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Dive into the research topics where Andrey Galkin is active.

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Featured researches published by Andrey Galkin.


Applied Microbiology and Biotechnology | 1995

Cloning of formate dehydrogenase gene from a methanol-utilizing bacterium Mycobacterium vaccae N10.

Andrey Galkin; Ljudmila Kulakova; V. I. Tishkov; Nobuyoshi Esaki; Kenji Soda

The gene of NAD+-dependent formate dehydrogenase (FDH) from Mycobacterium vaccae N10 was cloned into Escherichia coli by hybridization with digoxigenin-labeled DNA probes, which were prepared by amplification of the chromosomal DNA from the bacterium by the polymerase chain reaction with degenerate primers. The primers were designed on the basis of the most conserved parts of known sequences of FDH from different organisms. An open-reading frame of 1200 bp exhibited extremely high sequence similarity to the FDH gene of Pseudomonas sp. 101. The deduced amino acid sequence of FDH from Mycobacterium vaccae N10 (McFDH) was identical to that of Pseudomonas sp. 101 (PsFDH) except for two amino acid residues: isoleucine-35 (threonine in PsFDH) and glutamate-61 (lysine in PsFDH). The physicochemical properties of both enzymes appeared to be closely similar to each other, but the thermostability of McFDH was a little lower than that of PsFDH. To examine the role of the two amino acid residues in the thermostability of the enzymes, glutamate-61 of McFDH was replaced by glutaminyl, prolyl and lysyl residues by site-directed mutagenesis. All the mutant enzymes showed higher thermostability than the wild-type McFDH. The negative charge of glutamate-61 contributes to the stability of the wild-type enzyme being lower than that of PsFDH.


Applied Microbiology and Biotechnology | 1995

Gene cloning, purification, and characterization of thermostable and halophilic leucine dehydrogenase from a halophilic thermophile, Bacillus licheniformis TSN9

Shinji Nagata; S. Bakthavatsalam; Andrey Galkin; H. Asada; S. Sakai; Nobuyoshi Esaki; Kenji Soda; Toshihisa Ohshima; Susumu Nagasaki; Haruo Misono

A halophilic and thermophilic isolate from the sand of Tottori Dune was found to produce a thermostable and halophilic leucine dehydrogenase (EC 1.4.1.9). It was identified to be a new strain of Bacillus licheniformis. The enzyme gene was cloned into Escherichia coli JM109 with a vector plasmid pUC18. The enzyme was purified to homogeneity from the clone cell extract by ion-exchange column chromatography with a yield of 31%. The enzyme was found to be composed of eight subunits identical in relative molecular mass (43 000). The amino acid sequence of the enzyme, deduced from the nucleotide sequence of the gene, showed an identity of 84.6% with that of the B. stearothermphilus enzyme [Nagata S, Tanizawa K, Esaki N, Sakamoto Y, Ohshima T, Tanaka H, Soda K (1988) Biochemistry 27:9056–9062], although both enzymes were similar to each other in various enzymological properties such as thermostability, substrate and coenzyme specificities, and stereospecificity for hydrogen transfer from the C-4 of NADH. However, they were markedly distinct from each other in halophilicity: the B. licheniformis enzyme was much more stable than the other in the presence of high concentrations of salts.


Journal of Fermentation and Bioengineering | 1997

Conversion of α-keto acids to D-amino acids by coupling of four enzyme reactions

Andrey Galkin; Ljudmila Kulakova; Hiroaki Yamamoto; Katsuyuki Tanizawa; Hidehiko Tanaka; Nobuyoshi Esaki; Kenji Soda

Abstract We developed a new procedure for stereospecific conversion of various α-keto acids to the corresponding d -amino acids with four thermostable enzymes: d -amino acid aminotransferase, alanine racemase, l -alanine dehydrogenase and formate dehydrogenase. Optically pure d -enantiomers of glutamate, phenylalanine and tyrosine were obtained with high conversion rates.


Extremophiles | 2005

Cold-active DnaK of an Antarctic psychrotroph Shewanella sp. Ac10 supporting the growth of dnaK-null mutant of Escherichia coli at cold temperatures

Kazuaki Yoshimune; Andrey Galkin; Ljudmila Kulakova; Tohru Yoshimura; Nobuyoshi Esaki

Shewanella sp. Ac10 is a psychrotrophic bacterium isolated from the Antarctica that actively grows at such low temperatures as 0°C. Immunoblot analyses showed that a heat-shock protein DnaK is inducibly formed by the bacterium at 24°C, which is much lower than the temperatures causing heat shock in mesophiles such as Escherichia coli. We found that the Shewanella DnaK (SheDnaK) shows much higher ATPase activity at low temperatures than the DnaK of E. coli (EcoDnaK): a characteristic of a cold-active enzyme. The recombinant SheDnaK gene supported neither the growth of a dnaK-null mutant of E. coli at 43°C nor λ phage propagation at an even lower temperature, 30°C. However, the recombinant SheDnaK gene enabled the E. coli mutant to grow at 15°C. This is the first report of a DnaK supporting the growth of a dnaK-null mutant at low temperatures.


Applied and Environmental Microbiology | 1997

Synthesis of optically active amino acids from alpha-keto acids with Escherichia coli cells expressing heterologous genes.

Andrey Galkin; Ljudmila Kulakova; Tohru Yoshimura; Kenji Soda; Nobuyoshi Esaki


Biochimica et Biophysica Acta | 2004

Cold-active esterase from Psychrobacter sp. Ant300: gene cloning, characterization, and the effects of Gly-->Pro substitution near the active site on its catalytic activity and stability.

Ljudmila Kulakova; Andrey Galkin; Toru Nakayama; Tokuzo Nishino; Nobuyoshi Esaki


Applied and Environmental Microbiology | 1999

Cold-Active Serine Alkaline Protease from the Psychrotrophic Bacterium Shewanella Strain Ac10: Gene Cloning and Enzyme Purification and Characterization

Ljudmila Kulakova; Andrey Galkin; Tatsuo Kurihara; Tohru Yoshimura; Nobuyoshi Esaki


Biochemical and Biophysical Research Communications | 1993

Catalytic properties and stability of a Pseudomonas sp.101 formate dehydrogenase mutants containing Cys-255-Ser and Cys-255-Met replacements.

V. I. Tishkov; Andrey Galkin; Gn Marchenko; Egorova Oa; D.V. Sheluho; Ludmila Kulakova; L.A. Dementieva; A.M. Egorov


Applied and Environmental Microbiology | 1999

Cold-Adapted Alanine Dehydrogenases from Two Antarctic Bacterial Strains: Gene Cloning, Protein Characterization, and Comparison with Mesophilic and Thermophilic Counterparts

Andrey Galkin; Ljudmila Kulakova; Hiroyuki Ashida; Yoshihiro Sawa; Nobuyoshi Esaki


Protein Engineering | 1997

Construction of a new leucine dehydrogenase with preferred specificity for NADP+ by site-directed mutagenesis of the strictly NAD+-specific enzyme.

Andrey Galkin; Ljudmila Kulakova; Toshihisa Ohshima; Nobuyoshi Esaki; Kenji Soda

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Toshihisa Ohshima

Osaka Institute of Technology

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A.M. Egorov

Moscow State University

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Kazuaki Yoshimune

National Institute of Advanced Industrial Science and Technology

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