Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Reexamination Certificate
2001-10-30
2004-11-16
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
C435S157000, C435S440000, C435S325000, C536S023200
Reexamination Certificate
active
06818426
ABSTRACT:
This application claims priority under 35 USC §119 to Japanese Patent Application No. 2000-333363, filed Oct. 31, 2000, hereby incorporated by reference in its entirety.
TECHNICAL FIELD
The present invention relates to a novel nicotinamide adenine dinucleotide-dependent (R)-2,3-butanediol dehydrogenase. The present invention also relates to a polynucleotide encoding the enzyme protein, a method for producing the enzyme and a method for producing alcohol, particularly (2R,3R)-2,3-butanediol, by using the enzyme.
BACKGROUND
(R)-2,3-butanediol dehydrogenase is an enzyme which plays important roles in fermentation production of (2R,3R)-2,3-butanediol with microorganisms using glucose as raw material and in 2,3-butanediol metabolism in microorganisms. Further (2R,3R)-2,3-butanediol generated via the enzyme reaction is a useful compound as raw material for the synthesis of liquid crystal, pharmaceuticals, etc.
(R)-2,3-butanediol dehydrogenase is a dehydrogenase having the activity of selectively oxidizing the hydroxyl group of 2,3-butanediol in (R) configuration and also has the activity of oxidizing the hydroxyl group of meso-2,3-butanediol in (R) configuration as well as that of (2R,3R)-2,3-butanediol in (R) configuration.
Previously, regarding the enzyme having the activity of 2,3-butanediol dehydrogenation, it has been reported dehydrogenase activity toward (2R,3R)-2,3-butanediol is contained, for example, in the microorganisms listed below, based on studies concerning biosynthesis and metabolism of 2,3-butanediol (Arch. Microbiol., 116:197-203, 1978; J. Ferment. Technol., 61:467-471, 1983; J. Ferment. Technol., 62:551-559, 1984). However a variety of natures such as stereoselectivity and specific activity of 2,3-butanediol dehydrogenase was unclear in such previous studies because assays for the activity were conducted by using only cell-free extract and thus various enzymes coexisted:
Aeromonas hydrophila;
Bacillus cereus
IAM 1072;
Bacillus coagulans
ATCC 8038;
Micrococcus lysodeikticus
IAM 1056;
Micrococcus luteus
IAM 1097;
Micrococcus roseus
IAM 1295;
Pseudomonas saccharophila
IAM 1504;
Sarcina lutea
IAM 1099;
Staphylococcus aureus.
With respect to enzymes highly purified and having a variety of natures clarified, the following enzymes have been shown to have the activity of 2,3-butanediol dehydrogenase. However, these contain only the activity of catalyzing DL-form and there is no report on the stereoselectivity. Furthermore, the activities of the 2,3-butanediol dehydrogenases with the exception of
Pichia ofunaensis
are comparable to or lower than the activity of glycerol dehydrogenase and thus the specific activities are generally lower.
Glycerol dehydrogenase derived from
Achromobacter liquidum
(
Achromobacter liquidum
KY 3047) (Examined Published Japanese Patent Application No. (JP-B) Sho 58-40467);
Glycerol dehydrogenase derived from
Bacillus
sp. (
Bacillus
sp. G-1) (JP-B Hei 03-72272);
Glycerol dehydrogenase derived from
Bacillus stearothermophilus
(Biochim. Biophys. Acta., 994:270-279, 1989);
Glycerol dehydrogenase derived from
Citrobacter freundii
(
Citrobacter freundii
DSM 30040) (J. Bacteriol., 177:4392-4401, 1995);
Glycerol dehydrogenase derived from
Erwinia aroideae
(
Erwinia aroideae
IFO 3830) (Chem. Pharm. Bull., 26:716-721, 1978);
Glycerol dehydrogenase derived from
Geotrichum candidum
(
Geotrichum candidum
IFO 4597) (JP-B Hei 01-27715);
Dihydroxyacetone reductase derived from
Pichia ofunaensis
(
Pichia ofunaensis
AKU 4328) (J. Biosci. Bioeng., 88:148-152, 1999);
Glycerol dehydrogenase derived from
Schizosaccharomyces pombe
(J. Gen. Microbiol., 131:1581-1588, 1985).
A known enzyme highly purified and having a clarified high selectivity to (2R,3R) isomer of 2,3-butanediol is glycerol dehydrogenase produced by
Escherichia coli
(
Escherichia coli
W-1485) (J. Biol. Chem., 259:2124-2129, 1984). Because Vmax of this enzyme toward (2R,3R)-2,3-butanediol is 28.0 U/mg protein and Vmax toward racemic body is 21.2 U/mg protein, the enzyme is suggested to exhibit the stereoselectivity to (2R,3R) isomer. Here, 1 U of the enzyme is defined as an enzyme activity of reducing 1 &mgr;mol oxidized nicotinamide adenine dinucleotide (hereinafter abbreviated to NAD
+
) into reduced nicotinamide adenine dinucleotide (hereinafter abbreviated to NADH) for one minute in the presence of (2R,3R)-2,3-butanediol as a substrate.
Further it has been reported that (R)-2,3-butanediol dehydrogenase derived from
Saccharomyces cerevisiae
produces (2R,3R)-2,3-butanediol from 2,3-butanedione (Arch. Microbiol., 154:267-273, 1990), but the dehydrogenase activity to DL-2,3-butanediol is about 20.3 U/mg protein; all of the above exhibit merely low specific activities.
In addition, the gene encoding 2,3-butanediol dehydrogenase participating in the metabolism of 2,3-butanediol has been cloned from
Pseudomonas putida
and expressed in
E. coli
(FEMS Microbiol. Lett., 124(2): 141-150, 1994), but the stereoselectivity has not yet been reported. Further genomic analysis has identified a gene from
Pseudomonas aeruginosa,
which has high homology to the 2,3-butanediol dehydrogenase gene derived from
Pseudomonas putida.
However this gene has not yet been expressed recombinantly and thus neither enzyme activity nor stereoselectivity has been verified.
The followings are industrially important challenges; the discovery of (R)-2,3-butanediol dehydrogenase that is useful for producing optically active alcohols such as (2R,3R)-2,3-butanediol, high stereoselectivity and high specific activity; particularly, the isolation of gene encoding the enzyme and preparation of transformants capable of expressing the enzyme to make it possible to conveniently produce the enzyme on a large scale.
SUMMARY
An objective of the present invention is to provide (R)-2,3-butanediol dehydrogenase that can use NAD
+
as a coenzyme. Another objective of the present invention is to provide (R)-2,3-butanediol dehydrogenase capable of giving products of high optical purity in high yield when it is utilized in an enzymatic production process of optically active (2R,3R)-2,3-butanediol using 2,3-butanedione as a substrate.
Yet another objective of the present invention is to isolate a polynucleotide encoding (R)-2,3-butanediol dehydrogenase having desired properties and to obtain a recombinant thereof. In addition, still another objective is to provide a method for enzymatically producing optically active (2R,3R)-2,3-butanediol by using the novel (R)-2,3-butanediol dehydrogenase.
The present inventors have studied a group of enzymes participating in glycerol metabolism in
Pichia angusta
(previous name:
Hansenula polymorpha
) (Agri. Biol. Chem., 51:2401-2407, 1987). There are two glycerol metabolism pathways, namely phosphorylation pathway and oxidation pathway, in this fungal strain; thus it has been clarified that the strain has both glycerol dehydrogenase I (GDH-I) catalyzing reduction reaction using NADH and dibydroxyacetone as substrates at pH 6.0 as well as glycerol dehydrogenase II (GDH-II) catalyzing oxidation reaction using NAD
+
and glycerol as substrates at pH 9.0.
One of these two types of enzymes, GDH-I, was purified to a single band in electrophoresis and a variety of natures thereof have been clarified. The result showed that GDH-I is a novel (R)-2,3-butanediol dehydrogenase having the high activity as well as high selectivity to the hydroxyl group of 2,3-butanediol in (R) configuration.
Further, the present inventor isolated a polynucleotide encoding this enzyme and prepared recombinant bacteria overexpressing this enzyme, thereby completing the present invention. Specifically the present invention relates to the following (R)-2,3-butanediol dehydrogenase, a polynucleotide encoding this enzyme, a method for producing this enzyme and uses thereof.
[1] An (R)-2,3-butanediol dehydrogenase having the following physicochemical properties (1) to (3):
(1) Action
The dehydrogenase produces (R)-acetoin by acting on (2R,3R)-2,3-butanediol using nicotinamide adenine din
Onodera Keiko
Tani Yoshiki
Yamamoto Hiroaki
Achutamurthy Ponnathapu
Daicel Chemical Industries Ltd.
Fish & Richardson P.C.
Pak Yong D.
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