RU2545966C1 - Mutant formate dehydrogenase (versions) - Google Patents

Abstract

FIELD: biotechnologies.

SUBSTANCE: invention represents four new mutant shapes of plant formate dehydrogenase (FDG). In the first one of the proposed mutant proteins, natural residue of phenylalanine in a position corresponding to position 290 in a sequence of formate dehydrogenase from soya Glycine max has been replaced with alanine residue. In the second mutant shape of the ferment, the same natural residue of phenylalanine 290 has been replaced with tyrosine residue, in the third one with threonine residue and in the fourth one with glutamine residue.

EFFECT: invention allows obtaining formate dehydrogenases having improved properties in comparison to those both with a ferment of a wild type and a with its earlier described mutant shapes.

4 cl, 2 tbl, 5 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.

Four new mutant forms of plant formate dehydrogenase (FDH) were obtained, which have improved properties compared to both the wild-type enzyme and its previously described mutant forms. In the first of the proposed mutant proteins, the natural phenylalanine residue at the position corresponding to position 290 in the formate dehydrogenase sequence from soy Glycine max is replaced by an alanine residue. In the second mutant form of the enzyme, the same natural phenylalanine residue 290 is replaced by a tyrosine residue, in the third by a threonine residue and in the fourth by a glutamine 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 improved catalytic properties compared to the corresponding wild-type enzyme, to DNA encoding mutant forms of formate dehydrogenase, to the use of these enzymes in 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., Stingl 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 Mycobacterium vaccae N10 bacteria 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 S-1- (2-chlorophenyl) ethanol from o-chloroacetophenone (Mädje K., Schmölzer 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.11-17-17) . 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. 10, p.176-183.) Immobilization of formate dehydrogenase from Candida methylica yeast, as well as other enzymes on nanoparticles, was carried out with the synthesis of high optical purity S-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. 18, 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., Axarli 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 genes 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 dehydrogenases from Arabidopsis thaliana and soybean: expression in E. coli cells and kinetic properties of recombinant enzymes.Vestnik Mosk. Univ. Ser.2 Chemistry, 2006, v. 47, N1, p.31- 34), and it has the lowest values of Michaelis constants compared to 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).

The main disadvantages of the known natural and recombinant formate dehydrogenases from wild-type plants are the low thermal stability and low value of the catalytic constant k _{cat of} these enzymes, which also leads to low catalytic efficiency (the ratio of the catalytic constant to Michaelis constant, k _cat / K _M ). Thus, for the successful use of soy formate dehydrogenase in practice, it is necessary to increase the catalytic efficiency 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 formate dehydrogenases, the literature describes many examples of successful improvements in temperature and chemical stability using protein engineering methods. An analysis of such works is discussed in detail in the reviews of Tishkov VI and Popov VO Protein engineering of formate dehydrogenase. Biomol. Eng, 2006, v.23, p. 89-110 and Alekseeva A.A., Savin S.S., Tishkov V.I. NAD ⁺ -dependent plant formate dehydrogenase. Acta Naturae, 2011, vol. 3, No. 4 (11), pp. 40-56. Experiments to increase the thermal stability of recombinant FDG from soybeans are described in (Alekseeva A.A., Savin S.S., Kleimenov S.Yu., Uporov I.V., Pometun E.V., Tishkov V.I. Stabilization of recombinant formate dehydrogenase from soy Glycine max by the method of rational design. Biochemistry, 2012, vol. 77, No. 10, p. 1443-1456) and (Alekseeva A.A., Serenko A.A., Kargov IS, Savin SS, Kleymenov S.Yu. , and Tishkov VI Engineering catalytic properties and thermal stability of plant formate dehydrogenase by single-point mutations (Protein Eng. Des. SeL, 2012, v.25, No. 11, p. 781-788).

At the same time, there are very few successful examples of improving the catalytic efficiency of formate dehydrogenases. Known mutant PseFDH GAV, in which K _M for NAD ⁺ is improved to 35 uM compared to 65 uM at PseFDH wildtype (Tishkov VI and Popov VO Biomol. Eng, 2006, v.23, p.89-110). In the case of mutant PseFDH SM4 (Alekseeva AA et al. Protein Eng. Des. Sel., 2012, v.25, №11, p.781-788) _KM no NAD ⁺ is 41 uM, and K _M for formate - 3.2 mm (6.5 mm in the wild-type enzyme). The magnitude of the catalytic constant of these mutants is the same as that of wild-type PseFDH. In the dissertation (Felber, S. Optimierung der NAD-abhuengigen Formiatdehydrogenase aus Candida boidinii fuer den Einsatz in der Biokatalyse. Ph.D. dissertation. 1-176. 2001. Heinrich-Heine University of Duesseldorf. URL: http: // diss. ub.uniduesseldorf.de/ebib/diss/file?dissid=78. 2001) describes a mutant FDG from C. boidnii yeast, in which, due to the replacement of Phe285Ser, the catalytic constant increased from 3.7 to 6.1 s ^-1 , however, when this deteriorated (increased) Michaelis constants for NAD ⁺ and formate from 45 to 73 μM and from 5.9 to 14 mm, respectively. As a result, the catalytic efficiency of the mutant enzyme in formate decreased from 0.63 to 0.44 mM- ¹ s ^-1 , and the catalytic efficiency in NAD ^{+ was} not changed.

In the case of recombinant FDG from soy, mutants with the substitutions Leu24Asp, Val127Arg and Ala267Met (Alekseeva A.A. et al. Biochemistry, 2012, v.77, p.1443-1456) and Phe290Asp and Phe290Ser (Alekseeva A.A. et al. Protein Eng. Des. Sel., 2012, v.25, No. 11, p. 781-788), in which the catalytic constant was increased from 2.9 s ^-1 to 3.2-5.1 s ^-1 , however, these replacements were accompanied by a deterioration of the Michaelis constant in formate. As a result, the catalytic efficiency in formate of these mutants (except for the replacement of Ala267Met) became even worse than that of the starting enzyme. In the case of replacing Ala267Met, the catalytic efficiency in formate increased by 23% to 2.38 compared with 1.93 mM ^-1 s ^-1 in FDG of wild-type soybean.

The technical problem solved by the proposed technical solution is to obtain mutant forms of formate dehydrogenase from soy Glycine max with improved catalytic properties compared to the wild-type enzyme and its known mutants.

The technical result obtained by the implementation of the proposed technical solution consists in improving the catalytic properties of the obtained new mutant forms of formate dehydrogenase from soy Glycine max in relation to the properties of the wild-type enzyme and its known mutants.

To achieve the indicated technical result, it was proposed to use a recombinant plant formate dehydrogenase, in which the amino acid sequence of wild-type formate dehydrogenase from soy Glycine max was used as the basis. The complete cDNA sequence of this enzyme (access code in the GeneBank BT094321.1 database) contains at the beginning a nucleotide sequence encoding a signal peptide necessary for the transport of formate dehydrogenase precursor protein from the cytoplasm to mitochondria of plant cells. After the pro-protein enters the mitochondria, this peptide is cleaved to form active formate dehydrogenase. Due to the lack of mitochondria in bacteria, the above post-translational modification of the formate dehydrogenase precursor protein is not possible. Therefore, for expression in E. coli cells, a sequence encoding a signal peptide (31 amino acid residues) was deleted from the soybean formate dehydrogenase cDNA sequence. As a result, the wild-type active recombinant enzyme is synthesized without an additional stage of post-translational modification (Alekseeva A.A., Savin S.S., Tishkov V.I. NAD ⁺ -dependent plant formate dehydrogenase. Acta Naturae, 2011, v. 3, No. 4 ( 11), pp. 40-56).

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

Example 1

Obtaining a mutant formate dehydrogenase in which the Phe290 residue is replaced by the Ala 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 Glycine max soy formate dehydrogenase cDNA was inserted without restriction sites encoding a signal peptide at the NdeI and EcoRI restriction sites.

To introduce the Phe290Ala mutation into the formate dehydrogenase gene using PCR, universal T7for and T7rev 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 Ala residue at position 290:

SoyF290Afor 5′-GATGTTTGG GCC CCACAGCCAGCTCCAA-3 ′ SoyF290Arev 5′-GCTGTGG GGC CCAAACATCACCACTATAACCT-3 ′

Nucleotide substitutions for the Phe290Ala mutation are underlined 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 1):

Table 1. The conditions for the polymerase chain reaction (NDP). 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 + SoyF290Arev and 2) SoyF290Afor + T7Rev. Plasmid pSoyFDH was used as a matrix. The resulting PCR products, fragment 1 and fragment 2, were purified using preparative agarose gel electrophoresis using the appropriate kits for this, 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 KpnI and EcoRI for two hours at 37 ° C, then the restriction mixture was inactivated to inactivate restriction enzymes for 20 minutes at 65 ° C (used restriction enzymes and buffer solutions from Fermentas (Lithuania )). 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 an appropriate kit, for example, GeneJet Gel Extraction Kit (Fermentas, Lithuania) and ligated 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 wild-type G. max.

After ligation with the reaction mixture, E. coli TG1 cells (genotype supE hsdΔ5 thi Δ (lac-proAB) F ′ [traD36 proAB ⁺ lacI ^q lacZΔM15]) were transformed and grown overnight at 37 ° C on solid agarized LB 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, the mutant SoyFDH Phe290Ala gene was sequenced in the isolated plasmids. As a result, in 5 isolated plasmids, in all cases, only those mutations that provided the replacement of Phe290Ala were identified in the mutant enzyme gene. Thus, the plasmid pSoyFDH_F290A was obtained, which provided the production of the new formate dehydrogenase SoyFDH Phe290Ala with the replacement of Phe290Ala.

To obtain a mutant enzyme, E. coli BL21 (DE3) pLysS Codon Plus cells (E. coli genotype B F ^- ompT hsdS (rB ^- mB ^- ) dcm ⁺ Tet ^r gal λ (DE3) pLysS endA Hte [argU proL Cam ^r ) were transformed plasmid pSoyFDH_F290A was plated on Petri dishes with agar medium containing ampicillin (100 μg / ml) and chloramphenicol (25 μg / ml) and cultured (see below).

Example 2

Obtaining a mutant plant formate dehydrogenase in which the Phe290 residue is replaced by the Tyr residue.

This example is identical to example 1 except that the following deoxyribo oligonucleotides were used as the forward and reverse primers to allow the introduction of the Phe290Tyr substitution:

Soy_F290Yfor 5′-GATGTTTGG TAT CCACAGCCAGCTCCAAAG-3 ′ Soy_F290Yrev 5′-GGCTGTGG ATA CCAAACATCACCACTATAACCT-3 ′

Nucleotide substitutions for the Phe290Tyr mutation are underlined in bold.

Plasmid pSoyFDH with nucleotide substitutions that provide the Phe290Tyr mutation in the enzyme gene is called pSoyFDH_F290Y.

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 plasmid pSoyFDH_F290Y, plated on Petri dishes with agar medium containing ampicillin (100 μg / ml) and chloramphenicol (25 μg / ml) and cultured (see below).

Example 3

Obtaining a mutant plant formate dehydrogenase in which the Phe290 residue is replaced by a Thr residue.

This example is identical to example 1 except that the following deoxyribo oligonucleotides were used as the forward and reverse primers to allow the introduction of the Phe290Thr substitution:

Soy_F290TFor 5′-GATGTTTGG ACC CCACAGCCAGCTCCAA-3 ′ Soy_F290TRev 5′-GCTGTGG GGT CCAAACATCACCACTATAACCT-3 ′

Nucleotide substitutions for the Phe290Thr mutation are underlined in bold.

Plasmid pSoyFDH with nucleotide substitutions that provide the Phe290Thr mutation in the enzyme gene is called pSoyFDH_F290T.

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_F290T, seeded on Petri dishes with agar medium containing ampicillin (100 μg / ml) and chloramphenicol (25 μg / ml) and cultured (see below).

Example 4

Obtaining a mutant plant formate dehydrogenase in which the Phe290 residue is replaced by the Gln residue.

This example is identical to Example 1 except that the following deoxyribo oligonucleotides were used as the forward and reverse primers to allow the introduction of the Phe290Gln substitution:

Soy_F290QFor 5′-GATGTTTGG CAG CCACAGCCAGCTCCAAAG-3 ′ Soy_F290QRev 5′-GCTGTGG CTG CCAAACATCACCACTATAACCT-3 ′

Nucleotide substitutions for the mutation of Phe290Gln are highlighted in bold with underline.

The plasmid pSoyFDH with nucleotide substitutions providing the Phe290Gln mutation in the enzyme gene was called pSoyFDH_F290Q.

To obtain a mutant enzyme, E. coli BL21 (DE3) pLysS Codon Plus cells (E. coli genotype B F ^- ompT hsdS (rB ^- mB ^- ) dcm ⁺ Tet ^r gal λ (DE3) pLysS endA Hte [argU proL Cam ^r ) were transformed plasmid pSoyFDH_F290Q was plated on Petri dishes with agar medium containing ampicillin (100 μg / ml) and chloramphenicol (25 μg / ml) and cultured (see below).

Example 5

Determination of the catalytic parameters of mutant forms of soy formate dehydrogenase containing one of the amino acid substitutions - Phe290Ala, Phe290Tyr, Phe290Thr and Phe290Gln.

To determine the catalytic parameters, mutant SoyFDH Phe290Ala, SoyFDH Phe290Tyr, SoyFDH Phe290Thr, pSoyFDH Phe290Gln and wild-type soy formate dehydrogenase were isolated in highly purified form. The procedure for obtaining highly purified preparations was the same for the mutant SoyFDH Phe290Ala, SoyFDH Phe290Tyr, SoyFDH Phe290Thr, pSoyFDH Phe290Gln, and for the wild-type enzyme.

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 reached 600 nm, 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 Phe290Ala, SoyFDH Phe290Tyr, SoyFDH Phe290Thr, pSoyFDH Phe290Gln and the wild-type enzyme, a 20% cell suspension in 0.1 M sodium phosphate buffer, pH 8.0 was subjected to two freezing cycles and then milled disintegrator "Branson Sonifier 250" (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 technique 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 a 12% polyacrylamide gel in the presence of 0.1% sodium dodecyl sulfate on a MiniProtean II electrophoresis device from BioRad.

FDH activity was determined spectrophotometrically by the accumulation of NADH at 340 nm (ε ₃₄₀ = 6220 M ^-1 cm ^-1) spectrophotometer «Schimadzu UV 1800 PC» 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.

Michaelis constants for NAD ⁺ and formate were determined from the dependences of the enzyme activity on the concentration (0.4-6 K _M ) of the corresponding substrate. The concentration of the second substrate was saturating (> 30K _M ). The exact concentration of the initial NAD ⁺ solution was determined spectrophotometrically at a wavelength of 260 nm (ε ₂₆₀ = 17800 M ^-1 cm ^-1 ). A solution of sodium formate with a given concentration was prepared by dissolving the desired amount of substrate in 0.1 M sodium phosphate buffer, pH 7.0. The solution volume was adjusted in a volumetric flask. K _m values were calculated from experimental curves using the nonlinear regression method using the Origin Pro 8.5 software.

To find the catalytic constants, several samples of the studied enzyme with different concentrations were prepared. For each sample, the values of the maximum velocity and the concentration of active centers were determined. The concentration of active centers was determined from the dependences of quenching the intrinsic fluorescence of the enzyme in complex with NAD ⁺ with the addition of an azide ion as described in (Romanova E.G., Alekseeva A.A., Pometun E.V., Tishkov V.I. Determination of the concentration of active of centers and catalytic constant of recombinant formate dehydrogenase from soybean Glycine max. Vestnik of Moscow University, Ser. 2: Chemistry, 2010, v.51, No. 3, p.156-159). The measurements were carried out in 0.1 M sodium phosphate buffer, pH 7.0 on a Cary Eclipse spectrofluorimeter (Varian USA). Next, we plotted the dependence of the maximum velocity on the concentration of active centers and the value of the catalytic constant was determined as the slope of the straight line by linear regression using the Origin Pro 8.5 program.

) возросла по сравнению с исходной формиатдегидрогеназой сои на 51-102%. Кроме того, все четыре новые мутантные SoyFDH превосходят по каталитической эффективности по формиату и лучшую ранее описанную мутантную SoyFDH Ala267Ile (табл.2).The results of determining the catalytic parameters for the obtained mutant forms of SoyFDH Phe290Ala, SoyFDH Phe290Tyr, SoyFDH Phe290Thr, pSoyFDH Phe290Gln and wild-type protein are presented in table 2. For comparison, the data on the catalytic parameters of previously obtained mutant SoyFDH are also presented in this table. As follows from table 2, the replacement of the phenylalanine residue at position 290 with residues of alanine, tyrosine, threonine and glutamine leads to an increase in the value of the catalytic constant by 21-38% compared with the wild-type enzyme. In the case of K _m for NAD ⁺ , within the experimental error, this parameter did not change in the case of replacing Phe290Thr and slightly improved in the case of replacing Phe290Ala, Phe290Tyr, and Phe290Gln. The main important result is the fact that with the introduction of the substitutions Phe290Ala, Phe290Tyr, Phe290Thr and Phe290Gln, the Michaelis constant in formate in comparison with the wild-type enzyme not only does not deteriorate (which was observed for the other mutant SoyFDH described previously, Table 2), but also improves a bit. As a result, the catalytic efficiency of the obtained formate mutants (ratio $$k_{cat}/K_m^{HCO{O}^{-}}$$

) increased in comparison with the initial formate dehydrogenase of soybeans by 51-102%. In addition, all four new mutant SoyFDH are superior in catalytic efficiency in formate to the best previously described mutant SoyFDH Ala267Ile (Table 2).

table 2 Kinetic parameters of mutant SoyFDH and wild-type enzyme (0.1 M sodium phosphate buffer, pH 7.0) * Enzyme k _cat , s ^-1

, мкМ $$K_m^{NA{D}^{+}}$$

μm $$K_m^{HCO{O}^{-}}$$

mm $$k_{cat}/K_m^{NA{D}^{+}}$$

, (μM * s) ^-1 $$k_{cat}/K_m^{HCO{O}^{-}}$$

, (mm * s) ^-1- Wild type soyfdh 2.9 ± 0.1 13.3 ± 0.8 1.5 ± 0.1 0.20 1.93 Mutant SoyFDH of this application SoyFDH F290A 3.8 ± 0.1 8.6 ± 0.6 1.1 ± 0.1 0.44 3.45 SoyFDH F290Y 3.5 ± 0.1 10.9 ± 0.3 0.9 ± 0.1 0.32 3.89 SoyFDH F290T 4.0 ± 0.4 14.3 ± 0.9 1.3 ± 0.1 0.28 3.08 SoyFDH F290Q 3.5 ± 0.2 11.7 ± 0.6 1.2 ± 0.1 0.29 2.92 Mutant SoyFDH previously described ∗∗ SoyFDH F290N 2.8 ± 0.1 14.0 ± 1.0 4,5 ± 0,3 0.20 0.62 SoyFDH F290D 5.1 ± 0.3 12.8 ± 0.8 5.0 ± 0.2 0.40 1,02 SoyFDH F290S 4.1 ± 0.2 9.1 ± 0.7 4.1 ± 0.3 0.45 1.00 SoyFDH L24D 3.2 ± 0.2 13.7 ± 0.7 2.3 ± 0.1 0.23 1.39 SoyFDH V127R 3.6 ± 0.7 9.5 ± 0.5 3.4 ± 0.2 0.38 1.06 SoyFDH A267M 5.0 ± 0.9 9.9 ± 0.8 2.1 ± 0.2 0.51 2,38 ∗ The parameters that are superior to those for the wild-type enzyme are highlighted in bold. ∗∗ Alekseeva A.A., Savin S.S., Kleimenov S.Yu., Uporov I.V., Pometun E.V., Tishkov V.I. Stabilization of recombinant formate dehydrogenase from soy Glycine max by the rational design method. Biochemistry, 2012, vol. Engineering catalytic properties and thermal stability of plant formate dehydrogenase by single-point mutations. Protein Eng. Des. Sel., 2012, v.25, No. 11, p. 781-788.

Claims (4)

1. Mutant formate dehydrogenase with improved properties, characterized by the amino acid sequence corresponding to the amino acid sequence of the wild-type formate dehydrogenase from Glycine max, in which the phenylalanine residue at position 290 is replaced by an alanine residue. 2. Mutant formate dehydrogenase with improved properties, characterized by an amino acid sequence corresponding to the amino acid sequence of wild-type formate dehydrogenase from Glycine max, in which the phenylalanine residue at position 290 is replaced by a tyrosine residue. 3. Mutant formate dehydrogenase with improved properties, characterized by an amino acid sequence corresponding to the amino acid sequence of wild-type formate dehydrogenase from Glycine max, in which the phenylalanine residue at position 290 is replaced by a threonine residue. 4. Mutant formate dehydrogenase with improved properties, characterized by an amino acid sequence corresponding to the amino acid sequence of wild-type formate dehydrogenase from Glycine max, in which the phenylalanine residue at position 290 is replaced by a glutamine residue.