RU2312897C1 - Recombinant thermostable formate dehydrogenase - Google Patents

Abstract

FIELD: biotechnology, biochemistry, enzymes.

SUBSTANCE: invention represents a recombinant formate dehydrogenase showing amino acid sequence that corresponds to amino acid sequence of formate dehydrogenase of wild type and containing glutamic acid residue at position corresponding to position 106 in amino acid sequence of formate dehydrogenase from Mycobacterium vaccae N10 wherein glutamic acid residue is replaced for arginine residue. This substitution can be used in combination with other mutations providing acquiring novel and/or improved properties in formate dehydrogenases. Invention provides preparing novel formate dehydrogenase that possesses the enhanced stability as compared with other enzymes.

EFFECT: improved and valuable biochemical property of enzyme.

4 cl, 2 tbl, 5 ex

Description

The invention relates to the field of genetic and protein engineering and can be used in the analysis of formate in complex samples and in the processes of enzymatic synthesis of optically active compounds or physiologically active substances using dehydrogenases.

New mutant forms of formate dehydrogenase (FDH) bacteria have been obtained, characterized by increased thermal stability compared to the corresponding wild-type enzymes. All proposed muteins are characterized in that the natural amino acid residue of glutamic acid at the position corresponding to position 106 in the formate dehydrogenase sequence from Mycobacterium vaccae N10 is replaced with an arginine residue. This substitution can be used in combination with other mutations that provide formate dehydrogenases with new or improved properties.

The present invention relates to a new protein, in particular, mutant formate dehydrogenases from bacteria that have increased thermal stability compared to the corresponding wild-type enzyme, to DNA encoding mutant formate dehydrogenases, to the use of these enzymes in analytical tests for the presence of formate, to kits including them and in the processes of enzymatic synthesis of optically active compounds or physiologically active substances using dehydrogenases.

NAD ⁺ -dependent formate dehydrogenase catalyzes the oxidation of formate to carbon dioxide during the coupled reduction of NAD ⁺ to NADH.

The exceptionally high specificity of formate dehydrogenase to formate is used to determine formate in complex mixtures. The irreversibility of the reaction catalyzed by this enzyme and the wide pH optimum of the formate dehydrogenase activity allow it to be used very effectively for the regeneration of NADH in the synthesis of optically active compounds using dehydrogenases. An example is the large-scale process for producing tert-L-leucine from trimethylpyruvate using leucine dehydrogenase with a NADH regeneration system based on formate dehydrogenase from Candida boidinii yeast (Bommarius et al., Tetrahedron Asymmetry 1995, 6, 2851-2852).

Formate dehydrogenases are widespread in nature and can be obtained from bacteria, yeast, lower fungi and higher plants. A detailed list of known sources of formate dehydrogenases can be found in the review (Tishkov V., Popov V. Protein engineering of formate dehydrogenase. Biomolecular Engineering, 2006, v. 23, N2-3, p. 89-110). However, since many of the genes encoding these enzymes have been cloned and sequenced, they can also be obtained using recombinant DNA technology. Recombinant DNA sequences encoding enzymes are used to transform microorganisms such as E. coli, which then express the desired enzyme product.

The disadvantage of natural formate dehydrogenases and recombinant wild-type formate dehydrogenases is their low catalytic activity and relatively low chemical and temperature stability. This is especially true for enzymes from yeast, lower fungi and higher plants. Formate dehydrogenase from bacteria Pseudomonas sp. 101 has the highest chemical and temperature stability. For this enzyme, mutant forms are described in which the serine residues at positions 131, 160, 184 and 228 (all of which belong to the alpha-helical regions of the enzyme structure) are replaced with alanine residues (Rojkova et. Al. Bacterial formate dehydrogenase. Increasing the enzyme thermal stability by hydrophobization of alpha helices. FEBS Letters - 1999, v.445, N1, p. 183-188). The obtained mutant enzymes with one, two, and a total of four amino acid substitutions exceeded the thermal stability of FDG wild-type from Pseudomonas sp. 101 by 10-60%. Mutant forms are known for formate dehydrogenase from C. boidinii that surpass the corresponding wild-type enzyme in activity, chemical and temperature stability (Slusarczyk H., Felber S., Kula M. - R., Pohl M., 2003. Novel mutants of formate dehydrogenase from Candida boidinii. US Patent Application Publication US 2003/0157664, 09/21/2003), which, however, are inferior in all these parameters to the mutant PseFDG with the replacement Cys255Ala (Odintseva E.R., Popova A.S., Rozhkova AM, Tishkov V.I. The role of cysteine residues in the stability of bacterial formate dehydrogenase. Vestn. Mosk. Un. Ser. 2, Chemistry, 2002, v. 43, No. 6, pp. 356-359).

The technical problem solved by the proposed technical solution is to obtain a mutant form of formate dehydrogenase with improved characteristics.

The technical result obtained by the implementation of the proposed technical solution is to increase the thermal stability of the obtained form of formate dehydrogenase.

To achieve the indicated technical result, it was proposed to use a recombinant thermostable formate dehydrogenase characterized by an amino acid sequence that corresponds to the amino acid sequence of wild-type formate dehydrogenase with the substitution of the natural residue of glutamic acid at position corresponding to position 106 in the Mycobacterium vaccae N10 formate dehydrogenase sequence, the addition of which enzyme. The sequence of the indicated wild-type formate dehydrogenase preferably corresponds to the formate dehydrogenase Mycobacterium vaccae N10, Pseudomonas sp. 101, Thiobacillus sp.KNK65MA, Ancylobacter aquaticus sp.KNK607M, Moraxella sp.C-1, Paracoccus denimbritium. , Sinorhizobium meliloti 1021. Most preferably, the wild-type formate dehydrogenase sequence is an enzyme sequence derived from Mycobacterium vaccae N10, Pseudomonas sp. 101 or Moraxella sp.C-1. Preferably, the protein has at least 80% homology with respect to formate dehydrogenase from Mycobacterium vaccae N10, Pseudomonas sp. 101 or Moraxella sp. C-1 and contains a glutamic acid residue at position corresponding to position 106 in the amino acid sequence of formate dehydrogenase from Mycobacterium vaccae N10. In addition, in addition to replacing the natural glutamic acid residue with an arginine residue at the position corresponding to position 106 in the amino acid sequence of the formate dehydrogenase from Mycobacterium vaccae N10, it may contain at least one additional substitution, which provides the improvement of thermal stability, chemical stability, kinetic properties, pairwise or a simultaneous improvement of all of the above properties compared with the corresponding wild-type formate dehydrogenase.

The proteins of the invention include both wild-type and recombinant formate dehydrogenases. They usually have 70% homology to wild-type sequences, such as those of the enzyme Mycobacterium vaccae N10, Pseudomonas sp. 101 or Moraxella sp.C-1, in the sense that at least 70% of the amino acids present in the enzymes wild type found in the proteins of the invention. Such proteins may have a higher degree of homology, in particular at least 80%, and most preferably at least 90% with respect to the wild-type enzymes listed above. The homology of a particular sequence with respect to the sequences of the invention can be estimated using the method of multiple comparative analysis of the primary structure described by Lipman and Pearson (Lipman DJ & Pearson WR Rapid and Sensitive Protein Similarity Searches, Science, 1985, vol. 227, pp. 1435-1441 ) The “optimized” percentage scheme should be calculated according to the following parameters for the Lipman-Pearson algorithm: ktup = 1, space = 4 and space length = 12. Sequences for which homology should be evaluated should be used as the “test sequence”, which means that the main sequence for comparison, such as the sequence from Mycobacterium vaccae N10, Pseudomonas sp. 101 or Moraxella sp.C-1, should be entered into the algorithm first . The mutant formate dehydrogenase according to the invention can be obtained from Mycobacterium vaccae N10, Pseudomonas sp. 101, Thiobacillus sp.KNK65MA, Ancylobacter aquaticus sp.KNK607M, Moraxella sp.C-1, Paracoccus denitrificans PD1222, Paracoccus denitrificans PD12.12, Paracoccus denitrificans Hyphom. JC17, Sinorhizobium meliloti 1021, uncultivated marine gamma proteobacteria EBAC20E09.

It was experimentally established that recombinant bacterial formate dehydrogenases obtained from Mycobacterium vaccae N10, Pseudomonas sp. 101 and Moraxella sp.C-1 and containing the replacement Glu106Arg, have increased thermal stability compared to wild-type enzymes. It was also experimentally established that the introduction of the replacement of Glu106Arg into the mutant formate dehydrogenase from Pseudomonas sp.101 with other amino acid substitutions providing increased thermal stability of the enzyme results in a new mutant enzyme with even higher thermal stability.

In the future, the methods of preparation and advantages of the obtained modified formate dehydrogenase will be considered using examples of its implementation.

Example 1

Obtaining a mutant formate dehydrogenase Mycobacterium vaccae N10 with increased thermal stability using disordered mutagenesis.

All genetic engineering manipulations, unless otherwise specified, are carried out in accordance with Manniatis E., Fritsch. E., Sambrook J. Molecular Cloning, M. - Mir, 1984 or in accordance with the instructions of manufacturers of devices, kits and other reagents. All of these methods are well known to those skilled in the art.

The disordered mutagenesis of the full-sized FDH gene from Mycobacterium vaccae N10 was carried out using polymerase chain reaction (NDP) under conditions that caused errors in the DNA sequence (Caldwell R.C., Joyce GF Randomization of Genes Mutagenesis. PCR Methods Appl.2, 1992, 28 -33) (error-prone PCR). Plasmid pFDH6, in which the formate dehydrogenase gene is controlled by the tandem of two promoters, lac and tac, was used as a matrix for random mutagenesis. Plasmid pFDH6 was obtained from the commercially available plasmid pUC119, in which a 81-base DNA fragment was inserted from the HindIII and BamHI restriction sites, cut at the same restriction sites from another commercially available plasmid pDR540 and containing the tac promoter (for details, see Tishkov for obtaining this plasmid VI, Galkin AG, Fedorchuk VV, Savitsky PA, Rojkova AM, Gieren H., Kula M. - R. Pilot scale production and isolation of recombinant NAD ⁺ - and NADP ⁺ -specific formate dehydrogenase. Biotechnology & Bioengineering - 1999, v. 64, N2, p. 187-193). The FDG gene from Mycobacterium vaccae N10 was inserted into the pFDH6 plasmid at the BamHI and EcoRI restriction sites, which were introduced using PCR according to the well-known method, respectively, before the ribosomal binding site and immediately after the stop coding. As a result, the plasmid pMycFDH6 was obtained. It was isolated from E. coli cells using the NucleoSpin® Plus plasmid isolation kit according to the manufacturer's instructions (Macherey-Nagel GmbH & Co. KG).

For PCR with errors used a pair of the following primers:

pFDH6For 5′-CAT GAT TAC GCC AAG CTT ACT C-3 ′

pFDH6Rev 5′-AAC GAC GGC CAG T GA ATT C AC-3 ′

The sequences of these primers are part of the sequence of the pFDH6 plasmid and can be used to introduce random mutations using PCR into any gene cloned into this plasmid. The reaction mixture for PCR with errors had the following composition (table 1).

Table 1. The composition of the reaction mixture for PCR with errors amount Component 5 μl 10-fold mutagenic buffer (60 mM MgCl ₂ , 500 mM KCl, 0.1% (w / v) gelatin, 20 mM Tris-HCl, 50 mM (NH ₄ ) ₂ SO ₄ , pH 8.3 at 25 ° C) 5 μl 10-fold dNTP mutagenesis solution (2 mM dGTP and dATP, 10 mM dTTPu dCTP) 1.5-5 μl 10x MnCI ₂ solution (5 mM MnCl ₂ ) 2 feptamol DNA template (approximately 3.4 ng of a plasmid with a size of 4400 nucleotides, i.e. pMycFDH6) 25 picomole primer pFDH6For 25 picomole primer pFDH6Rev 1 μl Taq DNA polymerase (3 units / μl), Sibenzym, Russia up to 50 μl deionized water purified using Millipore MilliQ system

The reaction was carried out in a 0.5 ml thin-walled plastic tube (Eppendorf, Germany). To prevent evaporation of the reaction mixture and its condensation on the lid, 50 μl of mineral oil was added to the tube. The tube was heated for 5 min at 95 ° C and then the PCR reaction was performed with errors according to the following program: 1st stage - 95 ° C, 60 s; Stage 2 - 50 ° C, 45 s and Stage 3 - 72 ° C, 2 min, 25-35 cycles in total. After that, the reaction mixture was incubated for another 5 min at 72 ° C. The temperature in the second stage is chosen 3-5 degrees below Tm the melting point of the duplexes formed by the primers.

PCR products are purified either using suitable kits (e.g., NucleoSpin Extract (Macherey-Nagel GmbH & Co. KG)) or using preparative agarose gel electrophoresis. They are then treated with restriction enzymes BamHI and EcoRI for 6 hours at 37 ° C, purified from short fragments by preparative agarose gel electrophoresis, followed by extraction of the desired strip from agarose using a NucleoSpin Extract kit (Macherey-Nagel GmbH & Co. KG) and cloned into the original plasmid from which the FDH gene fragment from wild-type Mycobacterium vaccae N10 was previously removed from the same BamHI and EcoRI restriction sites. The resulting library of mutant clones was expressed in E. coli TG1 strain (genotype supE hsdΔ5 thi Δ (lac-proAB) F ′ [traD36 proAB ⁺ lacI ^q lacZΔM15]). Plasmid pMycFDH6 has a beta-lactamase gene and confers ampicillin resistance to E. coli cells containing this plasmid.

The E. coli TG1 strain was transformed with the electroporation library obtained using a BIORAD pulse generator for E. coli and grown overnight at 37 ° C on LB agar containing ampicillin at a concentration of 150 μg / ml and 0.1 mM isopropyl-β-D- thiogalactoside (IPTG). The diameter of the colonies at the end of the cultivation was about 1 mm.

At the end of the cultivation, the cells were poured with a 1.5% agarose solution with lysis buffer No. 1 (0.1 M phosphate buffer; pH 7.0, 0.2% Triton X-100, 10 mM EDTA) in an amount sufficient to form a layer about 2 mm thick. Before pouring, the temperature of the agarose should not exceed 55-60 ° C. After the agarose solidified, the plates were washed 5 times with lysis buffer No. 1 and 5 times with 0.1 M phosphate buffer, pH 7.0 (the thickness of the solution layer on the plate was 5 mm).

Next, the colonies were stained for activity. For this, a developing solution No. 1 was prepared (0.1 M phosphate buffer containing 1.5 M sodium formate, 0.2 g / L phenazine methasulfate, 2 g / L blue nitrotetrazolium, pH 7.0. The solution covers the cups with a 1- layer thickness 2 mm After incubation of the cups with the developing solution No. 1 for 10 minutes in the dark with constant stirring, they added the developing solution No. 2 (60 mg / ml NAD ⁺ in 0.1 M phosphate buffer, pH 7.0) at the rate of 1 part of the developing solution No. 2 to 50 parts of the developing solution No. 1. Next, the plates are incubated in the dark at 30 ° C. with stirring (about 15-30 minutes) until the formation of a black-brown halo around the colonies is noticeable, then the plates are washed twice with a solution of 0.1 M phosphate buffer, pH 7.0 and dried, so that the colonies can be selected for further cultivation.

Colonies containing active formate dehydrogenase were selected and cultured overnight at 37 ° C in 96-well microplates in 0.2 ml of 2YT medium containing ampicillin at a concentration of 150 μg / ml. The obtained solutions of colonies in microplates were used as spare for further work.

To search for clones containing formate dehydrogenase with increased thermal stability, re-cultivation was carried out. For this, 50 μl of cell suspension was taken from the wells of the spare microplates and transferred to a new microplate containing 0.2 ml of 2YT medium, ampicillin at a concentration of 150 μg / ml and 0.1 mM IPTG in each well. Microplates with cells were incubated overnight at 37 ° C with constant stirring. After cultivation, cells in microplates were pelleted by centrifugation (1600 g, 15 min, 20 ° C), the culture fluid was removed from the wells and resuspended in 200 μl of lysis buffer No. 2 (0.1 M phosphate buffer, pH 8.0, egg lysozyme 0.1 mg / ml and Triton X-100, 0.1 volume%). Cell lysis was performed for 2 hours at 35 ° C with constant stirring. Cell debris was besieged using centrifugation (5000 g, 15 min, 20 ° C). 25 μl of the supernatant was transferred to a new microplate to measure the initial activity (A _O ). To do this, another 175 μl of phosphate buffer, pH 7.0, containing 0.35 M sodium formate and 1.2 mg / ml NAD ^{+ was} added to each well of the new microplate ^. The reaction was recorded at 30 ° C by increasing the absorbance by 340 nm on the analyzer microplates (Spectramax® Plus, MWG Biotech). Another 50 μl of the supernatant was transferred to another microplate for PCR, containing 50 μl of 0.1 M phosphate buffer in each well, pH 7.0. The microplate was placed in the fuser of the PCR device in microplates (Techne 96, Techne) and incubated for 20 min at a certain temperature (usually 60-63 ° C). Then the microplate was cooled in a mixture of water and ice for 5 min, if necessary, the precipitate was removed by centrifugation and 50 μl were taken to determine the residual activity (A ₂₀ ).

Plasmid DNA was isolated from E. coli clones expressing MusFDH with enhanced thermal stability, as described above, and then sequenced to determine mutations responsible for the thermal stability of the enzyme. DNA sequencing was performed on an ABI PRISM ^™ Model 377 automated DNA sequencer and ABI PRISM ^™ BigDye ^™ terminator sequencing reaction kit (Perkin Elmer Applied Biosystems), which is based on the dideoxy termination method [F. Sanger et al., Proc. Natl. Acad. Sci. USA 1977, 74, 5463-5467].

As a result of this work, a new mutant MusFDG Glu106Arg was identified. The effect of such a substitution on the thermal stability of the enzyme was quantified according to the procedure described in Example 5. The resulting mutant formate dehydrogenase was 18% more stable than the wild-type enzyme (Table 2).

Example 2

Obtaining mutant MorPDH with the replacement of Glu106Arg using directed mutagenesis.

Directed mutagenesis was carried out according to the Kunkel method (Kunkel T.A. Rapid and efficient site-rectified mutagenesis without phenotypic selection. Proc. Natl. Acad. Sci. USA - 1985, v. 82, p. 488-492) using the pMorFDH6 uridine matrix in single-chain form using the MORE106R primer carrying the desired replacement

5′-CGC-CTT-GGC-GAT GCG NCG GGC GGT CAG ATA GGC-3 ′

The triplet providing the Glu106Arg mutation is highlighted in bold. The letter N means that in this position there can be any of 4 bases (A, T, G or C).

To obtain plasmid pMorFDH6 in single-stranded form with the substitution of base T for U, a special E. coli cell line RZ1023 was used. E. coli RZ1023 cells transformed with plasmid pMorFDH6 were inoculated in 4 ml of 2YT medium containing 100 μg / ml ampicillin and 5 μg / ml uridine, and grown at 37 ° C for 8-10 hours under aeration. Then the cells were infected with helper phage M13K07 (plaque-forming particles of CBE 10 ⁹ -10 ¹⁰ ) with a multiplicity of infection 10 and incubated at 37 ° C for 30 minutes. Then, the entire incubated mixture was placed in 20 ml of 2YT medium (containing 100 μg / ml ampicillin, 100 μg / ml kanamycin, 5 μg / ml uridine and 10 μg / ml thiamine) and grown at 37 ° C for 12 hours with strong aeration. Cells were centrifuged in an Eppendorff 5403 centrifuge at 10,000 rpm for 15 minutes. The supernatant was transferred to a clean centrifuge tube, 0.2 volumes of a 20% PEG-6000 solution and 2.5 M NaCI were added, incubated at 0 ° C for 1 hour and centrifuged at 10,000 rpm for 30 minutes. The phage particle pellet was resuspended in 400 μl of deionized water and the solution was transferred to a clean 1.5 ml tube. Then, 400 μl of a mixture of phenol with chloroform (in a 1: 1 ratio) was added to it, mixed and centrifuged at 14000 rpm for 15 minutes. The upper phase was taken and the extraction was repeated with a mixture of phenol and chloroform again. Then, 400 μl of chloroform was added to the upper phase, mixed and centrifuged at 14,000 rpm for 15 minutes. DNA was besieged by adding a double volume of isopropanol and centrifuged at 14,000 rpm for 30 minutes. The precipitate was washed with 1 ml of chilled 70% ethanol and centrifuged under the same conditions. DNA was dried in air for 10-15 minutes and dissolved in 30 μl of deionized water.

For the phosphorylation reaction, 2 μl of 10-fold phosphorylation buffer (700 mM Tris-HCl, 100 mM MgCl ₂ , pH 7.6), 2 μl of ATP solution (10 mM), 2 μl of DTT (50 mM) were added to 25 picomoles of dry primer ), 2 μl of T4 phage polynucleotide kinase (10 u / μl), 12 μl of deionized water and incubated for 2 hours at 37 ° C. Then, 2 μl (0.025 picomoles) of uridine matrix, 2 μl of 10-fold annealing buffer (200 mm Tris-HCl, 100 mm MgCl ₂ , 500 mm NaCl, pH 7.5) and 8 μl deionized were added to 10 μl of phosphorylated primer solution water. The resulting solution was placed in a glass of boiling water and left on the table to slowly cool to room temperature. To carry out the synthesis reaction of the second DNA strand, 3 μl of 10-fold synthesis buffer (100 mM Tris-HCl, 20 mM DTT, 5 of each dNTP, 10 mM ATP), 1.5 μl of T4 DNA polymerase were added to the reaction mixture ( 10 u / μl), 1.5 μl of T4 phage DNA ligase (400 u / μl), 4 μl of deionized water and incubated sequentially for 5 minutes at 0 ° C, 5 minutes at room temperature and 2-3 hours at 37 ° C .

The mutagenesis reaction mixture transformed E. coli TG1 cells, as described in Example 1. Plasmid isolation and sequencing were also carried out analogously to Example 1. Of the 6 selected plasmids, all 6 contained only the required Glu106Arg replacement. The change in the thermal stability of the mutant MorPDH Glu106Arg was quantified as described in Example 5. The mutant MorPDH Glu106Arg was 11% more stable than the wild-type enzyme (Table 2).

Example 3

Obtaining mutant PseFDG with replacement of Glu106Arg using directed mutagenesis.

This example is identical to example 2 except that the plasmid pPseFDH6 and primer were used as a matrix for mutagenesis

5′-GGC CTT GGC GAT GCG NCG GGG CGT CAG ATA G-3 ′

The same primer can be used to introduce the Glu106Arg substitution in the MusPDH gene.

The change in the thermal stability of the mutant PSeFDG Glu106Arg was quantitatively determined as described in Example 5. The resulting mutant PSeFDH with Glu106Arg substitution was 15% more stable than wild-type PseFDG (Table 2).

Example 4

Obtaining a multipoint mutant PSePDH with substitutions of Ser (131, 160, 184, 228) Ala and Glu106Arg.

This example is identical to Example 3 except that the replacement Glu106Arg was introduced into the mutant PSePDH gene, in which, compared to the wild-type PsePDG gene, Ser residues at positions 131, 160, 184 and 228 are replaced by Ala residues. Plasmid pPseFDH6T4, the preparation of which is described in Rojkova, A.M., et al., Was used as the initial matrix. Bacterial formate dehydrogenase. Increasing the enzyme thermal stability by hydrophobization of alpha helices. FEBS Letters - 1999, v.445, N1, p. 183-188.

The obtained mutant PseFDG with substitutions of Ser (131, 160, 184, 228) Ala and Glu106Arg was 85% more stable than wild-type PseFDG (Table 2).

Example 5

Accurate determination of the effect of stabilization of formate dehydrogenase by replacing Glu106Arg.

To accurately evaluate the stabilization effect of formate dehydrogenase due to the replacement of Glu106Arg, the mutant enzyme and wild-type formate dehydrogenase were isolated in a highly purified state and the thermal inactivation rate constants were determined at a certain temperature (above 54 ° C).

The purification procedure is exactly the same for all of the above recombinant formate dehydrogenases and does not depend on the source of the gene (Mycobacterium vaccae N10, Pseudomonas sp. 101 or Moraxella sp. C-1), or on the type of enzyme (mutant or wild type). The biomass of E. coli TG1 cells containing the target enzyme was obtained by culturing a single colony in 1 L flasks containing 250 ml of 2YT medium and 150 μg / ml ampicillin at 37 ° C with continuous stirring (110 rpm). Upon reaching the absorption of the medium at 600 nm, A ₆₀₀ values of 0.6-0.9 were added to the flasks with IPTG to a concentration of 0.5 mM and cultured for another 12 hours. Cells were harvested by centrifugation (2500g, 15 min, 4 ° C). Then they were resuspended in 0.1 M phosphate buffer, pH 7.0 to obtain a 20% suspension (total volume 10 ml). For preliminary destruction, the cells were subjected to freezing-thawing and then treated with ultrasound for 5 min under ice cooling. The precipitate was removed by centrifugation (10000 g, 30 min, 4 ° C), and 6.4 ml of a 4 M solution of ammonium sulfate in 0.1 M phosphate buffer, pH 7.0, was added to the supernatant (volume 8 ml). The resulting solution was left overnight at 4 ° C, the resulting precipitate was removed by centrifugation (10000g, 30 min, 4 ° C). The supernatant was applied to a 1 × 10 cm column with Phenylsepharose Fast Flow (Pharmacia Biotech, Germany) balanced with a 1.5 M solution of ammonium sulfate in 0.1 M phosphate buffer, pH 7.0. After the enzyme was applied to the column, it was washed with a 1.2 M solution of ammonium sulfate in 0.1 M phosphate buffer, pH 7.0 (solution A) until the absorbance of solution leaving the column (70-100 ml) at 280 nm was zero. The enzyme was eluted from the column by a linear downward gradient (50 ml of solution A — 50 ml of 0.1 M phosphate buffer, pH 7.0). Fractions containing the mutant enzyme or wild-type formate dehydrogenase were collected and concentrated on a PM10 membrane in a 10 ml concentration cell (membrane and cell manufactured by Amicon, USA) to a volume of 1 ml. The obtained enzyme was applied to a 1 × 100 cm column with Sephacryl S-200 (Pharmacia Biotech, Germany), equilibrated with 0.1 M phosphate buffer, pH 7.0. 2 ml fractions were collected, their absorption at 280 nm A _{280 was} determined on a Shimadzu 1601 PC spectrophotometer (Shimadzu GmBH, Germany), and the enzymatic activity was measured at 340 nm at 30 ° C on the same spectrophotometer (0.1 M phosphate buffer, pH 7,0, 03 M sodium formate, 1 mg / ml NAD ^+). Fractions having a constant ratio (activity / A ₂₈₀ ) were selected. The purity of the preparations obtained is at least 95% according to analytical electrophoresis in 12% polyacrylamide gel in the presence of sodium dodecyl sulfate. Electrophoresis was performed on a BioRad instrument according to the manufacturer's instructions.

To determine the rate constant of the thermal inactivation of wild-type formate dehydrogenase and mutant enzyme with Glu106Arg replacement for each type of enzyme, a series of 12-15 plastic tubes with a volume of 1.5 ml (Eppendorf, Germany) containing 60 μl of the enzyme solution in a concentration of 0.2-1 mg / ml in 0.1 M phosphate buffer, pH 7.0. The tubes were placed in a water thermostat at a certain temperature (54-64 ° C, thermostat accuracy ± 0.1 deg) and 1 tube was taken at specified intervals, cooled in cold water (0-10 ° C) for 5 min, centrifuged 3 min at 10000g and the residual enzymatic activity A was determined as described above. Then, we plotted the dependence of the natural logarithm of the residual activity ln (A / A ₀ ) on time and calculated the inactivation rate constant constant k _in from the slope. As an example, the dependences for wild-type PSePDH and its mutant form with the replacement of Glu106Arg are given. Table 2 shows the values of the inactivation rate constants for all studied FDG.

Table 2. Inactivation rate constants of mutant formate dehydrogenases with replacement of Glu106Arg and wild-type enzymes at 60 ° C in 0.1 M phosphate buffer, pH 7.0 Enzyme Inactivation rate constant, k _in · 10 ⁶ , s ^-1 The stabilization effect, (k _in ^mut / k _in ^wt ) · 100,% Formate dehydrogenase from Mycobacterium vaccae N10 MusFDG wild-type 157 ± 5.0 one hundred% MusFDG Glu106Arg 133 ± 5.0 118% Formate dehydrogenase from Moraxella sp.C-1 MorFDG wild-type 326 ± 6.0 one hundred% MorFDG Glu106Arg 295 ± 5.0 111% Formate dehydrogenase from Pseudomonas sp. 101 PseFDG wild-type 35.5 ± 0.5 one hundred% PseFDG Glu106Arg 31.0 ± 1.0 115% PseFDG Ser (131, 160, 184, 228) Ala / Glul06Arg 19.1 ± 0.3 185%

The obtained mutant form of formate dehydrogenase has increased thermal stability relative to the initial form.

Claims (4)

1. A recombinant thermostable formate dehydrogenase characterized by an amino acid sequence that corresponds to the amino acid sequence of a wild-type formate dehydrogenase containing a glutamic acid residue at position 106 in the Mycobacterium vaccae N10 formate dehydrogenase sequence in which said glutamic acid residue is replaced by arginine residue. 2. The recombinant formate dehydrogenase according to claim 1, characterized in that the sequence of said wild-type formate dehydrogenase corresponds to the formate dehydrogenase Mycobacterium vaccae N10, Pseudomonas sp. 101, Thiobacillus sp.KNK65MA, Ancylobacter aquaticus sp.KNx12Cnc60nc60cMcncfcnifica.nc60cnmcfcnifica , Paracoccus sp. 12-A, Hyphomicrobium sp. JC17, Sinorhizobium meliloti 1021. 3. The recombinant formate dehydrogenase according to claim 2, characterized in that the wild-type formate dehydrogenase sequence is an enzyme sequence obtained from Mycobacterium vaccae N10, Pseudomonas sp. 101 or Moraxella sp.C-1. 4. The recombinant formate dehydrogenase according to claim 1, characterized in that the protein has at least 80% homology with respect to formate dehydrogenase from Mycobacterium vaccae N10, Pseudomonas sp. 101 or Moraxella sp.C-1 and contains a glutamic acid residue at the position corresponding to position 106 in the amino acid sequence of formate dehydrogenase from Mycobacterium vaccae N10.