Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Reexamination Certificate
1997-06-11
2001-07-03
Achutamurthy, Ponnathapu (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
C435S069100, C435S189000, C435S252300, C435S320100, C536S063000
Reexamination Certificate
active
06255092
ABSTRACT:
A novel enzyme, a method to prepare said enzyme, a DNA segment encoding said enzyme, a transformant containing said DNA segment and a method of preparing optically active alcohol using said enzyme
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates to a method of producing a novel secondary alcohol dehydrogenase useful for the preparation of alcohol, aldehyde and ketone, especially for that of an optically active alcohol, a method of producing said enzyme, a DNA segment encoding said enzyme, a microorganism transformed with said DNA, and a method of producing alcohol, aldehyde and ketone, especially optically active alcohol using said enzyme.
2. Related Arts
Of the secondary alcohol dehydrogenase of the microbial origin requiring nicotinamide adenine dinucleotide phosphate (abbreviated as NADP
+
hereinafter), the one derived from
Thermoanaerobium brockii
is well documented (J. Am. Chem. Soc. 108, 162-169 (1986)). In addition, of the secondary alcohol dehydrogenase requiring nicotinamide adenine dinucleotide (abbreviated as NAD
+
hereinafter), there have been reported those derived from Pichia sp. NRRL-Y-11328 (Eur. J. Biochem. 101, 401-406 (1979)), Pseudomonas sp. SPD6 (Bioorg. Chem. 19, 398-417 (1991)),
Pseudomonas fluorescence
NRRL B-1244 (Tokkai Sho, 59-17982),
Pseudomonas maltophilia
MB11L (FEMS Microbiol. Lett. 93, 49-56 (1992)), Pseudomonas sp. PED (J. Org. Chem. 57, 1526-1532 (1992)), Pseudomonas sp. ATCC 21439 (Eur. J. Biochem. 119, 359-364 (1981)),
Candida boidinii
SAHM (Biochim. Biophys. Acta 716, 298-307 (1992)),
Mycobacterium vaccae
JOB-5 (J. Gen. Microbiol. 131, 2901-2907 (1985)),
Rhodococcus rhodochrous
PNKb1 (Arch. Microbiol. 153, 163-168 (1990)),
Comamonas terrigena
(Biochim. Biophys. Acta 661, 74-86 (1981)), and Arthrobacter sp. SBA (Tokkai Sho 51-57882).
However, the stereochemical substrate specificity of these secondary alcohol dehydrogenases is not satisfactory for the practical application. For example, as to 2-butanol, one of the most frequently reported substrates of the secondary alcohol dehydrogenase, there has not been reported the enzyme which will oxidize (S)-2-butanol stereospecifically to produce 2-butanone. (The enzymes derived from Pseudomonas sp. ATCC 21439, Pseudomonas sp. SPD6,
Comamonas terrigena, Candida boidinii
SAHM or Pichia sp. NRRL-Y-11328 oxidize (R)-isomer preferentially, while the one derived from
Pseudomonas fluorescens
NRRL B-1244 does not show any definite substrate stereochemical specificity, and the specificity of the enzyme derived from
Mycobacterium vaccae
JOB-5,
Rhodococcus rhodochrous
PNKb1, Pseudomonas sp. PED or
Pseudomonas maltophilia
MB11L has not been reported.) Furthermore, although the primary alcohol dehydrogenase (SADH-1) derived from baker's yeast (
Saccharomyces cerevisiae
) has been reported to oxidize 2-butanol with S configuration preferentially, the relative activity is as low as about 1% of that for ethanol, not suitable for practical use (Arch. Biochem. Biophys. 126, 933-944 (1968), J. Biol. Chem. 268, 7792-7798 (1993)).
Since the secondary alcohol dehydrogenase which will preferentially oxidize S-2-butanol has not been reported, there has been a strong demand for finding the enzyme with a high substrate stereochemical specificity.
There has been also a high demand for cloning DNA encoding said enzyme, because it will be possible to produce said enzyme on a large scale with a genetic engineering technique using the cloned gene of said enzyme.
SUMMARY OF THE INVENTION
During the wide-screening of microorganisms having the activity to preferentially oxidize (S)-2-butanol, the inventors of the present invention discovered that the microorganism belonging to genus Candida, especially
Candida parapsilosis
had the activity to preferentially oxidize (S)-2-butanol, further purified the enzyme to oxidize (S)-2-butanol from cells of cultured said microorganism, and studied its enzymatic properties finding that said enzyme has the ability to oxidize (S)-2-butanol with a high stereochemical specificity and also oxidize various other secondary alcohols stereospecifically.
It is one object of the present invention to provide an enzyme with the following physicochemical properties as defined in 1) to 9):
1) Functions
Said enzyme oxidizes alcohol with NAD
+
as the coenzyme to produce the corresponding ketone or aldehyde, and also reduces ketone or aldehyde with NADH as the coenzyme to produce the corresponding alcohol.
2) Substrate specificity
Said enzyme utilizes aliphatic alcohols including those with an aromatic substitution as its oxidizing substrate, has a relatively higher activity toward secondary alcohols than primary ones, and preferentially oxidizes 2-butanol with the S configuration. Said enzyme also utilizes aldehydes or aliphatic ketones with an aromatic substitution.
3) Molecular weight
The apparent molecular weight of said enzyme is estimated to be approximately 40,000 by SDS-PAGE. Physicochemical as well as enzymatic properties of said enzyme of the present invention are as follows:
4) Optimal pH and pH range for the enzyme stability
The optimal pH for the oxidation of (S)-2-butanol ranges from 8.5 to 9.5, and that for the reduction of 2-butanone from 5.5 to 6.5. Said enzyme is relatively stable in the pH range from 8.0 TO 10.0.
5) Optimal temperature range for the enzymatic reaction
Said enzyme shows the high activity at the temperature ranging from 25-55° C. with 50° C. as optimal for the enzymatic reaction.
6) Thermal inactivation
Said enzyme retains more than 90% of the original activity even after the heat treatment at 40° C. for 10 min.
7) Inhibition and stabilization
The activity of said enzyme is inhibited by various SH-reagents such as p-mercuribenzoic acid, mercuric chloride, zinc chloride and N-ethylmaleimide, and also by the reducing agents including 2-mercaptoethanol and dithiothreitol. Said enzyme activity is inhibited by o-phenanthroline but not by ethylenediaminetetraacetic acid.
8) Purification
Said enzyme can be purified to a single protein band on the sodium dodecyl sulfate polyacrylamide gel electrophoresis (abbreviated as SDS-PAGE hereinafter) by combining the conventional purification methods of ordinary proteins, comprising, for example, protamine sulfate precipitation after disrupting microbial cells, ammonium sulfate fractionation of the centrifuged supernatant, followed by a combination of anion exchange chromatography, hydrophobic chromatography and gel filtration.
9) Isoelectric point
Although said enzyme shows several bands on isoelectric focusing, the isoelectric point of the major protein band is located at pH 6.7.
The activity of all secondary alcohol dehydrogenases including said enzyme described in the preferred embodiments of the present specification was assayed as follows: (S)-2-butanol (50 &mgr;mol) and the enzyme were incubated in a reaction mixture containing Tris-HCl (50 &mgr;mol, pH 9.0) and NAD
+
(2.5 &mgr;mol) at 30° C., and the rate of NADH formation was followed at 340 nm. One unit of enzyme was defined as the amount of enzyme necessary to catalyze the formation of 1 &mgr;mol of NADH per min under the assay conditions.
It is another object of the present invention to provide a DNA segment encoding said secondary alcohol dehydrogenase. Inventors of the present invention digested the purified said enzyme with lysylendopeptidase, purified the digested fragments by reversed phase chromatography, and determined a portion of its amino acid sequence using a protein sequencer. PCR (polymerase chain reaction) was performed using primers synthesized based on said amino acid sequence determined above and the chromosomal DNA of
Candida parapsilosis
as the template. A portion of gene encoding said secondary alcohol dehydrogenase was amplified and its base sequence (core sequence) was determined. Then in order to elucidate the base sequence in the flanking region of said DNA sequence determined above (core sequence), the chromosomal DNA of
Candida parapsilosis
was digested with HaeII,
Kawada Naoki
Kojima Tomoko
Matsuyama Akinobu
Yamamoto Hiroaki
Achutamurthy Ponnathapu
Daicel Chemical Industries Ltd.
Moore William W.
Oblon & Spivak, McClelland, Maier & Neustadt P.C.
LandOfFree
Stereospecific alcohol dehydrogenase isolated from Candida... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Stereospecific alcohol dehydrogenase isolated from Candida..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Stereospecific alcohol dehydrogenase isolated from Candida... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2500192