U.S. patent application number 10/475150 was filed with the patent office on 2004-12-30 for enzymatic method for the enantioselective reduction of keto compounds.
Invention is credited to Bange, Gert, Breese, Klaus, Gupta, Antje, Neubauer, Peter.
Application Number | 20040265978 10/475150 |
Document ID | / |
Family ID | 7682020 |
Filed Date | 2004-12-30 |
United States Patent
Application |
20040265978 |
Kind Code |
A1 |
Gupta, Antje ; et
al. |
December 30, 2004 |
Enzymatic method for the enantioselective reduction of keto
compounds
Abstract
The inventon relates to an enzymatic method for the
enantioselective reduction of organic keto compounds to the
corresponding chiral hydroxy compounds, an alcohol dehydrogenase
from Lactobacillus minor and a method for the enantioselective
production of (S)-hydroxy compounds from a racemate.
Inventors: |
Gupta, Antje; (Wiesbaden,
DE) ; Breese, Klaus; (Halle, DE) ; Bange,
Gert; (Heidelberg, DE) ; Neubauer, Peter;
(Oulu, DE) |
Correspondence
Address: |
ProPat
Klause Schweitzer
2912 Crosby Road
Crosby Building
Charlotte
NC
28211-2815
US
|
Family ID: |
7682020 |
Appl. No.: |
10/475150 |
Filed: |
January 12, 2004 |
PCT Filed: |
April 15, 2002 |
PCT NO: |
PCT/EP02/04143 |
Current U.S.
Class: |
435/117 ;
435/155; 435/156 |
Current CPC
Class: |
C12P 7/04 20130101; C12P
7/62 20130101; C12R 2001/19 20210501; C12N 1/205 20210501; C12N
9/0006 20130101; C12P 41/002 20130101 |
Class at
Publication: |
435/117 ;
435/156; 435/155 |
International
Class: |
C12P 017/00; C12P
007/02; C12P 007/22 |
Foreign Application Data
Date |
Code |
Application Number |
Apr 20, 2001 |
DE |
101 19 274.6 |
Claims
1. A method for the enantioselective reduction of a keto compound
of the formula I R.sup.1--C(O)--R.sup.2 (I) where R.sup.1 and
R.sup.2 are, independently of one another, identical or different
and are 1. hydrogen, 2. --(C.sub.1-C.sub.20)-alkyl in which alkyl
is straight-chained or branched, 3. --(C.sub.2-C.sub.20)-alkenyl in
which alkenyl is straight-chained or branched and, optionally,
comprises one, two, three or four double bonds, 4.
--(C.sub.2-C.sub.20)-alkynyl in which alkynyl is straight-chained
or branched and, optionally, comprises one, two, three or four
triple bonds, 5. --(C.sub.6-C.sub.14)-aryl, 6.
--(C.sub.1-C.sub.8)-alkyl --(C.sub.6-C.sub.14)-aryl or 7. R.sup.1
and R.sup.2 form in combination with the --C(O) radical a
--(C.sub.6-C.sub.14)-aryl or a --(C.sub.5-C.sub.14)-hetercycle,
where the radicals defined above under 1. to 7. are unsubstituted
or, independently of one another, mono- to trisubstituted by a)
--OH, b) halogen such as fluorine, chlorine, bromine or iodine, c)
--NO.sub.2, d) --C(O)--O--(C.sub.1-C.sub.20)-alkyl in which alkyl
is linear or branched and unsubstituted or mono- to trisubstituted
by halogen, hydroxyl, amino or nitro, or e)
--(C.sub.5-C.sub.14)-heterocycle which is unsubstituted or mono- to
trisubstituted by halogen, hydroxyl, amino or nitro, said method
comprising a) a compound of the formula I with a proportion of
equal to/greater than 5% to 30%, based on the total volume of the
reaction mixture, alcohol dehydrogenase, water, cofactor NADPH or
NADH and an organic solvent immiscible with water and having a logP
of from 0.5 to 4.0; b) are incubated in a two-phase system of water
and organic solvent immiscible with water; c) the oxidized cofactor
produced by said alcohol dehydrogenase is steadily regenerated, and
d) the chiral hydroxy compound is isolated.
2. The method as claimed in claim 1, wherein a compound of the
formula I of the series ethyl 4-chloro-3-oxobutanoate,
acetophenone, methyl acetoacetate, ethyl 2-oxo-4-phenylbutyrate,
2,5-hexanedione, ethyl pyruvate or 2-octanone is used.
3. The method as claimed in claim 1, wherein an organic solvent
having a logP of from 0.6 to 3.0, is used.
4. The method as claimed in claim 3, wherein an organic solvent
having a logP of from 0.63 to 1.75 is used.
5. The method as claimed in claim 1, wherein the organic solvent
used is diethyl ether, tert-butyl methyl ether, diisopropyl ether
or ethyl acetate.
6. The method as claimed in claim 1, wherein an alcohol
dehydrogenases from yeast, equine liver, Thermoanaerobium brockii,
Rhodococcus erythropolis, Lactobacillus kefir, Lactobacillus
brevis, Lactobacillus minor or an alcohol dehydrogenase having the
amino acid sequence according to SEQ ID NO: 4 is used.
7. The method as claimed in claim 1, wherein a buffer selected from
potassium phosphate, Tris/HCl or triethanolamine buffer, having a
pH of from 5 to 10, is added.
8. The method as claimed in claim 7, wherein magnesium ions at a
concentration of from 0.2 mM to 10 mM are added to the buffer.
9. The method as claimed in claim 1, wherein the cofactor added is
NADPH or NADH in an amount of from 0.01 mM to 0.25 mM, based on the
aqueous phase.
10. The method as claimed in claim 1, wherein glycerol, sorbitol or
dimethyl sulfoxide is added as stabilizer for alcohol
dehydrogenase.
11. The method as claimed in claim 1, wherein isopropanol is
added.
12. The method as claimed in claim 1, wherein the compounds of the
formula I are used in an amount of from 5% to 30% based on the
total volume.
13. The method as claimed in claim 1, wherein the reaction is
carried out at a temperature of from about 10.degree. C. to
70.degree. C.
14. The method as claimed in claim 1, wherein the organic solvent
are used in an amount of from 1% to 90%, based on the total volume
of the reaction mixture.
15. The method as claimed in claim 1, wherein the ratio of organic
solvent to water is from 9 to 1 to 1 to 9.
16. The method as claimed in claim 10, wherein the stabilizer is
used in an amount of from 5% to 30%, based on the volume of the
total reaction mixture.
17. The method as claimed in claim 11, wherein isopropanol is used
in an amount of from 5% to 30%, based on the volume of the total
reaction mixture.
18. The method as claimed in claim 6, wherein the alcohol
dehydrogenase is used in an amount of from 20 000 U to 200 000 U
per kg of compound of the formula I to be reacted.
19. The method according to claim 18, comprising a Lactobacillus
minor alcohol dehydrogenase having the amino acid sequence
according to SEQ ID NO: 4.
20. The method according to claim 18, comprising a Lactobacillus
minor alcohol dehydrogenase having the amino acid sequence
according to SEQ ID NO: 3.
21. A method for obtaining the alcohol dehydrogenase from
Lactobacillus minor as claimed in claim 19, wherein the DNA coding
for Lactobacillus minor alcohol dehydrogenase is expressed in a
suitable prokaryotic or eukaryotic microorganism, in particular in
cells of Escherichia coli cell deposited under DSM 14196, and,
optionally, said alcohol dehydrogenase is purified.
22. A method for obtaining an enantioselective (S)-hydroxy compound
of the formula II R.sup.1--C(OH)--R.sup.2 (II) where R.sup.1 and
R.sup.2 are, independently of one another, identical or different
and are 1. hydrogen, 2. --(C.sub.1-C.sub.20)-alkyl in which alkyl
is straight-chained or branched, 3. --(C.sub.2-C.sub.20)-alkenyl in
which alkenyl is straight-chained or branched and, optionally,
comprises one, two, three or four double bonds, 4.
--(C.sub.2-C.sub.20)-alkynyl in which alkynyl is straight-chained
or branched and, optionally, comprises one, two, three or four
triple bonds, 5. --(C.sub.6-C.sub.14)-aryl, 6.
--(C.sub.6-C.sub.8)-alkyl--(C.sub.6-C.sub.14)-aryl or 7. R.sup.1
and R.sup.2 form in combination with the --C(O) radical a
--(C.sub.6-C.sub.14)-aryl or a --(C.sub.6-C.sub.14)-heterocycle,
where the radicals defined above under 1. to 7. are unsubstituted
or, independently of one another, mono- to trisubstituted by a)
--OH, b) halogen such as fluorine, chlorine, bromine or iodine, c)
--NO.sub.2, d) --C(O)--O--(C.sub.1-C.sub.20)-alkyl in which alkyl
is linear or branched and unsubstituted or mono- to trisubstituted
by halogen, hydroxyl, amino or nitro, or e)
--(C.sub.6-C.sub.14)-heterocycle which is unsubstituted or mono- to
trisubstituted by halogen, hydroxyl, amino or nitro, said method
comprising a) a racemic mixture comprising the compound of the
formula II, alcohol dehydrogenase, water, cofactor NADP or NAD and
an organic solvent having a logP of from 0.6 to 1.9, from the
series diethyl ether, tert-butyl methyl ether, diisopropyl ether,
ethyl acetate, b) is incubated in a two-phase system of water and
organic solvent immiscible with water and C) the enantiomerically
pure (S)-hydroxy compound is isolated.
23. The method as claimed in claim 22, wherein acetone is added.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to an enzymic method for the
enantioselective reduction of organic keto compounds to give the
corresponding chiral hydroxy compounds, to an alcohol dehydrogenase
from Lactobacillus minor and to an enzymic method for
enantioselectively obtaining (S)-hydroxy compound from a
racemate.
BACKGROUND OF THE INVENTION
[0002] Optically active hydroxy compounds are valuable synthetic
building blocks for the preparation of a multiplicity of
pharmacologically important compounds. These compounds are often
difficult to prepare by conventional chemical methods and only
rarely attain the enantiomeric purity required for pharmacological
applications. Therefore, biotechnological methods are usually
employed in preparing chiral compounds, the stereoselective
reaction being carried out either by whole microorganisms or using
isolated enzymes.
[0003] The use of isolated enzymes has often proved advantageous
here, since higher yields and a higher enantiomeric purity are
usually attainable by using such enzymes.
[0004] Dehydrogenases and in particular alcohol dehydrogenases are
valuable catalysts for obtaining chiral products by stereoselective
reduction of organic keto compounds to the corresponding chiral
alcohols. Known enzymes are essentially the corresponding enzymes
from yeast, equine liver or Thermoanaerobium brockii. These enzymes
require NADH (nicotine adenine dinucleotide) or NADPH (nicotine
adenine dinucleotide phosphate) as coenzyme. Other examples of
known alcohol dehydrogenases are an (S)-specific alcohol
dehydrogenase from Rhodococcus erythropolis and an (R)-specific
alcohol dehydrogenase from the genus Lactobacillus. Both enzyme
types have a broad spectrum of keto compound substrates and have
high enantioselectivity. The alcohol dehydrogenases from
Lactobacillus kefir (DE 40 14 573) and Lactobacillus brevis (DE 196
10 984) are particularly suitable for obtaining chiral
(R)-alcohols.
[0005] However, the disadvantages of employing alcohol
dehydrogenases are the low enzyme stability and enzyme activity of
alcohol dehydrogenases in organic solvents and the frequently only
poor water solubility of the keto compounds to be reduced. Another
limiting factor for employing alcohol dehydrogenases in organic
solvents is furthermore the necessary use of NADP or NAD as
cofactor requirement, since the cofactor (NADP, NAD) is
water-soluble and is regenerated by economical methods.
[0006] It is the object of the invention to improve said
disadvantages by modifying the method conditions. This object is
achieved according to the invention by using a two-phase system
comprising an organic solvent, alcohol dehydrogenase, water,
cofactor and keto compound.
[0007] The method of the invention has a long stability time due to
the enzyme-stabilizing action of the solvent, an enantiomeric
purity of more than 99.9% of the prepared chiral hydroxy compounds
and a high yield based on the amount of keto compound used.
BRIEF DESCRIPTION OF THE INVENTION
[0008] The method of the invention therefore relates to a method
for the enantioselective reduction of a keto compound of the
formula I
R.sup.1--C(O)--R.sup.2 (I)
[0009] where R.sup.1 and R.sup.2 are, independently of one another,
identical or different and are
[0010] hydrogen,
[0011] 1. --(C.sub.1-C.sub.20)-alkyl in which alkyl is
straight-chained or branched,
[0012] 2. --(C.sub.2-C.sub.20)-alkenyl in which alkenyl is
straight-chained or branched and, optionally, comprises one, two,
three or four double bonds,
[0013] 4. --(C.sub.2-C.sub.20)-alkynyl in which alkynyl is
straight-chained or branched and, optionally, comprises one, two,
three or four triple bonds,
[0014] 5. --(C.sub.6-C.sub.14)-aryl,
[0015] 6. --(C.sub.1-C.sub.8)-alkyl-(C.sub.6-C.sub.14)-aryl or
[0016] 7. R.sup.1 and R.sup.2 form in combination with the --C(O)
radical a --(C.sub.6-C.sub.14)-aryl or a
--(C.sub.5-C.sub.14)-heterocycle,
[0017] where the radicals defined above under 1. to 7. are
unsubstituted or, independently of one another, mono- to
trisubstituted by
[0018] a) --OH,
[0019] b) halogen such as fluorine, chlorine, bromine or
iodine,
[0020] c) --NO.sub.2,
[0021] d) --C(O)--O--(C.sub.1-C.sub.20)-alkyl in which alkyl is
linear or branched and unsubstituted or mono- to trisubstituted by
halogen, hydroxyl, amino or nitro, or
[0022] e) --(C.sub.5-C.sub.14)-heterocycle which is unsubstituted
or mono- to trisubstituted by halogen, hydroxyl, amino or
nitro,
[0023] characterized in that
[0024] a) the compound of the formula I, alcohol dehydrogenase,
water, cofactor and an organic solvent having a logP of from 0.5 to
4.0 are incubated
[0025] b) in a two-phase system and
[0026] c) the chiral hydroxy compound is isolated.
DETAILED DESCRIPTION OF THE INVENTION
[0027] Carbon atoms in the ring. Examples of
--(C.sub.6-C.sub.14)-aryl radicals are phenyl, naphthyl. The term
aryl means aromatic carbon radicals having from 6 to 14, for
example 1-naphthyl, 2-naphthyl, biphenylyl, for example
2-biphenylyl, 3-biphenylyl and 4-biphenylyl, anthryl or fluorenyl.
Preferred aryl radicals are biphenylyl radicals, naphthyl radicals
and in particular phenyl radicals. The term "halogen" means an
element of the series fluorine, chlorine, bromine or iodine. The
term "--(C.sub.1-C.sub.20)-alkyl" means a hydrocarbon radical whose
carbon chain is straight-chained or branched and comprises from 1
to 20 carbon atoms.
[0028] The term "--(C.sub.5-C.sub.14)-heterocycle" represents a
monocyclic or bicyclic 5-membered to 14-membered heterocyclic ring
which is partially or completely saturated. Examples of heteroatoms
are N, O and S. Examples of the
terms--(C.sub.5-C.sub.14)-heterocycle are radicals derived from
pyrrole, furan, thiophene, imidazole, pyrazole, oxazole, isoxazole,
thiazole, isothiazole, tetrazole, 1,2,3,5-oxathiadiazole 2-oxides,
triazolones, oxadiazolones, isoxazolones, oxadiazolidinediones,
triazoles, which are substituted by F, --CN, --CF.sub.3 or
--C(O)--O--(C.sub.1-C.sub.4)-alkyl, 3-hydroxypyrro-2,4-diones,
5-oxo-1,2,4-thiadiazoles, pyridine, pyrazine, pyrimidine, indole,
isoindole, indazole, phthalazine, quinoline, isoquinoline,
quinoxaline, quinazoline, cinnoline, carboline and benzo-fused,
cyclopenta-, cyclohexa- or cyclohepta-fused derivatives of these
heterocycles. Particular preference is given to the radicals 2- or
3-pyrrolyl, phenylpyrrolyl such as 4- or 5-phenyl-2-pyrrolyl,
2-furyl, 2-thienyl, 4-imidazolyl, methylimidazolyl, for example
1-methyl-2-, -4- or -5imidazolyl, 1,3-thiazol-2-yl, 2-pyridyl,
3-pyridyl, 4-pyridyl, 2-, 3- or 4-pyridyl N-oxide, 2-pyrazinyl, 2-,
4- or 5-pyrimidinyl, 2-, 3- or 5-indolyl, substituted 2-indolyl,
for example 1-methyl-, 5-methyl-, 5-methoxy-, 5-benzyloxy-,
5-chloro- or 4,5-dimethyl-2 -indolyl, 1-benzyl-2- or -3-indolyl,
4,5,6,7-tetrahydro-2-indolyl, cyclohepta[b]-5-pyrrolyl, 2-, 3- or
4-quinolyl, 1-, 3- or 4-isoquinolyl,
1-oxo-1,2-dihydro-3-isoquinolyl, 2-quinoxalinyl, 2-benzofuranyl,
2-benzothienyl, 2-benzoxazolyl or benzothiazolyl or
dihydropyridinyl, pyrrolidinyl, for example 2- or
3-(N-thiomorpholidinyl), piperazinyl, morpholinyl, thiomorpholinyl,
tetrahydrothienyl or benzodioxolanyl.
[0029] Preferred compounds of the formula I are ethyl
4-chloro-3-oxo-butanoate, acetophenone, methyl acetoacetate, ethyl
2-oxo-4-phenylbutyrate, 2,5-hexanedione, ethyl pyruvate and
2-octanone, preferably ethyl 4-chloro-3-oxobutanoate. The compounds
of the formula I are used in the method of the invention in an
amount of from 2% to 30%, based on the total volume, preferably
from 10% to 25%, in particular from 15% to 22%.
[0030] Preference is given to adding to the water a buffer, for
example potassium phosphate buffer, Tris/HCl buffer or
triethanolamine buffer, having a pH of from 5 to 10, preferably a
pH of from 6 to 9. The buffer concentration is from 10 mM to 150
mM, preferably from 90 mM to 110 mM, in particular 100 mM. The
buffer additionally also contains magnesium ions, for example
MgCl.sub.2, at a concentration of from 0.2 mM to 10 mM, preferably
from 0.5 to 2 mM, in particular 1 mM.
[0031] The temperature is, for example, from about 10.degree. C. to
70.degree. C., preferably from 30.degree. C. to 60.degree. C.
[0032] The organic solvents that can be used according to the
invention preferably have a logP of from 0.6 to 2.0, in particular
from 0.6 to 1.9, particularly preferably from 0.63 to 1.75.
Examples of preferred organic solvents are diethyl ether,
tert-butyl methyl ether, diisopropyl ether, dibutyl ether and ethyl
acetate, in particular ethyl acetate. Ethyl acetate may be used,
for example, in an amount of from 1% to 90%, based on the total
volume of the reaction mixture, preferably from 15% to 60%, in
particular from 20% to 50%.
[0033] The ratio of organic solvent to water is from 9 to 1 to 1 to
9, preferably from 1 to 1 to 1 to 3.
[0034] One liquid phase of the two-phase system of the invention is
water and the second liquid phase is the organic solvent.
Optionally, there may also still be a solid or another liquid phase
produced, for example, by incompletely dissolved alcohol
dehydrogenase or by the compound of the formula I. Preference,
however, is given to two liquid phases without solid phase. The two
liquid phases are preferably mixed mechanically so as to generate
large surfaces between the two liquid phases.
[0035] The concentration of the NADPH or NADH cofactor, based on
the aqueous phase, is from 0.05 mM to 0.25 mM, in particular from
0.06 mM to 0.2 mM.
[0036] Preference is given to using in the method of the invention
also another stabilizer of alcohol dehydrogenase. Examples of
suitable stabilizers are glycerol, sorbitol or dimethyl sulfoxide
(DMSO).
[0037] The amount of glycerol is from 5% to 30%, based on the
volume of the total mixture. Preferred amounts of glycerol are from
10% to 20%, in particular 20%.
[0038] It is possible to add in the method of the invention
additionally isopropanol in order to regenerate the NADH or NADPH
consumed. For example, alcohol dehydrogenase converts the
isopropanol and NADP to NADPH and acetone.
[0039] The amount of isopropanol used is from 5% to 30%, based on
the volume of the total mixture. Preferred amounts of isopropanol
are from 10% to 20%, in particular 10%.
[0040] Examples of suitable alcohol dehydrogenases are derived from
yeast, equine liver or Rhodococcus erythropolis, said enzymes
requiring NADH as coenzyme, or from Thermoanaerobium brockii,
Lactobacillus kefir or Lactobacillus brevis, the latter enzymes
requiring NADPH as coenzyme.
[0041] If, for example, an alcohol dehydrogenases of yeast, equine
liver, Thermoanaerobium brockii or Rhodococcus erythropolis is used
in the method of the invention, then the corresponding (S)-hydroxy
compound is obtained from the compound of the formula I. If, for
example, an alcohol dehydrogenases of Lactobacillus kefir or
Lactobacillus brevis is used in the method of the invention, then
the corresponding (R)-hydroxy compound is obtained from the
compound of the formula I.
[0042] The alcohol dehydrogenase may be used in the method of the
invention either in completely purified or in partially purified
form or when inside cells. The cells used here may be in native,
permeabilized or lysed form.
[0043] The volume activity of the alcohol dehydrogenase used is
from 100 units/ml (U/ml) to 2000 U/ml, preferably about 800 U/ml,
with a protein content of about 20 mg/ml to 22 mg/ml. The alcohol
dehydrogenase preferably used has a specific activity of from about
35 to 40 U/mg of protein. From 20 000 to 200 000 U, preferably
about 100 000 U, of alcohol dehydrogenase are used per kg of
compound of the formula I to be converted. The enzyme unit 1 U
corresponds to the amount of enzyme required in order to convert 1
.mu.mol of the compound of the formula I per minute (min).
[0044] The method of the invention is carried out, for example, in
a closed reaction vessel made of glass or metal. For this purpose,
the components are transferred individually into the reaction
vessel and stirred, for example, under a nitrogen or air atmosphere
stirring. The reaction time is from 1 day to 14 days, preferably
from 4 to 7 days, depending on the substrate and the compound of
the formula I used.
[0045] The reaction mixture is subsequently worked up. For this
purpose, the aqueous phase is removed and the ethyl acetate phase
is filtered. The aqueous phase can, optionally, be extracted once
more and worked up further like the ethyl acetate phase. This is
followed by evaporating the filtered phase under reduced pressure.
This results, for example, in the product ethyl
4-chloro-3(S)-hydroxybutsnoate which has an enantiomeric purity of
more than 99.9% and is essentially free of the reactant ethyl
4-chloro-3-oxo-butanoate. After distillation of the product, the
total yield of the processes is from 82% to 88%, based on the
amount of reactant used.
[0046] Surprisingly, the organic solvents having a logP of from 0
to 4 demonstrate a stabilizing action on alcohol dehydrogenase,
while the prior art advises against the use of the two-phase
systems with organic solvents (M. R. Kula, U. Kragel; chapter 28,
Dehydrogenases in Synthesis of Chiral Compounds; R. N. Patel,
Stereoselective Biocatalyses, 2000; Peters J. 9.
Dehydrogenases-Characteristics, Design of Reaction Conditions, and
Application, In: H. J. Rehm, G. Reed Biotechnology, Vol. 3,
Bioprocessing, V C H Weinheim, 1993; J. Lynda et al., Solvent
selection strategies for extractive Biocatalysis, Biotechnol. Prog.
1991, 7, pages 116-124). The organic phase used in the method of
the invention is ethyl acetate, said organic phase serving on the
one hand as reservoir for the compound of the formula I but also
the reaction product, the chiral hydroxy compound, being
simultaneously extracted from the aqueous phase.
[0047] In contrast to the prior art, the use of organic solvents
having a log-P value of from 0 to 3 results in an additional
stabilization of alcohol dehydrogenase, which increases with time.
In the prior art, organic solvents having a log-P value (logarithm
of the octanol/water distribution coefficient) of from 0 to 2, in
particular, have a particularly destabilizing action on enzymes and
are thus hardly considered as organic phase in the two-phase system
(K. Faber, Biotransformations in organic chemistry, 3.sup.rd
edition 1997, Springer Verlag, chapter 3.to 3.17).
[0048] The invention further relates to Lactobacillus minor alcohol
dehydrogenase which has a high temperature optimum. Lactobacillus
minor alcohol dehydrogenase has the DNA sequence according to SEQ
ID NO: 3 and the amino acid sequence according to SEQ ID NO: 4
according to the attached sequence protocol. Said Lactobacillus
minor alcohol dehydrogenase is R-specific, and it is possible, for
example, to obtain from a compound of the formula I the
corresponding (R)-hydroxy compound. Surprisingly, the
enantioselective alcohol dehydrogenase from Lactobacillus minor can
be overexpressed in Escherichia coli RB 791, while alcohol
dehydrogenases of other species of the genus Lactobacillus were
expressed only to a substantially lower extent. This is all the
more surprising, since the wild-type strain of Lactobacillus minor
itself expresses only very small amounts of alcohol dehydrogenase
which was therefore not detectable by common screening methods
(whole cell biotransformation, activity assay). It was therefore
very surprising that it was possible to clone an R-enantioselective
alcohol dehydrogenase from Lactobacillus minor and to overexpress
it in Escherichia coli to such an extraordinarily large extent (50%
of the cellular proteins of the clone, 20 000 units/g of wet
mass).
[0049] The purified enzyme from Lactobacillus minor is stable in a
pH range from about 5.5 to 8.5. The enzyme is stable to about
40.degree. C. and the pH optimum of the enzymic reaction is in the
range from pH 7 to pH 7.5. The temperature optimum of the enzymic
reaction is about 55.degree. C. The enzyme has a broad spectrum of
substrates.
[0050] The enzyme can be purified to a specific activity of from 35
to 40 U/mg of protein by means of hydrophobic interaction
chromatography.
[0051] The invention also relates to a method for obtaining alcohol
dehydrogenase from Lactobacillus minor. For this purpose, the DNA
coding for Lactobacillus minor alcohol dehydrogenase is expressed
in a suitable prokaryotic or eukaryotic microorganism.
Lactobacillus minor alcohol dehydrogenase is preferably transformed
into and expressed in an Escherichia coli strain, in particular in
Escherichia coli RB 791.
[0052] Lactobacillus minor alcohol dehydrogenase can be obtained,
for example, in such a way that the recombinant Escherichia coli
cells are cultured, expression of said alcohol dehydrogenase is
induced and then, after about 10 to 18 hours (h), the cells are
disrupted by ultrasound treatment or by means of a French press
(Gaullin, Siemens). The cell extract obtained may either be used
directly or be purified further. For this purpose, the cell extract
is centrifuged, for example, and the supernatant obtained is
subjected to a hydrophobic interaction chromatography. Said
chromatography is preferably carried out at pH 7.0 in an aqueous
buffer which also contains magnesium ions.
[0053] The invention further relates to a method for obtaining an
enantioselective (S)-hydroxy compound of the formula II
R.sup.1--C(OH)--R.sup.2 (II)
[0054] where R.sup.1 and R.sup.2 are, independently of one another,
identical or different and are
[0055] 1. hydrogen,
[0056] 2. --(C.sub.1-C.sub.20)-alkyl in which alkyl is
straight-chained or branched,
[0057] 3. --(C.sub.2-C.sub.20)-alkenyl in which alkenyl is
straight-chained or branched and, optionally, comprises one, two,
three or four double bonds,
[0058] 4. --(C.sub.2-C.sub.20)-alkynyl in which alkynyl is
straight-chained or branched and, optionally, comprises one, two,
three or four triple bonds,
[0059] 5. --(C.sub.6-C.sub.14)-aryl,
[0060] 6. --(C.sub.1-C.sub.8)-alkyl --(C.sub.6-C.sub.14)-aryl
or
[0061] 7. R.sup.1 and R.sup.2 form in combination with the --C(O)
radical a --(C.sub.6-C.sub.14)-aryl or a
--(C.sub.6-C.sub.14)-heterocycle,
[0062] where the radicals defined above under 1. to 7. are
unsubstituted or, independently of one another, mono- to
trisubstituted by
[0063] a) --OH,
[0064] b) halogen such as fluorine, chlorine, bromine or
iodine,
[0065] c) --NO.sub.2,
[0066] d) --C(O)--O--(C.sub.1-C.sub.20)-alkyl in which alkyl is
linear or branched and unsubstituted or mono- to tri-substituted by
halogen, hydroxyl, amino or nitro, or
[0067] e) --(C.sub.6-C.sub.14)-heterocycle which is unsubstituted
or mono- to tri-substituted by halogen, hydroxyl, amino or
nitro,
[0068] characterized in that
[0069] a) a racemic mixture comprising the compound of the formula
II, the alcohol dehydrogenase of the invention, water, cofactor and
an organic solvent an organic solvent having a logP of from 0.5 to
4.0, for example from the series diethyl ether, tert-butyl methyl
ether, diisopropyl ether or ethyl acetate, is incubated
[0070] b) in a two-phase system and
[0071] c) the enantiomerically pure (S)-hydroxy compound formed is
isolated.
[0072] The reaction conditions are essentially the same as in the
abovementioned method for the enantiospecific reduction of the keto
compound of the formula I. However, instead of enantioselectively
reducing the keto compound of the formula I, the method comprises
oxidizing the corresponding (R)-hydroxy compound of the formula II
to the corresponding keto compound. Furthermore, the method uses
acetone rather than isopropanol for regenerating NADP. For example,
the alcohol dehydrogenase of the invention converts acetone and
NADPH to NADP and isopropanol. The amount of acetone used is from
5% to 30%, based on the volume of the total mixture. Preferred
amounts of acetone are from 10% to 20%, in particular 10%.
[0073] The alcohol dehydrogenase of the invention may be present
for preparation of the compound of the formula II in either
completely or partially purified form or may also be used in said
method when inside cells. Said cells may be present here in a
native, permeabilized or lysed form.
[0074] The invention also relates to a recombinant Escherichia coli
clone, RB 791, which expresses Lactobacillus minor alcohol
dehydrogenase and which was deposited under the conditions of the
Budapest Treaty with the Deutsche Sammlung fur Mikroorganismen und
Zellkulturen, Mascheroder Weg 1b, 38124 Brunswick on Mar. 26, 2001
under the number DSM 14196.
[0075] The invention is illustrated by the following examples:
EXAMPLE 1
Screening for R-alcohol Dehydrogenases in Strains of the Genus
Lactobacillus by Means of Whole Cell Biotransformation
[0076] Various Lactobacillus strains were cultured for screening in
the following medium (numbers in each case in g/l): glucose (20),
yeast extract (5), meat extract (10), diammonium hydrogen citrate
(2), sodium acetate (5), magnesium sulfate (0.2), manganese sulfate
(0.05), dipotassium hydrogen phosphate (2).
[0077] The medium was sterilized at 121.degree. C. and the strains
of the genus Lactobacillus (abbreviated to L. hereinbelow) were
cultured without further pH regulation or addition of oxygen. The
cells were subesequently removed by centrifugation, and in each
case 4 g of cells were resuspended for whole cell biotransformation
in a final volume of 10 ml of potassium phosphate buffer (KPi
buffer) (50 mM, pH=7.0). After addition of in each case 0.1 g of
glucose, the cells were shaken at 30.degree. C. for 15 min. Ethyl
4-chloro-3-oxo-butanoate was added at a final concentration of 40
mM to the cell suspension, and the medium was analyzed by gas
chromatography in each case after 10 min and 120 min. For this
purpose, the cells were removed by centrifugation, the supernatant
was filtered and diluted in chloroform to a final concentration of
10-15 .mu.g/ml of ethyl 4-chloro-3-oxobutanoate.
[0078] The various Lactobacillus strains were used to convert ethyl
4-chloro-3-oxobutanoate, used as substrate, with the following
enantiomeric purity to ethyl(S)-4-chloro-3-hydroxybutyrate.
[0079] The enantiomeric excess is calculated as follows:
ee(%)=((R-alcohol-S-alcohol)/(R-alcohol+S-alcohol)).times.100.
1 TABLE 1 ee of ethyl 4-chloro-3- Lactobacillus strain
(S)-hydroxybutanoate in % L. reuteri 34.6 L. kandleri 90 L.
collinoides 71.3 L. bifermentans 53.6 L. oris 63.4 L. brevis 74 L.
halotolerans 67.2 L. minor 18.6 L. parabuchneri 78.5 L. kefir 87.8
L. fructosus 28.9
EXAMPLE 2
Obtaining Recombinant R-Specific Alcohol Dehydrogenases
[0080] A.) Preparation of Genomic DNA from Strains of the Genus
Lactobacillus
[0081] The cell pellet of approximately 2 ml of culture liquid of
the genus Lactobacillus was resuspended in 300 .mu.l of TE buffer
(containing 10 mM Tris/HCl, pH=8.1 mM EDTA), admixed with 20 mg/ml
lysozyme and incubated at 37.degree. C. for 10 min. This was
followed by adding 100 .mu.l of sodium dodecylsulfate (SDS) (10%),
100 .mu.l of sodium perchlorate (5M) and 500 .mu.l of
chloroform/isoamyl alcohol (24:1). After shaking vigorously, the
protein was removed by centrifugation and the aqueous phase
transferred to a new Eppendorf vessel and this was followed by
adding 800 .mu.l of ethanol (EtOH) (96%). The Eppendorf vessel was
inverted several times and the precipitated chromosomal DNA then
transferred to a new Eppendorf vessel and washed with 200 .mu.l of
EtOH. The DNA was again transferred to a new Eppendorf vessel,
dried under reduced pressure and dissolved in 100 .mu.l of TE
buffer.
[0082] B.) Oligonucleotides as 5' and 3' primers for PCR
(Polymerase Chain Reaction)
[0083] The primers used for the PCR were derived from the known
N-terminal and C-terminal sequence of L. kefir alcohol
dehydrogenase, with known preferences for particular codons in
lactobacilli being taken into account. Thus, the codon ATG (Met) as
start codon was put in front of each 5' primer, and furthermore the
cleavage site for the restriction enzyme Bam HI (GGATCC) was
located upstream of said start codon on the 5' primer, in order to
enable subsequent cloning into the expression sector. The stop
codon (TAG) and the cleavage site for Hind III (AAGCTT) were placed
downstream of the 3' primer. The primer constructs are listed
below:
[0084] N=A, C or G; Y=T or C; R=A or G
2 5' primer 5'GCGGATCCATGACNGAYCGNTTRAARGGNAARGTN (SEQ ID NO:1)
GC3' 3' primer 5'GGGAAGCTTCTAYTGNGCNGTRTANCCNCCRTCNA (SEQ ID NO:2)
C3'
[0085] The primers were prepared according to known methods.
[0086] C.) PCR (Polymerase Chain Reaction) with Genomic DNA from
Strains of the Genus Lactobacillus
[0087] PCR Mixture (100 .mu.l):
3 Amount used per reaction Concentration dNTP's 8 .mu.l per NTP 2.5
nmol/.mu.l Oligos per oligo 10 .mu.l: 20 .mu.l 2 pmol/.mu.l
Chromosomal DNA 3 .mu.l ca. 1 .mu.g/.mu.l 10 .times. buffer 10
.mu.l (Promega) Taq polymerase 1 .mu.l 2 U/.mu.l (Promega) H.sub.2O
58 .mu.l dNTPs are a mixture of deoxynucleotide triphosphates such
as dATP, dGTP, dCTP, dTTP
[0088] Cycle:
[0089] 95.degree. C. for 2 min, followed by maintaining 80.degree.
C.
[0090] hot start, followed by
[0091] 95.degree. C. for 30 sec, followed by
[0092] 40.degree. C. for 1 min 30.times.
[0093] followed by in each case 30 times 95.degree. C. for 30 s and
40.degree. C. for 1 min, then
[0094] 72.degree. C. for 2.5 min, followed by
[0095] 72.degree. C. for 2.5 min followed by
[0096] maintaining 10.degree. C.
[0097] For analysis, 10 .mu.l of the mixture were applied to a 1%
strength agarose gel and electrophoretically fractionated at a
constant 100 V. The PCR revealed distinct amplification of a DNA
piece of approximately 750 bp.
[0098] D.) Isolation of PCR Fragments from the Gel
[0099] In order to obtain the PCR fragment, the entire PCR mixture
was applied to a 1% strength agarose gel and electrophoretically
fractionated at a constant 100 V. For this purpose, the gel was
divided into two lanes, one containing the complete PCR mixture and
the other one containing only a sample of 5 .mu.l, so that the PCR
fragment was excised from the gel by staining with ethidium bromide
only the lane with the sample for orientation purposes, in order to
rule out damage due to ethidium bromide and UV light of the PCR
fragment to be isolated.
[0100] Isolation from the gel was carried out using the QIAquick
Gel Extraction Kit from Qiagen, Hilden, Germany.
[0101] A total concentration of 20 ng/.mu.l DNA was determined.
[0102] E.) Ligation
[0103] To prepare the ligation, the purified PCR fragment and the
cloning vector used, pQE30 or pQE70, both from Quiagen, were
cleaved with Bam HI and Hind III (4 .mu.l of DNA=200 ng of DNA, 1
.mu.l of 10.times. buffer, 1 .mu.l of enzyme, BSA and H.sub.2O
(Biolabs, New England)).
[0104] The cleaved plasmid was then purified again by means of the
QIAquick Gel Extraction Kit, taken up in water, dephosphorylated by
means of alkaline phosphatase (USB, Amersham Life Science).
[0105] For purification, the appropriate reaction mixtures were
again applied to a 1% strength agarose gel, and thus the digested
amplicon and the plasmid were isolated from the gel, as described
under D.). The concentration of plasmid and amplicon after
purification was approximately 20 ng/.mu.l.
[0106] For ligation, 3 .mu.l of pQE30 or pQE70 (60 ng), 2.5 .mu.l
of amplicon (50 ng), 2 .mu.l of ligase buffer (Boehringer;
Mannheim), 1.5 .mu.l of H.sub.2O and 1 .mu.l of T4 ligase
(Boehringer; Mannheim) were used. The mixture was incubated at
16.degree. C. overnight.
[0107] Subsequently, 40 .mu.l of electrocompetent Escherichia coli
RB791 cells were transformed with 1.5 .mu.l of ligation mixture by
electroporation. The cells were introduced to 500 .mu.l of SOC
medium, incubated at 37.degree. C. for 45 min and then in each case
250 .mu.l were plated out on LB.sub.amp agar plates. The SOC medium
contains per liter of water 20 g of tryptone, 5 g of yeast extract,
0.5 g of NaCl, 10 ml of 1 M MgSO.sub.4 and 10 ml of 1 M MgCl.sub.2.
LB.sub.ampagar plates contain per liter of water 10 g of tryptone,
5 g of yeast extract, 10 g of NaCl, 20 g of agar, pH 7.0, and 50 mg
of ampicillin.
[0108] Grown colonies were removed and cultured in 4 ml of liquid
culture (LB.sub.amp medium) at 37.degree. C. overnight. In each
case 2 ml of this cell suspension were used for plasmid preparation
(according to the Quiagen miniprep protocol (Quiagen, Hilden,
Germany)).
[0109] The plasmid was prepared starting with a Bam HI and Hind III
restriction digest. The complete digest was applied to a 1%
strength agarose gel and electrophoretically fractionated at 100 V
(detection of the 750 kp insert), followed by using the plasmids
for sequencing, optionally.
[0110] Clones having the 750 kp insert were then plated out on
LB.sub.amp agar plates.
[0111] F.) Sequencing of Plasmids
[0112] Sequencing was carried out by means of SequiThermEXCEL II
Long-Read DNA Sequencing Kit (Biozym, Oldendorf, Germany) on an
Li-Cor sequencer (MWG Biotech, Ebersberg, Germany), according to
the manufacturer's instructions. The primers used were the standard
sequencing primers for pQE vectors.
[0113] G.) Screening of Clones with Respect to Soluble R-ADH
Expression
[0114] Clones having inserts of 750 kp were studied with regard to
enzymic activity and stereoselectivity. For this purpose, the
clones were removed from the LB.sub.amp agar plates and cultured in
20 ml of liquid cultures (LB.sub.amp medium) at 25.degree. C. and
then, at a cell density (OD.sub.500) of 0.5, induced with 1 mM
isopropyl-.beta.-D-thiogalactopyra- noside (IPTG). After 18 h, the
cells were removed by centrifugation and in each case 40 mg of
cells were taken up in 350 .mu.l of Kpi buffer (50 mM, pH=7, 1 mM
MgCl.sub.2). The enzyme was liberated from the cells by wet
grinding with the aid of glass beads (0.5 g, 0.3 mm). In addition,
the cells were disrupted by means of a Retsch mill at 4.degree. C.
for 20 minutes.
[0115] The enzyme assay contained 870 .mu.l of triethanolamine
buffer (100 mM, pH=7.0, 1 mM MgCl.sub.2), 100 .mu.l of a 100 mM
solution of ethyl 4-chloro-acetoacetate, 10 .mu.l of NADPH (final
concentration 0.19 mM) and 20 .mu.l of enzyme solution.
[0116] Enzyme unit definition: 1 U corresponds to the amount of
enzyme required for converting 1 .mu.mol of substrate (ethyl
4-chloro-3-oxobutanoate) per 1 min.
[0117] Stereoselectivity was detected by incubating 480 .mu.l of
triethanolamine buffer (100 mM, pH=7.0, 1 mM MgCl.sub.2) with 1.0
mM ethyl 4-chloro-3-oxobutanoate, 1.9 mM NADPH (in each case final
concentration) and 20 .mu.l of enzyme solution. After incubating
for 15 min, the reaction mixture was filtered and diluted 1:10 in
chloroform, and a sample was analyzed by means of GC-MS.
[0118] Conditions of Gas Chromatography (GC):
[0119] Chiral column: Lipodex E, ID=0.25 mm, 1=25 m
(Macherey-Nagel)
[0120] 1. 2 min 60.degree. C.
[0121] 2. in 28 min from 60.degree. C. to 130.degree. C. with a
rate of 2.5.degree. C. per minute
[0122] 3. 15 min at 130.degree. C.
[0123] An (R)-specific alcohol dehydrogenase was able to be cloned
and actively overexpressed from the following Lactobacillus
strains:
4 Activity Clone in U/g of Strain Plasmid number cells* ee in % L.
parabuchneri pQE30 12 450 >99.9 L. parabuchneri pQE30 14 170
>99.9 L. kandleri pQE30 11 280 >99.9 L. kandleri pQE70 17 710
>99.9 L. minor pQE30 2 2 830 >99.9 L. minor pQE70 3 680
>99.9 L. minor pQE70 4 700 >99.9 *Activity calculated from
G.) (wet grinding); the activities are considerably higher after
fermentation and disruption by French press.
[0124] H.) Enzyme Obtainment and Purification
[0125] The enzyme was obtained by culturing the strain with the
highest enzymic activity in a fermenter (fed batch, 10 l) and
inducing it at 40 OD.sub.500 with 1 mM IPTG. After 18 h, the cells
were harvested, taking up 300 g of cells in 3 l of Kpi buffer (50
mM, pH=7, 1 mM MgCl.sub.2) and disrupted subsequently by means of a
French press (Gaullin, Siemens). The supernatant obtained after
centrifugation is referred to as crude extract hereinbelow and had
a volume activity of approximately 2000 U/ml (20 000 U/g of wet
mass).
[0126] To characterize the enzyme, a portion of the enzyme obtained
was purified by means of hydrophobic interaction chromatography on
Q-Sepharose ff (fast flow). For this purpose, the column used was
equilibrated with 50 mM Kpi buffer pH=7.0, 1 mM MgCl.sub.2. After
application of the crude extract to the column and brief washing
with equilibration buffer, the enzyme was eluted with an increasing
linear salt gradient (0-1M NaCl, 1 ml/min) at a salt concentration
of about 0.3 M NaCl. Combining the enzyme-containing fractions
resulted in approximately 25 ml of purified enzyme with a volume
activity of about 800 U/ml and a protein content of from 20 to 22
mg/ml. The enzyme purified in this way thus has a specific activity
of approximately 35 to 40 U/mg of protein.
[0127] All enzymic activities were determined at 25.degree. C. The
enzyme activity was calculated as follows:
[0128] Calculation: 1 unit=conversion of 1 .mu.mol of substrate
[0129] per min
[0130] Lambert-Beer law
[0131] NADPH decrease was monitored at 340 nm (see enzymic assay
mixture)=.DELTA.E/min
[0132] N=enzyme dilution factor
[0133] V=enzyme volume in ml (0.01)
[0134] V.sub.cuvette=cuvette volume=1 ml
[0135] d=cuvette light path=1 cm
[0136] e.sub.NADPH=NADPH extinction coefficient=6.22
[mM.sup.-1*cm.sup.-1]
Activity=(.DELTA.E/min*N*V.sub.cuvette)/(e.sub.NADPH*V*d)
[0137] Protein determination was carried out according to Bradford
(Bio-Rad Laboratories GmbH, Protein Assay).
EXAMPLE 3
Enzyme-Catalyzed Preparation of
ethyl(S)-4-chloro-3-hydroxybutyrate
[0138] A.) On a 5-Liter Scale
[0139] The alcohol dehydrogenase crude extract obtained in Example
2 and the coenzyme NADP were employed in the enzyme-catalyzed
synthesis of ethyl(S)-4-chloro-3-hydroxybutyrate from ethyl
4-chloro-3-oxobutanoate. The oxidized coenzyme was continuously
regenerated due to the concomitant presence of isopropanol so that
the reaction requires only catalytic amounts of coenzyme.
[0140] The mixture contained:
[0141] 2 l of triethanolamine buffer 100 mM pH=7.0, 1 mM
[0142] MgCl.sub.2, 10 % glycerol,
[0143] 400 mg of NADP,
[0144] 600 ml of isopropanol,
[0145] 800 ml of ethyl acetate,
[0146] 600 ml of ethyl 4-chloro-3-oxobutanoate and approximately
100 000 units of alcohol dehydrogenase.
[0147] After 3 days of stirring at room temperature, complete
conversion of ethyl 4-chloro-3-oxo-butanoate to
ethyl(S)-4-chloro-3-hydroxybutyrate with enantiomeric purity of
more than 99.9% was detected by gas chromatography.
[0148] After removing the aqueous phase, evaporating the solvent
and, optionally, distillation, purified
ethyl(S)-4-chloro-3-hydroxybutyrate is obtained with an
enantiomeric purity of more than 99.9%.
[0149] B.) On a 50 l Scale
[0150] The reaction mixture for converting 10 l of ethyl
4-chloro-3-oxo-butanoate is composed as follows:
[0151] 18 l of triethanolamine buffer 100 mM pH=7.0, 1 mM
[0152] MgCl.sub.2, 10% glycerol,
[0153] 4 g of NADP,
[0154] 10 l of isopropanol,
[0155] 10 l of ethyl acetate,
[0156] 10 l of ethyl 4-chloro-3-oxo-butanoate and approximately 2
million units of alcohol dehydrogenase (1.25 l of crude
extract).
[0157] After 7 days of stirring at room temperature, complete
conversion of ethyl 4-chloro-3-oxo-butanoate to
ethyl(S)-4-chloro-3-hydroxybutyrate with enantiomeric purity of
more than 99.9% was detected by gas chromatography.
EXAMPLE 4
Biochemical Characterization of Cloned Alcohol Dehydrogenase from
Lactobacillus Minor
[0158] A.) pH Stability
[0159] The activity of the enzyme as a function of storage in
buffers with different pH values in the range from pH 4 to 11 was
studied. For this purpose, various buffers (50 mM) in the range
from pH 4 to 11 were prepared and the enzyme purified in Example 2
was diluted 1:100 therein and incubated for 30 min. All buffers
contained 1 mM MgCl.sub.2. 10 .mu.l of this were then used in the
normal enzyme assay (triethanolamine buffer 100 mM pH=7.0, 1 mM
MgCl.sub.2, 10 mM ethyl 4-chloro-3-oxo-butanoate and 0.19 mM
NADPH). The reaction was monitored at 30.degree. C. and 340 nm for
1 min.
[0160] The starting value here is the measured value obtained
immediately after diluting the enzyme in triethanolamine buffer 50
mM pH=7.0. Under predefined conditions, this value corresponded to
a change in extinction of 0.20/min and was set as 100% value, with
all subsequent measured values being related to this value.
5TABLE 2 Activity in % Activity in % pH Buffer system (n = 2)
Buffer system (n = 2) 4 sodium acetate/ 87.5 .+-. 6.5 acetic acid
4.5 sodium acetate/ 94.5 .+-. 3.0 acetic acid 5 sodium acetate/
94.5 .+-. 1.5 MES/NaOH 55 .+-. 5 acetic acid 5.5
KH.sub.2PO.sub.4/K.sub.2PO.sub.4 96 .+-. 3 MES/NaOH 77.1 .+-. 2.1 6
KH.sub.2PO.sub.4/K.sub.2PO.sub.4 100 .+-. 0 triethanol- 100 .+-. 0
amine/NaOH 6.5 KH.sub.2PO.sub.4/K.sub.2PO.sub.4 97.5 .+-. 2.5
triethanol- 100 .+-. 0 amine/NaOH 7
KH.sub.2PO.sub.4/K.sub.2PO.sub.4 100 .+-. 0 triethanol- 97.9 .+-.
2.1 amine/NaOH 7.5 KH.sub.2PO.sub.4/K.sub.2PO.sub.4 97.5 .+-. 7.5
tris/HCl 94.6 .+-. 1.3 8 KH.sub.2PO.sub.4/K.sub.2PO.sub.4 93.0 .+-.
3.0 tris/HCl 89.2 .+-. 0 8.5 KH.sub.2PO.sub.4/K.sub.2PO.sub- .4
102.5 .+-. 2.5 tris/HCl 60 .+-. 4.2 9 glycine/NaOH 76.5 .+-. 1.5
tris/HCl 63.1 .+-. 4.8 9.5 glycine/NaOH 52.5 .+-. 7.5 10
glycine/NaOH 52.5 .+-. 7.5 11 glycine/NaOH 0.0 .+-. 0
[0161] Table 2 indicates that the enzyme has good pH stability, in
particular in the acidic range, the enzyme stability appearing to
be a function not only of the pH but also of the buffer system
used. When using, for example, TRIS and MES buffers, the enzyme is
found to be inactivated more strongly than in the KPi buffer with
the same pH. There was no significant inactivation in the KPi
buffer in the pH range from 5.5 to 8.5.
[0162] B.) Temperature Stability
[0163] The temperature stability for the range from 25.degree. C.
to 50.degree. C. was determined similarly to the manner described
in A.). For this purpose, in each case a 1:100 dilution of the
purified enzyme was incubated at the particular temperature for 30
min and then measured at 30.degree. C. using the above assay
procedure. Here too, the starting value used was the measured value
obtained immediately after diluting the enzyme in triethanolamine
buffer 50 mM pH=7.0. This value was also set here as 100% value. L.
minor alcohol dehydrogenase is stable up to a temperature of
40.degree. C. Thereafter, the activity rapidly declines.
6 TABLE 3 Activity in Activity in Temperature % (n = 4) Temperature
% (n = 4) 25 101 .+-. 3.2 40 33.4 .+-. 3.8 30 81.2 .+-. 5.8 42 0
.+-. 0 35 67.0 .+-. 1.6 45 0 .+-. 0 37 20.2 .+-. 2.4 50 0 .+-.
0
[0164] C.) pH Optimum
[0165] The pH optimum was determined by determining the enzymic
reaction in the relevant buffer listed in Table 3. As in the
standard assay, the concentration of ethyl 4-chloro-3-oxo-butanoate
and of NADPH was 10 mM and 0.19 mM, respectively. The reaction was
determined at 30.degree. C. The enzyme of the invention was found
to have a pH optimum between 7 and 7.5.
7TABLE 4 Activity in U/ml of undiluted pH Buffer system enzyme 4
sodium acetate/acetic acid 85 4.5 sodium acetate/acetic acid 132 5
MES/NaOH 218 5.5 MES/NaOH 240 6 triethanolamine/NaOH 381 6.5
triethanolamine/NaOH 349 7 triethanolamine/NaOH 510 7.5 tris/HCl
707 8 tris/HCl 585 8.5 tris/HCl 486 9 tris/HCl 488 10 glycine/NaOH
131 11 glycine/NaOH 0
[0166] D.) Temperature Optimum
[0167] The optimal assay temperature was determined by measuring
the enzyme activity from 25.degree. C. to 60.degree. C. The assay
mixture corresponded to the standard concentration of ethyl
4-chloro-3-oxo-butanoate and NADPH. As Table 5 demonstrates, the
optimal assay temperature of the enzyme is 55.degree. C., with the
activity declining rapidly thereafter.
8 TABLE 5 Activity in U/ml of undiluted Temperature enzyme 25 540
30 1235 35 1968 40 1621 45 2469 50 2469 55 2855 60 0
[0168] E.) Spectrum of Substrates
[0169] Furthermore, substrates other than ethyl
4-chloro-3-oxo-butanoate were also used in the enzymic assay
mixture. For this purpose, the following assay mixture was
used:
[0170] 970 .mu.l of triethanolamine buffer (100 mM, pH=7.0, 1
mM
[0171] MgCl.sub.2 containing 10 mM keto compound)
[0172] 20 .mu.l of NADPH (0.19 mM in assay mixture)
[0173] 10 .mu.l of enzyme (1:100)
[0174] The activity determined with ethyl 4-chloro-3-oxo-butanoate
was set to 100% and the enzyme activities of the other substrates
were related to this value.
9 TABLE 6 Substrate Activity in % (n = 2) Ethyl
4-chloro-3-oxo-butanoate 100 Ethyl pyruvate 192.3 .+-. 11.5
2-Octanone 90.8 .+-. 1.2 Methyl acetoacetate 120 .+-. 7.7 Ethyl
2-oxo-4-phenylbutyrate 62.7 .+-. 4.8
[0175] F.) Enzyme Stability in Organic Solvents
[0176] The stability of the enzyme when contacted with organic
solvents was studied by diluting L. minor alcohol dehydrogenase
1:100 in the solvent mixtures listed, followed by incubation at
room temperature (for organic solvents not miscible with water, the
dilution refers to the aqueous phase). Continuous mixing of both
phases was ensured (shaker, 200 rpm). 10 .mu.l of the enzyme
solution were then used in the standard assay mixture. Here too,
the starting value was set to 100% after dilution in the buffer
(triethanolamine buffer 100 mM, pH=7.0, 1 mM MgCl.sub.2), with all
other values being related to this value.
10TABLE 7 A.) Water-miscible solvents: Solvent logP t = 2 h t = 8 h
t = 24 h t = 48 h Buffer 86 70 3 0 10% isopropanol 0.28 32 34 16 0
20% isopropanol 0.28 16 17 7 0 10% DMSO -1.3 73 54 60 40 20% DMSO
-1.3 73 54 57 40 1M sorbitol 93 74 60 6 10% glycerol -3 120 64 62
28 20% glycerol -3 120 100 100 104
[0177] As Table 7A demonstrates, glycerol, DMSO and sorbitol have
an activating and, respectively, stabilizing action on the alcohol
dehydrogenase used. In contrast, the isopropanol to be used in the
process has an inactivating action.
11 B.) Solvent not miscible with water Solvent logP t = 2 h t = 8 h
t = 24 h t = 48 h Buffer 86 70 3 0 20% ethyl acetate 0.68 87 50 10
8 20% diethyl ether 0.85 53 42 37 23 20% tert-butyl methyl ether
1.21 67 51 38 24 20 diisopropyl ether 1.55 100 57 41 29 20% dibutyl
ether 2.9 92 71 23 6 20% pentane 3 74 55 7 6 20% hexane 3.5 80 39 2
5 20% heptane 4 51 49 7 6 20% octane 4.5 87 47 2 1
[0178] As Table 7B demonstrates, the alcohol dehydrogenase studied
exhibits considerable stability in a large number of organic
solvents. Surprisingly, solvents having logP values between 0 and 3
inhibit the alcohol dehydrogenase studied no more than those having
logP values between 3 and 4.5, in particular with regard to longer
incubation times (24 h and 48 h) solvents having logP values betwen
0 and 3 stabilize the ADH studied, compared to the corresponding
values in the buffer. The aliphatic solvents studied, pentane,
hexane, heptane and octane, do not exhibit this stabilizing action
with long-term incubation.
[0179] The logP value of a component X is the logarithm of the
[0180] distribution coefficient of X in the octanol/water two-phase
system (50/50)
[0181] P=concentration of X in octanol phase/concentration of X in
aqueous phase
[0182] G. Enzyme Stability Under Process Conditions
[0183] The stability of the enzyme under process conditions was
studied by diluting L. minor alcohol dehydrogenase 1:100 with the
solvent mixtures used in the two-phase system, followed by
incubation at room temperature. 10 .mu.l of the enzyme solution
were then used in the standard assay mixture.
[0184] Table 8 depicts the enzyme activities as a % of the starting
value.
12 TABLE 8 6 h 20 h 46 h 60 h 84 h Triethanolamine 100 75 0 0 0
buffer, 100 mM, 1 mM MgCl.sub.2 Mixture B 100 85 80 60 55 Mixture C
110 95 95 85 80 Mixture D 100 65 55 50 50 Mixture B: buffer, 10%
glycerol, 10% isopropanol Mixture C: buffer, 20% glycerol, 20%
isopropanol Mixture D: buffer, 10% glycerol, 10% isopropanol + 20%
ethyl acetate
[0185] It was found that recombinant L. minor alcohol dehydrogenase
is stable and active in the combination of solvents used in the
two-phase system for several days.
Sequence CWU 1
1
4 1 795 DNA Artificial Sequence Description of Artificial Sequence
DNA primer 1 atgagaggat cgcatcacca tcaccatcac ggatccatga ccgatcggtt
gaaggggaaa 60 gtagcaattg taactggcgg taccttggga attggcttgg
caatcgctga taagtttgtt 120 gaagaaggcg caaaggttgt tattaccggc
cgtcacgctg atgtaggtga aaaagctgcc 180 agatcaatcg gcggcacaga
cgttatccgt tttgtccaac acgatgcttc tgatgaaacc 240 ggctggacta
agttgtttga tacgactgaa gaagcatttg gcccagttac cacggttgtc 300
aacaatgccg gaattgcggt cagcaagagt gttgaagata ccacaactga agaatggcgc
360 aagctgctct cagttaactt ggatggtgtc ttcttcggta cccgtcttgg
aatccaacgt 420 atgaagaata aaggactcgg agcatcaatc atcaatatgt
catctatcga aggttttgtt 480 ggtgatccag ctctgggtgc atacaacgct
tcaaaaggtg ctgtcagaat tatgtctaaa 540 tcagctgcct tggattgcgc
tttgaaggac tacgatgttc gggttaacac tgttcatcca 600 ggttatatca
agacaccatt ggttgacgat cttgaagggg cagaagaaat gatgtcacag 660
cggaccaaga caccaatggg tcatatcggt gaacctaacg atatcgcttg gatctgtgtt
720 tacctggcat ctgacgaatc taaatttgcc actggtgcag aattcgttgt
cgacggaggg 780 tacaccgccc aatag 795 2 37 DNA Artificial Sequence
Description of Artificial Sequence DNA primer 2 gcggatccat
gacngaycgn ttraarggna argtngc 37 3 36 DNA Artificial Sequence
Description of Artificial Sequence DNA primer 3 gggaagcttc
taytgngcng trtanccncc rtcnac 36 4 264 PRT Lactobacillus sp. 4 Met
Arg Gly Ser His His His His His His Gly Ser Met Thr Asp Arg 1 5 10
15 Leu Lys Gly Lys Val Ala Ile Val Thr Gly Gly Thr Leu Gly Ile Gly
20 25 30 Leu Ala Ile Ala Asp Lys Phe Val Glu Glu Gly Ala Lys Val
Val Ile 35 40 45 Thr Gly Arg His Ala Asp Val Gly Glu Lys Ala Ala
Arg Ser Ile Gly 50 55 60 Gly Thr Asp Val Ile Arg Phe Val Gln His
Asp Ala Ser Asp Glu Thr 65 70 75 80 Gly Trp Thr Lys Leu Phe Asp Thr
Thr Glu Glu Ala Phe Gly Pro Val 85 90 95 Thr Thr Val Val Asn Asn
Ala Gly Ile Ala Val Ser Lys Ser Val Glu 100 105 110 Asp Thr Thr Thr
Glu Glu Trp Arg Lys Leu Leu Ser Val Asn Leu Asp 115 120 125 Gly Val
Phe Phe Gly Thr Arg Leu Gly Ile Gln Arg Met Lys Asn Lys 130 135 140
Gly Leu Gly Ala Ser Ile Ile Asn Met Ser Ser Ile Glu Gly Glu Val 145
150 155 160 Gly Asp Pro Ala Leu Gly Ala Tyr Asn Ala Ser Lys Gly Ala
Val Arg 165 170 175 Ile Met Ser Lys Ser Ala Ala Leu Asp Cys Ala Leu
Lys Asp Tyr Asp 180 185 190 Val Arg Val Asn Thr Val His Pro Gly Tyr
Ile Lys Thr Pro Leu Val 195 200 205 Asp Asp Leu Glu Gly Ala Glu Glu
Met Met Ser Gln Arg Thr Lys Thr 210 215 220 Pro Met Gly His Ile Gly
Glu Pro Asn Asp Ile Ala Trp Ile Cys Val 225 230 235 240 Tyr Leu Ala
Ser Asp Glu Ser Lys Phe Ala Thr Gly Ala Glu Phe Val 245 250 255 Val
Asp Gly Gly Tyr Thr Ala Gln 260
* * * * *