Lactate dehydrogenase, its encoding gene, recombinant vector, recombinant bacterium, starter culture, and their applications

Abstract

The present invention relates to genetic engineering and discloses a lactate dehydrogenase, its coding gene, recombinant vector, recombinant bacterium, starter and their applications. The lactate dehydrogenase is an enzyme having the amino acid sequence shown in SEQ ID NO.1, or an enzyme shown by an amino acid sequence in which the 247th amino acid of the amino acids shown in SEQ ID NO.1 is substituted, deleted or added with one or several amino acid residues and still has lactate dehydrogenase activity. The catalytic activity and thermal stability of this lactate dehydrogenase in the process of asymmetric reduction of sodium phenylpyruvate are significantly improved. Compared with the wild-type lactate dehydrogenase, the catalytic efficiency (k cat / K m ) is increased by about 2.4 times, and the thermal stability performance is significantly improved (the half-life t 1 / 2 at 50 °C is extended nearly 5 times; the half-inactivation temperature is increased by 19 °C), which is particularly suitable for catalyzing the asymmetric reduction of sodium phenylpyruvate to prepare D-phenyllactic acid and has good industrial application prospects.

Description

Technical Field

[0001] The present invention relates to genetic engineering, and particularly to a lactate dehydrogenase and its encoding gene, a recombinant vector, a recombinant strain, a starter, a method for preparing D-phenyllactic acid, and their applications in the preparation of D-phenyllactic acid. Background Art

[0002] Phenyllactic acid exists in honey, sourdough and lactic acid bacteria-fermented foods. It is a natural organic acid with broad-spectrum antibacterial activity. It can inhibit the growth of various microorganisms such as Gram-positive bacteria, Gram-negative bacteria, yeasts, and molds. It has good water solubility, high temperature resistance, a wide acting pH range, and is safe and non-toxic. It can prevent food spoilage, retain nutritional quality, and maintain the flavor of food at the same time. Therefore, it can be used as an antibacterial agent and preservative in the food industry. In addition, phenyllactic acid can also be used as a feed additive to increase the egg production rate and egg quality of poultry; or as an anti-wrinkle agent and antibacterial agent added to cosmetics. There is a chiral carbon atom in the phenyllactic acid molecule, so there is a pair of enantiomers. Among them, D-phenyllactic acid has stronger antibacterial activity and can also be used as a precursor to synthesize the hypoglycemic drug empagliflozin and the coronary heart disease treatment drug danshensu. Therefore, D-phenyllactic acid has received more attention. The broad application prospects of D-phenyllactic acid in industries such as food, feed, pharmaceuticals, and cosmetics have stimulated the research enthusiasm of the academic and industrial circles for its synthesis methods.

[0003] Multiple lactic acid bacteria can synthesize phenyllactic acid through intracellular metabolic pathways, but their fermentation cycle is long, the yield is low, and the optical purity of the product is poor, which restricts the large-scale fermentation production of D-phenyllactic acid. In the lactic acid bacteria metabolic pathway, phenylpyruvate is the direct precursor of phenyllactic acid, and D-phenyllactic acid is obtained through the asymmetric reduction of lactate dehydrogenase. Enzymes are biological macromolecules with biological catalytic functions. Biocatalytic reactions mediated by enzyme molecules have the characteristics of high catalytic activity, good stereoselectivity, mild reaction conditions, few by-products, and environmental friendliness, which conform to the development direction of green chemical synthesis and are increasingly becoming an important supplement to traditional chemical synthesis methods. They are widely used in the production of pharmaceuticals, pesticides, foods, cosmetics, and other fine chemicals. Using phenylpyruvate as a substrate and reducing it in one step under the catalysis of lactate dehydrogenase is the most commonly used method for preparing D-phenyllactic acid.

[0004] Through gene mining, a variety of lactate dehydrogenases and their encoding genes have been obtained, which can catalyze the asymmetric reduction of phenylpyruvate to synthesize D-phenyllactic acid, and the enantiomeric excess value (e.e.) of the product is greater than 99%. However, the existing lactate dehydrogenases have low catalytic activity and thermal stability, which limits their industrial application. Summary of the Invention

[0005] The object of the present invention is to overcome the problem of low asymmetric reduction activity of lactate dehydrogenase towards sodium phenylpyruvate existing in the prior art, and to provide a lactate dehydrogenase and its encoding gene, a recombinant vector, a recombinant strain, a starter, a method for preparing D-phenyllactic acid, and their applications in the preparation of D-phenyllactic acid. This lactate dehydrogenase has higher catalytic activity and better thermal stability.

[0006] To achieve the above object, in the first aspect of the present invention, a lactate dehydrogenase is provided, and the lactate dehydrogenase is an enzyme described in any one of (a)-(d):

[0007] (a) an enzyme having the amino acid sequence shown in SEQ ID NO.1;

[0008] (b) an enzyme shown by an amino acid sequence in which the 247th amino acid of the amino acids shown in SEQ ID NO.1 is substituted, deleted or added with one or several amino acid residues and still has lactate dehydrogenase activity;

[0009] (c) an enzyme shown by an amino acid sequence with a tag linked to the amino terminus and / or carboxyl terminus of the amino acid sequence described in (a) or (b);

[0010] (d) an enzyme shown by an amino acid sequence with a signal sequence linked to the amino terminus of the amino acid sequence described in (a) or (b).

[0011] Preferably, the lactate dehydrogenase is an enzyme shown by an amino acid sequence in which the 247th threonine of the amino acid sequence shown in SEQ ID NO.1 is substituted with isoleucine.

[0012] In the second aspect of the present invention, a gene encoding lactate dehydrogenase is provided, and this gene has a nucleotide sequence encoding the above-mentioned lactate dehydrogenase.

[0013] Preferably, the gene has a nucleotide sequence encoding an enzyme having the amino acid sequence shown in SEQ ID NO.1.

[0014] More preferably, the gene has the nucleotide sequence shown in SEQ ID NO.2.

[0015] In the third aspect of the present invention, a recombinant vector is provided, and the recombinant vector contains the above-mentioned gene.

[0016] Preferably, the expression vector of the recombinant vector is the pET28a plasmid.

[0017] In the fourth aspect of the present invention, a recombinant strain is provided, and the recombinant strain contains the above-mentioned gene or the above-mentioned recombinant vector.

[0018] Preferably, the recombinant strain is Escherichia coli and / or Bacillus subtilis.

[0019] More preferably, the recombinant strain is Escherichia coli.

[0020] The fifth aspect of the present invention provides a method for preparing a starter culture, which includes: inoculating the above-mentioned recombinant strain into a fermentation medium for fermentation to obtain a fermentation broth, and performing solid-liquid separation on the fermentation broth to obtain wet bacterial cells.

[0021] The sixth aspect of the present invention provides the use of at least one of the above-mentioned lactate dehydrogenase, the above-mentioned gene, the above-mentioned recombinant vector, the above-mentioned recombinant strain, and the starter culture prepared by the above-mentioned preparation method in the preparation of D-phenyllactic acid.

[0022] The seventh aspect of the present invention provides a method for preparing D-phenyllactic acid, which includes the following steps: contacting at least one of the above-mentioned lactate dehydrogenase, the above-mentioned gene, the above-mentioned recombinant vector, the above-mentioned recombinant strain, and the starter culture prepared by the above-mentioned preparation method with sodium phenylpyruvate.

[0023] Preferably, the contacting process includes: mixing the starter culture with sodium phenylpyruvate in the presence of a co-substrate and a reaction medium, and reacting at a temperature of 25-45 °C and a rotation speed of 150-250 rpm until the sodium phenylpyruvate reacts completely.

[0024] Preferably, the co-substrate is selected from at least one of glucose, glucose-6-phosphate, and isopropanol, and more preferably glucose; the reaction medium is a potassium phosphate buffer solution with a pH of 6.0-8.0.

[0025] Preferably, relative to 1 L of the reaction medium, the dosage of sodium phenylpyruvate is 1-100 g, the dosage of the co-substrate is 5-300 g, and the dosage of the starter culture is 10-250 g.

[0026] Through the above technical solutions, the beneficial effects of the present invention are as follows:

[0027] The catalytic activity and thermal stability of the lactate dehydrogenase provided by the present invention in the asymmetric reduction of sodium phenylpyruvate are significantly improved. Compared with the wild-type lactate dehydrogenase, the catalytic efficiency (k cat / K m ) is increased by about 2.4 times, and the thermal stability performance is significantly improved (the half-life t 1 / 2 at 50 °C is extended nearly 5 times; the half-inactivation temperature is increased by 19 °C). The lactate dehydrogenase provided by the present invention is particularly suitable for catalyzing the asymmetric reduction of sodium phenylpyruvate to prepare D-phenyllactic acid and has good industrial application prospects. Description of the Drawings

[0028] Figure 1It is the SDS-PAGE electrophoresis diagram in Example 3. Among them, M is the protein Marker; 1, 4, and 7 are the soluble parts of E. coli / pET28a-lrldh, E. coli / pET28a-lrldh-D249A, and E. coli / pET28a-lrldh-D249A / T247I after induced expression; 2, 5, and 8 are the insoluble parts of E. coli / pET28a-lrldh, E. coli / pET28a-lrldh-D249A, and E. coli / pET28a-lrldh-D249A / T247I after induced expression; 3, 6, and 9 are the purified enzymes of wild-type LrLDH, mutant D249A, and D249A / T247I.

[0029] Figure 2 It is the curve graph showing the change of residual enzyme activity of wild-type lactate dehydrogenase LrLDH (WT), mutant D249A, and mutant D249A / T247I over time during incubation at 50 °C in Example 5.

[0030] Figure 3 It is the curve graph showing the change of residual enzyme activity of wild-type lactate dehydrogenase LrLDH (WT), mutant D249A, and mutant D249A / T247I with the change of incubation temperature in Example 5.

[0031] Figure 4 It is the curve graph showing the change of relative enzyme activity of wild-type lactate dehydrogenase LrLDH (WT), mutant D249A, and mutant D249A / T247I with the change of temperature in Example 7.

[0032] Figure 5 It is the curve graph showing the change of the content of D-phenyllactic acid with the reaction time when the recombinant strains E. coli BL21(DE3) / pET28a-lrldh and E. coli / pET28a-lrldh-D249A / T247I catalyze the asymmetric reduction of sodium phenylpyruvate to synthesize D-phenyllactic acid in Example 8. Detailed implementation manners

[0033] In the ranges disclosed herein, the endpoints and any values are not limited to the exact ranges or values. These ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, between the endpoint values of each range, between the endpoint values of each range and individual point values, and between individual point values can be combined with each other to obtain one or more new numerical ranges, and these numerical ranges should be regarded as specifically disclosed herein.

[0034] In the present invention, unless otherwise specified, the enzyme activity of lactate dehydrogenase is determined by monitoring the consumption of NADH at 30°C. The standard reaction solution consists of a phosphate buffer (100 mM, pH 7.0) containing 0.5 mM NADH and 1 mM sodium phenylpyruvate, and 100 μL of appropriately diluted purified protein, with a total volume of 200 μL. The amount of enzyme required to consume 1 μmol of NADH per minute is defined as 1 enzyme activity unit, i.e., 1 U. "Specific enzyme activity" represents the catalytic ability per unit mass of protein, and the calculation formula for specific activity is: specific enzyme activity (U / mg) = total enzyme activity unit number / mg total protein; the unit M represents mol / L.

[0035] In the first aspect of the present invention, there is provided a lactate dehydrogenase, which is the enzyme described in any one of (a)-(d):

[0036] (a) An enzyme having the amino acid sequence shown in SEQ ID NO.1;

[0037] (b) An enzyme shown by an amino acid sequence in which the 247th amino acid of the amino acids shown in SEQ ID NO.1 is substituted, deleted, or added with one or several amino acid residues and still has lactate dehydrogenase activity;

[0038] (c) An enzyme shown by an amino acid sequence having a tag linked to the amino terminus and / or carboxyl terminus of the amino acid sequence described in (a) or (b);

[0039] (d) An enzyme shown by an amino acid sequence having a signal sequence linked to the amino terminus of the amino acid sequence described in (a) or (b).

[0040] In the present invention, in the amino acid sequence of lactate dehydrogenase, the 247th threonine can be substituted, deleted, or added with amino acid residues, and the role played by the residue in the protein domain (such as providing a positive charge or forming a hydrophobic pocket structure) remains unchanged, and it will not affect the three-dimensional structure of the protein. Therefore, the function of the protein can still be achieved. Preferably, in the present invention, the 247th threonine of the lactate dehydrogenase amino acid sequence shown in SEQ ID NO.1 is substituted with isoleucine to obtain an enzyme with the amino acid sequence shown in SEQ ID NO.3.

[0041] For the convenience of purification, common tags in the art can also be used to add modifications to (a) or (b). For example, it can be obtained by linking an amino acid sequence with a tag to the amino terminus and / or carboxyl terminus of (a). The tag will not affect the activity of the lactate dehydrogenase of the present invention, and whether to add a tag can be selected according to needs during actual application.

[0042] In the present invention, a signal sequence may also be linked to the amino terminus of the lactate dehydrogenase. The signal sequence may be derived from Escherichia coli and Bacillus subtilis, but is not limited thereto.

[0043] The above-mentioned lactate dehydrogenase can be obtained by artificial synthesis, or its coding gene can be synthesized first and then obtained through biological expression. Exemplarily, the lactate dehydrogenase provided in the present invention is obtained by mutating the coding gene of wild-type lactate dehydrogenase LrLDH using site-directed saturation mutagenesis technology, connecting an expression vector and then transforming the host Escherichia coli, and after inducing expression, the positive mutations with increased activity are detected by high-performance liquid chromatography detection method to obtain a lactate dehydrogenase mutant (i.e., the lactate dehydrogenase described in the present invention), and its catalytic activity and thermal stability are significantly improved during the asymmetric reduction of sodium phenylpyruvate. Compared with the wild-type lactate dehydrogenase LrLDH, while the catalytic efficiency is improved, the thermal stability performance is significantly enhanced. The specific method is as follows: The cloned wild-type lactate dehydrogenase LrLDH has been successfully constructed into an expression vector pET28a-lrldh, and the functional enzyme protein is overexpressed in Escherichia coli BL21(DE3); then through homology modeling and molecular docking, and combined with the online software HotSpot Wizard 3.0 for computer-aided design, the sites potentially affecting enzyme activity and thermal stability are selected; the site-directed saturation mutation sites are set, and appropriate primers are designed and synthesized. Using the recombinant expression plasmid containing the parental lactate dehydrogenase gene as a template, the plasmid of the full-length mutant gene is amplified by PCR. By transforming the plasmid containing the full-length mutation into an appropriate host cell, culturing, inducing expression, and screening out the positive mutants with high activity and thermal stability; finally, plasmid DNA is extracted from the positive mutants for DNA sequencing analysis to determine the mutations of the primers. In the preparation of the lactate dehydrogenase of the present invention, any suitable vector can be used.

[0044] The second aspect of the present invention provides a gene encoding lactate dehydrogenase, and this gene has a nucleotide sequence encoding the above-mentioned lactate dehydrogenase.

[0045] According to the present invention, preferably, the gene has a nucleotide sequence encoding an enzyme having the amino acid sequence shown in SEQ ID NO.1, or has a nucleotide sequence encoding an enzyme having the amino acid sequence shown in SEQ ID NO.3.

[0046] As described above, correspondingly, the 5'-end and / or 3'-end of the nucleotide sequence may also be linked to a coding sequence of a modified tag.

[0047] As is well known in the art, among the 20 different amino acids that make up proteins, except that Met (ATG) or Trp (TGG) is encoded by a single codon respectively, the other 18 amino acids are each encoded by 2 - 6 codons (Sambrook et al., Molecular Cloning, Cold Spring Harbor Laboratory Press, New York, USA, Second Edition, 1989, see Appendix D on page 950). That is, due to the degeneracy of the genetic codons, most codons that determine an amino acid are more than one, and the substitution of the third nucleotide in the triplet codon often does not change the amino acid composition. Therefore, the nucleotide sequences of genes encoding the same protein can be different.

[0048] More preferably, the gene encoding the enzyme having the amino acid sequence shown in SEQ ID NO.1 has the nucleotide sequence shown in SEQ ID NO.2, and the gene encoding the enzyme having the amino acid sequence shown in SEQ ID NO.3 has the nucleotide sequence shown in SEQ ID NO.4.

[0049] The nucleotide sequences provided by the present invention can generally be obtained by polymerase chain reaction (PCR) amplification, recombination, or artificial synthesis methods. Once the relevant nucleotide sequences are obtained, the relevant amino acid sequences can be obtained in large quantities by recombination methods. Usually, the obtained nucleotide sequences are cloned into a vector, then transferred into genetically engineered bacteria, and then the relevant nucleotide sequences are separated from the proliferated host cells by conventional methods.

[0050] In addition, the relevant nucleotide sequences can also be synthesized by known artificial chemical synthesis methods.

[0051] The third aspect of the present invention provides a recombinant vector, and the recombinant vector contains the above-mentioned gene.

[0052] The "vector" used in the recombinant vector can be various vectors known in the art, such as various commercially available plasmids, cosmids, phages, and retroviruses, etc. The preferred expression vector of the present invention is the pET28a plasmid. The construction of the recombinant vector can be carried out by using various restriction endonucleases capable of having cleavage sites at the multiple cloning sites of the vector (such as for pET28a, Nco I and Xho I, etc.) to obtain a linear plasmid by enzymatic digestion, and connecting it with the gene fragment cut by the same restriction endonuclease to obtain a recombinant plasmid. The present invention preferably uses double enzymatic digestion of pET28a and the gene fragment ligated thereto with Nco I and Xho I, and constructs the recombinant vector pET28a - D249A by ligation with a ligase.

[0053] The fourth aspect of the present invention provides a recombinant strain, and the recombinant strain contains the above-mentioned gene or the above-mentioned recombinant vector.

[0054] In the present invention, the recombinant vector can be transformed, transduced or transfected into a host cell (strain) by conventional methods in the art, such as chemical transformation by the calcium chloride method or transformation by high-voltage electroporation. The host cell can be a prokaryotic cell or a eukaryotic cell, preferably Escherichia coli and / or Bacillus subtilis, and more preferably, the host cell is Escherichia coli, such as Escherichia coli BL21(DE3).

[0055] In the present invention, lactate dehydrogenase can be used in the form of whole cells of the recombinant strain, or in the form of a crude enzyme or a purified enzyme isolated from the cells of the recombinant strain without purification. If necessary, the lactate dehydrogenase of the present invention can also be made into an immobilized enzyme or an immobilized cell using immobilization techniques known in the art.

[0056] In the present invention, the lactate dehydrogenase can be made into a corresponding enzyme preparation. Specifically, the enzyme preparation can exist in solid, semi-solid or liquid form, and the enzyme preparation can contain excipients or additives for preparing the enzyme preparation, etc., which can be selected by those skilled in the art according to needs and will not be elaborated herein.

[0057] The fifth aspect of the present invention provides a method for preparing a starter, which includes: inoculating the above-mentioned recombinant strain into a fermentation medium for fermentation to obtain a fermentation broth, and performing solid-liquid separation on the fermentation broth to obtain wet bacterial cells. The wet bacterial cells can be used as an enzyme preparation containing lactate dehydrogenase.

[0058] In the present invention, the starter can contain excipients commonly added in the preparation of bacterial agents in the art, which can be selected by those skilled in the art according to needs.

[0059] In the present invention, the solid-liquid separation can adopt conventional separation methods in the art, such as centrifugation, filtration, etc. Specifically, in the case of centrifugation, the conditions for centrifugation include: temperature of 0-10°C, rotation speed of 6000-10000 rpm, and time of 8-12 min.

[0060] In the present invention, there are no particular limitations on the conditions for fermenting the recombinant strain, as long as the recombinant strain can be proliferated in large quantities through the fermentation process. Preferably, the fermentation process includes: first preparing the recombinant strain into a seed solution, and then inoculating the seed solution into a fermentation medium containing kanamycin and culturing until the cell concentration OD 600 is 0.6-0.8, then adding lactose with a final concentration of 6-10 g / L, and culturing at a temperature of 20-35°C for 10-15 h to obtain a fermentation broth.

[0061] According to the present invention, preferably, the content of kanamycin in the fermentation medium is 40-60 mg / L.

[0062] Further preferably, the method for preparing the seed solution comprises: picking a single colony of the recombinant strain and inoculating it into a seed medium containing kanamycin for seed culture to obtain the seed solution. In the present invention, the single colony of the recombinant strain can be selected from the freshly prepared recombinant strain or the recombinant strain cryopreserved at low temperature (for example, the recombinant strain of synthetic lipid cryopreserved in a glycerol cryotube in an -80°C refrigerator).

[0063] The present invention has no particular limitation on the method of seed culture, as long as the recombinant strain can be activated and proliferated by this method. Preferably, the content of kanamycin in the seed medium is 40 - 60 mg / L; the parameters such as temperature, pH, rotation speed, and time used for seed culture can be conventional settings in the art. Preferably, the conditions for seed culture include: the temperature is 30 - 45°C and the time is 8 - 12 h.

[0064] In the present invention, there is no particular limitation on the seed medium and the fermentation medium, and they can be media conventionally used in the art. Preferably, the seed medium and the fermentation medium adopt LB liquid medium (tryptone 8 - 12 g / L, yeast extract 4 - 6 g / L, NaCl 8 - 12 g / L).

[0065] The sixth aspect of the present invention provides the use of at least one of the above-mentioned lactate dehydrogenase, the above-mentioned gene, the above-mentioned recombinant vector, the above-mentioned recombinant strain, and the starter prepared by the above-mentioned preparation method in the preparation of D-phenyllactic acid.

[0066] The seventh aspect of the present invention provides a method for preparing D-phenyllactic acid, which comprises the following steps: contacting at least one of the above-mentioned lactate dehydrogenase, the above-mentioned gene, the above-mentioned recombinant vector, the above-mentioned recombinant strain, and the starter prepared by the above-mentioned preparation method with sodium phenylpyruvate.

[0067] According to the present invention, preferably, the process of contacting comprises: in the presence of an auxiliary substrate and a reaction medium, mixing the starter (i.e., the wet cells separated from the fermentation broth) with sodium phenylpyruvate and reacting at a temperature of 25 - 35°C and a rotation speed of 150 - 250 rpm until the sodium phenylpyruvate reacts completely.

[0068] According to the present invention, preferably, the auxiliary substrate is at least one of glucose, glucose-6-phosphate, and isopropanol, and more preferably glucose. The reaction medium adopts a potassium phosphate buffer solution with a pH of 6.0 - 8.0, and the potassium phosphate buffer solution is obtained by mixing potassium dihydrogen phosphate and dipotassium hydrogen phosphate in different proportions and dissolving them, and its phosphate root concentration is 0.1 mol / L.

[0069] According to the present invention, preferably, relative to 1 L of the reaction medium, the dosage of sodium phenylpyruvate is 1-100 g, the dosage of the co-substrate is 5-300 g, and the dosage of the fermenting agent is 10-250 g.

[0070] The present invention will be described in detail below through examples.

[0071] In the following examples, Escherichia coli E.coli BL21(DE3) was purchased from Takara Bio Inc. (Dalian), with the number 9126; pET28a was purchased from Sangon Biotech (Shanghai) Co., Ltd., with the number B540183; sodium phenylpyruvate was purchased from Sangon Biotech (Shanghai) Co., Ltd., with the number A600877; the remaining reagents and raw materials were all conventional commercially available products.

[0072] LB liquid medium: 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of NaCl, the pH was adjusted to 7.0, and it was autoclaved at high pressure for 21 min for standby;

[0073] LB agar plate medium: 10 g / L of tryptone, 5 g / L of yeast extract, 10 g / L of NaCl, 12 g / L of agar, the pH was adjusted to 7.0, and it was autoclaved at high pressure for 21 min for standby.

[0074] Example 1 Preparation of lactate dehydrogenase mutants

[0075] The wild-type lactate dehydrogenase LrLDH gene (GenBank ID: AZFF01000004.1) was obtained by cloning, and the expression vector pET28a-lrldh was successfully constructed. The functional enzyme protein was overexpressed in Escherichia coli BL21(DE3). Then, through homology modeling and molecular docking, and combined with the online software HotSpot Wizard 3.0 for computer-aided design, the sites potentially affecting enzyme activity and thermal stability were selected; the site-directed saturation mutation sites were set, and appropriate primers were designed and synthesized. Using the recombinant expression plasmid containing the parental lactate dehydrogenase gene as a template, the plasmid of the full-length mutant gene was amplified by PCR; by transforming the plasmid containing the full-length mutation into an appropriate host cell, positive mutants with high activity and thermal stability were screened out through culture, induction expression. Finally, plasmid DNA was extracted from the positive mutants for DNA sequencing analysis to determine the mutations of the primers, and mutants D249A, D249K, D249A / T247I, D249A / T247W, and D249A / T247C were obtained;

[0076] Among them, D249A is a mutant enzyme with the amino acid sequence shown in SEQ ID NO.1, D249K is a mutant enzyme in which alanine at position 249 in the amino acid sequence shown in SEQ ID NO.1 is substituted with lysine, D249A / T247I is a mutant enzyme in which threonine at position 247 in the amino acid sequence shown in SEQ ID NO.1 is substituted with isoleucine, D249A / T247W is a mutant enzyme in which threonine at position 247 in the amino acid sequence shown in SEQ ID NO.1 is substituted with tryptophan, and D249A / T247C is a mutant enzyme in which threonine at position 247 in the amino acid sequence shown in SEQ ID NO.1 is substituted with cysteine.

[0077] Using the recombinant plasmid pET28b-lrldh as a template, full plasmid amplification of the mutant D249A was performed. The primer pairs were designed as D249-F and D249-R (see Table 1, and the nucleotide sequences are shown in SEQ ID NO.5 and SEQ ID NO.6 respectively), and the lactic acid dehydrogenase mutant D249A with the amino acid sequence shown in SEQ ID NO.1 (nucleotide sequence shown in SEQ ID NO.2) was obtained; then, using the gene of the lactic acid dehydrogenase mutant D249A (i.e., the nucleotide sequence shown in SEQ ID NO.2) recombinant plasmid as a template, full plasmid amplification was performed. The primer pairs were designed as T247 / D249A-F and T247 / D249A-R (see Table 1, and the nucleotide sequences are shown in SEQ ID NO.7 and SEQ ID NO.8 respectively), and the lactic acid dehydrogenase mutant D249A / T247I in which threonine at position 247 in the amino acid sequence shown in SEQ ID NO.1 was mutated to isoleucine was obtained (amino acid sequence shown in SEQ ID NO.3, nucleotide sequence shown in SEQ ID NO.4).

[0078] In Example 1, the PCR system was: 5×Prime STAR Buffer (Mg 2+ plus) 10 μL, dNTP Mix (2.5 mM for each nucleotide), 0.5 μL of each mutant primer, 0.5 μL of the template (recombinant plasmid), 0.5 μL of Prime STAR DNA polymerase, and water was added to 50 μL; the PCR reaction conditions were: pre-denaturation at 95°C for 5 min, followed by 25 cycles (95°C, 15 s; 55°C, 15 s; 72°C, 7 min), and finally extension at 72°C for 10 min.

[0079] After analyzing that the PCR reaction product was positive by 0.9% agarose gel electrophoresis, 20 μL of the PCR solution was taken, 1 μL of Dpn I was added, and the template plasmid was removed by digestion at 37°C for 3 h. It was inactivated at 65°C for 10 min, and then transformed into competent cells of Escherichia coli E. coli BL21(DE3). The cells were spread on an LB agar plate containing kanamycin (final concentration 50 μg / mL) and cultured overnight at 37°C. After sequencing confirmation by Sangon Biotech (Shanghai) Co., Ltd., the recombinant strains of lactate dehydrogenase E. coli BL21(DE3) / pET28a-lrldh-D249A (D249A) and E. coli BL21(DE3) / pET28a-lrldh-D249A / T247I (D249A / T247I) were obtained.

[0080] Table 1 Primers for Mutating Lactate Dehydrogenase

[0081] Primer Name Sequence (5’-3’) D249-F <![CDATA[ATACCGAT NNS CTGATCAAAGCGCTGGATT]]> D249-R <![CDATA[TTTGATCAG NNS ATCGGTATCAACCAGATC]]> T247 / D249A-F <![CDATA[CTGGTTGAT NNS GATGCACTGATCAAAGCGC]]> T247 / D249A-R <![CDATA[CAGTGCATC NNS ATCAACCAGATCCCCACG]]>

[0082] The underlined parts in Table 1 represent the mutated nucleotides.

[0083] Example 2 Induced Expression of Wild-Type Lactate Dehydrogenase and Mutants

[0084] The wild strain E. coli BL21(DE3) / pET28a-lrldh containing the wild-type lactate dehydrogenase gene and the recombinant strains of lactate dehydrogenase E. coli BL21(DE3) / pET28a-lrldh-D249A and E. coli BL21(DE3) / pET28a-lrldh-D249A / T247I obtained in Example 1 were respectively inoculated into LB liquid medium containing kanamycin (final concentration 50 mg / L), cultured at 37°C for 10 h, and then inoculated into fresh LB liquid medium containing kanamycin (final concentration 50 mg / L) at a volume concentration of 4%. They were cultured at 37°C until the cell concentration OD 600 was about 0.7. Then, lactose with a final concentration of 8 g / L was added to the culture medium, and it was continuously cultured at 28°C for 12 h to obtain the fermentation broth. The fermentation broth was centrifuged at 4°C and 8500 rpm for 10 min to collect the wet cells of E. coli BL21(DE3) / pET28a-lrldh, E. coli BL21(DE3) / pET28a-lrldh-D249A, and E. coli BL21(DE3) / pET28a-lrldh-T247I / D249A, which could be respectively used for the purification of wild-type lactate dehydrogenase and the biocatalytic preparation of D-phenyllactic acid.

[0085] Example 3 Purification of Wild-Type Lactate Dehydrogenase and Mutants

[0086] The wet cells of E. coli BL21(DE3) / pET28a-lrldh, E. coli BL21(DE3) / pET28a-lrldh-D249A, and E. coli BL21(DE3) / pET28a-lrldh-D249A / T247I obtained in Example 2 were respectively suspended in buffer A (20 mM phosphate buffer at pH 8.0 containing 500 mM NaCl and 20 mM imidazole) to make the wet cell concentration 50 g / L. The cell suspension was placed in an ice bath and sonicated for 10 min at a power of 300 W, then centrifuged (10000×g, 4 °C, 20 min) to remove cell debris. The supernatant was collected and loaded onto a pre-packed gravity Ni-NTA affinity chromatography column (5 mL) equilibrated with buffer A. The column was washed with 25 mL of buffer A to remove unbound proteins, and then the lactate dehydrogenase protein was eluted with buffer B (20 mM phosphate buffer at pH 8.0 containing 500 mM NaCl and 500 mM imidazole) and dialyzed overnight in phosphate buffer (20 mM, pH 8.0). All the above purification steps were carried out under the condition of 0 - 4 °C to obtain wild-type lactate dehydrogenase protein, mutant D249A protein, and mutant D249A / T247I protein. The obtained proteins were examined by SDS-PAGE with a 12% separating gel concentration under denaturing conditions, and the results are as Figure 1 shown.

[0087] Example 4 Determination of Specific Enzyme Activity and Heat Treatment Residue of Lactate Dehydrogenase and Its Mutants

[0088] The enzyme activities of the wild-type lactate dehydrogenase, mutant D249A, mutant D249A / T247I obtained in Example 3, and mutants D249K, D249A / T247W, and D249A / T247C obtained in Example 1 were determined: by monitoring the consumption of NADH at 30 °C. The standard reaction solution consisted of phosphate buffer (100 mM, pH 7.0) containing 0.5 mM NADH and 1 mM sodium phenylpyruvate and 100 μL of appropriately diluted purified protein, with a total volume of 200 μL. The amount of enzyme required to consume 1 μmol of NADH per minute was defined as 1 enzyme activity unit, i.e., 1 U. "Specific enzyme activity" represents the catalytic ability of per unit mass of protein, and the calculation formula for specific activity is: specific enzyme activity (U / mg) = total enzyme activity unit number / mg total protein; the unit M represents mol / L.

[0089] The protein purity was determined by the Bradford method with standard bovine serum albumin as a control.

[0090] Determination of residual enzyme activity: The purified enzyme solutions corresponding to the wild-type lactate dehydrogenase, mutant D249A, mutant D249A / T247I obtained in Example 3, and mutants D249K, D249A / T247W, and D249A / T247C obtained in Example 1 were respectively incubated at 50 °C for 30 min, sampled, quickly cooled to room temperature, and the specific enzyme activities were measured according to the method for measuring specific enzyme activity. Taking the enzyme activity of each untreated purified enzyme solution as 100%, the residual enzyme activity was calculated.

[0091] The enzyme activities and residual enzyme activities of wild-type lactate dehydrogenase, mutants D249A, D249A / T247I, D249K, D249A / T247W, and D249A / T247C are shown in Table 2. Compared with wild-type lactate dehydrogenase, the specific enzyme activity of mutant D249A increased significantly, reaching 3.10 U / mg, about 6 times that of wild-type lactate dehydrogenase LrLDH. After incubation at 50 °C for 30 min, the residual enzyme activity of mutant D249A was 70.03%, significantly higher than that of wild-type lactate dehydrogenase LrLDH (the residual enzyme activity after incubation under the same conditions was 37.70%). It can be seen that by mutation, both the specific enzyme activity and thermal stability of lactate dehydrogenase were improved simultaneously.

[0092] On the basis of mutant D249A, the threonine at position 247 of mutant D249A was further mutated to isoleucine to obtain mutant D249A / T247I. Its specific enzyme activity was 1.61 U / mg, and the residual enzyme activity after heat treatment was 89.07%. Compared with wild-type lactate dehydrogenase, its catalytic activity was about 2 times that of the wild-type, and the residual activity after heat treatment was about 1.4 times that of the wild-type (the results are shown in Table 2). Compared with the single mutant D249A, although mutant D249A / T247I sacrificed some catalytic activity, its enzyme thermal stability was significantly improved. Mutant D249A / T247I achieved a better balance, improving both catalytic activity and thermal stability. In addition, the SDS-PAGE results showed that both mutant D249A and mutant D249A / T247I improved the soluble expression of lactate dehydrogenase in Escherichia coli. The results are shown in Figure 1 .

[0093] Table 2 Specific enzyme activities of lactate dehydrogenase and its mutants and relative residual enzyme activities after heat treatment

[0094] Enzyme Specific Enzyme Activity (U / mg) Enzyme Activity Multiple Residual Enzyme Activity (%) Wild-Type Lactate Dehydrogenase 0.51±0.08 1.00 37.70±2.20 D249A 3.10±0.17 6.08 70.03±2.75 D249K 1.34±0.07 2.63 30.59±1.38 D249A / T247I 1.61±0.08 3.16 89.07±3.55 D249A / T247W 0.15±0.02 0.29 62.30±3.68 D249A / T247C 0.09±0.00 0.18 58.30±1.37

[0095] Example 5 Determination of the thermal stability of lactate dehydrogenase and its mutants

[0096] Half-life (t 1 / 2) Determination: The purified enzyme solutions of wild-type lactate dehydrogenase, mutant D249A, and mutant D249A / T247I obtained in Example 3 were respectively incubated at 50 °C for 30 min. Samples were taken at regular intervals, and the residual enzyme activity was measured according to the method described in Example 4. A thermal inactivation curve was plotted, as shown in Figure 2 shown. It can be seen from Figure 2 that the catalytic activities of wild-type lactate dehydrogenase, mutant D249A, and mutant D249A / T247I all decreased rapidly.

[0097] The half-life (t 1 / 2 ) was calculated by the first-order inactivation equation: ln (residual enzyme activity) = -k D t, where t 1 / 2 = ln2 / k D (where k D represents the inactivation constant and t represents the incubation time).

[0098] Determination of the half-inactivation temperature : The purified enzyme solutions of wild-type lactate dehydrogenase, mutant D249A, and mutant D249A / T247I obtained in Example 3 were respectively incubated at 20 °C, 25 °C, 30 °C, 35 °C, 40 °C, 45 °C, 50 °C, 55 °C, and 60 °C for 10 min. The residual enzyme activity was measured according to the method described in Example 4, and a curve was plotted, as shown in Figure 3 shown.

[0099] It can be seen from Figure 2 and Figure 3 that the t 1 / 2 values of wild-type lactate dehydrogenase LrLDH, mutant D249A, and mutant D249A / T247I were 3.70 min, 7.88 min, and 21.66 min respectively; the values were 38 °C, 44 °C, and 57 °C respectively. For details, see Table 3. It can be seen that the thermal stability of mutant D249A and mutant D249A / T247I was significantly enhanced by site-directed mutagenesis.

[0100] Example 6 Determination of the kinetic parameters of lactate dehydrogenase and its mutants

[0101] With the concentration of NADH fixed at 1 mM, the specific enzyme activities of wild-type lactate dehydrogenase, mutant D249A, and mutant D249A / T247I were measured at different substrate concentrations (the concentration of sodium pyruvate was 1 - 5 mM). Through non-linear fitting of the Michaelis equation, the kinetic parameters were obtained. The results are shown in Table 3. The V max values of wild-type lactate dehydrogenase, mutant D249A, and mutant D249A / T247I were 1.95 U / mg, 8.01 U / mg, and 5.28 U / mg respectively, and the Km were 2.83 mM, 1.67 mM and 2.32 mM, respectively. The V of mutant D249A max was more than 3-fold higher than that of wild-type lactate dehydrogenase, and the K m decreased significantly.

[0102] k was calculated by the following formula cat :

[0103] k cat = V max × relative molecular mass of enzyme protein / 6000,

[0104] Compared with wild-type lactate dehydrogenase, the catalytic efficiency (k cat / K m ) of mutant D249A was increased by more than 6-fold. Although the k cat / K m of mutant D249A / T247I decreased compared with that of mutant D249A, it was still increased by 2.4-fold compared with wild-type lactate dehydrogenase. Therefore, mutant D249A and mutant D249A / T247I maintained the catalytic activity of the enzyme while achieving an improvement in enzyme thermal stability.

[0105] Table 3 Kinetic parameters and t of wild-type lactate dehydrogenase, mutant D249A and mutant D249A / T247I 1 / 2 and values

[0106]

[0107] Example 7 Determination of the Optimum Temperature of Lactate Dehydrogenase and Its Mutants

[0108] The catalytic activities of the wild-type lactate dehydrogenase, mutant D249A and mutant D249A / T247I obtained in Example 3 towards sodium phenylpyruvate were measured at different temperatures (30 °C, 35 °C, 40 °C, 42 °C, 44 °C, 46 °C, 50 °C, 52 °C, 54 °C, 56 °C, 60 °C), and the results are as Figure 4 shown. Compared with wild-type lactate dehydrogenase, the optimum catalytic activity of mutant D249A increased slightly (from 40 °C to 44 °C); mutant D249A / T247I further increased the optimum catalytic temperature to 52 °C. This indicates that the thermal stability of mutant D249A and mutant D249A / T247I enzymes was significantly improved, and the thermal denaturation of enzyme molecules slowed down at higher temperatures. Therefore, the optimum catalytic temperature of lactate dehydrogenase was increased.

[0109] Example 8 Application of Lactate Dehydrogenase Mutant D249A / T247I in the Asymmetric Reduction of Sodium Phenylpyruvate to Synthesize D-Phenyllactic Acid

[0110] The concentrations of sodium phenylpyruvate and phenyl lactic acid were determined by a liquid chromatograph (LC-20A, Shimadzu, Japan) equipped with an ODS HYPERSIL liquid chromatography column (4.6×250 mm, 5 μm, Thermo, USA) and an ultraviolet detector (SPD-10A VP Plus, Shimadzu, Japan); the detector wavelength was set at 210 nm, the column temperature was controlled at 40 °C, the mobile phase was composed of acetonitrile and 0.1% formic acid aqueous solution mixed in a ratio of 1:4, and the flow rate was set at 1.0 mL / min.

[0111] The optical purity of D-phenyl lactic acid was determined by a liquid chromatograph equipped with a CHIRALCEL OJ-RH column (4.6×150 mm, 5 μm, Daicel, Japan), the ultraviolet detector wavelength was set at 210 nm, the column temperature was controlled at 40 °C, the mobile phase was composed of acetonitrile, methanol, trifluoroacetic acid and water mixed in a ratio of 50:50:1.5:898.5, and the flow rate was set at 0.6 mL / min.

[0112] The catalytic reaction was carried out in a 100 mL round-bottom flask. 10 g of the wet cells of E. coli BL21(DE3) / pET28a-lrldh and E. coli BL21(DE3) / pET28a-lrldh-D249A / T247I obtained in Example 2 were respectively taken and added to 50 mL of potassium phosphate buffer (100 mM, pH 7.0) dissolved with 3 g of sodium phenylpyruvate and 3 g of glucose, and were fully suspended. The reaction was carried out in a magnetic stirring reactor at a temperature of 45 °C and a rotation speed of 200 rpm. 1.0 M Na2CO3 solution was automatically added to control the pH to 6.5. Samples were taken every 0.5 h, and the content and optical purity of D-phenyl lactic acid were determined by the above liquid chromatography method. The results are as Figure 5 shown.

[0113] From Figure 5It can be seen that after 5.5 h of reaction, the reaction catalyzed by E. coli BL21(DE3) / pET28a-lrldh-D249A / T247I (the lactate dehydrogenase is LrLDH-D249A / T247I) was basically completed, the cumulative concentration of D-phenyllactic acid was 29.2 g / L, and the space-time yield was 127.4 g / (L / h). While for E. coli BL21(DE3) / pET28a-lrldh (the lactate dehydrogenase is LrLDH-WT), after 7 h of reaction under the same conditions, the cumulative concentration of D-phenyllactic acid was only 12.9 g / L, and the space-time yield was 44.2 g / (L / h). It was found by chiral chromatography detection that LrLDH-D249A / T247I did not affect the stereoselectivity of lactate dehydrogenase. Under the action of LrLDH-D249A / T247I and LrLDH-WT, the phenyllactic acid products were both of the D type, and the enantiomeric excess value e.e. was greater than 99%. Compared with the wild-type lactate dehydrogenase, the catalytic activity and stability of the mutant enzyme LrLDH-D249A / T247I were significantly improved, and it was more suitable for the industrial production of D-phenyllactic acid.

[0114] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited thereto. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solutions of the present invention, including any other suitable combination of each technical feature. These simple modifications and combinations should also be regarded as the content disclosed by the present invention and fall within the protection scope of the present invention.

Claims

1. A lactate dehydrogenase, characterized in that, The lactate dehydrogenase is the enzyme described in any one of (a)-(d): (a) an enzyme having the amino acid sequence shown in SEQ ID NO.1; (b) an enzyme having the amino acid sequence in which the threonine at position 247 of the amino acids shown in SEQ ID NO.1 is replaced with isoleucine; (c) an enzyme having the amino acid sequence with a tag attached to the amino terminus and / or carboxy terminus of the amino acid sequence described in (a) or (b); (d) an enzyme having the amino acid sequence with a signal sequence attached to the amino terminus of the amino acid sequence described in (a) or (b).

2. A gene encoding lactate dehydrogenase, characterized in that, The gene has a nucleotide sequence encoding the lactate dehydrogenase according to claim 1.

3. The gene according to claim 2, characterized in that, The gene has the nucleotide sequence shown in SEQ ID NO.

2.

4. A recombinant vector, characterized in that, The recombinant vector contains the gene according to claim 2 or 3.

5. The recombinant vector according to claim 4, wherein The expression vector of the recombinant vector is the pET28a plasmid.

6. A recombinant strain, characterized in that, The recombinant strain contains the gene according to claim 2 or 3 or the recombinant vector according to claim 4 or 5.

7. The recombinant strain according to claim 6, characterized in that, The recombinant strain is Escherichia coli.

8. A preparation method of a leavening agent, characterized in that, The preparation method includes: inoculating the recombinant strain according to claim 6 or 7 into a fermentation medium for fermentation to obtain a fermentation broth, and performing solid-liquid separation on the fermentation broth to obtain wet bacterial cells.

9. Use of at least one of the lactate dehydrogenase according to claim 1, the gene according to claim 2 or 3, the recombinant vector according to claim 4 or 5, the recombinant strain according to claim 6 or 7, and the starter prepared by the preparation method according to claim 8 in the preparation of D-phenyllactic acid.

10. A method for preparing D-phenyllactic acid, characterized in that, The method includes the following steps: contacting at least one of the lactate dehydrogenase according to claim 1, the gene according to claim 2 or 3, the recombinant vector according to claim 4 or 5, the recombinant strain according to claim 6 or 7, and the starter prepared by the preparation method according to claim 8 with sodium phenylpyruvate; The co-substrate is selected from at least one of glucose, glucose-6-phosphate, and isopropanol.

11. The method according to claim 10, wherein The contacting process includes: mixing the starter with sodium phenylpyruvate in the presence of a co-substrate and a reaction medium, and reacting at a temperature of 25-45°C and a rotation speed of 150-250 rpm until the sodium phenylpyruvate reacts completely.

12. The method according to claim 11, wherein The co-substrate is glucose; The reaction medium uses a potassium phosphate buffer with a pH of 6.0-8.

0.

13. The method according to claim 12, wherein Relative to 1 L of the reaction medium, the dosage of sodium phenylpyruvate is 1-100 g, the dosage of the co-substrate is 5-300 g, and the dosage of the starter is 10-250 g.

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