CN115975965A

CN115975965A - Lactate dehydrogenase and coding gene, recombinant vector, recombinant bacterium and leavening agent thereof, and application of lactate dehydrogenase and coding gene, recombinant vector, recombinant bacterium and leavening agent

Info

Publication number: CN115975965A
Application number: CN202210956379.8A
Authority: CN
Inventors: 罗希; 付永前; 呼高伟; 尹龙飞; 尹丰伟; 孙小龙
Original assignee: Taizhou University
Current assignee: Taizhou University
Priority date: 2022-08-10
Filing date: 2022-08-10
Publication date: 2023-04-18

Abstract

The invention relates to genetic engineering and discloses a lactate dehydrogenase, a coding gene, a recombinant vector, a recombinant bacterium, a fermentation agent and application thereof. The lactate dehydrogenase is an enzyme with an amino acid sequence shown in SEQ ID NO.1, or an enzyme with an amino acid sequence which is shown in the amino acid sequence shown in SEQ ID NO.1 and still has the activity of the lactate dehydrogenase, wherein the 247 th position of the amino acid shown in SEQ ID NO.1 is substituted, deleted or added with one or more amino acid residuesThe catalytic activity and the thermal stability of the dehydrogenase in the process of asymmetrically reducing the sodium propiophenonate are obviously improved, and compared with the wild-type lactate dehydrogenase, the catalytic efficiency (k) is higher _cat /K _m ) The thermal stability is improved by about 2.4 times, and the half-life period t at 50 ℃ is obviously improved _1/2 The elongation is nearly 5 times; the semi-inactivation temperature is increased by 19 ℃), is particularly suitable for catalyzing asymmetric reduction of sodium propiophenonate to prepare D-phenyl lactic acid, and has better industrial application prospect.

Description

Lactate dehydrogenase and coding gene, recombinant vector, recombinant bacterium and leavening agent thereof, and application of lactate dehydrogenase and coding gene, recombinant vector, recombinant bacterium and leavening agent

Technical Field

The invention relates to gene engineering, in particular to lactate dehydrogenase and a coding gene thereof, a recombinant vector, a recombinant strain, a leaven, a method for preparing D-phenyllactic acid and application thereof in preparing D-phenyllactic acid.

Background

The phenyl lactic acid exists in honey, sour dough and lactic acid bacteria fermented food, is a natural organic acid, has broad-spectrum antibacterial activity, can inhibit the growth of various microorganisms such as gram-positive bacteria, gram-negative bacteria, yeast, mould and the like, has good water solubility, high temperature resistance and wider action pH, is safe and nontoxic, can prevent food spoilage, maintains the flavor of food while retaining the nutritional quality, and can be applied to the food industry as an antibacterial agent and an antiseptic agent. In addition, the phenyllactic acid can also be used as a feed additive to increase the laying rate of poultry and the quality of poultry eggs; or can be used as anti-wrinkle agent and bacteriostatic agent for cosmetic. The phenyl lactic acid molecule contains a chiral carbon atom, so that a pair of enantiomers exist, wherein D-phenyl lactic acid has stronger antibacterial activity, and can be used as a precursor to synthesize a hypoglycemic agent, namely englitazone and a coronary heart disease treatment drug, namely danshensu, so that D-phenyl lactic acid has attracted much attention. The wide application prospect of the D-phenyl lactic acid in the industries of food, feed, pharmacy, cosmetics and the like stimulates the research enthusiasm of academic and industrial communities on the synthetic method of the D-phenyl lactic acid.

Many lactic acid bacteria can synthesize the phenyl lactic acid through intracellular metabolic pathways, but the fermentation period is long, the yield is low, the optical purity of the product is poor, and the large-scale fermentation production of the D-phenyl lactic acid is restricted. In the metabolic pathway of lactic acid bacteria, sodium propiophenonate is the direct precursor of phenyl lactic acid, and D-phenyl lactic acid is obtained by asymmetric reduction of lactate dehydrogenase. The enzyme is a biological macromolecule with a biological catalysis function, the enzyme molecule mediated biological catalysis reaction has the characteristics of high catalytic activity, good stereoselectivity, mild reaction conditions, few byproducts, environmental friendliness and the like, accords with the development direction of green chemical synthesis, increasingly becomes an important supplement of the traditional chemical synthesis method, and is widely applied to the production of medicines, pesticides, foods, cosmetics and other fine chemicals. Sodium propiophenonate is used as a substrate, and is reduced in the next step under the catalytic action of lactate dehydrogenase, which is the most commonly adopted method for preparing D-phenyl lactic acid.

Various lactate dehydrogenases and encoding genes thereof are obtained through gene mining, the asymmetric reduction of sodium propiophenonate can be catalyzed to synthesize D-phenyl lactic acid, the enantiomeric excess (e.e.) of a product is greater than 99%, but the catalytic activity and the thermal stability of the existing lactate dehydrogenase are low, so that the industrial application of the lactate dehydrogenase is limited.

Disclosure of Invention

The invention aims to overcome the problem of low asymmetric reduction activity of lactate dehydrogenase on sodium phenylpyruvate in the prior art, and provides the lactate dehydrogenase and a coding gene thereof, a recombinant vector, a recombinant strain, a leavening agent, a method for preparing D-phenyl lactic acid and application of the lactate dehydrogenase and the coding gene in preparation of the D-phenyl lactic acid.

In order to achieve the above object, a first aspect of the present invention provides a lactate dehydrogenase which is an enzyme according to any one of (a) to (d):

(a) An enzyme having an amino acid sequence shown in SEQ ID NO. 1;

(b) An enzyme represented by an amino acid sequence which is obtained by substituting, deleting or adding one or more amino acid residues at the 247 th position of the amino acid shown in SEQ ID No.1 and still has the activity of lactate dehydrogenase;

(c) An enzyme represented by an amino acid sequence wherein a tag is attached to the amino terminus and/or the carboxy terminus of the amino acid sequence of (a) or (b);

(d) An enzyme represented by an amino acid sequence wherein a signal sequence is linked to the amino terminus of the amino acid sequence of (a) or (b).

Preferably, the lactate dehydrogenase is an enzyme represented by an amino acid sequence in which threonine 247 in the amino acid sequence represented by SEQ ID NO.1 is substituted with isoleucine.

In a second aspect, the present invention provides a gene encoding lactate dehydrogenase having a nucleotide sequence encoding the lactate dehydrogenase described above.

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

More preferably, the gene has a nucleotide sequence shown in SEQ ID NO. 2.

In a third aspect, the present invention provides a recombinant vector comprising the above-described gene.

Preferably, the expression vector of the recombinant vector is a pET28a plasmid.

In a fourth aspect, the present invention provides a recombinant strain containing the above gene or the above recombinant vector.

Preferably, the recombinant strain is escherichia coli and/or bacillus subtilis.

More preferably, the recombinant strain is escherichia coli.

The fifth aspect of the present invention provides a method for preparing a fermentation agent, comprising: and inoculating the recombinant strain into a fermentation culture medium for fermentation to obtain a fermentation broth, and performing solid-liquid separation on the fermentation broth to obtain wet thalli.

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 fermentation product produced by the above-mentioned production method for producing D-phenyllactic acid.

The seventh aspect of the present invention provides a method for preparing D-phenyllactic acid, comprising the steps of: contacting at least one of the lactate dehydrogenase, the gene, the recombinant vector, the recombinant strain and the leavening agent prepared by the preparation method with sodium propiophenonate.

Preferably, the process of contacting comprises: under the condition that an auxiliary substrate and a reaction medium exist, the leavening agent and the sodium propiophenonate are mixed and then react under the conditions that the temperature is 25-45 ℃ and the rotating speed is 150-250rpm until the sodium propiophenonate completely reacts.

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

Preferably, the dosage of the sodium propiophenonate is 1-100g, the dosage of the auxiliary substrate is 5-300g, and the dosage of the leavening agent is 10-250g relative to 1L of the reaction medium.

Through the technical scheme, the invention has the beneficial effects that:

the catalytic activity and the thermal stability of the lactate dehydrogenase provided by the invention in the process of asymmetrically reducing sodium propiophenonate are obviously improved, and compared with wild-type lactate dehydrogenase, the catalytic efficiency (k) of the lactate dehydrogenase is higher _cat /K _m ) The thermal stability is improved by about 2.4 times, and the half-life period t at 50℃ is obviously improved _1/2 The elongation is nearly 5 times; the semi-inactivation temperature was increased by 19 ℃). The lactate dehydrogenase provided by the invention is particularly suitable for catalyzing asymmetric reduction of sodium propiophenonate to prepare D-phenyl lactic acid, and has a good industrial application prospect.

Drawings

FIG. 1 is an SDS-PAGE electrophoresis chart in example 3, wherein M is a protein Marker; 1.4 and 7 are 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 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 wild type LrLDH, and purified enzymes of mutants D249A and D249A/T247I;

FIG. 2 is a graph showing the change of the residual enzyme activity with time upon incubation at 50 ℃ of the wild-type lactate dehydrogenase LrLDH (WT), the mutant D249A and the mutant D249A/T247I in example 5;

FIG. 3 is a graph showing the residual enzyme activities of wild-type lactate dehydrogenase LrLDH (WT), mutant D249A and mutant D249A/T247I in example 5 as a function of incubation temperature;

FIG. 4 is a graph showing the relative activities of wild-type lactate dehydrogenase LrLDH (WT), mutant D249A and mutant D249A/T247I in example 7 with respect to enzyme activity as a function of temperature;

FIG. 5 is a graph showing the content of D-phenyllactic acid according to the reaction time when recombinant strains E.coli BL21 (DE 3)/pET 28 a-lrldhh and E.coli/pET28a-lrldh-D249A/T247I catalyze sodium propiophenone to synthesize D-phenyllactic acid through asymmetric reduction in the presence of sodium propiophenone in example 8.

Detailed Description

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

In the present invention, in the case where no reverse explanation is made, the enzyme activity of lactate dehydrogenase is determined by monitoring the consumption of NADH at 30 ℃ and a standard reaction solution is composed of a phosphate buffer (100mM, pH 7.0) containing 0.5mM NADH and 1mM sodium propiophenonate and 100. Mu.L of purified protein diluted appropriately, the total volume is 200. Mu.L, and the amount of enzyme required to consume 1. Mu. Mol of NADH per minute is defined as 1 enzyme activity unit, i.e., 1U. "specific activity" represents the catalytic ability per unit mass of protein, and the calculation formula of specific activity is as follows: specific enzyme activity (U/mg) = total enzyme activity unit number/mg total protein; the unit M represents mol/L.

In a first aspect, the present invention provides a lactate dehydrogenase which is an enzyme according to any one of (a) to (d):

(a) An enzyme having an amino acid sequence shown in SEQ ID NO. 1;

(c) An enzyme represented by an amino acid sequence wherein a tag is linked to the amino terminus and/or the carboxy terminus of the amino acid sequence of (a) or (b);

In the present invention, threonine at position 247 in the amino acid sequence of lactate dehydrogenase may be substituted, deleted or added with an amino acid residue, and the role of the residue in the protein domain (e.g., the role of providing positive charge or forming a hydrophobic pocket structure) is not changed, and the steric structure of the protein is not affected, so that the function of the protein can still be achieved. Preferably, the 247 th threonine of the lactate dehydrogenase amino acid sequence shown in SEQ ID NO.1 is substituted with isoleucine to obtain the enzyme having the amino acid sequence shown in SEQ ID NO. 3.

For ease of purification, additional modifications of (a) or (b) may be made using tags commonly used in the art, for example, obtained by attaching the amino acid sequence of the tag to the amino terminus and/or the carboxy terminus of (a). The label does not affect the activity of the lactate dehydrogenase of the invention, and whether the label is added or not can be selected according to requirements in the practical application process.

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

The lactate dehydrogenase can be obtained by artificial synthesis, or can be obtained by synthesizing the coding gene and then performing biological expression. Illustratively, the lactate dehydrogenase provided by the invention mutates a wild-type lactate dehydrogenase LrLDH encoding gene by using a site-directed saturation mutagenesis technology, transforms host escherichia coli after being connected with an expression vector, detects positive mutation with improved activity by using an efficient liquid phase detection method after induction expression to obtain a lactate dehydrogenase mutant (namely the lactate dehydrogenase provided by the invention), the catalytic activity and the thermal stability of the lactate dehydrogenase mutant are obviously improved in the process of asymmetrically reducing sodium propiophenote, and compared with the wild-type lactate dehydrogenase LrLDH, the catalytic efficiency is improved, and the thermal stability is obviously improved. The specific method comprises the following steps: the cloned wild-type lactate dehydrogenase LrLDH has successfully constructed an expression vector pET28a-LrLDH, and the enzyme protein with normal function realizes the over-expression in escherichia coli BL21 (DE 3); then carrying out computer aided design by homologous modeling and molecular docking and combining with online software HotSpot Wizard 3.0, and selecting potential sites which may influence enzymatic activity and thermal stability; setting a site of site-directed saturation mutation, designing and synthesizing a proper primer, carrying out PCR amplification on a plasmid of a full-length mutant gene by taking the recombinant expression plasmid containing the parental lactate dehydrogenase gene as a template, transforming the plasmid containing the full-length mutation into a proper host cell, and carrying out culture, induced expression and screening out a positive mutant with high activity and thermal stability; and finally, extracting plasmid DNA from the positive mutant, and performing DNA sequencing analysis to determine the mutation of the primer. In the preparation of the lactate dehydrogenase of the present invention, any suitable vector may be used.

According to the present invention, it is preferred that the gene has a nucleotide sequence encoding an enzyme having an amino acid sequence shown by SEQ ID NO.1 or a nucleotide sequence encoding an enzyme having an amino acid sequence shown by SEQ ID NO. 3.

As mentioned above, the 5 'end and/or the 3' end of the nucleotide sequence may be linked to the coding sequence of the modified tag, respectively.

It is well known in the art that 18 amino acids, other than Met (ATG) or Trp (TGG), each encoded by a single codon, of the 20 different amino acids that make up a protein 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 page 950). That is, due to the degeneracy of genetic code, there is usually more than one codon determining one amino acid, and the substitution of the third nucleotide in the triplet codon will not change the composition of the amino acid, so that the nucleotide sequences of genes encoding the same protein may differ.

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.

The nucleotide sequence provided by the present invention can be obtained by a Polymerase Chain Reaction (PCR) amplification method, a recombinant method, or an artificial synthesis method. Once the nucleotide sequence of interest is obtained, the amino acid sequence of interest can be obtained in large quantities by recombinant methods. The nucleotide sequence obtained is usually cloned into a vector, then transferred into genetically engineered bacteria, and then separated from the proliferated host cells by a conventional method to obtain the relevant nucleotide sequence.

In addition, the nucleotide sequence can be synthesized by a known artificial chemical synthesis method.

As the "vector" used in the recombinant vector, various vectors known in the art, such as various commercially available plasmids, cosmids, phages, retroviruses and the like can be used, and a preferred expression vector of the present invention is pET28a plasmid. The recombinant vector can be constructed by digesting with various endonucleases (for example, for pET28a, nco I, xho I and the like) having a cleavage site at the multiple cloning site of the vector to obtain a linear plasmid, and ligating the linear plasmid with a gene fragment cleaved with the same endonuclease to obtain a recombinant plasmid. The recombinant vector pET28a-D249A is preferably constructed by double digestion of pET28a and a gene fragment connected with the same through Nco I and Xho I and through ligase connection.

In the present invention, the recombinant vector may be transformed, transduced or transfected into a host cell (strain) by a method conventional in the art, such as chemical transformation by calcium chloride method, high-voltage shock transformation. The host cell may be a prokaryotic or eukaryotic cell, preferably escherichia coli and/or bacillus subtilis, more preferably the host cell is escherichia coli, such as e.coli BL21 (DE 3).

In the present invention, the lactate dehydrogenase may be used in the form of whole cells of the recombinant strain, or may be used in the form of crude enzyme or purified enzyme which has been isolated from the cells of the recombinant strain and has not been purified. If necessary, the lactate dehydrogenase of the present invention can also be made into immobilized enzymes or immobilized cells using immobilization techniques known in the art.

In the present invention, the lactate dehydrogenase may be prepared into a corresponding enzyme preparation, specifically, the enzyme preparation may exist in a solid, semisolid or liquid form, the enzyme preparation may contain an auxiliary material or an additive for preparing the enzyme preparation, and the like, and those skilled in the art may select the enzyme preparation according to needs, and details are not repeated herein.

The fifth aspect of the present invention provides a method for preparing a fermentation agent, comprising: inoculating the recombinant strain into a fermentation culture medium for fermentation to obtain a fermentation liquid, and performing solid-liquid separation on the fermentation liquid to obtain wet thalli. The wet cells can be used as an enzyme preparation containing lactate dehydrogenase.

In the invention, the leavening agent can contain auxiliary materials added in the conventional preparation of microbial inoculum in the field, and the skilled person can select the auxiliary materials according to the needs.

In the present invention, the solid-liquid separation may be performed by a separation means conventional in the art, such as centrifugation, filtration, and the like. Specifically, a centrifugation mode is adopted, and the centrifugation conditions comprise: the temperature is 0-10 deg.C, rotation speed is 6000-10000rpm, and time is 8-12min.

In the present invention, the conditions for fermentation of the recombinant strain are not particularly limited as long as the recombinant strain can be proliferated in a large amount through the process of fermentation. Preferably, the process of fermentation comprises: preparing the recombinant strain into a seed solution, inoculating the seed solution into a fermentation culture medium containing kanamycin, and culturing to obtain the thallus concentration OD ₆₀₀ 0.6-0.8, adding lactose with final concentration of 6-10g/L, and heating at 20-35 deg.CCulturing for 10-15h to obtain fermentation liquid.

According to the invention, the kanamycin content of the fermentation medium is preferably 40 to 60mg/L.

Further preferably, the preparation method of the seed liquid comprises the following steps: and selecting a single colony of the recombinant strain to inoculate in a seed culture medium containing kanamycin for seed culture, thus obtaining the seed solution. In the present invention, a single colony of the recombinant strain may be selected from the group consisting of the recombinant strain freshly prepared or the recombinant strain cryopreserved at a low temperature (e.g., a recombinant strain that synthesizes lipids cryopreserved in a-80 ℃ refrigerator in a glycerol cryopreservation tube).

The method of seed culture in the present invention is not particularly limited as long as the recombinant strain can be activated and propagated by the method, and preferably, the content of kanamycin in the seed culture medium is 40 to 60mg/L; the parameters of temperature, pH, rotation speed, time and the like used for seed culture can be set conventionally in the field. Preferably, the conditions of the seed culture include: the temperature is 30-45 ℃ and the time is 8-12h.

In the present invention, the seed medium and the fermentation medium are not particularly limited, and may be a medium conventionally used in the art. Preferably, LB liquid medium (8-12 g/L tryptone, 4-6g/L yeast extract, 8-12g/L NaCl) is adopted as the seed culture medium and the fermentation culture medium.

The seventh aspect of the present invention provides a method for preparing D-phenyllactic acid, comprising the steps of: at least one of the lactate dehydrogenase, the gene, the recombinant vector, the recombinant strain and the leavening agent prepared by the preparation method is contacted with sodium propiophenonate.

According to the present invention, preferably, the process of contacting comprises: under the condition that an auxiliary substrate and a reaction medium exist, the leavening agent (namely wet thalli obtained by separating fermentation liquor) and sodium propiophenote are mixed and react under the conditions that the temperature is 25-35 ℃ and the rotating speed is 150-250rpm until the sodium propiophenote completely reacts.

According to the present invention, preferably, the co-substrate is at least one of glucose, glucose-6-phosphate and isopropanol, more preferably glucose. The reaction medium adopts a potassium phosphate buffer solution with the pH value of 6.0-8.0, the potassium phosphate buffer solution is prepared by mixing and dissolving monopotassium phosphate and dipotassium phosphate according to different proportions, and the concentration of phosphate radical is 0.1mol/L.

According to the invention, preferably, the amount of sodium propiophenonate is 1-100g, the amount of co-substrate is 5-300g and the amount of leavening agent is 10-250g, relative to 1L of the reaction medium.

The present invention will be described in detail below by way of examples.

Coli BL21 (DE 3) was purchased from precious bioengineering (gangrene) ltd, no. 9126; pET28a was purchased from Biotechnology engineering (Shanghai) Inc. under the number B540183; sodium propiophenonate was purchased from Biotechnology engineering (Shanghai) Inc. under the number A600877; the rest reagents and raw materials are conventional commercial products.

LB liquid medium: 10g/L tryptone, 5g/L yeast extract and 10g/L NaCl, adjusting the pH to 7.0, and performing steam sterilization under high pressure for 21min for later use;

LB agar plate medium: 10g/L tryptone, 5g/L yeast extract, 10g/L NaCl, 12g/L agar, pH adjusted to 7.0, steam sterilized under high pressure for 21min, for use.

Example 1 preparation of lactate dehydrogenase mutants

Obtaining a wild type lactate dehydrogenase LrLDH gene (GenBank ID: AZFF 01000004.1) by cloning, successfully constructing an expression vector pET28a-LrLDH, realizing overexpression of enzyme protein with normal function in escherichia coli BL21 (DE 3), then performing computer-aided design by homologous modeling and molecular docking and combining with online software HotSpot Wizard 3.0, and selecting potential sites possibly influencing enzyme activity and thermal stability; setting site-directed saturated mutation sites, designing and synthesizing proper primers, and carrying out PCR amplification on the plasmid of the full-length mutant gene by taking the recombinant expression plasmid containing the parent lactate dehydrogenase gene as a template; transforming plasmids containing full-length mutation into appropriate host cells, culturing, carrying out induced expression, screening positive mutants with high activity and thermal stability, extracting plasmid DNA from the positive mutants, and carrying out DNA sequencing analysis to determine mutation of primers to obtain mutants D249A, D249K, D249A/T247I, D A/T247W and D249A/T247C;

wherein D249A is a mutant enzyme having an amino acid sequence shown in SEQ ID NO.1, D249K is a mutant enzyme in which lysine is substituted by alanine at position 249 in the amino acid sequence shown in SEQ ID NO.1, D249A/T247I is a mutant enzyme in which isoleucine is substituted by threonine at position 247 in the amino acid sequence shown in SEQ ID NO.1, D249A/T247W is a mutant enzyme in which tryptophan is substituted by threonine at position 247 in the amino acid sequence shown in SEQ ID NO.1, and D249A/T247C is a mutant enzyme in which cysteine is substituted by threonine at position 247 in the amino acid sequence shown in SEQ ID NO. 1.

Carrying out whole plasmid amplification on the mutant D249A by taking the recombinant plasmid pET28 b-lrldhh as a template, and designing primer pairs as D249-F and D249-R (shown in Table 1, and the nucleotide sequences are respectively shown as SEQ ID NO.5 and SEQ ID NO. 6) to obtain a lactate dehydrogenase mutant D249A (the nucleotide sequence is shown as SEQ ID NO. 2) with the amino acid sequence shown as SEQ ID NO. 1; then, using a gene (namely, a nucleotide sequence is shown as SEQ ID NO. 2) recombinant plasmid of the lactate dehydrogenase mutant D249A as a template to carry out whole plasmid amplification, designing primer pairs as T247/D249A-F and T247/D249A-R (see Table 1, the nucleotide sequences are respectively shown as SEQ ID NO.7 and SEQ ID NO. 8), and obtaining the lactate dehydrogenase mutant D249A/T247I (the amino acid sequence is shown as SEQ ID NO.3, and the nucleotide sequence is shown as SEQ ID NO. 4) in which the 247 threonine of the amino acid sequence shown as SEQ ID NO.1 is mutated into isoleucine.

In example 1, the PCR system was: 5 × Prime STAR Buffer (Mg) ²⁺ plus) 10. Mu.L, dNTP Mix (2.5 mM each nucleotide), 0.5. Mu.L each of the mutant primers, 0.5. Mu.L of the template (recombinant plasmid), 0.5. Mu.L of Prime STAR DNA polymerase, and water to 50. Mu.LL; the PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 5min, 25 cycles (95 ℃, 15s,55 ℃, 15s,72 ℃,7 min), and final extension at 72 ℃ for 10min.

After the PCR reaction product is analyzed to be positive by 0.9 percent agarose gel electrophoresis, 20 mu L of PCR solution is taken, 1 mu L of Dpn I is added, enzyme digestion is carried out at 37 ℃ for 3h to remove template plasmids, inactivation is carried out at 65 ℃ for 10min, escherichia coli E.coli BL21 (DE 3) competent cells are transformed, LB agar plate containing kanamycin (the final concentration is 50 mu g/mL) is coated, and culture is carried out overnight at 37 ℃; through the sequencing confirmation of the biological engineering (Shanghai) GmbH, recombinant strains E.coli BL21 (DE 3)/pET 28 a-lrldhh-D249A (D249A) and E.coli BL21 (DE 3)/pET 28 a-lrldhh-D249A/T247I (D249A/T247I) are obtained.

TABLE 1 lactate dehydrogenase mutant primers

Primer name	Sequence (5 '-3')
		D249-F	ATACCGATNNSCTGATCAAAGCGCTGGATT
D249-R	TTTGATCAGNNSATCGGTATCAACCAGATC
		T247/D249A-F	CTGGTTGATNNSGATGCACTGATCAAAGCGC
T247/D249A-R	CAGTGCATCNNSATCAACCAGATCCCCACG

The underlined parts in table 1 indicate the mutated nucleotides.

Example 2 inducible expression of wild-type lactate dehydrogenase and mutants

The wild type lactate dehydrogenase gene containing wild strains E.coli BL21 (DE 3)/pET 28 a-lrldhh and the lactate dehydrogenase recombinant strains E.coli BL21 (DE 3)/pET 28 a-lrldhh-D249A and E.coli BL21 (DE 3)/pET 28 a-lrldhh-D249A/T247I obtained in example 1 were inoculated into LB liquid medium containing kanamycin (50 mg/L final concentration), respectively, cultured at 37 ℃ for 10 hours, further inoculated into fresh LB liquid medium containing kanamycin (50 mg/L final concentration), cultured at 37 ℃ to OD ₆₀₀ And the concentration is about 0.7, adding lactose with the final concentration of 8g/L into the culture solution, continuously culturing for 12 hours at the temperature of 28 ℃ to obtain fermentation liquor, centrifuging the fermentation liquor for 10 minutes at the temperature of 4 ℃ and the rotating speed of 8500rpm, and collecting E.coli BL21 (DE 3)/pET 28 a-lrldhh wet thalli, E.coli BL21 (DE 3)/pET 28a-lrldh-D249A wet thalli and E.coli BL21 (DE 3)/pET 28a-lrldh-T247I/D249A wet thalli, wherein the culture liquor can be respectively used for purifying wild type lactate dehydrogenase and preparing D-phenyl lactic acid by biological catalysis.

Example 3 purification of wild-type lactate dehydrogenase and mutants

Suspending the E.coli BL21 (DE 3)/pET 28 a-lrldhh wet cells and E.coli BL21 (DE 3)/pET 28 a-lrldhh-D249A wet cells obtained in example 2 and E.coli BL21 (DE 3)/pET 28 a-lrldhh-D249A/T247I wet cells with buffer A (2 mM, pH8.0 phosphate buffer containing 500mM NaCl and 20mM imidazole), respectively, allowing the wet cell concentration to be 50g/L, placing the cell suspension in an ice bath, ultrasonically disrupting at a power of 300W for 10min, then centrifuging (10000 Xg, 4 ℃,20 min) to remove cell debris, collecting the supernatant, loading Ni-NTA affinity chromatography equilibrated with buffer A with a pre-packed heavy column (5 mL), washing the column with 25mL buffer A to remove unbound protein, then loading buffer B (buffer B, pH8.0 mM, naCl containing 500mM NaCl and 500mM imidazole containing phosphate dehydrogenase) with a buffer, dialyzing the protein dehydrogenase protein, eluting all the protein in a buffer containing 0.10 mM lactate dehydrogenase, 0.249D, and purifying the protein in a buffer containing 20mM lactate dehydrogenase (2 ℃ or higher concentration) to obtain a buffer containing 20mM lactate dehydrogenase protein, and purifying step D mutant protein, wherein the protein obtained in a buffer, and the buffer containing 0.20.5 mM buffer, 4 ℃ overnight; the resulting protein was examined under denaturing conditions using SDS-PAGE at a separation gel concentration of 12%, and the results are shown in FIG. 1.

Example 4 determination of specific enzyme Activity and Heat treatment residue of lactate dehydrogenase and its mutants

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

Protein purity was determined by the Bradford method using standard bovine serum albumin as a control.

Determination of residual enzyme activity: respectively placing purified enzyme solutions corresponding to the wild-type lactate dehydrogenase obtained in example 3, the mutants D249A and D249A/T247I and the mutants D249K, D A/T247W and D249A/T247C obtained in example 1 at 50 ℃ for incubation for 30min, sampling, rapidly cooling to room temperature, respectively measuring specific enzyme activity according to a specific enzyme activity measuring method, and calculating residual enzyme activity by taking the enzyme activity of each untreated purified enzyme solution as 100%.

The enzyme activities and residual enzyme activities of wild-type lactate dehydrogenase, mutants D249A, D249A/T247I, D249K, D A/T247W and D249A/T247C are shown in Table 2. Compared with wild type lactate dehydrogenase, the specific enzyme activity of the mutant D249A is obviously increased to 3.10U/mg, which is about 6 times of the specific enzyme activity of wild type lactate dehydrogenase LrLDH. After incubation for 30min at 50 ℃, the residual enzyme activity of the mutant D249A is 70.03%, which is obviously higher than that of wild-type lactate dehydrogenase LrLDH (the residual enzyme activity after incubation under the same condition is 37.70%), and therefore, the specific enzyme activity and the thermal stability of the lactate dehydrogenase are simultaneously improved through mutation.

On the basis of the mutant D249A, the 247 th threonine of the mutant D249A is further mutated into isoleucine to obtain a mutant D249A/T247I, the specific enzyme activity of the mutant D249A/T247I is 1.61U/mg, the residual enzyme activity after heat treatment is 89.07%, compared with the wild-type lactate dehydrogenase, the catalytic activity of the mutant D249A is about 2 times that of the wild-type lactate dehydrogenase, and the residual activity after heat treatment is about 1.4 times that of the wild-type lactate dehydrogenase (the result is shown in Table 2). Compared with the single mutation D249A, although the mutant D249A/T247I sacrifices partial catalytic activity, the thermal stability of the enzyme is remarkably improved. The mutant D249A/T247I enables the enzyme to reach better balance, and simultaneously improves the catalytic activity and the thermal stability. In addition, SDS-PAGE results showed that both mutant D249A and mutant D249A/T247I increased the soluble expression of lactate dehydrogenase in E.coli, and the results are shown in FIG. 1.

TABLE 2 specific enzyme activity and relative residual enzyme activity after heat treatment of lactate dehydrogenase and its mutants

Enzymes	Specific 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

Example 5 determination of the thermostability of lactate dehydrogenase and its mutants

Half life (t) _1/2 ) The determination of (1): the wild-type lactate dehydrogenase, the mutant D249A and the mutant D249A/T247I purified enzyme solutions obtained in example 3 were incubated at 50 ℃ for 30min, respectively, and samples were taken at intervals of a certain time, and the residual enzyme activity was measured by the method described in example 4, and a heat inactivation curve was plotted, as shown in FIG. 2. As can be seen from FIG. 2, the catalytic activities of the wild-type lactate dehydrogenase, the mutant D249A and the mutant D249A/T247I all decreased rapidly.

Half life (t) _1/2 ) By the first order deactivation equation: ln (residual enzyme activity) = -k _D t calculation, t _1/2 ＝ln2/k _D (where k is _D To indicate a lossActivity constant, t denotes incubation time).

Semi-inactivation temperature

The determination of (1): the purified enzyme solutions of the wild-type lactate dehydrogenase, the mutant D249A and the mutant D249A/T247I obtained in example 3 were incubated at 20 ℃, 25 ℃, 30 ℃, 35 ℃, 40 ℃, 45 ℃, 50 ℃,55 ℃ and 60 ℃ for 10min, respectively, and the residual enzyme activity was measured and plotted as described in example 4, as shown in FIG. 3.

As can be seen from FIGS. 2 and 3, T of the wild-type lactate dehydrogenase LrLDH, the mutant D249A and the mutant D249A/T247I _1/2 3.70min,7.88min and 21.66min respectively;

respectively at 38 deg.C, 44 deg.C and 57 deg.C, as shown in Table 3. It can be seen that the thermostability of mutant D249A and mutant D249A/T247I was significantly enhanced by site-directed mutagenesis.

Example 6 determination of kinetic parameters of lactate dehydrogenase and its mutants

The concentration of fixed NADH is 1mM, the specific activities of the wild-type lactate dehydrogenase, the mutant D249A and the mutant D249A/T247I under different substrate concentrations (the concentration of sodium propiophenonate is 1-5 mM) are respectively measured, and kinetic parameters are obtained by nonlinear fitting of a Mie equation, and the results are shown in Table 3. V of wild-type lactate dehydrogenase, mutant D249A and mutant D249A/T247I _max Respectively 1.95U/mg,8.01U/mg and 5.28U/mg, K _m 2.83mM,1.67mM and 2.32mM, respectively. V of mutant D249A _max Compared with wild lactate dehydrogenase, the increase is more than 3 times, and K is _m And is significantly reduced.

K is calculated by the following formula _cat ：

k _cat ＝V _max The relative molecular mass of the x enzyme protein/6000,

catalytic efficiency (k) of mutant D249A compared with wild-type lactate dehydrogenase _cat /K _m ) Increased by more than 6 times, although mutated compared to mutant D249AK for volume D249A/T247I _cat /K _m Reduced, but still increased by 2.4 times compared with wild-type lactate dehydrogenase. Therefore, the mutants D249A and D249A/T247I also retained the catalytic activity of the enzyme, provided that the improvement in the thermostability of the enzyme was achieved.

TABLE 3 kinetic parameters and T for wild-type lactate dehydrogenase, mutant D249A and mutant D249A/T247I _1/2 And

value of

Example 7 optimum temperature measurement of lactate dehydrogenase and its mutant

The catalytic activity of the wild-type lactate dehydrogenase obtained in example 3, the mutant D249A and the mutant D249A/T247I on sodium propiophenonate was measured at different temperatures (30 ℃, 35 ℃, 40 ℃, 42 ℃,44 ℃, 46 ℃, 50 ℃, 52 ℃, 54 ℃, 56 ℃ and 60 ℃), respectively, and the results are shown in FIG. 4. Compared with wild lactate dehydrogenase, the optimum catalytic activity of the mutant D249A is slightly improved (from 40 ℃ to 44 ℃); mutant D249A/T247I further increased the optimum catalytic temperature to 52 ℃. This indicates that the thermal stability of the mutant D249A and the mutant D249A/T247I enzymes is significantly improved, and the thermal denaturation of the enzyme molecules at higher temperatures is slowed, thereby increasing the optimum catalytic temperature of lactate dehydrogenase.

Example 8 application of lactate dehydrogenase mutant D249A/T247I in synthesis of D-phenyl lactic acid by asymmetric reduction of sodium phenylpropionate

The concentrations of sodium propiophenone and phenyllactic acid were determined by a liquid chromatograph (LC-20A, shimadzu, japan) equipped with an ODS HYPERSIL liquid chromatography column (4.6X 250mm,5 μm, thermo, USA) and an ultraviolet detector (SPD-10A VP plus, shimadzu, japan); the wavelength of the detector is set to 210nm, the column temperature is controlled to be 40 ℃, the mobile phase is formed by mixing acetonitrile and 0.1% formic acid aqueous solution according to the proportion of 1:4, and the flow rate is set to be 1.0mL/min.

The optical purity of D-phenyllactic acid was measured by a liquid chromatograph equipped with a CHIRALCEL OJ-RH column (4.6 × 150mm,5 μm, daicel, japan), the ultraviolet detector wavelength was set to 210nm, the column temperature was controlled to 40 ℃, the mobile phase was mixed from acetonitrile, methanol, trifluoroacetic acid and water at a ratio of 50.

The catalytic reaction was carried out in a 100mL round-bottomed flask, and 10g of each of the E.coli BL21 (DE 3)/pET 28a-lrldh and E.coli BL21 (DE 3)/pET 28a-lrldh-D249A/T247I wet cells obtained in example 2 were taken, added to 50mL of potassium phosphate buffer (100mM, pH 7.0) in which 3g of sodium propiophenonate and 3g of glucose were dissolved, sufficiently suspended, reacted in a magnetic stirring reactor at 45 ℃ and 200rpm, and automatically supplemented with 1.0M Na ₂ CO ₃ The solution was sampled every 0.5h with the pH of the solution being controlled to 6.5, and the content of D-phenyllactic acid and its optical purity were measured by the above-mentioned liquid chromatography method, and the results are shown in FIG. 5.

As can be seen from FIG. 5, the reaction catalyzed by E.coli BL21 (DE 3)/pET 28a-LrLDH-D249A/T247I (lactate dehydrogenase is LrLDH-D249A/T247I) was substantially complete after 5.5h of reaction, the cumulative concentration of D-phenyllactic acid was 29.2g/L, and the space-time yield was 127.4 g/(L/h), while E.coli BL21 (DE 3)/pET 28a-LrLDH (lactate dehydrogenase is LrLDH-WT) was reacted for 7h under the same conditions, the cumulative concentration of D-phenyllactic acid was only 12.9g/L, and the space-time yield was 44.2 g/(L/h). The chiral chromatographic detection shows that the LrLDH-D249A/T247I does not influence the stereoselectivity of lactate dehydrogenase, and under the action of the LrLDH-D249A/T247I and the LrLDH-WT, the phenyllactic acid products are D-type, and the e.e value of the enantiomeric excess is more than 99 percent. Compared with wild type lactate dehydrogenase, the mutant enzyme LrLDH-D249A/T247I has obviously raised catalytic activity and stability, and is suitable for industrial production of D-phenyl lactic acid.

The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including various technical features being combined in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.

Claims

1. A lactate dehydrogenase which is an enzyme according to any one of (a) to (d):

(a) An enzyme having an amino acid sequence shown in SEQ ID No. 1;

2. The lactate dehydrogenase according to claim 1, wherein the lactate dehydrogenase is an enzyme represented by an amino acid sequence in which threonine 247 in the amino acid sequence represented by SEQ ID No.1 is substituted with isoleucine.

3. A gene encoding lactate dehydrogenase having a nucleotide sequence encoding the lactate dehydrogenase of claim 1 or 2;

preferably, the gene has a nucleotide sequence encoding an enzyme having an amino acid sequence shown in SEQ ID NO. 1;

more preferably, the gene has a nucleotide sequence shown in SEQ ID NO. 2.

4. A recombinant vector comprising the gene of claim 3;

5. A recombinant strain comprising the gene of claim 2 or 3 or the recombinant vector of claim 4;

preferably, the recombinant strain is escherichia coli and/or bacillus subtilis;

more preferably, the recombinant strain is escherichia coli.

6. A preparation method of a leaven is characterized by comprising the following steps: inoculating the recombinant strain of claim 5 into a fermentation medium for fermentation to obtain a fermentation broth, and performing solid-liquid separation on the fermentation broth to obtain wet bacteria.

7. Use of at least one of the lactate dehydrogenase according to claim 1 or 2, the gene according to claim 3, the recombinant vector according to claim 4, the recombinant strain according to claim 5, and the fermentation product produced by the production method according to claim 6 for producing D-phenyllactic acid.

8. A method for preparing D-phenyl lactic acid, comprising the steps of: at least one of the lactate dehydrogenase according to claim 1 or 2, the gene according to claim 3, the recombinant vector according to claim 4, the recombinant strain according to claim 5, and the fermentation product obtained by the production method according to claim 6 is contacted with sodium phenylpropionate.

9. The method of claim 8, wherein the contacting comprises: under the condition that an auxiliary substrate and a reaction medium exist, the leavening agent and sodium propiophenonate are mixed and then react under the conditions that the temperature is 25-45 ℃ and the rotating speed is 150-250rpm until the sodium propiophenonate completely reacts.

10. The method according to claim 9, wherein the co-substrate is selected from at least one of glucose, glucose-6-phosphate and isopropanol, preferably glucose;

the reaction medium adopts potassium phosphate buffer solution with the pH value of 6.0-8.0;