S152D 90 Humicola insolens/K13Q
82 Humicola insolens/R37P 82 Humicola insolens/S45T 87 Humicola
insolens/E48D 86 Humicola insolens/R7I 83 Humicola insolens/P14A 84
Humicola insolens/A19G 90 Humicola insolens/A19T 90 Humicola
insolens/R4H, D161P, A162P, 88 R196E Humicola insolens/D133N 80
Humicola insolens/D40N 40 Humicola insolens/Y90F 72 Humicola
insolens/A63D 78 Humicola insolens/G127S, I131A, A162P, 25 Y280F,
R252L Humicola insolens/Y147S 39 Humicola insolens/Y147F 71
Humicola insolens/T6S 44 Humicola insolens/S55E 14 Humicola
insolens/N123D 35 Humicola insolens/N123Y 71 Humicola
insolens/R158E 78 Humicola insolens/T136D, G141R 57 Humicola
insolens/G127S, I131A, A162P 52 Humicola insolens/W62E, G127S,
I131A, 35 A162P, Y280F, R252L Humicola insolens/W62E, G127S, I131A,
58 A162P Humicola insolens/W62E, G127S, I131A 64 Humicola
insolens/W62E, G127S, I131A, 80 Y280F, R252L Humicola
insolens/H119Q 57 Humicola insolens/Y8F, W62E 61 Humicola
insolens/W62E, A162P 76 Humicola insolens/W62E, A162P 80 Humicola
insolens/R158E, A162P 80 Humicola insolens/Y8F, Y147S 63 Humicola
insolens/Y147R 54 Humicola insolens/Y147V 22 Humicola
insolens/Y147C 67 Humicola insolens/Y147D 60 Humicola
insolens/N154D 74 Humicola insolens/R158E, R196E 79 Humicola
insolens/R158E, D161P, A162P 70 Humicola insolens/D67T, *143NGT 65
Humicola insolens/Q36T, D67T, *143NGT 53 Humicola insolens/143*NGW,
Q145D 53 Humicola insolens/L142P, 143*NGW, 42 Q145E Humicola
insolens/N65R, D67R, S76K 60 Humicola insolens/A63R, N65R, D67R 77
Humicola insolens/T93R 80 Humicola insolens/S76R 70 Humicola
insolens/S117R, F120S 58 Humicola insolens/N123Q 63 Humicola
insolens/N123M 49 Humicola insolens/N123A 80 Humicola
insolens/E48D, P49* 66 Humicola insolens/S55Y 61 Humicola
insolens/S55M 48 Humicola insolens/W18F 54 Humicola insolens/S45N
71 Humicola insolens/R7W 58 Humicola insolens/K13R 72 Humicola
insolens/R7L 74 Humicola insolens/S15T 38 Humicola insolens/W18Y 37
Humicola insolens/C16M, C86G 67 Humicola insolens/K13L 59 Humicola
insolens/C12M, C47G 12 Humicola insolens/W9F 62 Humicola
insolens/C47G 58 Humicola insolens/C12G, C47M 0 Humicola
insolens/D10E 0 Humicola insolens/R7K 49
[0363] Accordingly, this example demonstrates that the relative
activity pH profile can be altered towards acidic or alkaline pH by
creation of variants involving potentially charged residues and/or
by altering residues in the binding cleft less that 5 .ANG. from
the substrate.
Example 10
Wash Performance of Cellulases Made Resistant to Anionic
Surfactants
[0364] Application effect of a cellulase made resistant to anionic
surfactants vs. application effect of the native cellulase was
measured as `color clarification` of worn black cotton swatches
laundered with cellulase in a 0.1 liter mini-Terg-o-Meter.
Laundering was done in varying concentrations of anionic
surfactant.
[0365] The reaction medium contained phosphate buffer pH 7.0 and
varying concentrations of LAS in the range 0.2-1.0 g/L. Two
swatches were laundered at 40.degree. C. for 30 minutes, rinsed and
then dried. This laundering cycle was repeated four times. All
enzymes were tested at each LAS concentration.
[0366] Finally the black cotton swatches were graded against a
standard of similar swatches washed with varying dosages of the
native cellulase, the fungal .about.43 kD endo-beta-1,4-glucanase
from Humicola insolens, DSM 1800, (commercially available under the
tradename Carezyme.RTM.), and the effect expressed in PSU (panel
score units).
TABLE-US-00032 LAS concentration Variant 0.2 g/l 0.4 g/l 0.6 g/l
0.8 g/l 1.0 g/l Humicola insolens 15 0 0 0 0 Humicola insolens/ 30
14 30 22 11 R158E Humicola insolens/ 20 18 20 33 28 R158G
Sequence CWU 1
1
261202PRTHumicola insolens 1Ala Asp Gly Arg Ser Thr Arg Tyr Trp Asp
Cys Cys Lys Pro Ser Cys1 5 10 15Gly Trp Ala Lys Lys Ala Pro Val Asn
Gln Pro Val Phe Ser Cys Asn 20 25 30Ala Asn Phe Gln Arg Ile Thr Asp
Phe Asp Ala Lys Ser Gly Cys Glu 35 40 45Pro Gly Gly Val Ala Tyr Ser
Cys Ala Asp Gln Thr Pro Trp Ala Val 50 55 60Asn Asp Asp Phe Ala Leu
Gly Phe Ala Ala Thr Ser Ile Ala Gly Ser65 70 75 80Asn Glu Ala Gly
Trp Cys Cys Ala Cys Tyr Glu Leu Thr Phe Thr Ser 85 90 95Gly Pro Val
Ala Gly Lys Lys Met Val Val Gln Ser Thr Ser Thr Gly 100 105 110Gly
Asp Leu Gly Ser Asn His Phe Asp Leu Asn Ile Pro Gly Gly Gly 115 120
125Val Gly Ile Phe Asp Gly Cys Thr Pro Gln Phe Gly Gly Leu Pro Gly
130 135 140Gln Arg Tyr Gly Gly Ile Ser Ser Arg Asn Glu Cys Asp Arg
Phe Pro145 150 155 160Asp Ala Leu Lys Pro Gly Cys Tyr Trp Arg Phe
Asp Trp Phe Lys Asn 165 170 175Ala Asp Asn Pro Ser Phe Ser Phe Arg
Gln Val Gln Cys Pro Ala Glu 180 185 190Leu Val Ala Arg Thr Gly Cys
Arg Arg Ala 195 2002202PRTAcremonium sp. 2Gly Ser Gly His Thr Thr
Arg Tyr Trp Asp Cys Cys Lys Pro Ser Cys1 5 10 15Ala Trp Asp Glu Lys
Ala Ala Val Ser Arg Pro Val Thr Thr Cys Asp 20 25 30Arg Asn Asn Ser
Pro Leu Ser Pro Gly Ala Val Ser Gly Cys Asp Pro 35 40 45Asn Gly Val
Ala Phe Thr Cys Asn Asp Asn Gln Pro Trp Ala Val Asn 50 55 60Asn Asn
Val Ala Tyr Gly Phe Ala Ala Thr Ala Phe Pro Gly Gly Asn 65 70 75
80Glu Ala Ser Trp Cys Cys Ala Cys Tyr Ala Leu Gln Phe Thr Ser Gly
85 90 95Pro Val Ala Gly Lys Thr Met Val Val Gln Ser Thr Asn Thr Gly
Gly 100 105 110Asp Leu Ser Gly Thr His Phe Asp Ile Gln Met Pro Gly
Gly Gly Leu 115 120 125Gly Ile Phe Asp Gly Cys Thr Pro Gln Phe Gly
Phe Thr Phe Pro Gly 130 135 140Asn Arg Tyr Gly Gly Thr Thr Ser Arg
Ser Gln Cys Ala Glu Leu Pro145 150 155 160Ser Val Leu Arg Asp Gly
Cys His Trp Arg Tyr Asp Trp Phe Asn Asp 165 170 175Ala Asp Asn Pro
Asn Val Asn Trp Arg Arg Val Arg Cys Pro Ala Ala 180 185 190Leu Thr
Asn Arg Ser Gly Cys Val Arg Ala 195 2003202PRTVolutella
collectotrichoides 3Gly Thr Gly Arg Thr Thr Arg Tyr Trp Asp Cys Cys
Lys Pro Ser Cys1 5 10 15Gly Trp Asp Glu Lys Ala Ser Val Ser Gln Pro
Val Lys Thr Cys Asp 20 25 30Arg Asn Asn Asn Pro Leu Ala Ser Thr Ala
Arg Ser Gly Cys Asp Ser 35 40 45Asn Gly Val Ala Tyr Thr Cys Asn Asp
Asn Gln Pro Trp Ala Val Asn 50 55 60Asp Asn Leu Ala Tyr Gly Phe Ala
Ala Thr Ala Phe Ser Gly Gly Ser65 70 75 80Glu Ala Ser Trp Cys Cys
Ala Cys Tyr Ala Leu Gln Phe Thr Ser Gly 85 90 95Pro Val Ala Gly Lys
Thr Met Val Val Gln Ser Thr Asn Thr Gly Gly 100 105 110Asp Leu Ser
Gly Asn His Phe Asp Ile Leu Met Pro Gly Gly Gly Leu 115 120 125Gly
Ile Phe Asp Gly Cys Thr Pro Gln Trp Gly Val Ser Phe Pro Gly 130 135
140Asn Arg Tyr Gly Gly Thr Thr Ser Arg Ser Gln Cys Ser Gln Ile
Pro145 150 155 160Ser Ala Leu Gln Pro Gly Cys Asn Trp Arg Tyr Asp
Trp Phe Asn Asp 165 170 175Ala Asp Asn Pro Asp Val Ser Trp Arg Arg
Val Gln Cys Pro Ala Ala 180 185 190Leu Thr Asp Arg Thr Gly Cys Arg
Arg Ala 195 2004201PRTSordaria fimicola 4Gly Ser Gly Lys Ser Thr
Arg Tyr Trp Asp Cys Cys Lys Pro Ser Cys1 5 10 15Ala Trp Ser Gly Lys
Ala Ser Val Asn Arg Pro Val Leu Ala Cys Asp 20 25 30Ala Asn Asn Asn
Pro Leu Asn Asp Ala Asn Val Lys Ser Gly Cys Asp 35 40 45Gly Gly Ser
Ala Tyr Thr Cys Ala Asn Asn Ser Pro Trp Ala Val Asn 50 55 60Asp Asn
Leu Ala Tyr Gly Phe Ala Ala Thr Lys Leu Ser Gly Gly Thr65 70 75
80Glu Ser Ser Trp Cys Cys Ala Cys Tyr Ala Leu Thr Phe Thr Ser Gly
85 90 95Pro Val Ser Gly Lys Thr Leu Val Val Gln Ser Thr Ser Thr Gly
Gly 100 105 110Asp Leu Gly Ser Asn His Phe Asp Leu Asn Met Pro Gly
Gly Gly Val 115 120 125Gly Leu Phe Asp Gly Cys Lys Arg Glu Phe Gly
Gly Leu Pro Gly Ala 130 135 140Gln Tyr Gly Gly Ile Ser Ser Arg Ser
Glu Cys Asp Ser Phe Pro Ala145 150 155 160Ala Leu Lys Pro Gly Cys
Gln Trp Arg Phe Asp Trp Phe Lys Asn Ala 165 170 175Asp Asn Pro Glu
Phe Thr Phe Lys Gln Val Gln Cys Pro Ser Glu Leu 180 185 190Thr Ser
Arg Thr Gly Cys Lys Arg Ala 195 2005201PRTThielavia terrestris 5Gly
Ser Gly Gln Ser Thr Arg Tyr Trp Asp Cys Cys Lys Pro Ser Cys1 5 10
15Ala Trp Pro Gly Lys Ala Ala Val Ser Gln Pro Val Tyr Ala Cys Asp
20 25 30Ala Asn Phe Gln Arg Leu Ser Asp Phe Asn Val Gln Ser Gly Cys
Asn 35 40 45Gly Gly Ser Ala Tyr Ser Cys Ala Asp Gln Thr Pro Trp Ala
Val Asn 50 55 60Asp Asn Leu Ala Tyr Gly Phe Ala Ala Thr Ser Ile Ala
Gly Gly Ser65 70 75 80Glu Ser Ser Trp Cys Cys Ala Cys Tyr Ala Leu
Thr Phe Thr Ser Gly 85 90 95Pro Val Ala Gly Lys Thr Met Val Val Gln
Ser Thr Ser Thr Gly Gly 100 105 110Asp Leu Gly Ser Asn Gln Phe Asp
Ile Ala Met Pro Gly Gly Gly Val 115 120 125Gly Ile Phe Asn Gly Cys
Ser Ser Gln Phe Gly Gly Leu Pro Gly Ala 130 135 140Gln Tyr Gly Gly
Ile Ser Ser Arg Asp Gln Cys Asp Ser Phe Pro Ala145 150 155 160Pro
Leu Lys Pro Gly Cys Gln Trp Arg Phe Asp Trp Phe Gln Asn Ala 165 170
175Asp Asn Pro Thr Phe Thr Phe Gln Gln Val Gln Cys Pro Ala Glu Ile
180 185 190Val Ala Arg Ser Gly Cys Lys Arg Ala 195
2006203PRTFusarium oxysporum 6Gly Ser Gly His Ser Thr Arg Tyr Trp
Asp Cys Cys Lys Pro Ser Cys1 5 10 15Ser Trp Ser Gly Lys Ala Ala Val
Asn Ala Pro Ala Leu Thr Cys Asp 20 25 30Lys Asn Asp Asn Pro Ile Ser
Asn Thr Asn Ala Val Asn Gly Cys Glu 35 40 45Gly Gly Gly Ser Ala Tyr
Ala Cys Thr Asn Tyr Ser Pro Trp Ala Val 50 55 60Asn Asp Glu Leu Ala
Tyr Gly Phe Ala Ala Thr Lys Ile Ser Gly Gly65 70 75 80Ser Glu Ala
Ser Trp Cys Cys Ala Cys Tyr Ala Leu Thr Phe Thr Thr 85 90 95Gly Pro
Val Lys Gly Lys Lys Met Ile Val Gln Ser Thr Asn Thr Gly 100 105
110Gly Asp Leu Gly Asp Asn His Phe Asp Leu Met Met Pro Gly Gly Gly
115 120 125Val Gly Ile Phe Asp Gly Cys Thr Ser Glu Phe Gly Lys Ala
Leu Gly 130 135 140Gly Ala Gln Tyr Gly Gly Ile Ser Ser Arg Ser Glu
Cys Asp Ser Tyr145 150 155 160Pro Glu Leu Leu Lys Asp Gly Cys His
Trp Arg Phe Asp Trp Phe Glu 165 170 175Asn Ala Asp Asn Pro Asp Phe
Thr Phe Glu Gln Val Gln Cys Pro Lys 180 185 190Ala Leu Leu Asp Ile
Ser Gly Cys Lys Arg Ala 195 2007205PRTMyceliophthora thermophila
7Gly Ile Gly Gln Thr Thr Arg Tyr Trp Asp Cys Cys Lys Pro Ser Cys1 5
10 15Ala Trp Pro Gly Lys Gly Pro Ser Ser Pro Val Gln Ala Cys Asp
Lys 20 25 30Asn Asp Asn Pro Phe Asn Asp Gly Gly Ser Thr Arg Ser Gly
Cys Asp 35 40 45Ala Gly Gly Ser Ala Tyr Met Cys Ser Ser Gln Ser Pro
Trp Ala Val 50 55 60Ser Asp Glu Leu Ser Tyr Gly Trp Ala Ala Val Lys
Leu Ala Gly Ser65 70 75 80Ser Glu Ser Gln Trp Cys Cys Ala Cys Tyr
Glu Leu Thr Phe Thr Ser 85 90 95Gly Pro Val Ala Gly Lys Lys Met Ile
Val Gln Ala Thr Asn Thr Gly 100 105 110Gly Asp Leu Gly Asp Asn His
Phe Asp Leu Ala Ile Pro Gly Gly Gly 115 120 125Val Gly Ile Phe Asn
Ala Cys Thr Asp Gln Tyr Gly Ala Pro Pro Asn 130 135 140Gly Trp Gly
Asp Arg Tyr Gly Gly Ile His Ser Lys Glu Glu Cys Glu145 150 155
160Ser Phe Pro Glu Ala Leu Lys Pro Gly Cys Asn Trp Arg Phe Asp Trp
165 170 175Phe Gln Asn Ala Asp Asn Pro Ser Val Thr Phe Gln Glu Val
Ala Cys 180 185 190Pro Ser Glu Leu Thr Ser Lys Ser Gly Cys Ser Arg
Ala 195 200 2058203PRTCrinipellis scabella 8Thr Ala Gly Val Thr Thr
Arg Tyr Trp Asp Cys Cys Lys Pro Ser Cys1 5 10 15Gly Trp Ser Gly Lys
Ala Ser Val Ser Ala Pro Val Arg Thr Cys Asp 20 25 30Arg Asn Gly Asn
Thr Leu Gly Pro Asp Val Lys Ser Gly Cys Asp Ser 35 40 45Gly Gly Thr
Ser Phe Thr Cys Ala Asn Asn Gly Pro Phe Ala Ile Asp 50 55 60Asn Asn
Thr Ala Tyr Gly Phe Ala Ala Ala His Leu Ala Gly Ser Ser65 70 75
80Glu Ala Ala Trp Cys Cys Gln Cys Tyr Glu Leu Thr Phe Thr Ser Gly
85 90 95Pro Val Val Gly Lys Lys Leu Thr Val Gln Val Thr Asn Thr Gly
Gly 100 105 110Asp Leu Gly Asn Asn His Phe Asp Leu Met Ile Pro Gly
Gly Gly Val 115 120 125Gly Leu Phe Thr Gln Gly Cys Pro Ala Gln Phe
Gly Ser Trp Asn Gly 130 135 140Gly Ala Gln Tyr Gly Gly Val Ser Ser
Arg Asp Gln Cys Ser Gln Leu145 150 155 160Pro Ala Ala Val Gln Ala
Gly Cys Gln Phe Arg Phe Asp Trp Met Gly 165 170 175Gly Ala Asp Asn
Pro Asn Val Thr Phe Arg Pro Val Thr Cys Pro Ala 180 185 190Gln Leu
Thr Asn Ile Ser Gly Cys Val Arg Ala 195 2009203PRTMacrophomina
phaseolina 9Thr Ser Gly Val Thr Thr Arg Tyr Trp Asp Cys Cys Lys Pro
Ser Cys1 5 10 15Ala Trp Thr Gly Lys Ala Ser Val Ser Lys Pro Val Gly
Thr Cys Asp 20 25 30Ile Asn Asp Asn Ala Gln Thr Pro Ser Asp Leu Leu
Lys Ser Ser Cys 35 40 45Asp Gly Gly Ser Ala Tyr Tyr Cys Ser Asn Gln
Gly Pro Trp Ala Val 50 55 60Asn Asp Ser Leu Ser Tyr Gly Phe Ala Ala
Ala Lys Leu Ser Gly Lys65 70 75 80Gln Glu Thr Asp Trp Cys Cys Gly
Cys Tyr Lys Leu Thr Phe Thr Ser 85 90 95Thr Ala Val Ser Gly Lys Gln
Met Ile Val Gln Ile Thr Asn Thr Gly 100 105 110Gly Asp Leu Gly Asn
Asn His Phe Asp Ile Ala Met Pro Gly Gly Gly 115 120 125Val Gly Ile
Phe Asn Gly Cys Ser Lys Gln Trp Asn Gly Ile Asn Leu 130 135 140Gly
Asn Gln Tyr Gly Gly Phe Thr Asp Arg Ser Gln Cys Ala Thr Leu145 150
155 160Pro Ser Lys Trp Gln Ala Ser Cys Asn Trp Arg Phe Asp Trp Phe
Glu 165 170 175Asn Ala Asp Asn Pro Thr Val Asp Trp Glu Pro Val Thr
Cys Pro Gln 180 185 190Glu Leu Val Ala Arg Thr Gly Cys Ser Arg Ala
195 20010235PRTPseudomonas fluorescens 10Cys Asn Gly Tyr Ala Thr
Arg Tyr Trp Asp Cys Cys Lys Pro His Cys1 5 10 15Gly Trp Ser Ala Asn
Val Pro Ser Leu Val Ser Pro Leu Gln Ser Cys 20 25 30Ser Ala Asn Asn
Thr Arg Leu Ser Asp Val Ser Val Gly Ser Ser Cys 35 40 45Asp Gly Gly
Gly Gly Tyr Met Cys Trp Asp Lys Ile Pro Phe Ala Val 50 55 60Ser Pro
Thr Leu Ala Tyr Gly Tyr Ala Ala Thr Ser Ser Gly Asp Val65 70 75
80Cys Gly Arg Cys Tyr Gln Leu Gln Phe Thr Gly Ser Ser Tyr Asn Ala
85 90 95Pro Gly Asp Pro Gly Ser Ala Ala Leu Ala Gly Lys Thr Met Ile
Val 100 105 110Gln Ala Thr Asn Ile Gly Tyr Asp Val Ser Gly Gly Gln
Phe Asp Ile 115 120 125Leu Val Pro Gly Gly Gly Val Gly Ala Phe Asn
Ala Cys Ser Ala Gln 130 135 140Trp Gly Val Ser Asn Ala Glu Leu Gly
Ala Gln Tyr Gly Gly Phe Leu145 150 155 160Ala Ala Cys Lys Gln Gln
Leu Gly Tyr Asn Ala Ser Leu Ser Gln Tyr 165 170 175Lys Ser Cys Val
Leu Asn Arg Cys Asp Ser Val Phe Gly Ser Arg Gly 180 185 190Leu Thr
Gln Leu Gln Gln Gly Cys Thr Trp Phe Ala Glu Trp Phe Glu 195 200
205Ala Ala Asp Asn Pro Ser Leu Lys Tyr Lys Glu Val Pro Cys Pro Ala
210 215 220Glu Leu Thr Thr Arg Ser Gly Met Asn Arg Ala225 230
23511211PRTUstilago maydis 11Gly Met Ala Thr Arg Tyr Trp Asp Cys
Cys Leu Ala Ser Ala Ser Trp1 5 10 15Glu Gly Lys Ala Pro Val Tyr Ala
Pro Val Asp Ala Cys Lys Ala Asp 20 25 30Gly Val Thr Leu Ile Asp Ser
Lys Lys Asp Pro Ser Gly Gln Ser Gly 35 40 45Cys Asn Gly Gly Asn Lys
Phe Met Cys Ser Cys Met Gln Pro Phe Asp 50 55 60Asp Glu Thr Asp Pro
Thr Leu Ala Phe Gly Phe Gly Ala Phe Thr Thr65 70 75 80Gly Gln Glu
Ser Asp Thr Asp Cys Ala Cys Phe Tyr Ala Glu Phe Glu 85 90 95His Asp
Ala Gln Gly Lys Ala Met Lys Arg Asn Lys Leu Ile Phe Gln 100 105
110Val Thr Asn Val Gly Gly Asp Val Gln Ser Gln Asn Phe Asp Phe Gln
115 120 125Ile Pro Gly Gly Gly Leu Gly Ala Phe Pro Lys Gly Cys Pro
Ala Gln 130 135 140Trp Gly Val Glu Ala Ser Leu Trp Gly Asp Gln Tyr
Gly Gly Val Lys145 150 155 160Ser Ala Thr Glu Cys Ser Lys Leu Pro
Lys Pro Leu Gln Glu Gly Cys 165 170 175Lys Trp Arg Phe Ser Glu Trp
Gly Asp Asn Pro Val Leu Lys Gly Ser 180 185 190Pro Lys Arg Val Lys
Cys Pro Lys Ser Leu Ile Asp Arg Ser Gly Cys 195 200 205Gln Arg Ala
2101230DNAArtificial SequencePrimer 12gaatgacttg gttgacgcgt
caccagtcac 301330DNAArtificial SequencePrimer 13gaatgacttg
gttgagtact caccagtcac 301434DNAArtificial SequencePrimer
14cactggcggc gacctgggat ctaaccactt cgat 341537DNAArtificial
SequencePrimer 15atcgaagtgg ttagatccca ggtcgccgcc tgtgctc
371620DNAArtificial SequencePrimer 16cgacttcaat gtccagtcgg
201726DNAArtificial SequencePrimer 17gcgctctaga ggattaaagg cactgc
261835DNAArtificial SequencePrimer 18cgacctggga tcgaacgact
tcgatatcgc catgc 351930DNAArtificial SequencePrimer 19cgactccagc
ttccccgtct tcacgccccc 302034DNAArtificial SequencePrimer
20cgagcttcta gatctcgact agaggcactg ggag 342127DNAArtificial
SequencePrimer 21ggatgccatg cttggaggat agcaacc 272229DNAArtificial
SequencePrimer 22gggggcgtga agacgggaag ctggagtcg
29231081DNAHumicola grisea 23ctcgtgaacc
agcctgtctt cacttgcgac gccaaattcc agcgcatcac cgaccccaat 60accaagtcgg
gctgcgatgg cggctcggcc ttttcgtgtg ctgaccagac cccctgggct
120ctgaacgacg atgtcgccta tggcttcgct gccacggcta tttcgggtgg
atcggaagcc 180tcgtggtgct gcgcatgcta cgctcttact ttcacctcgg
gccctgtggc cggcaagacc 240atggtcgtcc agtcgaccaa caccggcggc
gatctcggca gcaaccattt cgacctccag 300attccaggcg gcggtgtcgg
catctttgat gggtgcaccc cccagttcgg aggtctcgct 360ggcgaacgct
acggtggcat ctcagaccgc agctcctgcg actcgttccc tgcggcgctc
420aagcccggct gcctctggcg cttcgattgg ttcaagaacg ccgacaaccc
gacctttacc 480ttcaagcagg tgcagtgccc cgccgagctt gttgccagga
ccggctgcaa gcgcgaggat 540gacggcaact tccccgtctt cacgcccccc
gcgggtagca acaccggcgg tagccagtcg 600agctccacta tcgcttccag
ctcgacctcc aaggctcaga cttcggccgc cagctccacc 660tccaaggctg
tcgtgactcc cgtctccagc tccacctcga aggccgctga ggtccccaaa
720tccagctcga cctccaaggc tgccgaggtc gccaagccca gctcaacttc
gacctcgacc 780tcgacctcga ccaaggtcag ctgctctgcg accggtggct
cctgcgtcgc tcagaagtgg 840gcgcagtgcg gcggcaatgg cttcaccggc
tgcacgtcgt gcgtcagcgg caccacctgc 900cagaagcaaa atgactggta
ctcccagtgc ctctaagtcg tttgtagtag cagtttgaag 960gatgtcaggg
atgagggagg gaggagtggg ggaaaagtac gccgcagttt tttggtagac
1020ttactgtatt gttgagtaat tacccattcg cttcttgtac gaaaaaaaaa
aaaaaaaaaa 1080a 10812441DNAArtificial SequencePrimer 24gctgcaagcc
gtcctgtggc tggagcgcta acgtgcccgc g 412536DNAArtificial
SequencePrimer 25cgatgtttcc ggaggccact ttgacattct ggttcc
362630DNAArtificial SequencePrimer 26gaatgacttg gttgacgcgt
caccagtcac 30
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