U.S. patent application number 14/220301 was filed with the patent office on 2014-08-28 for enzymatically catalyzed method of preparing mono-acylated polyols.
This patent application is currently assigned to BASF SE. The applicant listed for this patent is Anders Hamberg, Bernhard Hauer, Karl Hult, Cecilia Kvarnstrom Branneby, Anders Magnusson. Invention is credited to Anders Hamberg, Bernhard Hauer, Karl Hult, Cecilia Kvarnstrom Branneby, Anders Magnusson.
Application Number | 20140242651 14/220301 |
Document ID | / |
Family ID | 43416948 |
Filed Date | 2014-08-28 |
United States Patent
Application |
20140242651 |
Kind Code |
A1 |
Hauer; Bernhard ; et
al. |
August 28, 2014 |
ENZYMATICALLY CATALYZED METHOD OF PREPARING MONO-ACYLATED
POLYOLS
Abstract
The present invention relates to a biocatalytic method of
preparing a mono-acylated polyol catalyzed by triacylglycerol
lipase mutants, as for example derived from Candida antarctica
lipase B (CALB); a biocatalytic method of enantioselectively
preparing an asymmetric mono-acylated polyol, catalyzed by the same
enzyme mutants; as well as the use of a mutated triacylglycerol
lipase in a method of preparing mono-acylated polyols. The
invention also provides novel mutants, coding sequences thereof,
and recombinant microorganisms carrying said coding sequences.
Inventors: |
Hauer; Bernhard;
(Fussgonheim, DE) ; Kvarnstrom Branneby; Cecilia;
(Stockholm, SE) ; Hult; Karl; (Stockholm, SE)
; Magnusson; Anders; (Frankfurt, DE) ; Hamberg;
Anders; (Bagarmossen, SE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hauer; Bernhard
Kvarnstrom Branneby; Cecilia
Hult; Karl
Magnusson; Anders
Hamberg; Anders |
Fussgonheim
Stockholm
Stockholm
Frankfurt
Bagarmossen |
|
DE
SE
SE
DE
SE |
|
|
Assignee: |
BASF SE
Ludwigshafen
DE
|
Family ID: |
43416948 |
Appl. No.: |
14/220301 |
Filed: |
March 20, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13496342 |
Mar 15, 2012 |
8715970 |
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PCT/EP2010/063645 |
Sep 16, 2010 |
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14220301 |
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61245298 |
Sep 24, 2009 |
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Current U.S.
Class: |
435/135 ;
435/198; 435/252.3; 435/252.31; 435/252.33; 435/320.1;
536/23.2 |
Current CPC
Class: |
C12P 7/62 20130101; C12P
41/004 20130101; C12N 9/20 20130101 |
Class at
Publication: |
435/135 ;
435/198; 536/23.2; 435/320.1; 435/252.3; 435/252.33;
435/252.31 |
International
Class: |
C12P 7/62 20060101
C12P007/62; C12N 9/20 20060101 C12N009/20 |
Foreign Application Data
Date |
Code |
Application Number |
Sep 16, 2009 |
EP |
09170458.5 |
Claims
1-30. (canceled)
31. A biocatalytic method of preparing a mono-acylated polyol of
the general formula (I): ##STR00006## wherein R.sub.1 represents an
optionally substituted, linear or branched, saturated or
non-saturated hydrocarbyl residue; and A represents an optionally
substituted, linear or branched hydrocarbylene residue having at
least two carbon atoms, which method comprises a) reacting a polyol
of the formula (II) and an acyl donor compound of the formula (III)
##STR00007## wherein R.sub.1 and A are as defined above, and Don
represents a donor molecule residue carrying the said acyl group;
in the presence of a mutated triacylglycerole lipase (EC 3.1.1.3)
until a mono-acylated polyol of the above formula (I) is formed;
and b) obtaining a monoacylated polyol product.
32. The method of claim 31, wherein said mutated lipase contains at
least one amino acid mutation, which increases the selectivity of
the lipase for the mono-acylation of said polyol, if compared to
the corresponding non-mutated lipase.
33. The method of claim 31, wherein said mutant comprises at least
one mutation, which removes a stabilizing functional amino acid
from that part of the reactive center of the enzyme which
stabilizes an oxyanion transition state of the carbonyl group of
the mono-acylated polyol of formula (I) to be formed.
34. The method of claim 31, wherein a) a maximum monoester yield is
obtained which is at last 1% above the maximum yield as obtained by
the corresponding wild-type enzyme; b) a 3:1 molar ratio of
monoester to polyester is reached at a conversion rate of the
polyol which is at last 1% above the corresponding conversion rate
as obtained by the corresponding wild-type enzyme; and/or c) the
ratio of reaction times (T.sub.90(mutant)/T.sub.90(wild-type)) to
reach 90% monoacylated polyol based on the total amount of polyol
is above 1.
35. The method of claim 31, wherein said enzyme is a mutant of
Candida antarctica lipase B (CALB) comprising an amino acid
sequence of SEQ ID NO: 2, mutated in at least one position.
36. The method of claim 35, wherein said mutant comprises an amino
acid sequence of SEQ ID NO:2 wherein the amino acid Thr40 is
mutated.
37. The method of claim 36, wherein the mutation is such that
substantially no stabilizing interaction between the oxyanion
intermediate and the amino acid residue in position 40 occurs.
38. The method of claim 37 wherein the mutation comprises the
single mutations Thr40Ala, Thr40Val or Thr40Ser.
39. The method of claim 38, wherein said mutant is selected from
mutants having an amino acid sequence of SEQ ID NO: 4 or variants
of said mutant having a sequence identity of at least 60%, which
variants still contain a mutation in an amino acid position
corresponding to position Thr40 of SEQ ID NO:4.
40. The method of claim 37, wherein the mutant additionally
comprises at least one mutation in one of the amino acid positions
Leu 278, Ile 285 and Pro 280 of SEQ ID NO: 2 or 4.
41. The method of claim 40, wherein the mutants and the variants
thereof are not mutated in other amino acid positions contributing
to the catalytic site of the enzyme.
42. The method of claim 41, wherein the mutants are not mutated in
amino acid positions Ser105, Asp187, His224 (catalytic triade) and
Gln106 and wherein the variants are not mutated in amino acid
positions corresponding thereto.
43. The method of claim 35, wherein in SEQ ID NO:2, or in SEQ ID
NO:2 comprising a mutation at amino acid Thr40 according to SEQ ID
NO:4, one or more of Leu278, Ala281, Ala282 or Ile285 are
mutated.
44. The method of claim 43, wherein the one or more mutations are
independently selected from Leu278Ser, Ala281Val or Ala281Glu, and
Ala282Leu, Ala282Thr, Ala282Cys, Ala282Pro, Ala282Ile, Ala282Asp,
Ala282Val, Ala282Met or Ala282Arg.
45. The method of claim 44, wherein SEQ ID NO:2 comprises one
mutation, selected from Ala281Val, Ala281Glu, Ala282Leu, Ala282Thr,
Ala282Cys, Ala282Pro, Ala282Ile, Ala282Asp, Ala282Val, Ala282Met,
Ala282Arg and Ile285Phe, or wherein SEQ ID NO:2 comprises the
double mutation Leu278Ser and Ala282Leu.
46. The method of claim 31, wherein the reaction is performed in
the presence of the isolated enzyme mutant or a recombinant
microorganism functionally expressing said mutant.
47. The method of claim 31, wherein the polyol is a compound of
formula (II) wherein A is selected from the groups
--(CH.sub.2).sub.n-- and
--(CH.sub.2).sub.m--CR.sub.2R.sub.3--(CH.sub.2).sub.m'-- wherein n
is an integer of 2-6, m and m' independently of each other are
integers of 1-3 R.sub.2 and R.sub.3 independently of each other are
selected from H, OH, SH, NH.sub.2, optionally substituted carbo- or
heterocyclic rings and hydrocarbyl-residues, provided that R.sub.2
and R.sub.3 are not simultaneously H.
48. The method of claim 31, wherein the donor of formula (III) is
selected from compounds wherein R1 is C1-C6-alkyl and Don is an
--OR residue, wherein R is selected from C1-C6-alkyl and
C2-C4-alkenyl.
49. An enzymatically catalyzed method of enantioselectively
preparing an asymmetric mono-acylated polyol of the general formula
(I): ##STR00008## wherein R.sub.1 represents an optionally
substituted, linear or branched, saturated or non-saturated
hydrocarbyl residue; and A* represents an optionally substituted,
linear or branched, asymmetric hydrocarbylene residue having at
least two carbon atoms; which method comprises a) reacting a
stereoisomeric mixture of a polyol of the formula (II') and an acyl
donor compound of the formula (III) ##STR00009## wherein R.sub.1
and A* are as defined above, and Don represents a donor molecule
residue carrying the said acyl group; in the presence of a mutated
triacylglycerole lipase (EC 3.1.1.3) until a mono-acylated polyol
of the above formula (I) is formed; and b) obtaining an asymmetric
monoacylated polyol product.
50. The method of claim 49 wherein an enzyme mutant as defined in
anyone of the claims 2 to 15 in the form of an isolated enzyme
mutant or a recombinant microorganism functionally expressing said
mutant is applied.
51. The method of claim 49, wherein the polyol is a compound of
formula (II') wherein A* is selected from the groups
--(CH.sub.2).sub.m--CHR.sub.2--(CH.sub.2).sub.m'-- wherein m, m'
and R.sub.2 are as defined above.
52. The use of a mutated triacylglycerol lipase (EC 3.1.1.3) in a
method of preparing a mono-acylated polyol of the general formula
(I) or (I') as defined above.
53. A Candida antarctica lipase B (CALB) mutant showing a pattern
of at least two mutation of the amino acid sequence of SEQ ID NO:2,
which pattern is selected from the pattern as shown in Table A.
54. The mutant of claim 53 showing additionally one mutation
selected from Val210Ile, Ala281Glu, Val221Asp.
55. A Candida antarctica lipase B (CALB) mutant, having one or more
mutations in the amino acid sequence of SEQ ID NO:2, which are
independently selected from Leu278Ser, Ala281Val or Ala281Glu, and
Ala282Leu, Ala282Thr, Ala282Cys, Ala282Pro, Ala282Ile, Ala282Asp,
Ala282Val, Ala282Met or Ala282Arg.
56. The mutant of claim 55, having one mutation in SEQ ID NO:2,
selected from Ala281Val, Ala281Glu, Ala282Leu, Ala282Thr,
Ala282Cys, Ala282Pro, Ala282Ile, Ala282Asp, Ala282Val, Ala282Met,
Ala282Arg and Ile285Phe, or having in SEQ ID NO:2 the double
mutation Leu278Ser and Ala282Leu.
57. The mutant of claim 53, additionally having at least one of the
mutations as defined in claim 55.
58. A nucleic acid molecule encoding a mutant of claim 50.
59. An expression vector, comprising, optionally under the control
of a regulatory nucleic acid sequence, at least one coding sequence
of claim 58.
60. A microbial host carrying at least one expression vector of
claim 59 or coding sequence of claim 58.
Description
RELATED APPLICATIONS
[0001] This application is a divisional of U.S. application Ser.
No. 13/496,342, filed Mar. 15, 2012, which is a national stage
application (under 35 U.S.C. .sctn.371) of PCT/EP2010/63645, filed
Sep. 16, 2010, which claims benefit of European Application No.
09170458.5, filed Sep. 16, 2009, and U.S. Provisional Patent
Application No. 61/245,298, filed Sep. 24, 2009, the entire
contents of each above-mentioned application are hereby
incorporated by reference in their entirety.
SUBMISSION OF SEQUENCE LISTING
[0002] The Sequence Listing associated with this application is
filed in electronic format via EFS-Web and hereby incorporated by
reference into the specification in its entirety. The name of the
text file containing the Sequence Listing is
Sequence_Listing.sub.--074012.sub.--0204.sub.--01. The size of the
text file is 19 KB and the text file was created on May 9,
2014.
[0003] The present invention relates to a biocatalytic method of
preparing a mono-acylated polyol catalyzed by triacylglycerol
lipase mutants, as for example derived from Candida antarctica
lipase B (CALB); a biocatalytic method of enantioselectively
preparing an asymmetric mono-acylated polyol, catalyzed by the same
enzyme mutants; as well as the use of a mutated triacylglycerol
lipase in a method of preparing mono-acylated polyols. The
invention also provides novel mutants, coding sequences thereof,
and recombinant microorganisms carrying said coding sequences.
BACKGROUND OF THE INVENTION
[0004] Triacylglycerol lipases (EC 3.1.1.3) are valued, efficient
catalysts for a great variety of industrial uses, for example in
the detergents industry, oil chemistry, the food industry and in
the production of fine chemicals (Schmid R. D., Verger, R., Angew.
Chem. Int. Ausg. 37: 1608-33 (1998)). Lipases are carboxylic ester
hydrolases, which catalyze both the hydrolysis and the synthesis of
triglycerides and other generally hydrophobic esters. All
triacylglycerol lipases, whose three-dimensional crystal structure
has been elucidated, belong to the .alpha./.beta.-hydrolase folding
protein family, which have a similar overall architecture (Ollis D.
L., Cheah, E., Cygler, M., Dijkstra, B., Frolow, F., Franken, S.
M., Harel, M., Remingon, S. M., Silman, L., Schrag, J. D., Protein
Eng. 5: 197-211 (1992)).
[0005] Candida antarctica-lipase B (also designated herein as CALB)
is an efficient catalyst for many reactions and is used for example
for stereoselective transformations and polyester synthesis
(Anderson E. M., Larsson, K. M., Kirk, O., Biocat. Biotransform.
16: 181-204 (1998)) CALB has a solvent-accessible active center
(Uppenberg J., Hansen, M. T., Patkar, S., Jones, A., Structure 2:
293-308 (1994)) and does not display interphase activation
(Martinelle M., Holmquist M., Hult K., Biochim. Biophys. Acta
1258(3): 272-6 (1995)). The active center is a narrow funnel and
for this reason CALB has a higher activity with respect to
carboxylic acid esters, for example ethyl octanoate, than with
respect to triglycerides (Martinelle 1995, supra). The fact that
the activity of CALB in organic media is comparable to that in
water, and in particular the high enantioselectivity of CALB for
secondary alcohols make this enzyme one of the most important
lipases currently in use in biotechnology.
[0006] Modification of CALB by random mutagenesis was described
recently (Chodrorge M., Fourage L., Ullmann C., Duvivier V., Masson
J. M., Lefevre F., Adv. Synth. Catal. 347: 1022-1026 (2005).
Several attempts to improve CALB for special applications through
rational enzyme design have also been reported in the literature.
Although some of these led to good results (Patkar S., Vind J.,
Kelstrup E., Christensen M. W., Svendsen A., Borch K., Kirk O.,
Chem. Phys. Lipids 93(1-2): 95-101 (1988); Rotticci D.
"Understanding and Engineering the Enantioselectivity of Candida
antarctica Lipase B towards sec-Alcohols". Stockholm: Royal
institute of Technology 1-61 (2000)), the possibilities for
rational enzyme design are still limited through insufficient
understanding of the catalytic properties of the enzyme.
[0007] Zhang et al. report in Protein Engineering, vol. 16, no. 8
(2003) 599 on experiments aiming at an improvement of the tolerance
of CALB to irreversible thermal inactivation. By applying directed
evolution techniques single mutants containing the mutation V2101,
V221D or A281E were prepared. The double mutant (V2101, A281E) and
the triple mutant (V2101, A281E, V221D) as well as the single
mutant A281E showed a significant improvement of their melting
point (T.sub.m) versus the T.sub.m of the wild-type enzyme.
[0008] Another approach for improving the activity and thermal
stability of CALB was described by Suen et al. in Protein
Engineering, Design & Selection, vol. 17, no. 2 (2004), 133.
The technique of DNA family shuffling was used to create chimeric
lipase B proteins derived from Candida antarctica and Crytococcus
tzukubaensis as well as Hyphozyma sp. By high-throughput screening
chimeras could be identified showing a higher activity towards the
substrate 3-(3',4'-dichlorophenyl)glutarate, an improved half-life
at 45.degree. C. and an improved melting point (T.sub.m).
[0009] Magnusson et al. describe in J. Am. Chem. Soc. 123 (2001),
4354 mono-mutants of CALB which differ in their enantioselectivity
with respect to the hydrolysis of the two ethylesters
ethyl-3-hydroxybutanoat and ethyl-2-hydroxypropanoat. In
particular, the mono-mutants T40A and T40V are described therein.
The preparation of monoacylated polyols is not taught or
suggested.
[0010] Rotticci et al. disclose in ChemBiochem. 2 (2001), 766
experiments for improving the enantioselectivity of CALB towards
different optically active secondary mono-alcohols. In particular,
the single mutants 547A, S47N, S47H, T42N, T42D, T42H, T42V, W104H,
as well as the double mutant (T42V, S47A) have been prepared via
rational design and further investigated.
[0011] Branneby et al. disclose in J. Am. Chem. Soc. 125 (2003),
874 the single mutants S105A and S105G of CALB and their behaviour
during aldol condensation reactions.
[0012] Magnussen et al. disclose in ChemBiochem. (2005) 1051
mutants of CALB having an enlarged substrate pocket, which mutants
have the ability to utilize larger secondary mono-alcohols. In
particular, the single mutants T42V, S47A, W104A, W104H, W104Q and
the double mutant (T42V, S47A) were investigated.
[0013] Consequently, none of the above-mentioned documents teaches
or suggests making use of CALB enzymes in methods for monoacylating
polyols, in particular non-cyclic polyols.
[0014] The selective preparation of monoacylated polyols is
considered to be difficult to achieve because of the fact that a
monoacylated intermediate is expected to be quickly further
esterified by the same enzyme, so that monoesters of polyols are
expected to be merely intermediary formed during the course of the
reaction and with the progress of the esterification reaction the
proportion of monoesterified products is more and more
diminished.
[0015] Therefore, there was a continued need for providing an
enzymatically catalyzed method for selectively monoacylating
polyols, such as diols, in particular non-cyclic diols. In
particular, there was a need fur methods allowing the improved,
preferential preparation of monoacylated polyols. An improvement in
this respect may be characterized by an increased maximum yield of
monoester, an improved molar ratio of monoester product to higher
or fully esterified products, as for example diesters, an improved
molar ratio of monoester product to total esterified products,
and/or a higher monoester yields at higher degrees of
conversion.
[0016] There was a further need for methods for enantioselectively
preparing such monoacylated polyols, in the case of enzyme
substrates having an asymmetric carbon atom.
SUMMARY OF THE INVENTION
[0017] The above-mentioned problems could, surprisingly, be solved
by providing an enzymatically catalyzed method of preparing a
mono-acylated polyol, which method makes use of mutated
triacylglycerole lipase enzymes, like mutants of CALB, which were
engineered to show improved selectivity, i.p. regioselectivity,
allowing the preferential formation of monoacylated polyols.
[0018] The present invention allows performing polyol
esterification reactions during the course of which the monoester
product is produced in a significant molar excess over fully
esterified products not only at low but, surprisingly, also at high
degrees of polyol conversion. For example, a product distribution
(defined as the ratio of monoester to the sum of all esterified
products) of at least 90% are observed at degrees of conversion up
to about 50 to 90%, thus allowing to proceed the reaction almost to
completion, and isolating of the desired monoester product in high
yields from the reaction mixture.
[0019] The invention will be further explained by making reference
to the attached drawings.
DESCRIPTION OF DRAWINGS
[0020] FIG. 1 represents a mechanistic scheme illustrating the
interaction of amino acid residues Asp 187, Ser 105 and His 224 of
the reactive centre of CALB during a transesterification reaction
transferring the acyl residue C(O)R.sub.1 of a first ester
ROC(O)R.sub.1 to an alcohol HOR.sub.2 forming a new second ester
compound R.sub.2OC(O)R.sub.1. The tetrahedral intermediates are
stabilized in the oxianion hole of the reactive centre thus
favouring the transesterification reaction.
[0021] FIGS. 2A to 2C illustrate schematically the participation of
amino acid residues in position 106 and 40 of the CALB oxianion
hole in the stabilization of the oxianion intermediate as formed
during transesterification reactions. FIG. 2A illustrates the
stabilization of the oxianion by formation of 3 hydrogen bonds
between the oxianion and amino acid residues Gln 106 and Thr 40.
The illustrated ester is of the formula R.sub.1OC(O)R. FIG. 2B
shows the stabilization of the same substrate as in FIG. 2A but now
in the single mutant T40A of CALB, wherein Thr 40 replaced by Ala
as a result of which one stabilizing hydrogen bond is getting lost,
thus destabilizing said transition state. FIG. 2C illustrates the
substrate assistance during stabilization of the transition state
occurring in the same mutant T40A as observed during the
transesterification reaction of a butanediol monoester HOButOC(O)R.
The free hydroxy group of the diol interacts with the oxianion via
a hydrogen bond so that stability of the transition state is
regained. Said substrate assistance by said diol molecule explains
the diol preference during the transesterification reactions
catalyzed by the T40A mutant.
[0022] FIG. 3 illustrates the experimental results observed for the
transesterification reaction catalyzed by the CALB wild type and
the CALB T40A mutant in a transesterification reaction with
1,4-butanediol as substrate dissolved in ethyl acetate. The yield
for the mono- and di-acetates is shown as functions of the %
conversion. (A) illustrates the results for the CALB wild type. (B)
illustrates the results obtained for the CALB mutant T40A. As can
be seen, over a wide % conversion range the monoacetate ester is
preferably obtained.
[0023] FIG. 4 illustrates the experimental results observed for the
transesterification reaction catalyzed by the CALB wild type and
the CALB T40A mutant in a transesterification reactions with
1,2-ethanediol as substrate dissolved in ethyl acetate. The yield
for the mono- and di-acetate is shown as a function of %
conversion. (A) illustrates the results obtained for the CALB wild
type. (B) illustrates the results obtained for the CALB mutant
T40A. As can be seen, with the T40A mutant the monoacetate ester is
preferentially obtained over a wide % conversion range. The maximum
monoester yield for the wild type enzyme is 43%; the maximum
monoester yield for the mutant is significantly improved to 77%
illustrating an improved selectivity of the mutant for the
monoester.
[0024] FIG. 5 shows differences between wild type and T40A CALB
regarding product distributions in transesterification reactions
with 1,2-ethanediol dissolved in ethyl acetate. Illustrated in (A)
and (B) are diol conversions at two given product distributions
from the same experiments. 17% of the diol was converted with wild
type and 99% with T40A CALB for a product distribution of 75% (see
(A)). The corresponding figures for a product distribution of 90%
are 9% with wild type and 78% with T40A CALB (see (B)).
[0025] FIG. 6 illustrates the data fitted to a model based on
equations 1-3. The dots showing diol (rhombi), monoester (squares)
and diester (triangles) are measured values. The error bars show
values calculated using the obtained k.sub.cat/K.sub.M-values in
equations 1-3. (A) and (B) corresponds to wild type and T40A CALB
respectively.
[0026] FIG. 7 illustrates the yields of monoester and diester in
transacylation reactions with 2-methyl-1,3-propanediol as substrate
and vinyl butyrate as acyl donor dissolved in MTBE. Wild type (A)
and T40A (B) CALB are used as catalysts. As can be seen, over a
wide % conversion range the monobutyrate ester is preferably
obtained.
[0027] FIG. 8 shows yields of mono- end diester expressed as
function of 1,4-butanediol conversion in reactions with vinyl
butyrate as an acyl donor dissolved in MTBE. Wild type (A) and T40A
(B) CALB are used as catalysts. As can be seen, over a wide %
conversion range the monobutyrate ester is preferably obtained.
[0028] FIG. 9 shows conversions of competing substrates.
Conversions of diols are expressed as functions of 1-butanol
conversion. Two different diols are displayed, 1,2-ethanediol (A)
and 1,4-butanediol (B). The reactions are carried out using ethyl
acetate as acyl donor and solvent. Reactions catalyzed by wild type
(rhombi) and T40A (squares) CALB are compared. In both A and B the
selectivity towards diol over 1-butanol is higher for the reactions
catalyzed by T40A than wild type CALB.
[0029] FIG. 10 shows conversions of competing substrates.
Conversions of diols are expressed as functions of 1-butanol
conversion. Two different diols are displayed, 1,2-ethanediol (A)
and 1,4-butanediol (B). The reactions are carried out using vinyl
butyrate as acyl donor and MTBE as solvent. Reactions catalyzed by
wild type (rhombi), T40A (squares) and T40V (triangles) CALB are
compared. In both A and B the selectivity towards diol over
1-butanol is higher for the reactions catalyzed by T40A and T40V
than wild type CALB.
[0030] FIG. 11 shows converted 1,2-ethanediol per gram enzyme over
time. Ethyl acetate is used as acyl donor and solvent in A. In B,
vinyl butyrate is used as acyl donor and MTBE as solvent. The
reaction rates differ between wild type and T40A CALB, showing that
the wild type (rhombi) is a more efficient catalyst than the T40A
(squares) variant. The difference in reaction rates is greater when
ethyl acetate is used as acyl donor than for vinyl butyrate.
[0031] FIG. 12 shows the excess of 4-hydroxybutanediol over
butanediole diacrylate at various rates of conversion after
enzymatic catalysis by a commercially available enzyme (Novo 435)
(represented by filled dots), the A282L mutant (open squares) or
the L278S mutant (open triangles). In the respective ranges tested,
both CALB mutants show a measurable excess of 4-hydroxybutanediol
over a wide range of achieved conversions.
[0032] FIG. 13 shows the dependency of product excess or conversion
rates from flow rates for CALB Novo 435 and CALB A282L. Filled or
open dots represent conversion by Novo 435 or A282L, resp., and
filled or open squares represent excess of 4-HBA achieved by Novo
435 or A282L, resp., at a given conversion.
[0033] FIGS. 14A and 14B show the excess of produced monoacrylate
versus the degree of conversion in comparison of CALB Novo 435 and
selected CALB mutants according to the invention. The results are
based on samples taken directly from the medium. FIG. 14A:
comprises results from A282C (labelled by crosses); A282P (open
triangles); A282I (open squares); A282D (open diamonds, dashed
line); FIG. 14B comprises results from A282L (open squares); A282V
(open triangles, dashed line); A282R (open diamonds) and
L278S/A282L (crosses), while both in FIG. 14A and FIG. 14B Novo 435
is represented by filled dots.
[0034] FIGS. 15A and 15B show the excess of produced monoacrylate
versus degree of conversion in comparison of CALB Novo 435 and
selected CALB mutants according to the invention. The results are
based on enzyme immobilized on Resindinon Diaion HP20L beads
(Resindion SRL, a subsidiary of Mitsubishi Chemical). FIG. 15 A
comprises results from: A282I (represented by open triangles),
A282R (open diamonds), and L278S/A282L (open squares, dashed line);
FIG. 15B comprises results from A282C (open diamonds), I285F (open
squares) and A282L/I285F (open triangles), while both in FIG. 15A
and FIG. 15B Novo 435 is represented by filled dots.
DETAILED DESCRIPTION OF THE INVENTION
1. General Definitions
[0035] In the absence of information to the contrary the following
general definitions shall apply:
[0036] According to the invention, "triacylglycerol lipases" means
enzymes of class E.C. 3.1.1.3 according to the IUBMB enzyme
nomenclature (iubmb.unibe.ch; chem.qmul.ac.uk/iubmb/enzyme/).
[0037] According to a special embodiment of the method according to
the invention, the functionally expressed triacylglycerol lipase is
lipase B, the gene product of CALB from Candida antarctica. The
CALB gene was described (Uppenberg et al., 1994) and its nucleotide
or protein sequence was deposited under the access numbers Z30645
and CAA83122.1 at GenBank. Unless designated more precisely, here
CALB means a nucleotide sequence with this access number. Another
example of a triacylglycerol lipase is lipase B from Pseudozyma
tsukubaensis (Suen, W. C., Zhang, N., Xiao, L, Madison, V., Zaks,
A. Protein Eng. Des. Sel. 17(2): 133-40 (2004)).
[0038] An "enzymatically catalyzed" or "biocatalytic" method means
that said method is performed under the catalytic action of an
enzyme, including enzyme mutants, as herein defined. Thus the
method can either be performed in the presence of said enzyme in
isolated (purified, enriched) or crude form or in the presence of a
cellular system, in particular, natural or recombinant microbial
cells containing said enzyme in active form, and having the ability
to catalyze the conversion reaction as disclosed herein.
[0039] The terms "selectively mono-acylating polyols" or
"increasing the selectivity for mono-acylating polyols" in general
means that a monoester of said polyol is produced in a higher
proportion or amount (compared on a molar basis) than at least one,
preferably all, higher esterified polyol components during the
course of the esterification reaction disclosed herein, i.e. either
during the entire course of said reaction (i.e. between initiation
and termination of the reaction), at a certain point of time of
said reaction, or during an "interval" of said reaction. In
particular, said selectivity may be observed during an "interval"
corresponding 1 to 99%, 2 to 95%, 3 to 90%, 5 to 85%, 10 to 80%, 15
to 75%, 20 to 70%, 25 to 65%, 30 to 60, or 40 to 50% conversion of
the initial amount of polyol substrate. Said higher proportion or
amount may, for example, be expressed in terms of: [0040] a higher
maximum yield of the monoacylated polyol observed during the entire
course of the reaction or said interval thereof; [0041] a higher
relative amount of the monoacylated product at a defined %
conversion value of the polyol; and/or [0042] an identical relative
amount of the monoacylated product at a higher % degree of
conversion value; each of which being observed relative to a
reference method, said reference method being performed under
otherwise identical condition with the corresponding non-mutated
lipase enzyme.
[0043] The term "product distribution" describes the proportion of
the partial amount of a certain reaction product formed at a
certain point of time or "interval" during the course of the enzyme
catalyzed method described herein relative to the total amount of
all products formed by of said method, expressed in percent. Thus
the "product distribution" for a monoester of a certain polyol
defines the proportion (in percent) of said monoester relative to
the total amount of esters (mono- and polyesters), which have been
produced under the influence of an enzyme as defined herein at a
certain point of time or within a defined interval after initiation
of said enzymatic esterification reaction.
[0044] "Optically active" compounds are those having at least one
certer of asymmetry, i.p. at least one asymmetric carbon atom.
[0045] The term "about" indicates a potential variation of .+-.25%
of the stated value, in particular .+-.15%, .+-.10%, .+-.5%, .+-.2%
or .+-.1%.
[0046] The term "substantially" indicates a potential variation of
.+-.10% of the stated value, in particular .+-.5%, .+-.1%,
.+-.0.5%, .+-.0.2% or .+-.0.1% or less.
[0047] "Stereoselectivity" or "enantioselectivity" describes the
ability to produce an optically active compound in a
stereoisomerically or enantiomerically pure form or to specifically
convert a particular stereoisomer or An enzymatically catalyzed
method of preparing mono-acylated polyols enantiomer out of a
plurality of stereisomers or enantiomers. More specifically, this
means that a product of the invention is enriched with respect to a
specific stereoisomer or enantiomer. This may be quantified via the
enantiomeric purity % ee-parameter calculated according to the
formula:
% ee=[X.sub.A-X.sub.B]/[X.sub.A+X.sub.B]*100,
wherein X.sub.A and X.sub.B represent the molar ratio (Molenbruch)
of the enantiomers A and B.
[0048] Enantiomeric purities of at least 90% ee, like at least 95%
ee, or at lest 98% ee, or at east 99% ee or more may be
obtained.
[0049] The term "capable of forming a hydrogen bridge" refers to
the ability of an amino acid residue of an enzyme molecule of the
invention to form a hydrogen bridge with another molecule, as for
example with another amino acid residue of said enzyme or with a
substrate molecule or an intermediate state thereof located at or
within said enzyme molecule.
[0050] An condition "which stabilizes" an oxyanion transition state
means a condition which makes said oxyanion state energetically
more favorable if compared to the non-stabilized state.
Stabilization may, for example, be effected by a delocalization of
the negative charge of the anion.
[0051] The "substrate pocket" of an enzyme as defined herein, or
its "reactivity centre" harbours during the reaction to be
catalyzed a substrate molecule of and converts it to a product of
formula I. Said substrate pocket is composed of certain "structural
elements" i.e. portions of said substrate pocket with different
function. Said structural elements are in "functional arrangement"
to each other, i.e. they cooperate during the reaction to be
catalyzed.
[0052] A "sequence motif" or "pattern" represents a characteristic
arrangement or "fingerprint" of a plurality of amino acid residues
which are either adjacent to each other within a specific amino
acid sequence or are separated from each other in a defined manner,
i.e. by intermediate spacer sequences of characteristic length.
[0053] An "extended substrate specificity" refers to enzymes which,
if compared to a reference enzyme, convert additional structurally
different substrates.
[0054] An "altered/modified substrate specificity" refers to
enzymes which, if compared to a reference enzyme, convert a
partially or completely different set of substrate molecules. Thus,
the term "altered substrate specificity" may describes a situation
where a lipase enzyme, for example effected by mutation, is better
adapted for the acylation of a specific substrate molecule than a
reference enzyme (as for example the non-mutated enzyme). For
example, a higher preference or specificity may be caused by a
higher substrate affinity of the binding pocked formed by the
enzyme variants.
[0055] An "altered/modified regioselectivity" refers to enzymes
which, if compared to a reference enzyme, convert the one or more
potential reactive sites of a substrate molecule with altered
preference. Thus, the term "altered regioselectivity" may describes
a situation where a lipase enzyme, for example effected by
mutation, is better adapted for the mono-acylation of a specific
substrate molecule having more than one acylatable functional
groups than a reference enzyme (as for example the non-mutated
enzyme).
[0056] Due to the reversibility of enzymatic reactions the present
invention may also relate to the corresponding reverse (i.e.
deacylation) reaction of the biocatalytic acylation reactions
described herein.
2. Particular Embodiments of the Invention
[0057] The present invention provides the following particular
embodiments: [0058] 1. A biocatalytic, enzymatically catalyzed
method of preparing a mono-acylated polyol, in particular
mono-acylated diol, of the general formula (I):
[0058] ##STR00001## [0059] wherein [0060] R.sub.1 represents an
optionally substituted, linear or branched, saturated or
non-saturated hydrocarbyl residue; and [0061] A represents an
optionally substituted, linear or branched, non-cyclic,
hydrocarbylene residue, in particular having at least two carbon
atoms, and, in particular, wherein the oxygen atoms are linked to
different carbon atoms; [0062] which method comprises [0063] a)
reacting a polyol of the formula (II) and an acyl donor compound of
the formula (III)
[0063] ##STR00002## [0064] wherein R.sub.1 and A are as defined
above, in particular, wherein the hydroxyl groups are linked to
different carbon atoms, and [0065] Don represents a donor molecule
residue carrying the said acyl group, and is preferably selected
from --OR groups, wherein R represents an optionally substituted,
linear or branched, saturated or non-saturated, hydrocarbyl
residue; [0066] in the presence of a mutated triacylglycerole
lipase (EC 3.1.1.3) until a mono-acylated polyol of the above
formula (I) is formed; and [0067] b) obtaining a monoacylated
polyol product. [0068] 2. The method of embodiment 1, wherein said
mutated lipase contains at least one amino acid mutation, which
increases the selectivity of the lipase for the mono-acylation of
said polyol, if compared to the corresponding non-mutated lipase.
[0069] 3. The method of one of the preceding embodiments, wherein
said mutant comprises at least one mutation, which removes a
stabilizing functional amino acid group, in particular an amino
acid side chain capable of forming a hydrogen bridge, from that
part of the reactive center of the enzyme which stabilizes an
oxyanion transition state of the carbonyl group of the
mono-acylated polyol of formula (I) to be formed. [0070] 4. The
method of one of the preceding embodiments, wherein the enzyme is
mutated such that [0071] a) a maximum monoester yield is obtained
which is at last 1%, as for example 2 to 1000, 5 to 500, 10 to 200,
15 to 100, 10 to 80 or 15 to 50%, above the maximum yield as
obtained by the corresponding wild-type enzyme; [0072] b) a 3:1
molar ratio of monoester to polyester is reached at a conversion
rate of the polyol which is at last 1% as for example 2 to 1000, 5
to 500, to 200, 15 to 100, 10 to 80 or 15 to 50%, above the
corresponding conversion rate as obtained by the corresponding
wild-type enzyme; and/or [0073] c) the ratio of reaction times
(T.sub.90(mutant)/T.sub.90(wild-type)) to reach 90% (on molar
basis) monoacylated polyol based on the total amount of polyol is
above 1, as for example in the range of about 2 to 1000, 5 to 500,
10 to 200, 15 to 100, 10 to 80 or 15 to 50. [0074] 5. The method of
one of the preceding embodiments, wherein said enzyme is a mutant
of Candida antarctica lipase B (CALB) comprising an amino acid
sequence of SEQ ID NO: 2, mutated in at least one position, as for
example 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 positions. [0075] 6. The
method of embodiment 5, wherein said mutant comprises an amino acid
sequence of SEQ ID NO:2 wherein at least the amino acid Thr40 is
mutated. [0076] 7. The method of embodiment 6, wherein the mutation
is such that substantially no stabilizing interaction between the
oxyanion intermediate and the amino acid residue in position 40
occurs. [0077] 8. The method of embodiment 7 wherein the mutation
comprises the single mutations Thr40Ala, Thr40Val or Thr40Ser.
[0078] 9. The method of embodiment 8, wherein said mutant is
selected from mutants having an amino acid sequence of SEQ ID NO: 4
or variants of said mutant having a sequence identity of at least
60%, which variants still contain a mutation in an amino acid
position corresponding to position Thr40 of SEQ ID NO:2 or 4.
[0079] 10. The method of one of the embodiments 7 to 9, wherein the
mutant additionally comprises at least one mutation in one of the
amino acid positions Leu 278, Ile 285 and Pro 280 of SEQ ID NO: 2
or 4. [0080] 11. The method of embodiment 10, wherein the mutants
and the variants thereof are not mutated in other amino acid
positions contributing to the catalytic site of the enzyme. [0081]
12. The method of embodiment 11, wherein the mutants are not
mutated in amino acid positions Ser105, Asp187, His224 (catalytic
triade) and Gln106 and wherein the variants are not mutated in
amino acid positions corresponding thereto. [0082] 13. The method
of any one of embodiments 5 to 12, wherein in SEQ ID NO:2, or in
SEQ ID NO:2 comprising a mutation at amino acid Thr40 according to
SEQ ID NO:4, one or more of Leu278, Ala281, Ala282 or Ile285 are
mutated. [0083] 14. The method of embodiment 13, wherein the one or
more mutations are independently selected from: [0084] Leu 278Ser
for Leu278, [0085] Ala281Val or Ala281 Glu for Ala281, and [0086]
Ala282Leu, Ala282Thr, Ala282Cys, Ala282Pro, Ala28211e, Ala282Asp,
Ala282Val, Ala282Met or Ala282Arg for Ala282. [0087] 15. The method
of embodiment 14, wherein SEQ ID NO:2 comprises one mutation,
selected from Ala281Val, Ala281Glu, Ala282Leu, Ala282Thr,
Ala282Cys, Ala282Pro, Ala282Ile, Ala282Asp, Ala282Val, Ala282Met,
Ala282Arg and Ile285Phe, or wherein SEQ ID NO:2 comprises the
double mutation Leu278Ser and Ala282Leu. [0088] 16. The method of
one of the preceding embodiments, wherein the reaction is performed
in the presence of the isolated enzyme mutant or a recombinant
microorganism functionally expressing said mutant. [0089] 17. The
method of one of the preceding embodiments, wherein the polyol is a
compound of formula (II) wherein A is selected from the groups
[0089] (CH.sub.2).sub.n-- and
--(CH.sub.2).sub.m--CR.sub.2R.sub.3--(CH.sub.2).sub.m'-- [0090]
wherein [0091] n is an integer of 2-6, [0092] m and m'
independently of each other are integers of 1-3 [0093] R.sub.2 and
R.sub.3 independently of each other are selected from H, OH, SH,
NH.sub.2, optionally substituted carbo- or heterocyclic rings and
hydrocarbyl-residues, provided that R.sub.2 and R.sub.3 are not
simultaneously H. [0094] 18. The method of one of the preceding
embodiments, wherein the donor of formula (III) is selected from
compounds wherein R.sub.1 is C.sub.1-C.sub.6-alkyl and Don is an
--OR residue, wherein R is selected from C.sub.1-C.sub.6-alkyl and
C.sub.2-C.sub.4-alkenyl. [0095] 19. An biocatalytic, enzymatically
catalyzed, method of enantioselectively preparing an asymmetric
mono-acylated polyol of the general formula (I):
[0095] ##STR00003## [0096] wherein [0097] R.sub.1 represents an
optionally substituted, linear or branched, non-cyclic, saturated
or non-saturated hydrocarbyl residue; and [0098] A* represents an
optionally substituted, linear or branched, asymmetric
hydrocarbylene residue, having at least two carbon atoms, and, in
particular, wherein the oxygen atoms are linked to different carbon
atoms; which method comprises [0099] a) reacting a stereoisomeric
mixture of a polyol of the formula (II') and an acyl donor compound
of the formula (III)
[0099] ##STR00004## [0100] wherein R.sub.1 and A* are as defined
above, in particular, wherein the hydroxyl groups are linked to
different carbon atoms, and [0101] Don represents a donor molecule
residue carrying the said acyl group, and is preferably selected
from --OR groups, wherein R represents an optionally substituted,
linear or branched, saturated or non-saturated hydrocarbyl residue;
[0102] in the presence of a mutated triacylglycerol lipase (EC
3.1.1.3) until a mono-acylated polyol of the above formula (I) is
formed; and [0103] b) obtaining an asymmetric monoacylated polyol
product. [0104] 20. The method of embodiment 19 wherein an enzyme
mutant as defined in anyone of the embodiments 2 to 15 in the form
of an isolated enzyme mutant or a recombinant microorganism
functionally expressing said mutant is applied. [0105] 21. The
method of embodiment 19 or 20, wherein the polyol is a compound of
formula (II') wherein A* is selected from the groups
[0105] --(CH.sub.2).sub.m--CHR.sub.2--(CH.sub.2).sub.m'-- [0106]
wherein m, m' and R.sub.2 are as defined above. [0107] 22. The use
of a mutated triacylglycerol lipase (EC 3.1.1.3) in a method of
preparing a mono-acylated polyol of the general formula (I) or (I')
as defined above. [0108] 23. A Candida antarctica lipase B (CALB)
mutant showing a pattern of at least two mutations of the amino
acid sequence of SEQ ID NO:2 or 4, which pattern is selected from
the pattern as shown in Table A below. [0109] 24. The mutant of
embodiment 23 showing additionally one mutation selected from
Val210 Ile, Ala281Glu, Val221Asp in a sequence of SEQ ID NO:2 or 4.
[0110] 25. A Candida antarctica lipase B (CALB) mutant, having one
or more mutations in the amino acid sequence of SEQ ID NO:2, which
are independently selected from [0111] Leu278Ser for Leu278, [0112]
Ala281Val or Ala281 Glu for Ala281, and [0113] Ala282Leu,
Ala282Thr, Ala282Cys, Ala282Pro, Ala282Ile, Ala282Asp, Ala282Val,
Ala282Met or Ala282Arg for Ala282. [0114] 26. The mutant of
embodiment 25, having one mutation in SEQ ID NO:2, selected from
Ala281Val, Ala281Glu, Ala282Leu, Ala282Thr, Ala282Cys, Ala282Pro,
Ala282Ile, Ala282Asp, Ala282Val, Ala282Met, Ala282Arg and
Ile285Phe, or having in SEQ ID NO:2 the double mutation Leu278Ser
and Ala282Leu. [0115] 27. The mutant of embodiment 23 or 24,
additionally having at least one of the mutations as defined in any
one of embodiments 25 or 26. [0116] 28. A nucleic acid molecule
encoding a mutant of one of the embodiments 20 to 27. [0117] 29. An
expression vector, comprising, optionally under the control of a
regulatory nucleic acid sequence, at least one coding sequence of
embodiment 28. [0118] 24. A microbial host carrying at least one
expression vector of embodiment 23 or coding sequence of embodiment
22.
TABLE-US-00001 [0118] TABLE A Specific Mutation pattern: Thr40
Leu278 Ile285 Pro280 2-fold mutants Ala Phe Ala Trp Ala Ala Ala Ser
Ala Asn Ala Leu Ala Ser Ala Phe Ala Gln Ala Ala Val Phe Val Trp Val
Ala Val Ser Val Asn Val Leu Val Ser Val Phe Val Gln Val Ala Ser Trp
Ser Ala Ser Ser Ser Asn Ser Leu Ser Ser Ser Phe Ser Gln Ser Ala
3-fold mutants Ala Phe Leu Ala Phe Ala Phe Phe Ala Phe Gln Ala Phe
Ala Ala Trp Leu Ala Trp Ser Ala Trp Phe Ala Trp Gln Ala Trp Ala Ala
Ala Leu Ala Ala Ser Ala Ala Phe Ala Ala Gln Ala Ala Ala Ala Ser Leu
Ala Ser Ser Ala Ser Phe Ala Ser Gln Ala Ser Ala Ala Asn Leu Ala Asn
Ser Ala Asn Phe Ala Asn Gln Ala Asn Ala Val Phe Leu Val Phe Ser Val
Phe Phe Val Phe Gln Val Phe Ala Val Trp Leu Val Trp Ser Val Trp Phe
Val Trp Gln Val Trp Ala Val Ala Leu Val Ala Ser Val Ala Phe Val Ala
Gln Val Ala Ala Val Ser Leu Val Ser Ser Val Ser Phe Val Ser Gln Val
Ser Ala Val Asn Leu Val Asn Ser Val Asn Phe Val Asn Gln Val Asn Ala
Ser Phe Leu Ser Phe Ser Ser Phe Phe Ser Phe Gln Ser Phe Ala Ser Trp
Leu Ser Trp Ser Ser Trp Phe Ser Trp Gln Ser Trp Ala Ser Ala Leu Ser
Ala Ser Ser Ala Phe Ser Ala Gln Ser Ala Ala Ser Ser Leu Ser Ser Ser
Ser Ser Phe Ser Ser Gln Ser Ser Ala Ser Asn Leu Ser Asn Ser Ser Asn
Phe Ser Asn Gln Ser Asn Ala 4-fold mutants Ala Phe Leu Ala Ala Phe
Ser Ala Ala Phe Phe Ala Ala Phe Gln Ala Ala Trp Leu Ala Ala Trp Ser
Ala Ala Trp Phe Ala Ala Trp Gln Ala Ala Ala Leu Ala Ala Ala Ser Ala
Ala Ala Phe Ala Ala Ala Gln Ala Ala Ser Leu Ala Ala Ser Ser Ala Ala
Ser Phe Ala Ala Ser Gln Ala Ala Asn Leu Ala Ala Asn Ser Ala Ala Asn
Phe Ala Ala Asn Gln Ala Val Phe Leu Ala Val Phe Ser Ala Val Phe Phe
Ala Val Phe Gln Ala Val Trp Leu Ala Val Trp Ser Ala Val Trp Phe Ala
Val Trp Gln Ala Val Ala Leu Ala Val Ala Ser Ala Val Ala Phe Ala Val
Ala Gln Ala Val Ser Leu Ala Val Ser Ser Ala Val Ser Phe Ala Val Ser
Gln Ala Val Asn Leu Ala Val Asn Ser Ala Val Asn Phe Ala Val Asn Gln
Ala Ser Phe Leu Ala Ser Phe Ser Ala Ser Phe Phe Ala Ser Phe Gln Ala
Ser Trp Leu Ala Ser Trp Ser Ala Ser Trp Phe Ala Ser Trp Gln Ala Ser
Ala Leu Ala Ser Ala Ser Ala Ser Ala Phe Ala Ser Ala Gln Ala Ser Ser
Leu Ala Ser Ser Ser Ala Ser Ser Phe Ala Ser Ser Gln Ala Ser Asn Leu
Ala Ser Asn Ser Ala Ser Asn Phe Ala Ser Asn Gln Ala
3. Substrates
a) Polyols
[0119] Polyols to be acylated according to the present invention
arte generally of the general Formula II
HO-A-OH (II)
wherein A represents an optionally substituted, linear or branched,
non-cyclic, hydrocarbylene residue, having at least two carbon
atoms, and wherein the hydroxyl groups are linked to different
carbon atoms;
[0120] "Non-cyclic" in this context means that the HO-Groups are
not linked to a carbo- or heterocyclic mono- or polynuclear
ring.
[0121] A is in particular formed by a hydrocarbylene-backbone B
carrying both HO-groups according to the following general
formula
HO--CR.sub.4R.sub.4-B-CR.sub.4R.sub.4--OH
wherein the R.sub.4 residues independently of each other may be
selected from H and linear or branched, in particular linear,
C.sub.1-C.sub.6-alkyl, and B represents a group of the formula
--(CR.sub.5R.sub.5).sub.z-- wherein z is an integer of 0, 1, 2, 3,
4, 5 or 6 and the R.sub.5 residues independently of each other are
selected from H, linear or branched, in particular linear,
C.sub.1-C.sub.6-alkyl, optionally substituted
C.sub.3-C.sub.7-cycloalkyl and optionally substituted aryl or
heteroaryl.
[0122] In another embodiment the hydroxyl groups of the diol to be
acylated independently of each other may be tertiary, secondary or
primary hydroxyl groups.
[0123] Particular examples of A groups are selected from the
following groups, adapted to carrying two primary hydroxyl
groups:
--(CH.sub.2).sub.n-- and
--(CH.sub.2).sub.m--CR.sub.2R.sub.3--(CH.sub.2).sub.m'--
wherein n is an integer of 1, 2, 3, 4, 5, or 6, m and m'
independently of each other are integers of 1, 2 or 3 R.sub.2 and
R.sub.3 independently of each other are selected from H, OH, SH,
NH.sub.2, carbo- or heterocyclic ring, in particular, optionally
substituted C.sub.3-C.sub.7-cycloalkyl, aryl or heterorayl, and,
linear or branched, in particular linear, C.sub.1-C.sub.6-alkyl,
provided that R.sub.2 and R.sub.3 are not simultaneously H.
b) Acyl Donors
[0124] The "acyl donor" molecule has the ability to provide an acyl
group for the biocatalytic acylation reaction of the invention.
[0125] Generally the donor is of the above formula (III)
wherein
R.sub.1 represents an optionally substituted, linear or branched,
saturated or non-saturated hydrocarbyl residue, in particular
C.sub.1-C.sub.30-hydrocarbyl; and Don represents a donor molecule
residue carrying the said acyl group, and is preferably selected
from --OR groups wherein R represents an optionally substituted,
linear or branched, saturated or non-saturated, hydrocarbyl
residue, in particular C.sub.1-C.sub.6-alkyl or
C.sub.2-C.sub.4-alkenyl, like methyl, ethyl or vinyl.
c) Definitions
[0126] A "hydrocarbylene" residue of the present invention
particularly comprises a linear --(CH.sub.2).sub.n-- backbone with
n=1, 2, 3, 4, 5, 6, 7, or 8, which may be further substituted as
further defined herein.
[0127] A linear or branched, saturated or non-saturated,
"hydrocarbyl" residue according to the preset invention
particularly refers to linear or branched, alkyl or alkenyl
residues.
[0128] An "alkyl" residue comprises C.sub.1-C.sub.8-alkyl radicals
which are linear or branched radicals having from 1 to 8 carbon
atoms. Examples thereof are: [0129] C.sub.1-C.sub.4-alkyl radicals
selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, 2-butyl,
isobutyl or tert-butyl, [0130] C.sub.1-C.sub.6-alkyl radicals
selected from C.sub.1-C.sub.4-alkyl radicals as defined above and
additionally pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl,
2,2-dimethylpropyl, 1-ethylpropyl, hexyl, 1,1-dimethylpropyl,
1,2-dimethylpropyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl,
4-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl,
1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl,
3,3-dimethylbutyl, 1-ethylbutyl, 2-ethylbutyl,
1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl,
1-ethyl-1-methylpropyl, 1-ethyl-2-methylpropyl, [0131]
C.sub.1-C.sub.8-alkyl radicals selected from C.sub.1-C.sub.6-alkyl
radicals as defined above and additionally heptyl, octyl and their
constitutional isomers such as 2-ethylhexyl; and [0132]
C.sub.8-C.sub.30-alkyl radicals which are linear or branched
radicals having from 8 to 30 carbon atoms; examples thereof being
selected from octyl, nonyl, decyl, undecyl, dodecyl, tridecyl,
tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl,
nonadecyl, eicosyl, hencosyl, docosyl, tricosyl, tetracosyl,
pentacosyl, hexacosyl, heptacosyl, octacosyl, nonacosyl, squalyl,
their constitutional isomers, higher homologs and constitutional
isomers thereof.
[0133] An "alkenyl" residue comprises C.sub.2-C.sub.30-alkenyl
radicals which are monounsaturated linear or branched hydrocarbon
radicals having from 2 to 30 carbon atoms.
[0134] Particular examples are: [0135] C.sub.2-C.sub.4-alkenyl
radicals, like ethenyl or vinyl, 1- or 2-propenyl, 1-, 2- and
3-butenyl, [0136] C.sub.2-C.sub.8-alkenyl radicals comprising
C.sub.2-C.sub.4-alkenyl radicals as defined above as well as
2-methylpropen-3-yl, 2-methylpropen-1-yl, 1-, 2-, 3- and
4-pentenyl, 1-, 2-, 3-, 4- and 5-hexenyl, 1-, 2-, 3-, 4-, 5- and
6-heptenyl 1-, 2-, 3-, 4-, 5-, 6- and 7-octenyl and also their
constitutional isomers; as well as [0137] C.sub.8-C.sub.30-alkenyl
residues which are monounsaturated linear or branched hydrocarbon
radicals having from 8 to 30 carbon atoms. Examples thereof are
octenyl, nonenyl, decenyl, undecenyl, dodecenyl, tridecenyl,
tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl,
nonadecenyl, eicosenyl, hencosenyl, docosenyl, tricosenyl,
tetracosenyl, pentacosenyl, hexacosenyl, heptacosenyl, octacosenyl,
nonacosenyl, squalenyl, their constitutional isomers, higher
homologs and constitutional isomers thereof.
[0138] "Carbo- and heterocyclic" residues comprise, optionally
condensed, aromatic or non-aromatic ring groups, having 3 to 12
carbon atoms and optionally 1, 2, 3 or 4 same or different
ring-heteroatoms, like N, S and O.
[0139] As examples there may be mentioned: [0140] aromatic
carbocyclic rings like phenyl and naphthyl. [0141] non-aromatic
carbocyclic rings, in particular C.sub.3-C.sub.7-cycloalkyl
residues like cyclopropyl, cyclopropyl-methyl, cyclopropyl-ethyl,
cyclobutyl, cyclobutyl-methyl, cyclopenty, cyclopentyl-methyl,
cyclohexyl, cyclohexyl-methyl, Cycloheptyl, as well as the one- or
two-fold unsaturated analogues thereof, like for example
cyclobutenyl, cyclopentenyl, cyclohexenyl, cycloheptenyl,
cyclohexadienyl, cycloheptadienyl; [0142] 5- to 7-membered
saturated or unsaturated, aromatic or nonaromatic heterocyclic
residues having 1 to 4 ring-heteroatoms, selected from O, N and S,
wherein said heterocyclic group may be condensed with a further
hetero or carbocyclic residue. As examples there may be mentioned
residues of pyrrolidine, tetrahydrofurane, piperidine, morpholine,
pyrrole, furan, thiophene, pyrazole, imidazole, oxazole, thiazole,
pyridine, pyran, pyrimidine, pyridazine, pyrazine, cumarone, indole
and quinoline.
[0143] Non-limiting examples of "optionally substituted" residues
as defined herein comprise 1, 2, 3, 4, 5 or 6 identical or
different substituents like, HO, SH, NH.sub.2, NO.sub.2, halogen,
like F, Cl, Br, J; lower alkyl, lower alkoxy, lower alkylthio,
lower alkyl, lower alkenyl, lower alkynyl or hydroxyl-lower alkyl,
as defined above.
[0144] "Lower alkyl" refers to C.sub.1-C.sub.8-alkyl radicals as
defined above.
[0145] "Lower alkoxy" preferably refers to the
C.sub.1-C.sub.8-alkoxy analogues of the above-mentioned lower alkyl
radicals.
[0146] "Lower alkylthio" preferably refers to the
C.sub.1-C.sub.8-alkthio analogues of the above-mentioned lower
alkyl radicals. Examples are methylthio, ethylthio, propylthio,
isopropylthio, butylthio, sec-butylthio, isobutylthio and
tert-butylthio.
[0147] "Lower alkenyl" comprises C.sub.2-C.sub.8-alkenyl radicals
as defined above.
[0148] "Lower alkynyl" comprises the alkynyl homologues of the
above "lower alkeny" radicals.
[0149] The term "hydroxy lower-alkyl" refers to
C.sub.1-C.sub.8-hydroxyalkyl which is a linear or branched alkyl
radical having from 1 to 8, in particular from 1 to 4 carbon atoms,
in which at least one hydrogen atom, for example 1 or 2 of the
hydrogen atoms, is/are replaced by a hydroxyl group. Examples
thereof are, hydroxymethyl, 2-hydroxy-1-ethyl, 2- and
3-hydroxy-1-propyl, 2-, 3- and 4-hydroxy-1-butyl, 2-, 3-, 4- and
5-hydroxy-1-pentyl, 2-, 3-, 4-, 5- and 6-hydroxy-1-hexyl, 2-, 3-,
4-, 5-, 6- and 7-hydroxy-1-heptyl, 2-, 3-, 4-, 5-, 6-, 7- and
8-hydroxy-1-octyl, 2,3-dihydroxy-1-propyl and their constitutional
isomers.
[0150] The above definitions provided under a) b) and c)
simultaneously apply to the compounds of formulae (I'), (II') and
(III').
4. Enzymes and Enzyme Mutants According to the Invention
[0151] The present invention is not limited to the use of the
specifically disclosed triacylglycerole lipases and mutants, but
also extends to functional equivalents thereof.
[0152] "Functional equivalents" or analogs of the concretely
disclosed enzymes are, within the scope of the present invention,
various polypeptides thereof, which moreover possess the desired
biological function or activity, e.g. enzyme activity.
[0153] For example, "functional equivalents" means enzymes, which,
in a test used for enzymatic activity, display at least a 1 to 10%,
or at least 20%, or at least 50%, or at least 75%, or at least 90%
higher or lower activity of an enzyme, as defined herein.
[0154] "Functional equivalents", according to the invention, also
means in particular mutants, which, in at least one sequence
position of the amino acid sequences stated above, have an amino
acid that is different from that concretely stated, but
nevertheless possess one of the aforementioned biological
activities. "Functional equivalents" thus comprise the mutants
obtainable by one or more, as for example 1, 2, 3, 4, 5, 6, 7, 8,
9, 10, 11, 12, 13, 14, or 15, amino acid additions, substitutions,
insertions, deletions and/or inversions, where the stated changes
can occur in any sequence position, provided they lead to a mutant
with the profile of properties according to the invention.
Functional equivalence is in particular also provided if the
reactivity patterns coincide qualitatively between the mutant and
the unchanged polypeptide, i.e. if for example the same substrates
are converted at a different rate. Examples of suitable amino acid
substitutions are shown in the following table:
TABLE-US-00002 Original residue Examples of substitution Ala Ser
Arg Lys Asn Gln; His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His
Asn; Gln Ile Leu; Val Leu Ile; Val Lys Arg; Gln; Glu Met Leu; Ile
Phe Met; Leu; Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile;
Leu
[0155] "Functional equivalents" in the above sense are also
"precursors" of the polypeptides described, as well as "functional
derivatives" and "salts" of the polypeptides.
[0156] "Precursors" are in that case natural or synthetic
precursors of the polypeptides with or without the desired
biological activity.
[0157] The expression "salts" means salts of carboxyl groups as
well as salts of acid addition of amino groups of the protein
molecules according to the invention. Salts of carboxyl groups can
be produced in a known way and comprise inorganic salts, for
example sodium, calcium, ammonium, iron and zinc salts, and salts
with organic bases, for example amines, such as triethanolamine,
arginine, lysine, piperidine and the like. Salts of acid addition,
for example salts with inorganic acids, such as hydrochloric acid
or sulfuric acid and salts with organic acids, such as acetic acid
and oxalic acid, are also covered by the invention.
[0158] "Functional derivatives" of polypeptides according to the
invention can also be produced on functional amino acid side groups
or at their N-terminal or C-terminal end using known techniques.
Such derivatives comprise for example aliphatic esters of
carboxylic acid groups, amides of carboxylic acid groups,
obtainable by reaction with ammonia or with a primary or secondary
amine; N-acyl derivatives of free amino groups, produced by
reaction with acyl groups; or O-acyl derivatives of free hydroxy
groups, produced by reaction with acyl groups.
[0159] "Functional equivalents" naturally also comprise
polypeptides that can be obtained from other organisms, as well as
naturally occurring variants. For example, areas of homologous
sequence regions can be established by sequence comparison, and
equivalent enzymes can be determined on the basis of the concrete
parameters of the invention.
[0160] "Functional equivalents" also comprise fragments, preferably
individual domains or sequence motifs, of the polypeptides
according to the invention, which for example display the desired
biological function.
[0161] "Functional equivalents" are, moreover, fusion proteins,
which have one of the polypeptide sequences stated above or
functional equivalents derived there from and at least one further,
functionally different, heterologous sequence in functional
N-terminal or C-terminal association (i.e. without substantial
mutual functional impairment of the fusion protein parts).
Non-limiting examples of these heterologous sequences are e.g.
signal peptides, histidine anchors or enzymes.
[0162] "Functional equivalents" that are also included according to
the invention are homologues of the concretely disclosed proteins.
These possess percent identity values as stated above. Said values
refer to the identity with the concretely disclosed amino acid
sequences, and may be calculated according to the algorithm of
Pearson and Lipman, Proc. Natl. Acad, Sci. (USA) 85(8), 1988,
2444-2448.
[0163] The % identity values may also be calculated from BLAST
alignments, algorithm blastp (protein-protein BLAST) or by applying
the Clustal setting as given below.
[0164] A percentage identity of a homologous polypeptide according
to the invention means in particular the percentage identity of the
amino acid residues relative to the total length of one of the
amino acid sequences concretely described herein.
[0165] In the case of a possible protein glycosylation, "functional
equivalents" according to the invention comprise proteins of the
type designated above in deglycosylated or glycosylated form as
well as modified forms that can be obtained by altering the
glycosylation pattern.
[0166] Such functional equivalents or homologues of the proteins or
polypeptides according to the invention can be produced by
mutagenesis, e.g. by point mutation, lengthening or shortening of
the protein.
[0167] Such functional equivalents or homologues of the proteins
according to the invention can be identified by screening
combinatorial databases of mutants, for example shortening mutants.
For example, a variegated database of protein variants can be
produced by combinatorial mutagenesis at the nucleic acid level,
e.g. by enzymatic ligation of a mixture of synthetic
oligonucleotides. There are a great many methods that can be used
for the production of databases of potential homologues from a
degenerated oligonucleotide sequence. Chemical synthesis of a
degenerated gene sequence can be carried out in an automatic DNA
synthesizer, and the synthetic gene can then be ligated in a
suitable expression vector. The use of a degenerated genome makes
it possible to supply all sequences in a mixture, which code for
the desired set of potential protein sequences. Methods of
synthesis of degenerated oligonucleotides are known to a person
skilled in the art (e.g. Narang, S. A. (1983) Tetrahedron 39:3;
Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al.,
(1984) Science 198:1056; Ike et al. (1983) Nucleic Acids Res.
11:477).
[0168] In the prior art, several techniques are known for the
screening of gene products of combinatorial databases, which were
produced by point mutations or shortening, and for the screening of
cDNA libraries for gene products with a selected property. These
techniques can be adapted for the rapid screening of the gene banks
that were produced by combinatorial mutagenesis of homologues
according to the invention. The techniques most frequently used for
the screening of large gene banks, which are based on a
high-throughput analysis, comprise cloning of the gene bank in
expression vectors that can be replicated, transformation of the
suitable cells with the resultant vector database and expression of
the combinatorial genes in conditions in which detection of the
desired activity facilitates isolation of the vector that codes for
the gene whose product was detected. Recursive Ensemble Mutagenesis
(REM), a technique that increases the frequency of functional
mutants in the databases, can be used in combination with the
screening tests, in order to identify homologues (Arkin and Yourvan
(1992) PNAS 89:7811-7815; Delgrave et al. (1993) Protein
Engineering 6(3):327-331).
5. Coding Nucleic Acid Sequences
[0169] The invention also relates to nucleic acid sequences that
code for enzymes and mutants as defined herein.
[0170] The present invention also relates to nucleic acids with a
certain degree of "identity" to the sequences specifically
disclosed herein. "Identity" between two nucleic acids means
identity of the nucleotides, in each case over the entire length of
the nucleic acid.
[0171] For example the identity may be calculated by means of the
Vector NTI Suite 7.1 program of the company Informax (USA)
employing the Clustal Method (Higgins D G, Sharp P M. Fast and
sensitive multiple sequence alignments on a microcomputer. Comput
Appl. Biosci. 1989 April; 5(2):151-1) with the following
settings:
[0172] Multiple Alignment Parameter:
TABLE-US-00003 Gap opening penalty 10 Gap extension penalty 10 Gap
separation penalty range 8 Gap separation penalty off % identity
for alignment delay 40 Residue specific gaps off Hydrophilic
residue gap off Transition weighing 0
[0173] Pairwise Alignment Parameter:
TABLE-US-00004 FAST algorithm on K-tuple size 1 Gap penalty 3
Window size 5 Number of best diagonals 5
[0174] Alternatively the identity may be determined according to
Chema, Ramu, Sugawara, Hideaki, Koike, Tadashi, Lopez, Rodrigo,
Gibson, Toby J, Higgins, Desmond G, Thompson, Julie D. Multiple
sequence alignment with the Clustal series of programs. (2003)
Nucleic Acids Res 31 (13):3497-500, the web page:
ebi.ac.uk/Tools/clustalw/index.html# and the following settings
TABLE-US-00005 DNA Gap Open Penalty 15.0 DNA Gap Extension Penalty
6.66 DNA Matrix Identity Protein Gap Open Penalty 10.0 Protein Gap
Extension Penalty 0.2 Protein matrix Gonnet Protein/DNA ENDGAP -1
Protein/DNA GAPDIST 4
[0175] All the nucleic acid sequences mentioned herein
(single-stranded and double-stranded DNA and RNA sequences, for
example cDNA and mRNA) can be produced in a known way by chemical
synthesis from the nucleotide building blocks, e.g. by fragment
condensation of individual overlapping, complementary nucleic acid
building blocks of the double helix. Chemical synthesis of
oligonucleotides can, for example, be performed in a known way, by
the phosphoamidite method (Voet, Voet, 2nd edition, Wiley Press,
New York, pages 896-897). The accumulation of synthetic
oligonucleotides and filling of gaps by means of the Klenow
fragment of DNA polymerase and ligation reactions as well as
general cloning techniques are described in Sambrook et al. (1989),
see below.
[0176] The invention also relates to nucleic acid sequences
(single-stranded and double-stranded DNA and RNA sequences, e.g.
cDNA and mRNA), coding for one of the above polypeptides and their
functional equivalents, which can be obtained for example using
artificial nucleotide analogs.
[0177] The invention relates both to isolated nucleic acid
molecules, which code for polypeptides or proteins according to the
invention or biologically active segments thereof, and to nucleic
acid fragments, which can be used for example as hybridization
probes or primers for identifying or amplifying coding nucleic
acids according to the invention.
[0178] The nucleic acid molecules according to the invention can in
addition contain non-translated sequences from the 3' and/or 5' end
of the coding genetic region.
[0179] The invention further relates to the nucleic acid molecules
that are complementary to the concretely described nucleotide
sequences or a segment thereof.
[0180] The nucleotide sequences according to the invention make
possible the production of probes and primers that can be used for
the identification and/or cloning of homologous sequences in other
cellular types and organisms. Such probes or primers generally
comprise a nucleotide sequence region which hybridizes under
"stringent" conditions (see below) on at least about 12, preferably
at least about 25, for example about 40, 50 or 75 successive
nucleotides of a sense strand of a nucleic acid sequence according
to the invention or of a corresponding antisense strand.
[0181] An "isolated" nucleic acid molecule is separated from other
nucleic acid molecules that are present in the natural source of
the nucleic acid and can moreover be substantially free from other
cellular material or culture medium, if it is being produced by
recombinant techniques, or can be free from chemical precursors or
other chemicals, if it is being synthesized chemically.
[0182] A nucleic acid molecule according to the invention can be
isolated by means of standard techniques of molecular biology and
the sequence information supplied according to the invention. For
example, cDNA can be isolated from a suitable cDNA library, using
one of the concretely disclosed complete sequences or a segment
thereof as hybridization probe and standard hybridization
techniques (as described for example in Sambrook, J., Fritsch, E.
F. and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd
edition, Cold Spring Harbor Laboratory, Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., 1989). In addition, a
nucleic acid molecule comprising one of the disclosed sequences or
a segment thereof, can be isolated by the polymerase chain
reaction, using the oligonucleotide primers that were constructed
on the basis of this sequence. The nucleic acid amplified in this
way can be cloned in a suitable vector and can be characterized by
DNA sequencing. The oligonucleotides according to the invention can
also be produced by standard methods of synthesis, e.g. using an
automatic DNA synthesizer.
[0183] Nucleic acid sequences according to the invention or
derivatives thereof, homologues or parts of these sequences, can
for example be isolated by usual hybridization techniques or the
PCR technique from other bacteria, e.g. via genomic or cDNA
libraries. These DNA sequences hybridize in standard conditions
with the sequences according to the invention.
[0184] "Hybridize" means the ability of a polynucleotide or
oligonucleotide to bind to an almost complementary sequence in
standard conditions, whereas nonspecific binding does not occur
between non-complementary partners in these conditions. For this,
the sequences can be 90-100% complementary. The property of
complementary sequences of being able to bind specifically to one
another is utilized for example in Northern Blotting or Southern
Blotting or in primer binding in PCR or RT-PCR.
[0185] Short oligonucleotides of the conserved regions are used
advantageously for hybridization. However, it is also possible to
use longer fragments of the nucleic acids according to the
invention or the complete sequences for the hybridization. These
standard conditions vary depending on the nucleic acid used
(oligonucleotide, longer fragment or complete sequence) or
depending on which type of nucleic acid--DNA or RNA--is used for
hybridization. For example, the melting temperatures for DNA:DNA
hybrids are approx. 10.degree. C. lower than those of DNA:RNA
hybrids of the same length.
[0186] For example, depending on the particular nucleic acid,
standard conditions mean temperatures between 42 and 58.degree. C.
in an aqueous buffer solution with a concentration between 0.1 to
5.times.SSC (1.times.SSC=0.15 M NaCl, 15 mM sodium citrate, pH 7.2)
or additionally in the presence of 50% formamide, for example
42.degree. C. in 5.times.SSC, 50% formamide. Advantageously, the
hybridization conditions for DNA:DNA hybrids are 0.1.times.SSC and
temperatures between about 20.degree. C. to 45.degree. C.,
preferably between about 30.degree. C. to 45.degree. C. For DNA:RNA
hybrids the hybridization conditions are advantageously
0.1.times.SSC and temperatures between about 30.degree. C. to
55.degree. C., preferably between about 45.degree. C. to 55.degree.
C. These stated temperatures for hybridization are examples of
calculated melting temperature values for a nucleic acid with a
length of approx. 100 nucleotides and a G+C content of 50% in the
absence of formamide. The experimental conditions for DNA
hybridization are described in relevant genetics textbooks, for
example Sambrook et al., 1989, and can be calculated using formulae
that are known by a person skilled in the art, for example
depending on the length of the nucleic acids, the type of hybrids
or the G+C content. A person skilled in the art can obtain further
information on hybridization from the following textbooks: Ausubel
et al. (eds), 1985, Current Protocols in Molecular Biology, John
Wiley & Sons, New York; Hames and Higgins (eds), 1985, Nucleic
Acids Hybridization: A Practical Approach, IRL Press at Oxford
University Press, Oxford; Brown (ed), 1991, Essential Molecular
Biology: A Practical Approach, IRL Press at Oxford University
Press, Oxford.
[0187] "Hybridization" can in particular be carried out under
stringent conditions. Such hybridization conditions are for example
described in Sambrook, J., Fritsch, E. F., Maniatis, T., in:
Molecular Cloning (A Laboratory Manual), 2nd edition, Cold Spring
Harbor Laboratory Press, 1989, pages 9.31-9.57 or in Current
Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),
6.3.1-6.3.6.
[0188] "Stringent" hybridization conditions mean in particular:
Incubation at 42.degree. C. overnight in a solution consisting of
50% formamide, 5.times.SSC (750 mM NaCl, 75 mM tri-sodium citrate),
50 mM sodium phosphate (pH 7.6), 5.times.Denhardt Solution, 10%
dextran sulfate and 20 g/ml denatured, sheared salmon sperm DNA,
followed by washing of the filters with 0.1.times.SSC at 65.degree.
C.
[0189] The invention also relates to derivatives of the concretely
disclosed or derivable nucleic acid sequences.
[0190] Thus, further nucleic acid sequences according to the
invention can be derived from the sequences specifically disclosed
herein and can differ from it by addition, substitution, insertion
or deletion of individual or several nucleotides, and furthermore
code for polypeptides with the desired profile of properties.
[0191] The invention also encompasses nucleic acid sequences that
comprise so-called silent mutations or have been altered, in
comparison with a concretely stated sequence, according to the
codon usage of a special original or host organism, as well as
naturally occurring variants, e.g. splicing variants or allelic
variants, thereof.
[0192] It also relates to sequences that can be obtained by
conservative nucleotide substitutions (i.e. the amino acid in
question is replaced by an amino acid of the same charge, size,
polarity and/or solubility).
[0193] The invention also relates to the molecules derived from the
concretely disclosed nucleic acids by sequence polymorphisms. These
genetic polymorphisms can exist between individuals within a
population owing to natural variation. These natural variations
usually produce a variance of 1 to 5% in the nucleotide sequence of
a gene.
[0194] Derivatives of nucleic acid sequences according to the
invention mean for example allelic variants, having at least 60%
homology at the level of the derived amino acid, preferably at
least 80% homology, quite especially preferably at least 90%
homology over the entire sequence range (regarding homology at the
amino acid level, reference should be made to the details given
above for the polypeptides). Advantageously, the homologies can be
higher over partial regions of the sequences.
[0195] Furthermore, derivatives are also to be understood to be
homologues of the nucleic acid sequences according to the
invention, for example animal, plant, fungal or bacterial
homologues, shortened sequences, single-stranded DNA or RNA of the
coding and noncoding DNA sequence. For example, homologues have, at
the DNA level, a homology of at least 40%, preferably of at least
60%, especially preferably of at least 70%, quite especially
preferably of at least 80% over the entire DNA region given in a
sequence specifically disclosed herein.
[0196] Moreover, derivatives are to be understood to be, for
example, fusions with promoters. The promoters that are added to
the stated nucleotide sequences can be modified by at least one
nucleotide exchange, at least one insertion, inversion and/or
deletion, though without impairing the functionality or efficacy of
the promoters. Moreover, the efficacy of the promoters can be
increased by altering their sequence or can be exchanged completely
with more effective promoters even of organisms of a different
genus.
6. Constructs According to the Invention
[0197] The invention also relates to expression constructs,
containing, under the genetic control of regulatory nucleic acid
sequences, a nucleic acid sequence coding for a polypeptide or
fusion protein according to the invention; as well as vectors
comprising at least one of these expression constructs.
[0198] "Expression unit" means, according to the invention, a
nucleic acid with expression activity, which comprises a promoter
as defined herein and, after functional association with a nucleic
acid that is to be expressed or a gene, regulates the expression,
i.e. the transcription and the translation of this nucleic acid or
of this gene. In this context, therefore, it is also called a
"regulatory nucleic acid sequence". In addition to the promoter,
other regulatory elements may be present, e.g. enhancers.
[0199] "Expression cassette" or "expression construct" means,
according to the invention, an expression unit, which is
functionally associated with the nucleic acid that is to be
expressed or the gene that is to be expressed. In contrast to an
expression unit, an expression cassette thus comprises not only
nucleic acid sequences, which regulate transcription and
translation, but also the nucleic acid sequences, which should be
expressed as protein as a result of the transcription and
translation.
[0200] The terms "expression" or "overexpression" describe, in the
context of the invention, the production or increase of
intracellular activity of one or more enzymes in a microorganism,
which are encoded by the corresponding DNA. For this, it is
possible for example to insert a gene in an organism, replace an
existing gene by another gene, increase the number of copies of the
gene or genes, use a strong promoter or use a gene that codes for a
corresponding enzyme with a high activity, and optionally these
measures can be combined.
[0201] Preferably such constructs according to the invention
comprise a promoter 5'-upstream from the respective coding
sequence, and a terminator sequence 3'-downstream, and optionally
further usual regulatory elements, in each case functionally
associated with the coding sequence.
[0202] A "promoter", a "nucleic acid with promoter activity" or a
"promoter sequence" mean, according to the invention, a nucleic
acid that, functionally associated with a nucleic acid that is to
be transcribed, regulates the transcription of this nucleic
acid.
[0203] "Functional" or "operative" association means, in this
context, for example the sequential arrangement of one of the
nucleic acids with promoter activity and of a nucleic acid sequence
that is to be transcribed and optionally further regulatory
elements, for example nucleic acid sequences that enable the
transcription of nucleic acids, and for example a terminator, in
such a way that each of the regulatory elements can fulfill its
function in the transcription of the nucleic acid sequence. This
does not necessarily require a direct association in the chemical
sense. Genetic control sequences, such as enhancer sequences, can
also exert their function on the target sequence from more remote
positions or even from other DNA molecules. Arrangements are
preferred in which the nucleic acid sequence that is to be
transcribed is positioned behind (i.e. at the 3' end) the promoter
sequence, so that the two sequences are bound covalently to one
another. The distance between the promoter sequence and the nucleic
acid sequence that is to be expressed transgenically can be less
than 200 bp (base pairs), or less than 100 bp or less than 50
bp.
[0204] Apart from promoters and terminators, examples of other
regulatory elements that may be mentioned are targeting sequences,
enhancers, polyadenylation signals, selectable markers,
amplification signals, replication origins and the like. Suitable
regulatory sequences are described for example in Goeddel, Gene
Expression Technology: Methods in Enzymology 185, Academic Press,
San Diego, Calif. (1990).
[0205] Nucleic acid constructs according to the invention comprise
in particular sequences selected from those, specifically mentioned
herein or derivatives and homologues thereof, as well as the
nucleic acid sequences that can be derived from amino acid
sequences specifically mentioned herein which are advantageously
associated operatively or functionally with one or more regulating
signal for controlling, e.g. increasing, gene expression.
[0206] In addition to these regulatory sequences, the natural
regulation of these sequences can still be present in front of the
actual structural genes and optionally can have been altered
genetically, so that natural regulation is switched off and the
expression of the genes has been increased. The nucleic acid
construct can also be of a simpler design, i.e. without any
additional regulatory signals being inserted in front of the coding
sequence and without removing the natural promoter with its
regulation. Instead, the natural regulatory sequence is silenced so
that regulation no longer takes place and gene expression is
increased.
[0207] A preferred nucleic acid construct advantageously also
contains one or more of the aforementioned enhancer sequences,
functionally associated with the promoter, which permit increased
expression of the nucleic acid sequence. Additional advantageous
sequences, such as other regulatory elements or terminators, can
also be inserted at the 3' end of the DNA sequences. One or more
copies of the nucleic acids according to the invention can be
contained in the construct. The construct can also contain other
markers, such as antibiotic resistances or auxotrophy-complementing
genes, optionally for selection on the construct.
[0208] Examples of suitable regulatory sequences are contained in
promoters such as cos-, tac-, trp-, tet-, trp-tet-, lpp-, lac-,
lpp-lac-, lacI.sup.q-, T7-, T5-, T3-, gal-, trc-, ara-, rhaP
(rhaP.sub.BAD)SP6-, lambda-P.sub.R- or in the lambda-P.sub.L
promoter, which find application advantageously in Gram-negative
bacteria. Other advantageous regulatory sequences are contained for
example in the Gram-positive promoters ace, amy and SPO2, in the
yeast or fungal promoters ADC1, MFalpha, AC, P-60, CYC1, GAPDH,
TEF, rp28, ADH. Artificial promoters can also be used for
regulation.
[0209] For expression, the nucleic acid construct is inserted in a
host organism advantageously in a vector, for example a plasmid or
a phage, which permits optimum expression of the genes in the host.
In addition to plasmids and phages, vectors are also to be
understood as meaning all other vectors known to a person skilled
in the art, e.g. viruses, such as SV40, CMV, baculovirus and
adenovirus, transposons, IS elements, phasmids, cosmids, and linear
or circular DNA. These vectors can be replicated autonomously in
the host organism or can be replicated chromosomally. These vectors
represent a further embodiment of the invention.
[0210] Suitable plasmids are, for example in E. coli, pLG338,
pACYC184, pBR322, pUC18, pUC19, pKC30, pRep4, pHS1, pKK223-3,
pDHE19.2, pHS2, pPLc236, pMBL24, pLG200, pUR290,
pIN-III.sup.113-B1, .lamda.gt11 or pBdCl; in nocardioform
actinomycetes pJAM2; in Streptomyces pIJ101, pIJ364, pIJ702 or
pIJ361; in bacillus pUB110, pC194 or pBD214; in Corynebacterium
pSA77 or pAJ667; in fungi pALS1, pIL2 or pBB116; in yeasts 2alphaM,
pAG-1, YEp6, YEp13 or pEMBLYe23 or in plants pLGV23, pGHlac.sup.+,
pBIN19, pAK2004 or pDH51. The aforementioned plasmids represent a
small selection of the possible plasmids. Other plasmids are well
known to a person skilled in the art and will be found for example
in the book Cloning Vectors (Eds. Pouwels P. H. et al. Elsevier,
Amsterdam-New York-Oxford, 1985, ISBN 0 444 904018).
[0211] In a further embodiment of the vector, the vector containing
the nucleic acid construct according to the invention or the
nucleic acid according to the invention can be inserted
advantageously in the form of a linear DNA in the microorganisms
and integrated into the genome of the host organism through
heterologous or homologous recombination. This linear DNA can
comprise a linearized vector such as plasmid or just the nucleic
acid construct or the nucleic acid according to the invention.
[0212] For optimum expression of heterologous genes in organisms,
it is advantageous to alter the nucleic acid sequences in
accordance with the specific codon usage employed in the organism.
The codon usage can easily be determined on the basis of computer
evaluations of other, known genes of the organism in question.
[0213] The production of an expression cassette according to the
invention is based on fusion of a suitable promoter with a suitable
coding nucleotide sequence and a terminator signal or
polyadenylation signal. Common recombination and cloning techniques
are used for this, as described for example in T. Maniatis, E. F.
Fritsch and J. Sambrook, Molecular Cloning: A Laboratory Manual,
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1989) as
well as in T. J. Silhavy, M. L. Berman and L. W. Enquist,
Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold
Spring Harbor, N.Y. (1984) and in Ausubel, F. M. et al., Current
Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley
Interscience (1987).
[0214] The recombinant nucleic acid construct or gene construct is
inserted advantageously in a host-specific vector for expression in
a suitable host organism, to permit optimum expression of the genes
in the host. Vectors are well known to a person skilled in the art
and will be found for example in "Cloning Vectors" (Pouwels P. H.
et al., Publ. Elsevier, Amsterdam-New York-Oxford, 1985).
7. Hosts that can be Used According to the Invention
[0215] Depending on the context, the term "microorganism" means the
starting microorganism (wild-type) or a genetically modified
microorganism according to the invention, or both.
[0216] The term "wild-type" means, according to the invention, the
corresponding starting microorganism, and need not necessarily
correspond to a naturally occurring organism.
[0217] By means of the vectors according to the invention,
recombinant microorganisms can be produced, which have been
transformed for example with at least one vector according to the
invention and can be used for production of the polypeptides
according to the invention. Advantageously, the recombinant
constructs according to the invention, described above, are
inserted in a suitable host system and expressed. Preferably,
common cloning and transfection methods that are familiar to a
person skilled in the art are used, for example co-precipitation,
protoplast fusion, electroporation, retroviral transfection and the
like, in order to secure expression of the stated nucleic acids in
the respective expression system. Suitable systems are described
for example in Current Protocols in Molecular Biology, F. Ausubel
et al., Publ. Wiley Interscience, New York 1997, or Sambrook et al.
Molecular Cloning: A Laboratory Manual. 2nd edition, Cold Spring
Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring
Harbor, N.Y., 1989.
[0218] In principle, all prokaryotic organisms can be considered as
recombinant host organisms for the nucleic acid according to the
invention or the nucleic acid construct. Bacteria are used
advantageously as host organisms. Preferably they are selected from
native or recombinant bacteria having the ability to produce
inclusion bodies of the PHA-, TAG- or WE-type, as in particular the
TAG-producing nocardioform actinomycetes, in particular of the
genus Rhodococcus, Mycobacterium, Nocardia, Gordonia, Skermania and
Tsukamurella; as well as TAG-producing Streptomycetes; WE-producing
genera Acinetobacter and Alcanivorax; as well as recombinant
strains of the genus Escherichia, especially E. coli,
Corynebacterium, especially C. glutamicum and Bacillus, especially
B. subtilis.
[0219] The host organism or host organisms according to the
invention then preferably contain at least one of the nucleic acid
sequences, nucleic acid constructs or vectors described in this
invention, which code for an enzyme activity according to the above
definition.
[0220] The organisms used in the method according to the invention
are grown or bred in a manner familiar to a person skilled in the
art, depending on the host organism. As a rule, microorganisms are
grown in a liquid medium, which contains a source of carbon,
generally in the form of sugars, a source of nitrogen generally in
the form of organic sources of nitrogen such as yeast extract or
salts such as ammonium sulfate, trace elements such as iron,
manganese and magnesium salts and optionally vitamins, at
temperatures between 0.degree. C. and 100.degree. C., preferably
between 10.degree. C. to 60.degree. C. with oxygen aeration. The pH
of the liquid nutrient medium can be maintained at a fixed value,
i.e. regulated or not regulated during growing. Growing can be
carried out batchwise, semi-batchwise or continuously. Nutrients
can be supplied at the start of fermentation or can be supplied
subsequently, either semi-continuously or continuously.
8. Recombinant Production of Enzymes of the Invention
[0221] The invention also relates to methods for production of
proteins according to the invention by cultivating a microorganism
which expresses said protein, and isolating the desired product
from the culture.
[0222] The microorganisms as used according to the invention can be
cultivated continuously or discontinuously in the batch process or
in the fed batch or repeated fed batch process. A review of known
methods of cultivation will be found in the textbook by Chmiel
(Bioprocesstechnik 1. Einfuhrung in die Bioverfahrenstechnik
(Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by
Storhas (Bioreaktoren and periphere Einrichtungen (Vieweg Verlag,
Braunschweig/Wiesbaden, 1994)).
[0223] The culture medium that is to be used must satisfy the
requirements of the particular strains in an appropriate manner.
Descriptions of culture media for various microorganisms are given
in the handbook "Manual of Methods for General Bacteriology" of the
American Society for Bacteriology (Washington D.C., USA, 1981).
[0224] These media that can be used according to the invention
generally comprise one or more sources of carbon, sources of
nitrogen, inorganic salts, vitamins and/or trace elements.
[0225] Preferred sources of carbon are sugars, such as mono-, di-
or polysaccharides. Very good sources of carbon are for example
glucose, fructose, mannose, galactose, ribose, sorbose, ribulose,
lactose, maltose, sucrose, raffinose, starch or cellulose. Sugars
can also be added to the media via complex compounds, such as
molasses, or other by-products from sugar refining. It may also be
advantageous to add mixtures of various sources of carbon. Other
possible sources of carbon are oils and fats such as soybean oil,
sunflower oil, peanut oil and coconut oil, fatty acids such as
palmitic acid, stearic acid or linoleic acid, alcohols such as
glycerol, methanol or ethanol and organic acids such as acetic acid
or lactic acid.
[0226] Sources of nitrogen are usually organic or inorganic
nitrogen compounds or materials containing these compounds.
Examples of sources of nitrogen include ammonia gas or ammonium
salts, such as ammonium sulfate, ammonium chloride, ammonium
phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea,
amino acids or complex sources of nitrogen, such as corn-steep
liquor, soybean flour, soybean protein, yeast extract, meat extract
and others. The sources of nitrogen can be used separately or as a
mixture.
[0227] Inorganic salt compounds that may be present in the media
comprise the chloride, phosphate or sulfate salts of calcium,
magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc,
copper and iron.
[0228] Inorganic sulfur-containing compounds, for example sulfates,
sulfites, dithionites, tetrathionates, thiosulfates, sulfides, but
also organic sulfur compounds, such as mercaptans and thiols, can
be used as sources of sulfur.
[0229] Phosphoric acid, potassium dihydrogenphosphate or
dipotassium hydrogenphosphate or the corresponding
sodium-containing salts can be used as sources of phosphorus.
[0230] Chelating agents can be added to the medium, in order to
keep the metal ions in solution. Especially suitable chelating
agents comprise dihydroxyphenols, such as catechol or
protocatechuate, or organic acids, such as citric acid.
[0231] The fermentation media used according to the invention may
also contain other growth factors, such as vitamins or growth
promoters, which include for example biotin, riboflavin, thiamine,
folic acid, nicotinic acid, pantothenate and pyridoxine. Growth
factors and salts often come from complex components of the media,
such as yeast extract, molasses, corn-steep liquor and the like. In
addition, suitable precursors can be added to the culture medium.
The precise composition of the compounds in the medium is strongly
dependent on the particular experiment and must be decided
individually for each specific case. Information on media
optimization can be found in the textbook "Applied Microbiol.
Physiology, A Practical Approach" (Publ. P. M. Rhodes, P. F.
Stanbury, IRL Press (1997) p. 53-73, ISBN 0 19 963577 3). Growing
media can also be obtained from commercial suppliers, such as
Standard 1 (Merck) or BHI (Brain heart infusion, DIFCO) etc.
[0232] All components of the medium are sterilized, either by
heating (20 min at 1.5 bar and 121.degree. C.) or by sterile
filtration. The components can be sterilized either together, or if
necessary separately. All the components of the medium can be
present at the start of growing, or optionally can be added
continuously or by batch feed.
[0233] The temperature of the culture is normally between
15.degree. C. and 45.degree. C., preferably 25.degree. C. to
40.degree. C. and can be kept constant or can be varied during the
experiment. The pH value of the medium should be in the range from
5 to 8.5, preferably around 7.0. The pH value for growing can be
controlled during growing by adding basic compounds such as sodium
hydroxide, potassium hydroxide, ammonia or ammonia water or acid
compounds such as phosphoric acid or sulfuric acid. Antifoaming
agents, e.g. fatty acid polyglycol esters, can be used for
controlling foaming. To maintain the stability of plasmids,
suitable substances with selective action, e.g. antibiotics, can be
added to the medium. Oxygen or oxygen-containing gas mixtures, e.g.
the ambient air, are fed into the culture in order to maintain
aerobic conditions. The temperature of the culture is normally from
20.degree. C. to 45.degree. C. Culture is continued until a maximum
of the desired product has formed. This is normally achieved within
10 hours to 160 hours.
[0234] The cells can be disrupted optionally by high-frequency
ultrasound, by high pressure, e.g. in a French pressure cell, by
osmolysis, by the action of detergents, lytic enzymes or organic
solvents, by means of homogenizers or by a combination of several
of the methods listed.
9. Reaction Conditions
[0235] The at least one enzyme which is present during the method
for producing a mono-acylated polyol, can be present in living
cells naturally or recombinantly producing the enzyme or enzymes,
in harvested cells, in dead cells, in permeabilized cells, in crude
cell extracts, in purified extracts, or in essentially pure or
completely pure form. The at least one enzyme may be present in
solution or as an enzyme immobilized on a carrier. One or several
enzymes may simultaneously be present in soluble and immobilised
form.
[0236] The method according to the invention can be performed in
common reactors, which are known to those skilled in the art, and
in different ranges of scale, e.g. from a laboratory scale (few
millilitres to dozens of liters of reaction volume) to an
industrial scale (several liters to thousands of cubic meters of
reaction volume). If the lipase is used in a form encapsulated by
non-living, optionally permeabilized cells, in the form of a more
or less purified cell extract or in purified form, a chemical
reactor can be used. The chemical reactor usually allows
controlling the amount of the at least one enzyme, the amount of
the at least one substrate, the pH, the temperature and the
circulation of the reaction medium. When the at least one enzyme is
present in living cells, the process will be a fermentation. In
this case the biocatalytic production will take place in a
bioreactor (fermenter), where parameters necessary for suitable
living conditions for the living cells (e.g. culture medium with
nutrients, temperature, aeration, presence or absence of oxygen or
other gases, antibiotics, and the like) can be controlled. Those
skilled in the art are familiar with chemical reactors or
bioreactors, e.g. with procedures for up-scaling chemical or
biotechnological methods from laboratory scale to industrial scale,
or for optimizing process parameters, which are also extensively
described in the literature (for biotechnological methods see e.g.
Crueger and Crueger, Biotechnologie--Lehrbuch der angewandten
Mikrobiologie, 2. Ed., R. Oldenbourg Verlag, Munchen, Wien,
1984).
[0237] Cells containing the at least one lopase can be
permeabilized by physical or mechanical means, such as ultrasound
or radiofrequency pulses, French presses, or chemical means, such
as hypotonic media, lytic enzymes and detergents present in the
medium, or combination of such methods. Examples for detergents are
digitonin, n-dodecylmaltoside, octylglycoside, Triton.RTM. X-100,
Tween.RTM. 20, deoxycholate, CHAPS
(3-[(3-Cholamidopropyl)dimethylammonio]-1-propansulfonate),
Nonidet.RTM. P40 (Ethylphenolpoly(ethyleneglycolether), and the
like.
[0238] If the at least one enzyme is immobilised, it is attached to
an inert carrier. Suitable carrier materials are known in the art
and are, e.g., disclosed in EP-A-1149849, EP-A-1 069 183 and DE-OS
100193773 as well as the literature references cited therein (all
of which are specifically enclosed with regard to carrier
materials). Examples for suitable carrier materials are clays, clay
minerals such as kaolinite, diatomeceous earth, perlite, silica,
alumina, sodium carbonate, calcium carbonate, cellulose powder,
anion exchanger materials, synthetic polymers, such as polystyrene,
acrylic resins, phenol formaldehyde resins, polyurethanes and
polyolefins, such as polyethylene and polypropylene. For preparing
carrier-bound enzymes the carrier materials usually are used in the
form of fine powders, wherein porous forms are preferred. The
particle size of the carrier material usually does not exceed 5 mm,
in particular 2 mm. In case the at least one enzyme is present in a
whole-cell-preparation, said whole-cell-preparation may be present
in a free or immobilised form. Suitable carrier materials are e.g.
Ca-alginate or Carrageenan. Enzymes as well as cells may directly
be linked by glutaraldehyde. A wide range of immobilisation methods
is known in the art (e.g. J. Lalonde and A.
Margolin--Immobilization of Enzymes" in K. Drauz and H. Waldmann,
Enzyme Catalysis in Organic Synthesis 2002, Vol. III, 991-1032,
Wiley-VCH, Weinheim).
[0239] The conversion reaction can be carried out batch wise,
semi-batch wise or continuously. Reactants (and optionally
nutrients) can be supplied at the start of reaction or can be
supplied subsequently, either semi-continuously or
continuously.
[0240] The reaction may be performed in an aqueous or non-aqueous
reaction medium.
[0241] An aqueous medium may contain a suitable buffer in order to
adjust the pH to a value in the range of 5 to 9, like 6 to 8.
[0242] The non-aqueous medium may contain is substantially free of
water, i.e. will contain less that about 1 wt.-% (weight percent)
or 0.5 wt.-% of water.
[0243] In particular, the present method is performed in an organic
non-aqueous medium. As suitable organic solvents there may be
mentioned aliphatic carbohydrates having for example 5 to 8 carbon
atoms, like pentane, cyclopentane, hexane, cyclohexane, heptane,
octane or cyclooctane, halogenated aliphatic carbohydrates like
dichloromethane, chloroformate, CCl.sub.4, dichloroethane or
tetrachloroethane, aromatic carbohydrates, like benzene, toluene,
xyloles, chlorobenzene or dichlorobenzene, aliphatic acyclic and
cyclic ethers. Like diethylether, methyl-tert.-butylether,
ethyl-tert.-butylether, dipropylether, ddiisopropylether,
dibutylether, tetrahydrofuran or esters like ethylacetate or
n-butylacetate or ketones like methylisobutylketone or dioxan or
mixtures thereof.
[0244] Particularly useful are solvents, which may also function as
reactants, like the acyl donor ethyl acetate.
[0245] The concentration of the reactants may be adapted to the
optimum reaction conditions, which may depend on the specific
enzyme applied. For example, the initial substrate concentration
may be in the range of 0.01 to 0.5 M, as for example 10 to 100
mM.
[0246] If appropriate one reactant, as for example the acyl donor
may be used in molar excess in order to shift the reaction
equilibrium to the side of the product.
[0247] The reaction temperature may be adapted to the optimum
reaction conditions, which may depend on the specific enzyme
applied. For example, the reaction may be performed at a
temperature in a range of from 0 to 70.degree. C., as for example
20 to 50 or 25 to 40.degree. C. Examples for reaction temperatures
are about 30.degree. C., about 35.degree. C., about 37.degree. C.,
about 40.degree. C., about 45.degree. C., about 50.degree. C.,
about 55.degree. C. and about 60.degree. C.
[0248] The process may proceed until equilibrium between the
substrate and then product(s) is achieved, but may be stopped
earlier. Usual process times are in the range from 1 minute to 25
hours, in particular 10 min to 6 hours, as for example in the range
from 1 hour to 4 hours, in particular 1.5 hours to 3.5 hours.
10. Product Isolation
[0249] The methodology of the present invention can further include
a step of recovering an acylated product, optionally in
stereoisomerically or enantiomerically substantially pure form. The
term "recovering" includes extracting, harvesting, isolating or
purifying the compound from culture or reaction media. Recovering
the compound can be performed according to any conventional
isolation or purification methodology known in the art including,
but not limited to, treatment with a conventional resin (e.g.,
anion or cation exchange resin, non-ionic adsorption resin, etc.),
treatment with a conventional adsorbent (e.g., activated charcoal,
silicic acid, silica gel, cellulose, alumina, etc.), alteration of
pH, solvent extraction (e.g., with a conventional solvent such as
an alcohol, ethyl acetate, hexane and the like), distillation,
dialysis, filtration, concentration, crystallization,
recrystallization, pH adjustment, lyophilization and the like.
[0250] Identity and purity of the isolated product may be
determined by known techniques, like High Performance Liquid
Chromatography (HPLC), gas chromatography (GC), Spektroskopy (like
IR, UV, NMR), Colouring methods, TLC, NIRS, enzymatic or microbial
assays. (see for example: Patek et al. (1994) Appl. Environ.
Microbiol. 60:133-140; Malakhova et al. (1996) Biotekhnologiya 11
27-32; und Schmidt et al. (1998) Bioprocess Engineer. 19:67-70.
Ullmann's Encyclopedia of Industrial Chemistry (1996) Bd. A27, VCH:
Weinheim, S. 89-90, S. 521-540, S. 540-547, S. 559-566, 575-581 und
S. 581-587; Michal, G (1999) Biochemical Pathways: An Atlas of
Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A.
et al. (1987) Applications of HPLC in Biochemistry in: Laboratory
Techniques in Biochemistry and Molecular Biology, Bd. 17.)
[0251] The following examples only serve to illustrate the
invention. The numerous possible variations that are obvious to a
person skilled in the art also fall within the scope of the
invention.
EXPERIMENTAL PART
A. Materials and Methods
1. Enzyme Immobilisation
[0252] 5 g of Accurel <1500 micron MP1000/pp powder was used.
The powder was soaked in 95% ethanol during 5 days. The powder was
washed again in fresh ethanol, which was removed, prior to
incubation. 21 ml of T40A mutated CALB of an approximate
concentration of 2.7 mg/ml, dissolved in 100 mM PO.sub.4 buffer, pH
7.2 were added.
[0253] Enzyme-containing supernatant from P. pastoris cultivation
was concentrated to 3 g/l protein concentration. The pH was
adjusted to 7.5. 100 g Resindion Diaoin HP 20L beads were added per
litre of enzyme solution. The slurry was incubated under shaking
for 4 h. Then the solid was filtered off and dried in a slight air
stream.
[0254] For the T40V mutated CALB, 5 g of Accurel <1500 micron
MP1000/pp powder was used. The powder was soaked in 95% ethanol for
>1 hour and then washed with fresh ethanol. The powder was
washed with buffer, 100 mM K.sub.xH.sub.yPO.sub.4, pH 7.2, to
remove ethanol before immobilization. 70 ml of protein solution of
a concentration of approximately 30-50 mg/l. The immobilization
solution was left on a IKA.RTM. KS basic shaker at 80 rpm in
4.degree. C. for 3 days. The supernatant was filtered off and the
beads were washed by adding 3.times.100 ml of 50 mM NH.sub.4Ac
(pH7) over a vacuum manifold. The 50 mM NH.sub.4Ac was used because
it evaporates, so that there are no large amounts of salts left on
the beads.
2. GC Parameters for the Analysis of Different Constituents
2.1 Glycol
[0255] The temperature for the inlet was set at 200.degree. C. and
the detector was set to 270.degree. C. The temperature program
started at 40.degree. C. for 5 minutes, followed by a gradient of
10.degree. C./min to 200.degree. C., where the temperature was kept
constant for a final 10 minutes.
2.2 1,3-Propanediol
[0256] Temperatures for inlet and detector were both 200.degree. C.
The temperature program started at 40.degree. C. for 5 minutes,
followed by a gradient of 5.degree. C./min to 160.degree. C.,
followed by a gradient of 10.degree. C./min to 200.degree. C.,
where the temperature was held constant for a final 5 minutes.
2.3 1,4-Butanediol
[0257] The temperature for the inlet was switched off and the
detector was set to 270.degree. C. The temperature program started
at 30.degree. C. for 5 minutes, followed by a gradient of 2.degree.
C./min to 40.degree. C., followed by a gradient of 10.degree.
C./min to 200.degree. C., where the temperature was held constant
for a final 5 minutes.
2.4 2-Methyl-1,3-propanediol and 2-phenyl-1,3-propanediol
[0258] The temperature for the inlet was switched off and the
detector was set to 270.degree. C. The temperature program started
at 40.degree. C. for 5 minutes, followed by a gradient of
10.degree. C./min to 200.degree. C., where the temperature was held
constant for a final 10 minutes. 2.5. 1,2-Ethanediol and
1,4-butanediol
[0259] The following parameters were used for mutants and
determinations as described in the Examples 5, 6 or 7. The
temperature for the inlet was set at 200.degree. C. and the
detector was set to 200.degree. C. The temperature program started
at 40.degree. C. for 5 minutes, followed by a gradient of
10.degree. C./min to 200.degree. C., where the temperature was kept
constant for a final 10 minutes.
2.6. 1,4-Butanediol, 4-hydroxybutane acrylate and butanediol
diacrylate
[0260] The following parameters were used for mutants and
determinations as described in Examples 8 and 9. The temperature
for the inlet was set at 220.degree. C. and the detector was set to
225.degree. C. The temperature program started at 50.degree. C. for
5 minutes, followed by a gradient of 10.degree. C./min to
290.degree. C., where the temperature was kept constant for a final
10 minutes.
B. Examples
Example 1
Preparation of CALB Wild Type and T40A and T40V Mutants and
Immobilization
[0261] Wild type and T40A-mutated CALB were obtained from in-house
productions from earlier experiments according to previously
defined protocol [Magnusson, A., K. Hult, and M. Holmquist,
Creation of an enantioselective hydrolase by engineered
substrate-assisted catalysis. Journal of the American Chemical
Society, 2001. 123(18): p. 4354-4355. Rotticci-Mulder, J. C., et
al., Expression in Pichia pastoris of Candida antarctica lipase B
and lipase B fused to a cellulose-binding domain. Protein
Expression and Purification, 2001. 21(3): p. 386-392.]
[0262] The primers used for T40A mutated CALB were
TABLE-US-00006 (SEQ ID NO: 5) 5'-GACTGGTTCCAATTGACAAGC-3' and (SEQ
ID NO: 6) 5'-GCAAATGGCATTCTGACATCC-3'
[0263] The T40V mutated CALB was prepared using pGAPZaB CALB
construct obtained from earlier studies [Larsen M. W., et al.,
Expression of Candida antarctica lipase B in Pichia pastoris and
various Escerichia coli systems, Protein Expression and
Purification, 2008, 62(1): p. 90-97].
[0264] 25 ng template was used together with 100 ng of the
primers
TABLE-US-00007 (SEQ ID NO: 7)
5'-CCCATCCTTCTCGTCCCCGGAGTCGGCACCACAGGTCCA-3' and (SEQ ID NO: 8)
5'-GTCGAACGACTGTGGACCTGTGGTGCCGACTCCGGGGAC-3'.
[0265] 6% DMSO was used in the PCR reaction. The reaction was
carried out at 98.degree. C. for 1 min, 25 cycles with 10 s at
98.degree. C., 50 s at 60.degree. C. and 3 min at 72.degree. C. The
template DNA was digested with FastDigest Dpnl. Competent cells,
Echerichia coli strain XL 10 GOLD from Stratagene, were transformed
with 3 .mu.l of the mutagenesis reaction mixture. The mutation was
confirmed by sequencing by DNA cycle sequencing with
BigDye-terminators. The plasmid was extracted using QIAprep Spin
Miniprep Kit and linearised with FastDigest AvrII from Fermentas.
Transformation of Picchia pastoris X33 from invitrogen was done by
electroporation following the manual: pGAPZ A, B, C and
pGAPZ.alpha. A, B, C using a BIORAD Gene Pulser. Cultivation was
done by inoculating a single cell colony in 10 ml of media, BMGY
(10 g yeast extract, 20 g peptone, 100 mL 1 M
K.sub.2HPO.sub.4/KH.sub.2PO.sub.4 pH 6.0 per litre, 13.4 g yeast
nitrogen base with ammonium sulphate without amino acids (YNB), 0.4
mg biotin, and 10 mL glycerol in 1 L water), which was cultivated
in 30.degree. C. over night with an orbital agitation of 260 rpm.
500 .mu.l was then used to inoculate 0.5 I BMGY in 5 I shake flask.
Cultivation was done at 30.degree. C. with an orbital agitation of
260 rpm for 3 days. Harvest was done by centrifugation on a Sorvall
Super T21 centrifuge and removal of the cell pellet.
[0266] Protein expressions were done in recombinant yeast Picchia
pastoris.
[0267] Purifications of wild type and T40A CALB were done by
hydrophobic interactions chromatography followed by gel filtration.
A previously defined protocol were used for purification of wild
type and T40A CALB [Rotticci-Mulder, J. C., et al., Expression in
Pichia pastoris of Candida antarctica lipase B and lipase B fused
to a cellulose-binding domain. Protein Expression and Purification,
2001. 21(3): p. 386-392.]. The T40V CALB was purified by ion
exchange chromatography. A protocol was followed [Trodler P. et al,
Rational design of a one-step purification strategy for Candida
Antarctica lipase B by ion exchange chromatography. Journal of
Chromatography A 2008 1179(2) p. 386-392] with the exception that
buffer exchange was carried out by dialysis instead of crossed-flow
filtration. A dialysis bag from Spectra/Por.RTM., dialysis membrane
with molecular weight cut off 14,000 Da was filled with 0.5 l of
filtered culture media. The media was equilibrated with 4.times.1.5
l of buffer, 10 mM sodium formate, 10 mM sodium citrate and 10 mM
sodium acetate, pH 3.0, which was used for the ion exchange
chromatography. The purification was done using 7 ml Source 15S
matrix packed in a 16/20 column connected to an AKTA explorer.
[0268] The enzyme was immobilized on Accurel MP1000 carrier beads
(see general method, above), dried and equilibrated over saturated
lithium chloride. Equilibration of water activity could be achieved
by storing the enzyme over lithium chloride for a few days. This
T40A and wild type CALB had been stored for 3-4 years over lithium
chloride since it is suitable to store immobilized CALB in this
environment for a longer period of time to prevent loss of
activity. The T40V CALB was stored for 3 days over lithium chloride
before use.
[0269] The amount of enzyme loaded on the carriers was confirmed by
active site titration using the inhibitor methyl
4-methylumbelliferyl hexylphosphonate. Inhibitor from an ampoule
stored in a freezer (-20.degree. C.) was dissolved to a
concentration of 50 .mu.M in acetonitrile. The inhibitor was
previously synthesized and used for active site titration of CALB
as described in Magnusson, A. O., et al., Creating space for large
secondary alcohols by rational redesign of Candida antarctica
lipase B. Chembiochem, 2005. 6(6): p. 1051-1056 and Rotticci, D.,
et al., An active-site titration method for lipases. Biochimica Et
Biophysica Acta-Molecular and Cell Biology of Lipids, 2000.
1483(1): p. 132-140.
[0270] Inhibition reactions were run in three separate vials each
containing 50 mg enzyme carrier beads with immobilized CALB wild
type and T40A-mutant. The wild type was run in one of the reaction
vials, and the other two contained T40A-mutant. The enzymes were
incubated in 1 ml of 50 .mu.M inhibitor dissolved in acetonitrile.
Each vial were sealed and covered in aluminium foil, to prevent
light from damaging the fluorophore, and stored in continuous end
over end. Aliquots were taken after 2, 5 and 9 days. Each aliquot
contained of 100 .mu.l reaction solution from incubation dispensed
in 900 .mu.l buffer (100 mM Tris, 1 mM CaCl.sub.2, pH 8.0) in a
Perkin Elmer 10/4 mm quarts cuvette. Analysis was preformed by
fluorescence using a Perkin Elmer LS 50 B fluorimeter. The
wavelengths of excitation and emission were 360 nm and 445 nm
respectively. Fluorescence was also measured in two different
control samples. Inhibitor, 50 .mu.M in acetonitrile, was incubated
without enzyme for control of background fluorescence from
spontaneous hydrolysis of the methyl 4-methylumbelliferyl
hexylphosphonate. In addition to this, 50 mg of each enzyme was
incubated in acetonitrile without inhibitor for control of any
background fluorescence from the immobilized enzyme. The total
fluorescence from these samples was subtracted from the
fluorescence measured in the inhibition reactions. The same method
was used for active site titration of immobilized CALB T40V. A
sample taken and measured after two weeks of inhibition showed low
levels of inhibition.
[0271] Remaining activity was measured after inhibition. The
immobilized enzyme washed in 1 ml acetonitrile and stored over
saturated LiCl for 3 days. A solution containing 100 mM 1-butanol,
.gtoreq.99.5% from Riedel-deHaen, 20 mM decane, .gtoreq.98% from
Fluka and 1 M vinyl butyrate, .gtoreq.99% from Fluka dissolved in
MTBE, .gtoreq.99.5% from Labscan, were used as reaction solution
for activity measurements. 4 ml of the reaction solution was added
to inhibited enzyme. For comparison, the reaction was repeated with
the enzyme used for control of background fluorescence. The amounts
of enzyme used for activity measurements were adjusted to prevent
high conversion of the substrate, which may affect reaction rates.
The inhibited immobilized enzymes used in the reactions were 19 mg
wild type and 20 mg T40A. The amounts of immobilized enzymes taken
from the fluorescence controls used in the reactions were 2.8 mg of
the wild type and 9.0 T40A. 5 Aliquots were taken from each
reaction at reaction times between 50 and 350 seconds. Analyses
were performed on GC as defined above, under the section
analysis.
[0272] The activity measurements showed that 83% of the wild type
and 21% of the T40A-mutant was inhibited. Remaining activity was to
low to measure for CALB T40A. Taking these activities into
considerations, 4.6 mg active enzyme was immobilized per g carrier
beads for the T40A-mutant and 12 mg enzyme was immobilized per g
carrier beads for the wild type. The amount of active CALB T40V
immobilized was around 0.3 mg per g carrier beads.
Example 2
Enzyme-Catalyzed Acylation Reactions with Different Diols as
Substrate and Ethyl Acetate as Acyl Donor
[0273] Reactions were run with three different straight chain
primary diols: [0274] 1,2-ethanediol, .gtoreq.99.5% from MERCK,
[0275] 1,3-propanediol, 98% from Aldrich and [0276] 1,4-butanediol,
.gtoreq.99% from Aldrich.
[0277] Additionally, reactions with 1,3-propanediol with different
substitutions were run: [0278] 2-methyl-1,3-propanediol and [0279]
2-phenyl-1,3-propanediol,
[0280] 3-Hexanone, .gtoreq.98% from Aldrich, was used as internal
standard.
[0281] Each diol was dissolved to a concentration of 20 mM together
with 5 mM of 3-hexanone in ethyl acetate, .gtoreq.99% from Fluka,
which was used as acyl donor and solvent. Reactions were started by
adding 4 ml reaction solution to 20 mg enzyme carrier beads loaded
with either wild-type or T40A mutated CALB. Reactions were run at
29.degree. C. on a HLC rotating thermo block. Aliquots of 50 .mu.l
were taken, filtered through wool in a Pasteur pipette and diluted
with 50 .mu.l ethyl acetate prior to analysis. The 3-hexanon used
as internal standard were present in the reaction solution
throughout both reaction and analysis.
[0282] 5 mM 3-hexanone was used as internal standard in the
reaction mixtures together with 20 mM diol. Aliquots were taken
during the reaction by taking 50 .mu.l of reaction sample. The
samples were filtered though wool together with 50 .mu.l ethyl
acetate, thereby any remaining enzyme were removed and the reaction
samples were diluted by a factor of 2 prior to analysis.
[0283] The reactions catalyzed by the T40A-mutant were rerun with
100 mg enzyme carrier beads for determination of maximum monoester
yield. The reactions with 2-methyl-1,3-propanediol and
2-phenyl-1,3-propanediol were rerun at 100 mM concentration of diol
and 50 mg enzyme carrier beads, both with CALB wild type and CALB
T40A-mutant. This was done in order to determine the enantiomeric
excess.
[0284] All reactions were analyzed on GC, Hewlett Packard 5890
series II. Two different 25 m.times.0.32 mm WCOT fused silica
columns were used, depending on the different substrates and
products that were analyzed. A polar GC-column with CP Chirasil-Dex
CB coating was used for the reactions with 1,2-ethanediol and
1,3-propanediol. A non-polar GC-column with CP-SIL 5CB coating was
used for the reactions with 1,4-butanediol,
2-methyl-1,3-propanediol and 2-phenyl-1,3-propanediol.
[0285] The response factor for each diol was determined in
relationship to 3-hexanone. When all diol and mono-ester had
converted to di-ester, the response factor for the di-ester could
be determined in relationship to decane. The concentration of
mono-ester in each reaction was determined by subtracting the
concentration of di-ester from the starting concentration of
diol.
Example 3
Enzyme-Catalyzed Acylation Reactions with Different Diols as
Substrate and Vinyl Butyrate as Acyl Donor
[0286] Reactions were run with two different diols: [0287]
1,4-butanediol, .gtoreq.99% from Aldrich. [0288]
2-methyl-1,3-propanediol.
[0289] Each diol was dissolved to a concentration of 10 mM together
with 2 mM of decane .gtoreq.98% from Fluka and 200 mM vinyl
butyrate >99% from Fluka in MTBE 99.5% from Lab-Scan, which was
used as acyl donor and solvent. Reactions were started by adding 4
ml reaction solution to 20 mg enzyme carrier beads loaded with
either wild-type or T40A mutated CALB. Reactions were run at
29.degree. C. on a HLC rotating thermo block. Aliquots of 50 .mu.l
were taken, filtered through wool in a Pasteur pipette and diluted
with 50 .mu.l ethyl acetate prior to analysis. The decane used as
internal standard were present in the reaction solution throughout
both reaction and analysis.
[0290] All reactions were analyzed on GC, Hewlett Packard 5890
series II. A 25 m.times.0.32 mm WCOT fused silica non-polar
GC-column with CP-SIL 5CB coating was used for the reactions.
[0291] The response factor for each diol was determined in
relationship to decane. When all diol and mono-ester had converted
to di-ester, the response factor for the di-ester could be
determined in relationship to decane. The concentration of
mono-ester in each reaction was determined by subtracting the
concentration of di-ester from the starting concentration of
diol.
Example 4
Enzyme-Catalyzed Acylation Reactions with Different Diols as
Substrate in Competition with 1-Butanol
[0292] Reactions were run with two different straight chain primary
diols: [0293] 1,2-ethanediol, .gtoreq.99.5% from MERCK, [0294]
1,4-butanediol, .gtoreq.99% from Aldrich.
[0295] Additionally, 1-butanol, .gtoreq.99.5% from Riedel-de Haen,
was used as a competing substrate.
[0296] 25 mM decane .gtoreq.98% from Fluka, was used as internal
standard.
[0297] Two different acyl donors were used:
[0298] Ethyl acetate, .gtoreq.99% from Fluka, used as both solvent
and acyl donor 1 M Vinyl butyrate, >99% from Fluka, together
with in MTBE, 99.5% from Lab-Scan, used as solvent.
[0299] Each diol was dissolved to a concentration of 100 mM
together with 25 mM of decane in ethyl acetate, .gtoreq.99% from
Fluka, used as acyl donor and solvent. Reactions were started by
adding 3 ml reaction solution to 10 mg enzyme carrier beads loaded
with wild-type CALB or 20 mg carrier beads loaded with T40A mutated
CALB. Reactions were run at 29.degree. C. in a temperature bath
consisting of a HETO thermostat coupled to a Bom-roerder combined
temperature bath and magnetic stirrer. 25 mM decane was used as
internal standard were present in the reaction solution throughout
both reaction and analysis. Aliquots of 60 .mu.l were taken,
filtered through wool in a Pasteur pipette together with 540 .mu.l
ethyl acetate prior to analysis. The filtration through wool
removed any remaining enzyme from the reaction solution, and the
reaction samples were diluted by a factor of 10 prior to
analysis.
[0300] All reactions were analyzed on GC, Hewlett Packard 5890
series II. Two different 25 m.times.0.32 mm WCOT fused silica
columns were used, depending on the different substrates and
products that were analyzed. A polar GC-column with CP Chirasil-Dex
CB coating was used for the reactions with 1,2-ethanediol. A
non-polar GC-column with CP-SIL 5CB coating was used for the
reactions with 1,4-butanediol.
[0301] The response factor for each diol was determined in
relationship to decane. When all diol and mono-ester had converted
to di-ester, the response factor for the di-ester could be
determined in relationship to decane. The concentration of
mono-ester in each reaction was determined by subtracting the
concentration of di-ester from the starting concentration of
diol.
Example 5
Preparation of CALB A282L Mutant and Immobilization
[0302] Freeze dried A282L mutated CALB was obtained from in-house
productions from earlier experiments [Z. Marton, V. Leonard-Nevers,
P.-O, Syren, C. Bauer, S. Lamare, K. Hult, V. Tranc and M. Graber,
Mutations in the stereospecificity pocket and at the entrance of
the active site of Candida antarctica lipase B enhancing enzyme
enantioselectivity. Journal of Molecular Catalysis B:
Enzymatic].
A282L was proposed from structure-based enzyme engineering. The
primer pair to introduce the mutation was
TABLE-US-00008 (SEQ ID NO: 9) 5'-CCTGGCGCCGGCATTGGCAGCC-3',
and the corresponding reverse complementary sequence:
TABLE-US-00009 (SEQ ID NO: 10) 5'-GGCTGCCAATGCCGGCGCCAGG-3'.
After the enzyme carrying this mutation showed enhanced
selectivity, the position 282 was subjected to saturation
mutagenesis. For saturation mutagenesis the primers
TABLE-US-00010 (SEQ ID NO: 11) 5'-CTGGCGCCGGCGNNNGCAGCCAT-3' and
(SEQ ID NO: 12) 5'-ATGGCTGCNNNCGCCGGCGCCAG-3,
indicated in a generic form, wherein N represents A, T, C or G,
were used. Consequently, NNN represents the random use of any
possible codon triplett, because in the PCR a corresponding primer
mix was used. 900 transformation positive colonies were picked,
cultivated and the respective proteins expressed in microtitre
plate scale. Screening the selectivity in the transesterification
reaction ethyl acrylate+butane diol showed that position 282 is
important for the selectivity in diol transesterification. All
mutations in position 282 were created by saturation mutagenesis as
indicated above. 10 microtitre plates including 8 control wells
each (expression of wild type (WT), empty P. pastoris X33) were
picked with positive transformants. Enzyme expression was induced
by addition of methanol. The cells and supernatant were
freeze-dried. Freeze-dried pellets were used in transesterification
assays. GC was used for detection of the reaction products. For
construction the mutant I285F the following pair of primers was
used:
TABLE-US-00011 forward primer: (SEQ ID NO: 13)
5'-GCAGCCTTTGTGGCG-3' reverse primer: (SEQ ID NO: 14)
5'-CGCCACAAAGGCTGC-3'
The double mutation A282L/I285F was simulataneously introduced by
the following pair of primers:
TABLE-US-00012 forward primer: (SEQ ID NO: 15)
5'-CCGGCGCTTGCAGCCTTTGTGGCGGGTCCAAAG-3' reverse primer: (SEQ ID NO:
16) 5'-CTTTGGACCCGCCACAAAGGCTGCAAGCGCCGG-3'
Using the primers
TABLE-US-00013 forward: (SEQ ID NO: 17) 5'-GCTCTCTGCGCCGGC-3'
reverse: (SEQ ID NO: 18) 5'-GCCGGCGCAGCGACG-3',
the double mutant L278S/A282L was prepared by first separately
introducing the L278S mutant and adding this to the A282L
mutant.
[0303] Approximately 2 mg of the freeze dried A282L CALB or other
mutants as described above were dissolved in 10 ml 100 mM
K.sub.xH.sub.yPO.sub.4, pH 7.0. The enzyme was immobilized on 1 g
of Accurel beads according to the general protocol described under
A. Materials and Methods, 1. Enzyme Immobilisation, with the
following exceptions: The immobilisation solution was left in end
over end in room temperature for 24 hours. After immobilisation,
wash with NH4Ac and drying, the enzyme beads were subjected to an
additional wash in 50 ml 10 mM MOPS, pH 7.5, for 24 hours in end
over end in room temperature. The beads were dried under vacuum
overnight and stored over lithium chloride.
The amount of enzyme loaded on the beads was analyzed by active
site titration, as described for T40A and wild type CALB. An enzyme
load of 0.17% (w/w) was confirmed.
Example 6
Preparation of CALB A281V and A281E Mutants and Immobilization
[0304] The A281V and A281E mutated CALB variants were prepared by
overlapping extension PCR using pET22b+ vector obtained from
earlier studies [Larsen M. W., et al., Expression of Candida
antarctica lipase B in Pichia pastoris and various Escerichia coli
systems, Protein Expression and Purification, 2008, 62(1): p.
90-97]. In the first step forward and reverse PCR reactions were
done for the A281V and A281E mutated CALB. For the forward
reaction, reverse Not primer was used together with
5'-CGGCTGCGCTCCTGGCTCCTGTAGCTG-3' (SEQ ID NO:19) for the A281V
variant and 5'-CGGCTGCGCTCCTGGCTCCTGAGGCTG-3' (SEQ ID NO:21) for
the A281E variant. For the reverse reaction, forward NocI primer
was used together with 5'-CTGCAGCTACAGGAGCCAGGAGCGCAG-3' (SEQ ID
NO:20) for the A281V variant and 5'-CAGCCTCAGGAGCCAGGAGCGCAGCCG-3'
(SEQ ID NO:22) for the A281E variant. Second PCR reactions were
carried out for the A281V and A281E mutated CALB. 1 .mu.l from the
forward and 1 .mu.l from the reverse reaction were used together
with forward NcoI and reverse Not primers. The PCR temperature
program was identical for both steps. 98.degree. C. for 30s, 30
cycles with 10 s at 98.degree. C., 15 s at 65.degree. C. and 20 s
at 72.degree. C. and 5 min 72.degree. C. Forward NcoI and reverse
Not primers were from Thermo Scientific. The PCR products and
pET22b+ were digested using FastDigest restriction enzymes NotI and
NcoI, from Fermentas. The PCR products and linearised pET22b+ were
purified on a 1% agarose gel and extracted using the QIAquick Gel
Extraction Kit from QIAGEN. The PCR products were ligated back into
the linearised pET22b+ using T4 DNA ligase from Fermentas.
Incubations were done in room temperature for 1 hour and 20 min.
Electro competent Rosetta strain Escherichia coli cells were
transformed with the ligation solution. 1 .mu.l of the solution
containing A281V CALB and 0.5 .mu.l of the solution containing
A281E CALB were added to the cells. The mutation was confirmed by
sequencing by Eurofins MWG Operon (Ebersberg, Germany).
[0305] Expression and purification of the protein was done
according to a predefined protocol for periplasmic expression of
CALB using Escherichia coli strain Rosetta [Larsen M. W., et al.,
Expression of Candida antarctica lipase B in Pichia pastoris and
various Escerichia coli systems, Protein Expression and
Purification, 2008, 62(1): p. 90-97]. The purified enzymes were
subjected to buffer exchange using PD-10 columns from GE
healthcare. The buffer was changed into 10 mM MOPS, pH7.2, which
was used as immobilisation buffer.
[0306] For the A281V and A281E mutated CALB, 1.5 g and 2 g Accurel
enzyme carrier beads were used, respectively. Prior to the
immobilisation, he beads were washed for 2 hours in 95% ethanol and
then washed in fresh ethanol. The ethanol was washed off with
3.times.10 ml immobilisation buffer, 10 mM MOPS, pH 7.2 The
immobilisation solution was done in 10 ml 10 mM MOPS, pH 7.2, left
in end over end in room temperature overnight.
[0307] The amount of enzyme loaded on the beads could not be
analyzed by active site titration, the amounts were too low.
Immobilisation of the A281V and A281E CALB were confirmed by a
comassie stained gel with samples of the solution before and after
the immobilisation.
Example 7
Determination of Enzyme Selectivities and Specificities of A282L,
A281V and A281E
[0308] Reactions using the CALB mutants as described in Example 5
and Example 6 were run with two different straight chain primary
diols: 1,2-ethanediol, .gtoreq.99.5% from MERCK and 1,4-butanediol,
.gtoreq.99% from Aldrich. Two different acyl donors were used:
vinyl acetate, .gtoreq.99% from Fluka and vinyl butyrate, >99%
from Fluka. Decane, .gtoreq.99% from Fluka, was used as internal
standard. MTBE, 99.5% from Lab-Scan was used as solvent.
[0309] Each diol was dissolved to a concentration of 100 mM
together with 1 M of the acyl donor and 20 mM decane in MTBE.
Reactions were started by adding 3 ml reaction solution to a
specific amount of enzyme carrier beads. Different amounts of
enzyme carrier beads were used for the different reactions. For
reactions with 1,2-ethanediol and vinyl acetate, 19.7 mg wild type,
22.5 mg A282L, 50.5 mg A281V, and 45.5 A281E CALB were used. For
reactions with 1,2-ethanediol and vinyl butyrate, 19.3 mg wild
type, 19.7 mg A282L, 40.5 mg A281V, and 40.6 A281E CALB were used.
For reactions with 1,4-butanediol and vinyl acetate, 17.6 mg wild
type, 22.5 mg A282L, 69 mg A281V, and 54 A281E CALB were used. For
reactions with 1,4-butanediol and vinyl butyrate, 20.3 mg wild
type, 25.4 mg A282L, 52.2 mg A281V, and 53 A281E CALB were used.
The enzymes were evenly distributed in the reaction vials by
magnetic stirrers. The vials were kept in a 29.degree. C. water
bath during the reactions for temperature control. Aliquots of 10
.mu.l were taken, filtered through wool in a Pasteur pipette and
diluted with 90 .mu.l MTBE prior to analysis. The decane used as
internal standard was present in the reaction solution throughout
both reaction and analysis.
[0310] 20 mM decane was used as internal standard in the reaction
mixtures together with 100 mM diol. Aliquots were taken during the
reaction by taking 10 .mu.l of reaction sample. The samples were
filtered though wool together with 90 .mu.l MTBE, thereby any
remaining enzyme was removed and the reaction samples were diluted
by a factor of 10 prior to analysis.
[0311] The reactions catalyzed by the A281V-mutant were rerun with
higher amounts of enzyme carrier beads to test selectivity
determinations. For the reactions with 1,2-ethanediol and vinyl
acetate, 121 mg beads with A281V CALB were used. For the reactions
with 1,2-ethanediol and vinyl butyrate, 122.9 mg beads with A281V
CALB were used. For the reactions with 1,4-butanediol and vinyl
butyrate, 127.8 mg beads with A281V CALB were used.
[0312] All reactions were analyzed on GC, Hewlett Packard 5890
series II. Two different 25 m.times.0.32 mm WCOT fused silica
columns were used, depending on the different substrates and
products that were analyzed. A polar GC-column with CP Chirasil-Dex
CB coating was used for the reactions with 1,2-ethanediol. A
non-polar GC-column with CP-SIL 5CB coating was used for the
reactions with 1,4-butanediol. The response factor for each diol
was determined in relationship to decane. When all diol and
mono-ester had converted to di-ester, the response factor for the
di-ester could be determined in relationship to decane. The
concentration of mono-ester in each reaction was determined by
subtracting the concentration of di-ester from the starting
concentration of diol.
Example 8
Enzymatic Catalysis in Mini-Reactor Scale
[0313] The enzymatic conversion of butandiole and ethylacrylate
into 4-hydroxybutyl acrylate (4-HBA, butanediole monoacrylate) and
butanediole diacrylate (BDDA) was examined in a 750 ml mini plant
reactor with stream circulation over the enzyme column and a
partial stream over the column (50 ml column) as stabilizer.
[0314] 3.75 g immobilized CALB (Novozym.RTM. 435, herein also
referred to as Novo 435 or Novo435; Novozymes A/S, Denmark) or
A282L (as prepared in Example 5 and immobilized on Diaion HP 20L
according to the procedure described in Materials and Methods,
protein load: 3%), 50.0 g 1,4-butanediole (0.555 moles) and 555.7 g
ethylacrylate (5.55 moles) were used, resulting in molar ratio of
butanediole:ethylacrylate of 1:10. The total weight of the reaction
mix was 0.606 kg. 200 ppm hydroquinone monomethylether (MeHQ, 121
mg) and 200 ppm phenothiazine (121 mg) were included as
stabilizers. The educts were filled into the reactor. Prior to
allowing the passage of the reaction mix over the immobilized
enzyme the reaction mix was heated to reflux at 100 mbar (100 hPa).
Samples were taken at various time points and analyzed by gas
chromatography (gradient of 50.degree. C. to 290.degree. C.
temperature increase: 10.degree. C./min, injection temperature:
220.degree. C.).
Example 9
Dependency of Substrate Conversion and Excess of Product from Flow
Rate
[0315] For determining the impact of various flow rates on
substrate conversion and product excess a double shell glass column
of 49 ml total volume (volume of heated part: 26 ml, volume of part
containing enzyme: 23 ml, filling height of enzyme material: 167
mm), attached to a Desaga pump and a Ministat was used. The column
was filled with either CALB Novo 435 as described in Example 8 or
4.4 g CALB A282L (immobilized on Diaion HP 20L, 3% protein load).
The reaction mix comprised 90.0 g (1.0 mole) 1,4-butanediol and
1400 g (14 moles) ethylacrylate. The reactor was operated at flow
rates ranging from 10 ml/h to 1000 ml/h, samples were taken after
passage of 135-280 ml reaction mix, depending on the flow rate, and
analyzed by gas chromatography as described in Example 8.
Example 10
Determination of Enzyme Selectivities and Specificities of A282T,
A282C, A282P, A282I, A282D, A282V, A282M, A282R, I285, A282L/I285F
and L278S/A282L
[0316] 300 mg or another specified amount of the respective enzyme
(on a HP20L carrier) in a 50 ml Duran glas bottle were incubated
together with 440 .mu.l 1,4-butanediol, 5.4 ml ethylacrylate and 2
g molecular sieve (0.5 nm) at 40.degree. C. in a shaking water bath
(200 rpm). Samples were taken at 2, 4, 6, 24 and 48 hours. Prior to
gas chromatography 160 .mu.l sample and 240 .mu.l dioxane were
mixed and filtered.
C. Results
[0317] Results of Example 2: Enzyme-Catalyzed Acylation Reactions
with Different Diols as Substrate and Ethyl Acetate as Acyl
Donor
TABLE-US-00014 TABLE 1 Maximum monoester yield: Substance T40A WT
1,2-ethanediol 77% 43% 1,3-propanediol 50% 33% 1,4-butanediol 49%
25% 2-Me-1,3-propanediol 55% 42% 2-Ph-1,3-propanediol 55-65%
52%
TABLE-US-00015 TABLE 2 Conversion at 3:1 (monoester:diester)
Substance T40A WT 1,2-ethanediol 99% 17% 1,3-propanediol 22% 19%
1,4-butanediol 64% <5% 2-Me-1,3-propanediol 55% 30%
2-Ph-1,3-propanediol >95% 46%
TABLE-US-00016 TABLE 3 Conversion at 9:1 (monoester:diester)
Substance T40A WT 1,2-ethanediol 78% <9% 1,3-propanediol 19% 10%
1,4-butanediol 26% <5%
[0318] Tables 1-3 shows the difference in selectivity towards diol
over monoester between wild type and T40A-mutated CALB. The
presented were obtained at 20 mM starting concentration of the
diols dissolved in ethyl acetate, which was used as both acyl donor
and solvent. The reaction conditions were chosen for comparison
between the two catalysts. Further optimisation of the reaction
conditions could give higher yields. As diol is consumed in the
reaction the formed monoester will be more favoured to react and
form diester, due to differences in concentrations. Table 1 shows
that the maximum yield of monoacetylated diol increased for all
tested diols by using the T40A CALB mutant when compared to wild
type CALB. Tables 2 and 3 demonstrates which maximum conversions
can be acquired at a given purity of the product; 3:1 and 9:1
(monoester:diester). The figures in tables 2 and 3 shows that more
diol can be converted before the product purity drops under the
given levels when comparing T40A mutated to wild type CALB.
[0319] A great difference in concentration between acyl donor and
diol was accomplished in the reactions by using ethyl acetate as
solvent and acyldonor and a diol concentration of 20 mM. Therefore
the reactions was considered to irreversible, following the
reaction scheme:
##STR00005##
Irreversible reaction conditions validate the following
equations:
[ A ] = [ A ] 0 - k 1 t ( eq . 1 ) [ B ] = [ A ] 0 k 1 k 2 - k 1 (
- k 1 t - - k 2 t ) ( eq . 2 ) [ C ] = [ A ] 0 { 1 + 1 k 1 - k 2 (
k 2 - k 1 t - k 1 - k 2 t ) } ( eq . 3 ) ##EQU00001##
A, B and C corresponds to diol, monoester and diester,
respectively. k.sub.1 and k.sub.2 respectively corresponds to the
reaction rates of the acylation of diol and monoester.
[0320] The reaction rates are dependent on enzyme concentrations
and the k.sub.cat/K.sub.M towards the substrates. By fitting the
concentrations measured over time into the equations and using the
enzyme concentration determined by active site titration, the
following results were obtained.
TABLE-US-00017 TABLE 4 Specificity and selectivities towards diols
and monoesters for wild type CALB Selectivity .DELTA..DELTA.G,
diol- k.sub.cat/K.sub.M (s.sup.-1M.sup.-1) diol/ monoester Diol
diol monoester monoester (kJ/mol) 1,4-butanediol 7.7 12 0.6 -1.2
1,3-propanediol 12 13 0.9 -0.17 1,2-ethanediol 15 8.0 1.9 1.6
2-methyl-1,3-propanediol 5.8 3.8 1.5 1.1
TABLE-US-00018 TABLE 5 Specificities and selectivities towards
diols and monoesters for T40A CALB .DELTA..DELTA.G, diol-
K.sub.cat/K.sub.M (s.sup.-1M.sup.-1) Selectivity monoester Diol
diol monoester diol/monoester (kJ/mol) 1,4-butanediol 0.25 0.13 1.9
1.5 1,3-propanediol 0.46 0.19 2.4 2.2 1,2-ethanediol 0.76 0.10 7.6
5.1 2-methyl-1,3-propanediol 0.15 0.06 2.5 2.4
TABLE-US-00019 TABLE 6 Differences between T40A and wild type CALB
in selectivities towards diols over monoesters Selectivity diol/
Selectivity ratio .DELTA..DELTA..DELTA.G, monoester diol/monoester
T40A-WT Diol wild type T40A T40A/WT (kJ/mol) 1,4-butanediol 0.63
1.8 2.9 2.7 1,3-propanediol 0.94 2.4 2.6 2.4 1,2-ethanediol 1.9 7.7
4.0 3.5 2-methyl-1,3-propanediol 1.5 2.6 1.7 1.3
[0321] Values of k.sub.cat/K.sub.M towards diols and monoesters for
wild type and T40A CALB are presented in tables 4-6. Table 4
presents k.sub.cat/K.sub.M, for wild type and table 5 presents
k.sub.cat/K.sub.M for T40A CALB. Additionally, the selectivities
towards diols over monoesters,
(k.sub.cat/K.sub.M).sub.diol/(k.sub.cat/K.sub.M).sub.monoester are
presented. The differences in activation energies, .DELTA..DELTA.G,
are calculated from these selectivities. The selectivities in
tables 4 and 5 have been transferred to table 6 for comparison
between wild type and T40A mutant. The figures presenting the ratio
between the selectivities of T40A/wild type CALB, and the related
difference in activation energies, .DELTA..DELTA..DELTA.G, shows
the effect of the T40A mutation in CALB. The tested diols are
favoured over their corresponding monoesters as an effect of the
T40A mutation.
[0322] Further results are graphically summarized in FIGS. 3, 4, 5
and 6:
[0323] Examples of monoester and diester yield comparing reactions
catalyzed with wild type and T40A CALB are illustrated in FIGS. 3
and 4. Reactions with 1,2-ethanediol and 1,4-butanediol are
presented in FIGS. 3 and 4 respectively. In both figures the
maximum monoester yields are improved by using T40A instead of wild
type CALB.
[0324] FIG. 5 shows examples of product distribution for reactions
with 1,2-ethanediol catalyzed by wild type and T40A CALB. The
product distribution is calculated by the following equation:
product distribution = [ monoester ] [ monoester ] + [ diester ] (
eq . 4 ) ##EQU00002##
[0325] A product distribution of 75% corresponds to 3:1,
monoester:diester, and 90% to 9:1, monoester:diester.
[0326] FIG. 6 gives a view of how concentrations of diols, mono-
and diesters change over time for the reaction of 1,2-ethanediol
and ethyl acetate catalyzed by wild type or T40A CALB. The error
bars represent models from equations 1-3, fitted to the measured
values. The models give values for k.sub.cat/K.sub.M presented in
tables 4-6.
Results of Example 3: Enzyme-Catalyzed Acylation Reactions with
Different Diols as Substrate and Vinyl Butyrate as Acyl Donor
TABLE-US-00020 TABLE 7 Maximum monoester yields: Diol T40A WT
1,4-butanediol 44 25 2-methyl-1,3-propanediol 56 34
[0327] Table 7 shows differences in measured maximum yields between
wild type and T40A CALB. The selectivity towards the diols over
their corresponding monobutyrate esters is higher for T40A than
wild type CALB. These results agree with results presented in table
1.
[0328] The results are summarized in FIGS. 7 and 8
[0329] FIGS. 7 and 8 shows yields of mono- and diesters from
2-methyl-1,3-propanediol and 1,4-butanediol in reactions with vinyl
butyrate. MTBE was used as solvent. The difference in selectivity
between wild type (7A, 8A) and T40A (7B, 8B) towards diol over its
corresponding monoester is illustrated in the figures.
Results of Example 4: Competition Experiments Comparing Electivity
of Wild Type, T40A and T40V CALB.
[0330] 100 mM diol 100 mM 1-butanol 25 mM decane as internal
standard Ethyl acetate used as solvent and acyldonor. Alternatively
1 M vinyl butyrate and MTBE.
[0331] The results are summarized in FIGS. 9 and 10:
[0332] In FIGS. 9 and 10 the conversion of diols are expressed as
functions of 1-butanol conversion. Ethyl acetate was used as acyl
donor and solvent for the experiments presented in FIG. 9. Vinyl
butyrate was used as acyl donor and MTBE as solvent for the
experiments presented in FIG. 10. Comparisons between wild type and
T40A CALB are done in the graphs in FIGS. 9 and 10. Additionally,
the graphs in FIG. 10 show reactions catalyzed by T40V CALB. A
significant difference between wild type and T40A CALB can be
observed in all presented graphs. Diols are converted quicker than
1-butanol when using T40A instead of wild type CALB. Thereby a
higher selectivity towards diol over 1-butanol is accomplished with
T40A than with wild type CALB.
[0333] The higher selectivity towards diol over 1-butanol is
observed when 1,2-ethanediol is used as a substrate than for
1,4-butanediol. These observations agree with results presented in
table 6, where selectivities towards diols over monoesters are
compared between wild type and T40A CALB. The highest selectivity
was observed towards 1,2-ethanediol both in FIGS. 9 and 10 and in
table 6.
[0334] Similar results were obtained when comparing T40V and wild
type CALB. Low enzyme expression levels combined with low activity
towards the tested substrates gives low reaction rates. Hence, only
the reactions containing vinyl butyrate could be accurately
measured for the T40V variant. For these reactions, the reaction
with 1,4-butanediol were run for a longer period of time than the
reaction containing 1,2-ethanediol. Therefore, higher conversions
were measured in the reaction containing 1,4-butanediol. Here, it
can be seen that the selectivity towards 1,4-butanediol over
1-butanol is higher for T40V than for wild type CALB. The results
from the reaction with 1,2-ethanediol suggest that the selectivity
is higher also in this case.
[0335] FIG. 11 shows differences in reaction rates for reactions
with 1,2-ethanediol catalyzed with wild type and T40A CALB. The
amounts of converted moles 1,2-ethanediol per gram enzyme are
expressed as functions of time. In 11A ethyl acetate was used as
acyl donor and solvent. Vinyl butyrate was used as acyl donor and
MTBE as solvent in 11B.
TABLE-US-00021 TABLE 8 Initial reaction rates Initial reaction
rates (mol/(g min) Rate ratio, Substrates Wild type T40A T40A/WT
(%) 1,2-ethanediol 0.081 0.002 2.5 ethyl acetate 1,2-ethanediol
0.41 0.065 16 vinyl butyrate 1,4-butanediol 0.065 0.0008 1.2 ethyl
acetate 1,4-butanediol 0.16 0.016 9.6 vinyl butyrate
[0336] In table 8 the initial reaction rates are presented for
reactions catalyzed by wild type and T40A CALB. Two different
diols, 1,2-ethanediol and 1,4-butanediol were used in
transacylation reactions with two different acyl donors, ethyl
acetate and vinyl butyrate. The rate ratios presented in the third
column are a measure of remaining activity in T40A compared to wild
type CALB. The rate ratio for T40A over wild type CALB is higher
towards vinyl butyrate than towards ethyl acetate. A shift if the
rate determining step from acylation to deacylation when shifting
from ethyl acetate to vinyl butyrate can explain these differences.
There is no substrate assistance in the acylation step which would
slow down the acylation from ethyl acetate more than from vinyl
butyrate, since the vinyl butyrate is a more activated ester.
[0337] The differences in rate ratios for T40A over wild type CALB
towards vinyl butyrate and ethyl acetate together with the
selectivity towards diol over 1-butanol gives support to the
hypothesis of substrate assisted catalysis for diols in T40A.
Results of Example 7: Comparison of Selectivity of Wild Type,
A282L, A281V and A281E CALB
[0338] A different approach to increase monoester yield in
transacylation reactions catalyzed by CALB was tested. Mutations
A282L, A281V and A281E were made to prevent monoacylated diol to
react. The mutations are thought to sterically hinder larger
substrates selectively, but not smaller substrates. Thus, diols are
better substrates than their corresponding monoester. The results
are presented in tables 9 and 10. The reactions were run with a 10
times excess of the acyl donor over diol, and considered
irreversible. The reaction scheme 1 and equations 1-3, discussed
under Results of example 2, are valid. MTBE was used as
solvent.
[0339] Table 9 shows selectivities for wild type, A282L, A281V and
A281E towards diol over monoester. The selectivities are calculated
directly from the rate constants obtained by fitting experimental
data to equations 1-3. As the enzyme concentration is identical for
both acylation steps, the k.sub.1/k.sub.2 is equal to
(k.sub.cat/K.sub.M).sub.diol/(k.sub.cat/K.sub.M).sub.monoester.
Furthermore, the selectivities have been recalculated into energy
differences, .DELTA..DELTA.G. The .DELTA..DELTA.G corresponds to
the difference in energy in transition state. The tested diols are
1,2-ethanediol and 1,4-butanediol, reacting with either vinyl
acetate or vinyl butyrate. Both diols and acyl donors influence the
selectivity towards diol over monoester. All three mutants have
higher selectivity than the wild type towards all tested
substrates. The biggest difference between CALB variants is for
A282L and wild type CALB towards 1,2-ethanetiol and vinyl
butyrate.
TABLE-US-00022 TABLE 9 Selectivities and the correlating
differences in activation energies. 1,2-ethanediol 1,4-butanediol
vinyl acetate vinyl butyrate vinyl acetate vinyl butyrate PalB
.DELTA..DELTA.G** .DELTA..DELTA.G** .DELTA..DELTA.G**
.DELTA..DELTA.G** Variant k.sub.1/k.sub.2* (kJ/mol)
k.sub.1/k.sub.2* (kJ/mol) k.sub.1/k.sub.2* (kJ/mol)
k.sub.1/k.sub.2* (kJ/mol) WT 1.2 0.6 0.5 -1.7 0.5 -1.9 0.5 -1.7
A282L 2.6 2.4 5.8 4.4 1.6 1.2 3.0 2.8 A281V 5.2 4.1 5.0 4.0 1.7 1.4
2.0 1.7 A281E 5.1 4.1 5.0 4.0 1.8 1.4 2.2 2.0 *k.sub.1/k.sub.2 are
the constants in equations 1-3 and corresponds to
(k.sub.cat/K.sub.M).sub.diol/(k.sub.cat/K.sub.M).sub.monoester.
**Values for .DELTA..DELTA.G are calculated from the presented
k.sub.1/k.sub.2, and correspond to the difference in transition
state energy between the substrates.
[0340] Table 10, contains more detailed information about some of
the reactions presented in table 9. Specificity constants for wild
type and A282L CALB towards 1,2-ethanediol and 1,4-butanediol and
their corresponding monoesters, formed using vinyl acetate and
vinyl butyrate as acyl donor, are shown. In the tested cases, the
specificities are increased towards the diols by the A282L
mutation. The specificity towards 1,2-ethanediol in reaction with
vinylbutyrate increased from 1.6 to 8.1 s.sup.-1 mM.sup.-1. In most
cases the specificity towards the monoester decreased as a result
of the A282L mutation. The exception is towards monoacetylated
1,2-ethanediol, which may be too small for the sterical hindrance
caused by the A282L mutation. As presented in table 9, the
selectivity towards diols over their corresponding monoesters
increased as a result of the A282L mutation. Changes in
specificities towards both diols and monoesters presented in table
10 contributes to the increased selectivity.
TABLE-US-00023 TABLE 10 Individual specificities for wild type and
A282L CALB towards various diols and monoesters, formed with
various acyl donors. Specificities, k.sub.cat/K.sub.M (s-1 mM-1)
1,2-ethanediol 1,4-butanediol vinyl vinyl vinyl vinyl CALB acetate
butyrate acetate butyrate variant diol ester* diol ester* diol
ester* diol ester* WT 1.6 1.2 1.6 3.2 0.9 1.9 1.4 2.8 A282L 3.2 1.2
8.1 1.4 1.6 1.0 1.7 0.6 *The ester is the corresponding diol
monoacylated by the corresponding acyldonor.
[0341] Table 11 shows initial reaction rates for reactions
catalyzed wild type and A282L CALB. The tested diols are
1,2-ethanediol and 1,4-butanediol. The acyl donors used for
transacylation reactions were vinyl acetate and vinyl butyrate.
Comparison of initial reaction rates of A282L over wild type CALB
is presented in the last column as percentages. No significant
decrease in initial reaction rates can be observed as a result of
the A282L mutation. In fact, a significant increase can be seen in
3 out of 4 cases. A possible explanation for the increased initial
reaction rates is that the selectivity towards diol over monoester
is higher for A282L CALB than for the wild type, as discussed above
in tables 9 and 10. As soon as monoester is formed it competes with
the diol as substrate for the enzyme. Consequently, the diol
conversion rate is reduced. Another possibility for the increased
initial reaction rates is that the V.sub.max, and thus k.sub.cat,
is higher towards the substrates for A282L CALB than for the wild
type. An increased k.sub.cat means that the A282L-mutant is a
better catalyst for the monoacylation of the diols than wild type
CALB.
TABLE-US-00024 TABLE 11 Initial reaction rates Initial reaction
rates (mol/(g min) Rate ratio Substrates wild type A282L A282L/WT
(%) 1,2-ethanediol 0.21 0.35 173 vinyl acetate 1,2-ethanediol 0.36
1.0 287 vinyl butyrate 1,4-butanediol 0.12 0.21 178 vinyl acetate
1,4-butanediol 0.24 0.22 92 vinyl butyrate
Results of Example 8: Excess of Monoacrylate Using A282L Under Mini
Reactor Conditions
[0342] During the experiment the temperature of the reactor shell
and the bottom of the reactor were kept at 55.degree. C. and
41.degree. C., respectively. The column temperature was fairly
constant at 39.degree. C. during the main part of the reactor runs.
From the experimentally determined amounts of butanediol,
4-hydroxybutanediol and butanediol diacrylate the degree of
conversion and the excess of 4-hydroxybutanediol over butanediole
diacrylate were calculated. As shown in FIG. 12, a higher excess of
4-hydroxybutanediol over butanediole diacrylate within a wide range
of substrate conversions after enzymatic catalysis by the A282L
mutant or L278S is achieved. The excess obtained after enzymatic
catalysis by Novo 435 is measurably lower.
Results of Example 9: Dependency of Substrate Conversion and Excess
of Product from Flow Rate
[0343] After initiation of the reactor run, samples were taken as
indicated in Table 12:
TABLE-US-00025 TABLE 12 Flow rate Time of Sampling Volume passed
over [ml/h] [hh:mm] column at sampling [ml] 1000 00:00 0 1000 00.17
280 400 00:00 0 400 00:35 230 200 00:00 0 200 01:23 277 100 00:00 0
100 02:00 200 50 00:00 0 50 02:43 135 10 00:00 0 10 15:48 155
[0344] Table 13 shows the contents of 1,4-butanediol,
4-hydroxybutanediol and butanediol diacrylate of the samples taken
at the respective end time points of table 12 after passage over a
CALB A282L loaded column.
TABLE-US-00026 TABLE 13 Flow rate Percentage Percentage Percentage
Conversion Excess of Ratio [ml/h] BD 4-HBA BDDA [%] 4HBA 4HBA:BDDA
1000 89.6 9.4 1.0 10.4 80.8 9.4 400 68.8 27.9 3.3 31.12 78.8 8.5
200 51.7 41.1 7.2 48.3 70.2 5.7 100 35.0 50.6 14.3 65.0 55.9 3.5 50
19.7 53.7 26.6 80.3 33.7 2.0 10 3.1 32.2 64.7 96.9 -33.5 0.0
[0345] The dependency of product excess from conversion rates from
flow rates are shown in FIG. 13. The excess of the desired product
was comparable after conversion by CALB Novo 435 and CALB A282L at
flow rates of 400 ml/h and higher. Due to a considerably higher
conversion of substrate by CALB A282L at all flow rates, the mutant
according to the invention is superior to CALB Novo 435 in these
ranges. When comparing Novo 435 and A282L at approximately the same
conversion, the excess of monacrylate produced by A282L is
higher.
Results of Example 10: Determination of Enzyme Selectivities and
Specificities of A282T, A282C, A282P, A282I, A282Asp, A282V, A282M,
A282R, I285, A282L/I285F and L278S/A282L
[0346] The results of FIG. 14A and FIG. 14B are based on reactions
containing 300 mg enzyme immobilized on HP20L Diaion, those of
FIGS. 15A and 15B on 134 mg enzyme (A282I), 117 mg enzyme (A282R),
140 mg enzyme (A282C), 300 mg enzyme (A282L/I285F) and 115 mg
enzyme (I285F). From the results shown in FIG. 14A, FIG. 14B, and
FIG. 15A, FIG. 15B it is clear that the mutations A282C, A282P,
A282I, A282Asp, A282L, A282V, A282R, I285F as well as the double
mutant L282S/A282L cause a measurable increase monoacrylate excess
when compared to CALB Novo 435. The double mutant A282L/I285F leads
to a slight deterioration of monoacrylate excess versus degree of
conversion.
[0347] The references as cited herein and the attached sequence
listing are explicitly referred to.
Sequence CWU 1
1
221954DNACandida antarcticaCDS(1)..(954) 1cta cct tcc ggt tcg gac
cct gcc ttt tcg cag ccc aag tcg gtg ctc 48Leu Pro Ser Gly Ser Asp
Pro Ala Phe Ser Gln Pro Lys Ser Val Leu 1 5 10 15 gat gcg ggt ctg
acc tgc cag ggt gct tcg cca tcc tcg gtc tcc aaa 96Asp Ala Gly Leu
Thr Cys Gln Gly Ala Ser Pro Ser Ser Val Ser Lys 20 25 30 ccc atc
ctt ctc gtc ccc gga acc ggc acc aca ggt cca cag tcg ttc 144Pro Ile
Leu Leu Val Pro Gly Thr Gly Thr Thr Gly Pro Gln Ser Phe 35 40 45
gac tcg aac tgg atc ccc ctc tct gcg cag ctg ggt tac aca ccc tgc
192Asp Ser Asn Trp Ile Pro Leu Ser Ala Gln Leu Gly Tyr Thr Pro Cys
50 55 60 tgg atc tca ccc ccg ccg ttc atg ctc aac gac acc cag gtc
aac acg 240Trp Ile Ser Pro Pro Pro Phe Met Leu Asn Asp Thr Gln Val
Asn Thr 65 70 75 80 gag tac atg gtc aac gcc atc acc acg ctc tac gct
ggt tcg ggc aac 288Glu Tyr Met Val Asn Ala Ile Thr Thr Leu Tyr Ala
Gly Ser Gly Asn 85 90 95 aac aag ctt ccc gtg ctc acc tgg tcc cag
ggt ggt ctg gtt gca cag 336Asn Lys Leu Pro Val Leu Thr Trp Ser Gln
Gly Gly Leu Val Ala Gln 100 105 110 tgg ggt ctg acc ttc ttc ccc agt
atc agg tcc aag gtc gat cga ctt 384Trp Gly Leu Thr Phe Phe Pro Ser
Ile Arg Ser Lys Val Asp Arg Leu 115 120 125 atg gcc ttt gcg ccc gac
tac aag ggc acc gtc ctc gcc ggc cct ctc 432Met Ala Phe Ala Pro Asp
Tyr Lys Gly Thr Val Leu Ala Gly Pro Leu 130 135 140 gat gca ctc gcg
gtt agt gca ccc tcc gta tgg cag caa acc acc ggt 480Asp Ala Leu Ala
Val Ser Ala Pro Ser Val Trp Gln Gln Thr Thr Gly 145 150 155 160 tcg
gca ctc act acc gca ctc cga aac gca ggt ggt ctg acc cag atc 528Ser
Ala Leu Thr Thr Ala Leu Arg Asn Ala Gly Gly Leu Thr Gln Ile 165 170
175 gtg ccc acc acc aac ctc tac tcg gcg acc gac gag atc gtt cag cct
576Val Pro Thr Thr Asn Leu Tyr Ser Ala Thr Asp Glu Ile Val Gln Pro
180 185 190 cag gtg tcc aac tcg cca ctc gac tca tcc tac ctc ttc aac
gga aag 624Gln Val Ser Asn Ser Pro Leu Asp Ser Ser Tyr Leu Phe Asn
Gly Lys 195 200 205 aac gtc cag gca cag gct gtg tgt ggg ccg ctg ttc
gtc atc gac cat 672Asn Val Gln Ala Gln Ala Val Cys Gly Pro Leu Phe
Val Ile Asp His 210 215 220 gca ggc tcg ctc acc tcg cag ttc tcc tac
gtc gtc ggt cga tcc gcc 720Ala Gly Ser Leu Thr Ser Gln Phe Ser Tyr
Val Val Gly Arg Ser Ala 225 230 235 240 ctg cgc tcc acc acg ggc cag
gct cgt agt gca gac tat ggc att acg 768Leu Arg Ser Thr Thr Gly Gln
Ala Arg Ser Ala Asp Tyr Gly Ile Thr 245 250 255 gac tgc aac cct ctt
ccc gcc aat gat ctg act ccc gag caa aag gtc 816Asp Cys Asn Pro Leu
Pro Ala Asn Asp Leu Thr Pro Glu Gln Lys Val 260 265 270 gcc gcg gct
gcg ctc ctg gcg ccg gcg gct gca gcc atc gtg gcg ggt 864Ala Ala Ala
Ala Leu Leu Ala Pro Ala Ala Ala Ala Ile Val Ala Gly 275 280 285 cca
aag cag aac tgc gag ccc gac ctc atg ccc tac gcc cgc ccc ttt 912Pro
Lys Gln Asn Cys Glu Pro Asp Leu Met Pro Tyr Ala Arg Pro Phe 290 295
300 gca gta ggc aaa agg acc tgc tcc ggc atc gtc acc ccc tga 954Ala
Val Gly Lys Arg Thr Cys Ser Gly Ile Val Thr Pro 305 310 315
2317PRTCandida antarctica 2Leu Pro Ser Gly Ser Asp Pro Ala Phe Ser
Gln Pro Lys Ser Val Leu 1 5 10 15 Asp Ala Gly Leu Thr Cys Gln Gly
Ala Ser Pro Ser Ser Val Ser Lys 20 25 30 Pro Ile Leu Leu Val Pro
Gly Thr Gly Thr Thr Gly Pro Gln Ser Phe 35 40 45 Asp Ser Asn Trp
Ile Pro Leu Ser Ala Gln Leu Gly Tyr Thr Pro Cys 50 55 60 Trp Ile
Ser Pro Pro Pro Phe Met Leu Asn Asp Thr Gln Val Asn Thr 65 70 75 80
Glu Tyr Met Val Asn Ala Ile Thr Thr Leu Tyr Ala Gly Ser Gly Asn 85
90 95 Asn Lys Leu Pro Val Leu Thr Trp Ser Gln Gly Gly Leu Val Ala
Gln 100 105 110 Trp Gly Leu Thr Phe Phe Pro Ser Ile Arg Ser Lys Val
Asp Arg Leu 115 120 125 Met Ala Phe Ala Pro Asp Tyr Lys Gly Thr Val
Leu Ala Gly Pro Leu 130 135 140 Asp Ala Leu Ala Val Ser Ala Pro Ser
Val Trp Gln Gln Thr Thr Gly 145 150 155 160 Ser Ala Leu Thr Thr Ala
Leu Arg Asn Ala Gly Gly Leu Thr Gln Ile 165 170 175 Val Pro Thr Thr
Asn Leu Tyr Ser Ala Thr Asp Glu Ile Val Gln Pro 180 185 190 Gln Val
Ser Asn Ser Pro Leu Asp Ser Ser Tyr Leu Phe Asn Gly Lys 195 200 205
Asn Val Gln Ala Gln Ala Val Cys Gly Pro Leu Phe Val Ile Asp His 210
215 220 Ala Gly Ser Leu Thr Ser Gln Phe Ser Tyr Val Val Gly Arg Ser
Ala 225 230 235 240 Leu Arg Ser Thr Thr Gly Gln Ala Arg Ser Ala Asp
Tyr Gly Ile Thr 245 250 255 Asp Cys Asn Pro Leu Pro Ala Asn Asp Leu
Thr Pro Glu Gln Lys Val 260 265 270 Ala Ala Ala Ala Leu Leu Ala Pro
Ala Ala Ala Ala Ile Val Ala Gly 275 280 285 Pro Lys Gln Asn Cys Glu
Pro Asp Leu Met Pro Tyr Ala Arg Pro Phe 290 295 300 Ala Val Gly Lys
Arg Thr Cys Ser Gly Ile Val Thr Pro 305 310 315 3408PRTArtificial
SequenceT40 Mutant with alpha-factor 3Met Arg Phe Pro Ser Ile Phe
Thr Ala Val Leu Phe Ala Ala Ser Ser 1 5 10 15 Ala Leu Ala Ala Pro
Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln 20 25 30 Ile Pro Ala
Glu Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe 35 40 45 Asp
Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu 50 55
60 Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val
65 70 75 80 Ser Leu Glu Lys Arg Glu Ala Glu Ala Glu Phe Leu Pro Ser
Gly Ser 85 90 95 Asp Pro Ala Phe Ser Gln Pro Lys Ser Val Leu Asp
Ala Gly Leu Thr 100 105 110 Cys Gln Gly Ala Ser Pro Ser Ser Val Ser
Lys Pro Ile Leu Leu Val 115 120 125 Pro Gly Xaa Gly Thr Thr Gly Pro
Gln Ser Phe Asp Ser Asn Trp Ile 130 135 140 Pro Leu Ser Ala Gln Leu
Gly Tyr Thr Pro Cys Trp Ile Ser Pro Pro 145 150 155 160 Pro Phe Met
Leu Asn Asp Thr Gln Val Asn Thr Glu Tyr Met Val Asn 165 170 175 Ala
Ile Thr Thr Leu Tyr Ala Gly Ser Gly Asn Asn Lys Leu Pro Val 180 185
190 Leu Thr Trp Ser Gln Gly Gly Leu Val Ala Gln Trp Gly Leu Thr Phe
195 200 205 Phe Pro Ser Ile Arg Ser Lys Val Asp Arg Leu Met Ala Phe
Ala Pro 210 215 220 Asp Tyr Lys Gly Thr Val Leu Ala Gly Pro Leu Asp
Ala Leu Ala Val 225 230 235 240 Ser Ala Pro Ser Val Trp Gln Gln Thr
Thr Gly Ser Ala Leu Thr Thr 245 250 255 Ala Leu Arg Asn Ala Gly Gly
Leu Thr Gln Ile Val Pro Thr Thr Asn 260 265 270 Leu Tyr Ser Ala Thr
Asp Glu Ile Val Gln Pro Gln Val Ser Asn Ser 275 280 285 Pro Leu Asp
Ser Ser Tyr Leu Phe Asn Gly Lys Asn Val Gln Ala Gln 290 295 300 Ala
Val Cys Gly Pro Leu Phe Val Ile Asp His Ala Gly Ser Leu Thr 305 310
315 320 Ser Gln Phe Ser Tyr Val Val Gly Arg Ser Ala Leu Arg Ser Thr
Thr 325 330 335 Gly Gln Ala Arg Ser Ala Asp Tyr Gly Ile Thr Asp Cys
Asn Pro Leu 340 345 350 Pro Ala Asn Asp Leu Thr Pro Glu Gln Lys Val
Ala Ala Ala Ala Leu 355 360 365 Leu Ala Pro Ala Ala Ala Ala Ile Val
Ala Gly Pro Lys Gln Asn Cys 370 375 380 Glu Pro Asp Leu Met Pro Tyr
Ala Arg Pro Phe Ala Val Gly Lys Arg 385 390 395 400 Thr Cys Ser Gly
Ile Val Thr Pro 405 4317PRTArtificial SequenceT40 Mutant 4Leu Pro
Ser Gly Ser Asp Pro Ala Phe Ser Gln Pro Lys Ser Val Leu 1 5 10 15
Asp Ala Gly Leu Thr Cys Gln Gly Ala Ser Pro Ser Ser Val Ser Lys 20
25 30 Pro Ile Leu Leu Val Pro Gly Xaa Gly Thr Thr Gly Pro Gln Ser
Phe 35 40 45 Asp Ser Asn Trp Ile Pro Leu Ser Ala Gln Leu Gly Tyr
Thr Pro Cys 50 55 60 Trp Ile Ser Pro Pro Pro Phe Met Leu Asn Asp
Thr Gln Val Asn Thr 65 70 75 80 Glu Tyr Met Val Asn Ala Ile Thr Thr
Leu Tyr Ala Gly Ser Gly Asn 85 90 95 Asn Lys Leu Pro Val Leu Thr
Trp Ser Gln Gly Gly Leu Val Ala Gln 100 105 110 Trp Gly Leu Thr Phe
Phe Pro Ser Ile Arg Ser Lys Val Asp Arg Leu 115 120 125 Met Ala Phe
Ala Pro Asp Tyr Lys Gly Thr Val Leu Ala Gly Pro Leu 130 135 140 Asp
Ala Leu Ala Val Ser Ala Pro Ser Val Trp Gln Gln Thr Thr Gly 145 150
155 160 Ser Ala Leu Thr Thr Ala Leu Arg Asn Ala Gly Gly Leu Thr Gln
Ile 165 170 175 Val Pro Thr Thr Asn Leu Tyr Ser Ala Thr Asp Glu Ile
Val Gln Pro 180 185 190 Gln Val Ser Asn Ser Pro Leu Asp Ser Ser Tyr
Leu Phe Asn Gly Lys 195 200 205 Asn Val Gln Ala Gln Ala Val Cys Gly
Pro Leu Phe Val Ile Asp His 210 215 220 Ala Gly Ser Leu Thr Ser Gln
Phe Ser Tyr Val Val Gly Arg Ser Ala 225 230 235 240 Leu Arg Ser Thr
Thr Gly Gln Ala Arg Ser Ala Asp Tyr Gly Ile Thr 245 250 255 Asp Cys
Asn Pro Leu Pro Ala Asn Asp Leu Thr Pro Glu Gln Lys Val 260 265 270
Ala Ala Ala Ala Leu Leu Ala Pro Ala Ala Ala Ala Ile Val Ala Gly 275
280 285 Pro Lys Gln Asn Cys Glu Pro Asp Leu Met Pro Tyr Ala Arg Pro
Phe 290 295 300 Ala Val Gly Lys Arg Thr Cys Ser Gly Ile Val Thr Pro
305 310 315 521DNAArtificial SequencePCR Primer 5gactggttcc
aattgacaag c 21621DNAArtificial SequencePCR Primer 6gcaaatggca
ttctgacatc c 21739DNAArtificial SequencePCR Primer 7cccatccttc
tcgtccccgg agtcggcacc acaggtcca 39839DNAArtificial SequencePCR
Primer 8gtcgaacgac tgtggacctg tggtgccgac tccggggac
39922DNAArtificial SequencePrimer 9cctggcgccg gcattggcag cc
221022DNAArtificial SequencePrimer 10ggctgccaat gccggcgcca gg
221123DNAArtificial SequenceGeneric forward primer for mutating
position 282 11ctggcgccgg cgnnngcagc cat 231223DNAArtificial
SequenceGeneric reverse primer for mutating position 282
12atggctgcnn ncgccggcgc cag 231315DNAArtificial SequenceForward
primer for I285F mutation 13gcagcctttg tggcg 151415DNAArtificial
SequenceReverse primer for I285F mutation 14cgccacaaag gctgc
151533DNAArtificial SequenceForward primer for A282/I285F mutation
15ccggcgcttg cagcctttgt ggcgggtcca aag 331633DNAArtificial
SequenceReverse primer for A282L/I285F mutation 16ctttggaccc
gccacaaagg ctgcaagcgc cgg 331715DNAArtificial SequenceForward
primer for L278S mutation 17gctctctgcg ccggc 151815DNAArtificial
SequenceReverse primer for L278S mutation 18gccggcgcag cgacg
151927DNAArtificial SequenceForward primer for A281V mutation
19cggctgcgct cctggctcct gtagctg 272027DNAArtificial SequenceReverse
primer for A281V mutation 20ctgcagctac aggagccagg agcgcag
272127DNAArtificial SequenceForward primer for A281E mutation
21cggctgcgct cctggctcct gaggctg 272227DNAArtificial SequenceReverse
primer for A281E mutation 22cagcctcagg agccaggagc gcagccg 27
* * * * *