RU2557299C1 - Mutant plant formate dehydrogenase (versions) - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention is two new mutant forms of plant formate dehydrogenase (FDH) having improved thermostability compared to the initial protein. In the mutant proteins the natural residue of alanine in the position corresponding to position 267 in the initial sequence of formate dehydrogenase of soybean Glycine max, is replaced by the residue of methionine. In addition, in one of the mutants the natural residue of isoleucine in the position 272 is additionally replaced by the residue of valine.

EFFECT: invention enables to obtain formate dehydrogenase with increased temperature stability.

2 cl, 2 tbl, 4 ex

Description

The invention relates to the field of genetic and protein engineering and can be used in the synthesis of optically active compounds or physiologically active substances, detection of formate, as well as to obtain stress-resistant plants.

Two new mutant forms of plant formate dehydrogenase (FDH) have been obtained, which have enhanced thermal stability compared to the starting enzyme. In all of the proposed mutant proteins, the natural alanine residue at the position corresponding to position 267 in the sequence of the initial formate dehydrogenase from Glycine max soybean was replaced with a methionine residue. In addition, in one of the mutants, the natural isoleucine residue at position 272 is replaced by a valine residue. These substitutions can be used in combination with other mutations that provide formate dehydrogenases with new or improved properties.

This invention relates to a new protein, in particular to mutant formate dehydrogenases from plants that have enhanced thermal stability compared to the corresponding wild-type enzyme, to DNA encoding mutant forms of formate dehydrogenase, to the use of these enzymes in systems for the synthesis of optically active compounds or physiologically active substances, for the detection of formate, as well as for obtaining stress-resistant plants.

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

FDG is highly specific for formate; therefore, this enzyme is successfully used to determine formate in complex mixtures.

The oxidation reaction of the formate ion is irreversible, the product is carbon dioxide, which can be easily removed from the reaction sphere. FDG also has a wide pH optimum of activity. Therefore, formate dehydrogenase can be effectively used 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 yeast Candida boidinii (Bommarius AS, Schwarm M., Stingi K., Kottenhahn M., Huthmacher K. and Huthmacher K. and Huthmacher K. and K., Synthesis and Use of Enantiomerically Pure tert-Leucine, Tetrahedron Asymmetry, 1995, v. 6, p. 2851-2888). A method is also used in which the synthesis process and regeneration are carried out directly in a bacterial cell. In one of the first studies, the FDH gene from bacteria Mycobacterium vaccae N10 was expressed in E. coli cells together with one of the enzymes, either leucine dehydrogenase, or alanine dehydrogenase, or phenylalanine dehydrogenase. Such E. coli cells were used for the synthesis of L-leucine, L-valine, L-norleucine, L-methionine, L-phenylalanine and L-tyrosine with a yield of more than 80% and optical purity up to 100% (Galkin, A., Kulakova , L., Yoshimura, T., Soda, K., & Esaki, N. Synthesis of optically active amino acids from alpha-keto acids with Escherichia coli cells expressing heterologous genes. Appl. Environ. Microbiol., 1997, v. 63 , p. 4651-4656). Recently, the FDH gene from Candida boidini yeast was expressed in E. coli together with ketoreductase and this biocatalyst was used in the synthesis of xylitol from xylose and 5-1- (2-chlorophenyl) ethanol from o-chloroacetophenone (Madje K., Schmolzer K. , Nidetzky B., Kratzer R. Host cell and expression engineering for development of an E. coli ketoreductase catalyst: Enhancement of formate dehydrogenase activity for regeneration of NADH. Microb. Cell Fact. 2012 v.11, p.11-17). One of the common methods for working with enzymes is their immobilization on solid carriers. Authors (Demir AS, Talpur FN, Betui Sopaci S., Kohring GW, Celik A. Selective oxidation and reduction reactions with cofactor regeneration mediated by galactitol-, lactate-, and formate dehydrogenases immobilized on magnetic nanoparticles. J Biotechnol., 2011, v, 2011 .10, p.176-183.) Formate dehydrogenase from Candida methylica yeast, as well as other enzymes, was immobilized onto nanoparticles with the synthesis of high optical purity 5-1,2-propanediol.

Formate dehydrogenase is found in bacteria, yeast, microscopic fungi, and also plants. Formate dehydrogenase activity in plants was first discovered in 1921 (Thunberg T. Sur la presence de certains ferments oxydants dans les grains de Phaseolus vulgaris. Arch.int.PhysioL, 1921, v.l8, pp. 601-606). In plants, FDG is a stress enzyme, the synthesis of which increases dramatically under stressful effects such as drought, an increased dose of ultraviolet radiation, sudden temperature jumps, lack of oxygen, etc. (Hourton-Cabassa S., Ambard-Bretteville F., Moreau F., Davy de V., Remy R., and Francs-Small CC Stress Induction of Mitochondrial Formate Dehydrogenase in Potato Leaves. Plant Physiol, 1998, v. 116, pp. 627-635; Suzuki K., Itai R., Suzuki K., Nakanishi H., Nishizawa NK, Yoshimura E., and Mori S. Formate dehydrogenase, an enzyme of anaerobic metabolism, is induced by iron deficiency in barley roots Plant Physiol, 1998, v. 116, pp. 725-732; Thompson, P., Bowsher, CG, and Tobin, AK, Heterogeneity of mitochondrial protein biogenesis during primary leaf development in barley. Plant Physiol, 1998, v. 118, p. 1089 -1099; Andreadeli A., Flemetakis E., Axarii I., Dimou M., Udvardi MK, Katinakis P., and Labrou NE Cloning and characterization of Lotus japonicus formate dehydrogenase: a possible correlation with hypoxia. Biochim. Biophys.Acta, 2009, v. 1794, pp. 976-984; David P., des Francs-Small CC, Sevignac M., Thareau V., Macadre C., Langin T., and Geffroy V. Three highly similar formate dehydrogenase ge nes located in the vicinity of the B4 resistance gene cluster are differentially expressed under biotic and abiotic stresses in Phaseolus vulgaris. Theor. Appl Genet., 2010, v. 121, pp. 87-103). A detailed review of the physiological role, production and properties of FDG from plants was published in 2011 (Alekseeva A.A., Savin CC, Tishkov V.I. NAD ⁺ -dependent formate dehydrogenase of plants. Acta Naturae, 2011, v. 3, No. 4 (11), pp. 40-56).

Many of the genes encoding these enzymes were cloned and sequenced, so they can be obtained using recombinant DNA technology. The cDNA sequence encoding formate dehydrogenase is inserted into a genetic engineering vector (plasmid, phage, etc.), which is used to transform microorganisms, for example, E. coli bacteria. The resulting recombinant strain is used to express the desired enzyme product.

Plant formate dehydrogenases are characterized by low Michaelis constants compared to enzymes from other sources. One of the most promising for practical use of formate dehydrogenases from plants is formate dehydrogenase from soy Glycine max (SoyFDH). This enzyme was obtained in active and soluble form (Sadykhov E.G., Serov A.E., Yasny I.E., Voinova N.S., Alekseeva A.A., Petrov A.S., Tishkov V.I. NAD ⁺ -dependent formate dehydrogenase from Arabidopsis thaliana and soybean: expression in E. coli cells and kinetic properties of recombinant enzymes.Vestn. Mosk. Un. Ser. 2. Chemistry, 2006, v. 47, N1, p.31- 34) and it has the lowest values of Michaelis constants in comparison with all studied formate dehydrogenases at the moment (Alekseeva A.A., Savin S.S., Tishkov V.I. NAD ⁺ -dependent formate dehydrogenase plants. Acta Naturae, 2011, t .3, No.4 (11 ), pp. 40-56). A characteristic feature of plant formate dehydrogenases is the presence of several isoenzymes that are encoded by individual genes. In the case of soybean formate dehydrogenase, 6 isoenzymes are described in the literature (Alekseeva A.A., Savin S.S., Tishkov V.I. NAD ⁺ -dependent formate dehydrogenase of plants. Acta Naturae, 2011, v. 3, No. 4 (11), p .40-56). At our disposal there is another soy isoenzyme gene. The amino acid sequence of this isoenzyme is presented in this application as the sequence Seq_ID No. 1.

The main disadvantages of the known natural and recombinant formate dehydrogenases from wild-type plants are the low temperature stability of these enzymes. Thus, for the successful use of formate dehydrogenase from soy in practice, it is necessary to increase the stability of this enzyme. One approach to solving this problem is the mutagenesis of the amino acid residues of the target protein.

For bacterial and yeast enzymes in the literature there are many examples of successful improvement of properties by methods of protein engineering. To increase the thermal stability of formate dehydrogenase from bacteria Pseudomonas sp. 101 (PseFDH), approaches based on hydrophobization of α-helices (replacing Ser residues in α-helices with Ala) were used (Rojkova AM, Galkin AG, Kulakova LB, Serov AE, Savitsky PA, Fedorchuk VV, and Tishkov VI Bacterial formate dehydrogenase. Increasing the enzyme thermal stability by hydrophobization of alpha-helices. FEBS Lett., 1999, v.445, pp.183-188), increasing the hydrophobicity of the protein globule (replacing Ala residues with Val in contact regions of α-helices) (Fedorchuk VV, Galkin AG, Yasny IE, Kulakova LB, Rojkova AM, Filippova AA, and Tishkov VI Influence of interactions between amino acid residues 43 and 61 on thermal stability of bacterial formate dehydrogena ses. Biochemistry (Mosc.), 2002, v. 67, pp. 1145-1151) and the removal of conformational stresses in the polypeptide chain (replacement of bulk Asn and Tyr by Gly) (Serov AE and Tishkov VI Role of Pro residues in stability of prokaryotic and euacaryotic formate dehydrogenases. Vestn.Mosk.Un-ta. ser.2. Chemistry, 2002, v. 43, pp. 345-349; Serov A.E., Odintzeva E.R., Uporov I.V., and Tishkov V.I. Use of Ramachandran plot for increasing thermal stability of bacterial formate dehydrogenase. Biochemistry (Mosc.), 2005, v. 70, pp. 804-808). The search for positions for mutagenesis was based on the data of X-ray diffraction analysis and comparison of the amino acid sequences of FDG from different sources. As a result of a single replacement, the stabilization effect was small - from 10 to 50%, but in almost all cases - additive (Rojkova AM, Galkin AG, Kulakova LB, Serov AE, Savitsky PA, Fedorchuk VV, and Tishkov VI Bacterial formate dehydrogenase. Increasing the enzyme thermal stability by hydrophobization of alpha-helices. FEBS Lett., 1999, v.445, p. 183-188; Serov AE, Odintzeva ER, Uporov IV, and Tishkov VI Use of Ramachandran plot for increasing thermal stability of bacterial formate dehydrogenase Biochemistry (Mosc.), 2005, v. 70, pp. 804-808).

Mutations that enhance operational and temperature stability were combined in the PseFDH GAV mutant. As a result of this association, the affinity for NAD ⁺ increased by 2 times, and thermal stability - by 2.5 times, and operating - more than 1000 times compared with wild-type PseFDH. The preparation of mutant PseFDHs with improved thermal stability made it possible to introduce a heat treatment step into the purification of a recombinant enzyme. Heating the cell-free extract at 60 ° C for 20-30 min leads to an increase in the purity of the PseFDH GAV preparation from 50 to 80-85% without loss of enzymatic activity (Tishkov VI and Popov VO Protein engineering of formate dehydrogenase. Biomol. Eng, 2006, v .23, pp. 89-110).

Also in the literature there is evidence of an increase in the stability of FDG from C. boidini yeast. In the work (Slusarczyk N., Felber S., Kula MR, and Pohl M. Novel mutants of formate dehydrogenase from Candida boidinii. US Patent Application Publication, 2003, US 2003/0157664), a chemically more stable mutant CboFDH SM with the replacement Cys23Ser, which was much more stable than the wild-type enzyme in solutions of 150 mM H ₂ O ₂ and 50 μM CuSO ₄ , but its thermal stability decreased by 6.7 times (T _m decrease by 5 ° C). To compensate for the loss of stability, the method of directed evolution was used. Four mutations were found that give the greatest stabilization effect (Glu151Asp, Arg178Ser, Lys306Arg, Thr315Asn), which increased the thermal stability of the CboFDH_SM mutant by 36 times (increase in T _m by 10 ° С), and by 5.7 times in comparison with the wild-type enzyme .

In the case of recombinant soybean formate dehydrogenase (SoyFDH), mutants are described with point substitutions of the Phe290 residue with Asp, Asn and Ser residues (Alekseeva AA et al. Protein Eng. Des. Sel., 2012, v.25, N11, p. 781-788 ), in which the inactivation rate constant at 56 ° C was reduced from 2.5 (Ala267Met replacement) to 42 times (Phe290Asp replacement) compared to that for the wild-type enzyme. At the same time, the initial recombinant soybean FDH has the same chemical stability during incubation with hydrogen peroxide as the mutant FDH from bacteria Pseudomonas sp. 101 with the replacement of two cysteine residues Cys145Ser and Cys255Ala (Savin S.S., Tishkov V.I. Inactivation hydrogen peroxide as a method for assessing the stress stability of formate dehydrogenase in vivo Acta Naturae, 2010, v. 2, No. 1 (4), pp. 80-84. However, the obtained mutant SoyFDHs are inferior in stability to FDG from bacteria and yeast.

The technical problem solved by the proposed technical solution is to obtain a mutant form of formate dehydrogenase from soy Glycine max with increased temperature stability compared with the stability of the wild-type enzyme.

The technical result obtained by the implementation of the proposed technical solution consists in increasing the temperature stability of the obtained mutant form of formate dehydrogenase from soy Glycine max in relation to the thermal stability of the wild-type enzyme.

To achieve the technical result, it is proposed to use a recombinant plant formate dehydrogenase, in which the amino acid sequence of formate dehydrogenase from soy Glycine max, specified in Seq_ID No. 1, is used. A distinctive feature of this sequence is the presence in it of 1 position at the N-terminus of the Ala residue, while in the active wild-type enzyme, the Ser residue is in this position. This replacement allows to increase the biosynthesis rate and stability of recombinant formate dehydrogenase when expressed in E. coli cells.

In the future, the preparation and advantages of the obtained mutant plant formate dehydrogenases will be examined using examples of the preparation and sale of this enzyme.

Example 1

Obtaining a mutant plant formate dehydrogenase in which the Ala267 residue is replaced by the Met residue.

Directed mutagenesis was performed using polymerase chain reaction (PCR). Plasmid pSoyFDH was used as a matrix for targeted mutagenesis. The plasmid pSoyFDH was obtained from the commercially available plasmid pET21a (Novagen, USA), into which the cDNA of the original soy formate dehydrogenase Glycine max was inserted at the NdeI and EcoRI restriction sites.

To introduce the Ala267Met mutation into the plant formate dehydrogenase gene using PCR, universal T7 for and T7 rev primers were used:

T7For 5′-TAATACGACTCACTATAGGG-3 ′

T7rev 5′-GCTAGTTATTGCTCAGCGG-3 ′,

as well as direct and reverse primers carrying the required substitution in a triplet encoding in the soyfdh gene the residue Met at position 267:

SoyA267Mfor 5′-GAGGGGCAATTATGGACACCCAAGCAATTGCA-3 ′

SoyA267Mrev 5′-TTGGGTGTCCATAATTGCCCCTCGAGCATTGTT-3 ′.

Nucleotide substitutions providing the Ala267Met mutation are highlighted in bold with underline.

The PCR reaction was carried out in a 0.5 ml thin-walled plastic tube (SSI, USA). To prevent evaporation of the reaction mixture and its condensation on the lid, 30 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 carried out according to the following program: 1st stage - 95 ° C, 60 s; Stage 2 - 56 ° C, 60 s and Stage 3 - 72 ° C, 2 min, 25-35 cycles in total. After that, the reaction mixture was kept for another 10 min at 72 ° C. The temperature in the second stage was chosen 3-5 ° below T _{m the} melting point of the duplexes formed by the primers.

The reaction mixture for PCR had the following composition (table I):

Table 1 Polymerase Chain Reaction (PCR) Conditions amount Component 2.5 μl 10x PFU Turbo DNA Polymerase Buffer (Fermentas, Lithuania) 2 μl dNTP mixture (dATP, dGTP, dTTP, dCTP), concentration 2.5 mM 1 μl DNA matrix, concentration ~ 10 ng / μl 2 μl primer 1, concentration 10 nmol / ml (Synthol, Russia) 2 μl primer 2, concentration of 10 nmol / ml (Synthol, Russia) 0.5 μl PFU Turbo DNA Polymerase (2.5 U / μl) (Fermentas, Lithuania) up to 25 μl deionized water purified using the MilliQ system (Millipore, USA)

At the first stage, two polymerase chain reactions were carried out using the following primer pairs: 1) T7For + SoyA267Mrev and 2) SoyA267Mfor + T7Rev. Plasmid pSoyFDH was used as a matrix. The resulting PNR products, fragment 1 and fragment 2 were purified using preparative agarose gel electrophoresis using appropriate kits, for example, GeneJet Gel Extraction Kit (Fermentas, Lithuania). Then, a third PCR combination was performed with T7For and T7Rev primers, where previously obtained fragments 1 and 2 were used as the DNA template.

The third PCR product was purified similarly to fragments 1 and 2 and was treated with restriction endonucleases Kpnl and EcoRI for two hours at 37 ° C, then the restriction mixture was incubated to restrict restriction enzymes for 20 minutes at 65 ° C (restriction enzymes and buffer solutions from Fermentas (Lithuania were used) )). Then, the obtained DNA fragments were purified from short fragments by preparative agarose gel electrophoresis followed by extraction of the desired strip from agarose using a special kit, for example, GeneJet Gel Extraction Kit (Fermentas, Lithuania), and doped with a fragment of the original plasmid pSoyFDH, from which it was digested according to the same the KpnI and EcoRI restriction sites removed the soyfdh gene fragment from G. max wild-type.

The ligation reaction mixture transformed E. coli TG1 cells (genotype supE hsdΔ5 thi Δ (lac-proAB) F ′ [traD36 proAB ⁺ lac ^q lacZΔM15]) and grown overnight at 37 ° C on LB solid agar medium containing ampicillin at a concentration of 100 μg / ml (plasmid pSoyFDH contains the gene for resistance to ampicillin).

The grown colonies were inoculated in 6-8 ml of 2YT medium containing 150 μg / ml ampicillin and grown at 37 ° C for 8-10 hours with aeration. Plasmid DNA was isolated from the obtained cell culture using the kit intended for this, for example, GeneJet Plasmid Miniprep Kit (Fermentas, Lithuania). To control the introduction of the required mutations, plasmid DNA was sequenced at the Genome Collective Use Center (VA Engelhardt Institute of Molecular Biology, Russian Academy of Sciences). As a result, out of 5 isolated plasmids, all 5 contained only the required Ala267Met replacement. Thus, the plasmid pSoyFDH_A267M was obtained, which provided the production of the new formate dehydrogenase SoyFDH Ala267Met with the replacement of Ala267Met.

For mutant enzyme E.coli BL21 cells (DE3) pLysS Codon Plus (genotype E. coli B ^{F - ompT hsdS (rB - mB} -) dcm + Tet r gal λ (DE3) pLysS endA Hte [argU proL Cam r]) transformed with the plasmid pSoyFDH_A267M, seeded on Petri dishes with agar medium containing ampicillin (150 μg / ml) and chloramphenicol (25 μg / ml) and cultured (see below).

Example 2

Study of the temperature stability of the mutant SoyFDH formate dehydrogenase with the replacement of Ala267Met and the wild-type enzyme.

To accurately assess the effect of changes in temperature stability due to the introduced substitutions, the mutant enzyme SoyFDH Ala267Met and wild-type formate dehydrogenase were isolated in a highly purified state and the dependence of the residual activity on the incubation time at 52, 54, and 56 ° C was determined.

The procedure for obtaining highly purified preparations was the same for both the mutant SoyFDH Ala267Met and the wild-type enzyme.

The expression of wild-type SoyFDH and its mutants was performed in E. coli BL21 (DE3) CodonPlus / pLysS cells. To prepare the seed, a single colony was taken from the cup and cultured for 7–9 h at 30 ° C and 180 rpm until the absorbance at a wavelength of 600 nm was reached A ₆₀₀ ≈0.6-0.8 in 5 ml of 2YT medium (yeast extract 10 g / l, bactotriptone 16 g / l, sodium chloride 5 g / l, pH 7.0) in the presence of 150 μg / ml ampicillin and 25 μg / ml chloramphenicol. Then, the contents of the tubes were transferred to conical rocking flasks with 1 L chippers containing 200 ml of 2YT medium and 150 μg / ml ampicillin and the cells were cultured at 30 ° C and 80-90 rpm until the absorbance at 600 nm was reached, A ₆₀₀ = 0.6-0.8. Then, cell induction was carried out by adding a lactose solution (300 g / l) to the final culture medium to a final inducer concentration of 20 g / l. After induction, the culture temperature was reduced to 20 ° C and the cells were cultured for 17 hours at 120 rpm. The resulting biomass was besieged in a Beckman J-21 centrifuge (USA) at 7500 rpm for 20 min at 4 ° C and after removal of the culture fluid, the cells were resuspended in 0.1 M sodium phosphate buffer, pH 8.0 in a ratio of 1: 4 (mass.). The resulting suspension was frozen and stored at -20 ° C.

To isolate the mutant SoyFDH Ala267Met and the wild-type enzyme, a 20% suspension of cells in 0.1 M sodium phosphate buffer, pH 8.0 was subjected to two freeze-thaw cycles, and then the cells were destroyed using a Branson Sonifier 25 O ultrasonic disintegrator (Germany ) with constant cooling. The precipitate was removed by centrifugation on an Eppendorf 5804 R centrifuge (11000 rpm, 30 min).

For purification, we used a modified method developed to obtain recombinant FDG from bacteria Pseudomonas sp. 101 (Rojkova AM, Galkin AG, Kulakova LB, Serov AE, Savitsky PA, Fedorchuk VV, and Tishkov VI Bacterial formate dehydrogenase. Increasing the enzyme thermal stability by hydrophobization of alpha-helices. FEBS Lett., 1999, v. 445, pp. 183-188). The enzyme purification procedure included the precipitation of ballast proteins with ammonium sulfate (45% of saturation), precipitation of the target protein at a concentration of ammonium sulfate 85% of saturation, and its subsequent re-dissolution in a solution containing 45% ammonium sulfate in 0.1 M phosphate buffer, pH 7.0 (buffer A). The obtained supernatant was used for hydrophobic chromatography on Phenyl Sepharose Fast Flow (Pharmacia Biotech) in a downward gradient of 45-0% ammonium sulfate concentration in buffer A and desalination on a Sephadex G-25 column in the same buffer. The purity of the obtained preparations was controlled using analytical electrophoresis in 12% polyacrylamide gel in the presence of 0.1% sodium dodecyl sulfate on a MiniProtean II electrophoresis apparatus from BioRad.

FDH activity was determined spectrophotometrically from the accumulation of NADH at a wavelength of 340 nm (ε ₃₄₀ = 6220 M ^-1 cm ^-1 ) on a Schimadzu UV 1800 PC spectrophotometer at 30 ° C in 0.1 M sodium phosphate buffer, pH 7.0 . The concentration of sodium formate and NAD ⁺ in the cuvette was 0.6 M and 0.2 mg / ml, respectively.

Thermostability of the enzyme was measured in 0.1 M sodium phosphate buffer, pH 7.0. For each experiment, a series of 0.5 ml plastic tubes of 100 μl of an enzyme solution (0.2 mg / ml) in each was prepared. The tubes were placed in a water thermostat preheated to the required temperature (52-56 ° С, thermostating accuracy ± 0.1 ° С). At certain points in time, one tube was taken and transferred to ice for 5 min, after which the tube was centrifuged for 3 min at 12,000 rpm on an Eppendorf 5415D centrifuge. The residual activity of FDG was measured as described above. The thermal inactivation rate constant km was determined as the slope of the straight line from the graph of the natural logarithm of the residual activity versus time (semi-logarithmic coordinates ln (A / A ₀ ) -t) by linear regression using the Origin Pro 8.5 program.

As a parameter characterizing thermal stability, the rate constant of the thermal inactivation process was used. The results are presented in table 2. As follows from table 2, the introduction of the Ala267Met mutation increases the stability of formate dehydrogenase from soybeans by 4.9, 3.0 and 2.5 times at temperatures of 52, 54 and 56 ° C, respectively.

Example 3

Obtaining a mutant plant formate dehydrogenase in which the residue A1a267 is replaced by the residue Met and the residue 11e272 is replaced by the residue Val.

This example is identical to example 1 except that the plasmid pSoyFDH_A267M (for the preparation see example 1) was used as a template for targeted mutagenesis, and the following deoxyribo oligonucleotides were used as direct and reverse primers for the introduction of the Ile272Val substitution:

Soy_I272Vfor 5′-ACCCAAGCA GTT GCAGATGCTTGCTCCAGT-3 ′

Soy_I272Vrev 5′-AAGCATCTGC AAC TGCTTGGGTGTCCATAATTGC-3 ′

Nucleotide substitutions providing the Ile272Val mutation are highlighted in bold with underline.

The plasmid pSoyFDH with nucleotide substitutions providing the Ala267Met and Ile272Val mutations in the gene of the enzyme was called pSoyFDH_ (A267M, I272V).

For mutant enzyme E.coli BL21 cells (DE3) pLysS Codon Plus (genotype E. coli B ^{F - ompT hsdS (rB - mB} -) dcm + Tet r gal λ (DE3) pLysS endA Hte [argU proL Cam r]) transformed with the plasmid pSoyFDH_ (A267M, I272V), seeded on Petri dishes with agar medium containing ampicillin (150 μg / ml) and chloramphenicol (25 μg / ml) and cultured (see above).

Example 4

Study of the temperature stability of the mutant SoyFDH formate dehydrogenase with substitutions of Ala267Met and Ile272Val and the wild-type enzyme.

To accurately assess the effect of changes in temperature stability due to the introduced substitutions, the mutant enzyme SoyFDH (Ala267Met, Ile272Val) and wild-type formate dehydrogenase were isolated in a highly purified state and the dependence of the residual activity on incubation time at 52, 54, and 56 ° С was determined.

The cultivation of cells containing the plasmid pSoyFDH_ (A267M, I272V) was carried out according to the procedure described in example 2.

Isolation, purification, as well as measurement of the activity and thermal stability of the mutant form of formate dehydrogenase from SoyFDH soybean (Ala267Met, Ile272Val) was carried out according to the methods described in example 2.

As a parameter characterizing thermal stability, the rate constant of the thermal inactivation process was used. The results are presented in table 2. As follows from table 2, the introduction of mutations (Ala267Met, Ile272Val) increases the stability of formate dehydrogenase from soybeans by 15, 8 and 5.6 times at temperatures of 52, 54 and 56 ° C, respectively.

table 2 Thermal inactivation rate constants of formate dehydrogenase from wild-type soybean SoyFDH, as well as mutant forms SoyFDH Ala267Met and SoyFDH (Ala267Met, Ile272Val) Enzyme form temperature 52 54 56 k _in , min ^-1 k _in (wt) / k _in (mut) k _in , min ^-1 k _in (wt) / k _in (mut) km, min ^-1 k _in (wt) / k _{in (} mut) Wild type soyfdh (3.0 ± 0.1) * 10 ^-2 one (5.3 ± 0.2) * 10 ^-2 one (12.8 ± 0.2) * 10 ^-2 SoyFDH Ala267Met (0.63 ± 0.02) * 10 ^-2 4.9 (1.78 ± 0.04) * 10 ^-2 3.0 (5.16 ± 0.07) * 10 ^-2 2,5 SoyFDH (Ala267Met, Ile272Val) (0.21 ± 0.01) * 10 ^-2 fifteen (0.65 ± 0.01) * 10 ^-2 8 (2.26 ± 0.08) * 10 ^-2 5,6

Claims (2)

1. Mutant plant formate dehydrogenase with increased temperature stability, characterized by an amino acid sequence corresponding to the amino acid sequence of formate dehydrogenase from Glycine max (SEQ_ID No. 1), in which the alanine residue at position 267 is replaced by a methionine residue (SEQ_ID No. 2). 2. Mutant plant formate dehydrogenase with increased temperature stability, characterized by the amino acid sequence corresponding to the amino acid sequence of formate dehydrogenase from Glycine max (SEQ_ID No. 1), in which the alanine residue at position 267 is replaced by a methionine residue, and the isoleucine residue at position 272 is replaced by a valine residue (SEQ_ID No. 3).

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