Chemistry: molecular biology and microbiology – Enzyme – proenzyme; compositions thereof; process for... – Oxidoreductase
Reexamination Certificate
2001-11-28
2004-12-14
Achutamurthy, P. (Department: 1652)
Chemistry: molecular biology and microbiology
Enzyme , proenzyme; compositions thereof; process for...
Oxidoreductase
C435S189000, C435S252300, C435S090000, C435S132000, C435S440000, C536S023200
Reexamination Certificate
active
06830907
ABSTRACT:
TECHNICAL FIELD
The present invention relates to mutants of
Mycobacterium vaccae
-derived formate dehydrogenase, polynucleotides encoding the mutants, and a method for producing reduced form of &bgr;-nicotinamide adenine dinucleotide (NADH) from oxidized form of &bgr;-nicotinamide adenine dinucleotide (NAD
+
) by using them.
BACKGROUND
Previously there has been a known method for producing optically active (S)-4-halo-3-hydroxybutyrate ester, which is an asymmetric reduction method (Unexamined Published Japanese Patent Application No. (JP-A) Sho 61-146191; JP-A Hei 6-209782, etc.) using microorganisms such as baker's yeast. However, the method has been industrially unusable, because multiple types of reductases are present in microbial cells and thus the optical purity and yield of the product are low. The optically active (S)-4-halo-3-hydroxybutyrate ester can be used as an intermediate for pharmaceuticals and such, and thus methods for obtaining optically pure enantiomer (synthesis or resolution) have been an industrially important challenge.
Kluyveromyces aestuarii
-derived carbonyl reductase (JP-A 2000-236883) is known to generate (S)-4-halo-3-hydroxybutyrate ester from 4-haloacetoacetate ester. A method has been reported, for synthesizing (S)-4-halo-3-hydroxybutyrate ester by using this enzyme. However, a stoichiometric amount of reduced form of &bgr;-nicotinamide adenine dinucleotide (NADH) as a co-enzyme is required in the production of optically active alcohols by using this enzyme. The co-enzyme is extremely expensive, and therefore, on an industrial scale, it is economically disadvantageous to utilize the method where a required amount of the co-enzyme is just used. Thus it is important to repeatedly reuse the co-enzyme by reducing oxidized form of &bgr;-nicotinamide adenine dinucleotide (NAD
+
) to NADH, to construct an economically advantageous process.
So far, there have been reports in which formate dehydrogenase (Methods in Enzymology 136:9-21, 1987) or glucose dehydrogenase (JP-A 2000-236883) is used to reduce the co-enzyme NAD
+
into NADH. However, glucose dehydrogenase converts glucose to gluconic acid, and as a result, there is a problem in which an equal amount of gluconic acid and optically active alcohol of interest is generated.
On the other hand, formate dehydrogenase converts formic acid into carbonic acid, and the generated carbonic acid is efficiently eliminated from the system being converted to a carbon dioxide. Thus the method can be an economically advantageous process. However, there is also a disadvantage in the use of formate dehydrogenase, i.e., the stability of this enzyme is not high enough and as a consequence it has a tendency to be inactivated. It is known that the inactivation depends on various factors, pH value, temperature, mechanical stress, ionic strength and type of ion in the substrate solution, heavy metals, oxidation of thiol group by oxygen, etc. (JP-A Hei 11-225784). In this context, there are reports on methods in which the following mutations are used to enhance the stability.
Tishkov et al. have shown that mutants of formate dehydrogenase from
Pseudomonas
sp. 101, in which the cysteine at position 256 has been substituted with serine or methionine by site-directed mutagenesis, have enhanced stability to mercury but reduced thermal stability (Biochem. Biophys. Res. Commun. 192:4480-4485, 1993). They have also reported mutants showing enhanced thermal stability, which were similarly created by substituting serine with alanine, valine, or leucine at position 131, 160, 168, 184, or 228 (FEBS Letters 445:183-188, 1999).
Slusarczyk et al. have shown that mutants of formate dehydrogenase from
Candida boidinii
created by site-directed mutagenesis, in which the cysteine at position 23 has been substituted with serine as well as cysteine at position 262 with valine or alanine, exhibit enhanced stability to copper, enhanced pH stability in the range of weak alkaline pH, but reduced thermal stability (Eur. J. Biochem. 267:1280-1289, 2000).
Despite these research efforts, there has been a problem to be solved, which is lower yield due to decreased activity of formate dehydrogenase during the production of the reduced products such as alcohols from the oxidized substrates such as ketones in conjunction with the regeneration of co-enzyme NADH by using the above enzyme.
SUMMARY
With consideration given to the situation, the present invention was achieved, and an objective of the present invention is to provide formate dehydrogenase of which activity is not lowered during the process of producing the reduced product from the oxidized substrate while co-enzyme NADH is being regenerated. In addition, another objective of the present invention is to efficiently produce reduced product from oxidized substrate by using such an enzyme.
In order to achieve the above objectives, the inventors first investigated causes of the decrease of formate dehydrogenase activity during the production process of the above-mentioned reduced product. Then the inventors found that formate dehydrogenase was rapidly inactivated in the presence of organic solvents, such as ketones, as raw materials. Thus the inventors made an effort to search mutants of formate dehydrogenase for those resistant to organic solvents or those of which activity is enhanced by organic solvents. The inventors eventually succeeded in the construction of mutants of formate dehydrogenase having the nature in which the activity has been enhanced in the presence of organic solvents as compared with that in the absence of organic solvent by modifying the cysteine residue at position 146 in
Mycobacterium vaccae
-derived formate dehydrogenase (SEQ ID NO:2), which were found through constructing a variety of mutants of formate dehydrogenase and searching them.
Furthermore, the inventors found that mutants of formate dehydrogenase showing the resistance to organic solvents were obtainable by modifying the cysteine at position 256.
Further the inventors have succeeded in the coexpression of formate dehydrogenase and carbonyl reductase in
E. coli
by constructing expression vectors containing polynucleotides encoding these mutant enzymes and polynucleotide encoding carbonyl reductase which reduces ketones into alcohols. The use of these expressed enzymes have made it possible to efficiently produce reduced product from oxidized substrate, e.g., ketones, and for example, to efficiently produce alcohols from the substrate, while co-enzyme NADH is being regenerated.
As described above, the inventors created formate dehydrogenase mutants that is resistant to organic solvents and of which activity is enhanced in the presence of an organic solvent, and found a method for efficiently producing reduced product of oxidized substrate from the substrate by coexpressing the enzyme and carbonyl reductase; thus the inventors completed the present invention.
Specifically, the present invention relates to the following polypeptides and a method for efficiently producing reduced product from oxidized substrate using the polypeptides.
1. A polypeptide having a strong activity of formate dehydrogenase in the presence of an organic solvent, said polypeptide comprising a mutation in which amino acids other than cysteine are substituted at least for cysteine residues at positions 146 and/or 256 in the amino acid sequence of SEQ ID NO:2.
2. The polypeptide of 1, wherein the substituted amino acid at position 146 is serine or valine.
3. The polypeptide of 1, wherein the substituted amino acid at position 256 is serine, alanine, or valine.
4. The polypeptide of 1, wherein said polypeptide comprises a mutation in which amino acids other than cysteine are substituted at least for cysteine residues at positions 146 and 256 in the amino acid sequence of SEQ ID NO:2.
5. The polypeptide of 4, wherein the substituted amino acid at position 146 is serine or valine, and the substituted amino acid at position 256 is serine, alanine, or valine.
6. The polypeptide of 1, wherein said polypeptide further compri
Kimoto Norihiro
Mitsuhashi Kazuya
Yamamoto Hiroaki
Achutamurthy P.
Daicel Chemical Industries Ltd.
Pak Yong D.
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