Leucine dehydrogenase mutants and their applications

Abstract

This application relates to the field of biotechnology, and more particularly to a leucine dehydrogenase mutant and its applications. The leucine dehydrogenase mutant and its applications provided in this application are obtained by screening wild-type leucine dehydrogenase for mutations, resulting in a leucine dehydrogenase mutant with the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. This mutant exhibits improved catalytic efficiency and thermal stability for the asymmetric reductive amination of prochiral α-keto acid compounds, which is beneficial for improving the synthesis efficiency of L-amino acids.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to a leucine dehydrogenase mutant and its applications. Background Technology

[0002] With the increasing prominence of chiral non-natural amino acids such as L-tert-leucine in various fields, the exploration of green and efficient synthetic routes for them has gradually become a focus of researchers' attention. The biocatalytic method for synthesizing chiral non-natural amino acids uses α-keto acid compounds as substrates and inorganic ammonia, catalyzed by amino acid dehydrogenases, to prepare them.

[0003] Leucine dehydrogenase (LeuDH) belongs to the amino acid dehydrogenase superfamily and is a type of NAD+ dehydrogenase. + NADH-dependent oxidoreductases are an important biological pathway for the synthesis of L-amino acids. In the background, leucine dehydrogenases exhibit poor thermostability and conversion efficiency, which hinders the improvement of L-amino acid synthesis efficiency. Summary of the Invention

[0004] In view of the above problems, this application provides a leucine dehydrogenase mutant and its application to solve the technical problems that are detrimental to the improvement of L-amino acid synthesis efficiency due to the poor thermal stability and conversion efficiency of leucine dehydrogenase.

[0005] In a first aspect, embodiments of this application provide a leucine dehydrogenase mutant having an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0006] Secondly, embodiments of this application provide a polynucleotide that encodes the aforementioned leucine dehydrogenase mutant.

[0007] Thirdly, embodiments of this application provide a recombinant vector comprising the aforementioned polynucleotides.

[0008] Fourthly, embodiments of this application provide a host cell, the host cell comprising the recombinant vector described above.

[0009] Fifthly, embodiments of this application provide the application of the above-described leucine dehydrogenase mutant in the asymmetric reductive amination of prochiral α-keto acid compounds.

[0010] Optionally, in the presence of coenzyme NAD+ and a coenzyme regeneration system, the leucine dehydrogenase mutant of claim 1 is used to catalyze the asymmetric reductive amination of the prochiral α-keto acid compound to generate L-amino acids.

[0011] Optionally, the coenzyme regeneration system includes a cosubstrate and an oxidoreductase, wherein the cosubstrate is isopropanol, formate, or glucose, and the oxidoreductase is isopropanol dehydrogenase, formate dehydrogenase, or glucose dehydrogenase.

[0012] Optionally, the prochiral α-keto acid compound is trimethylpyruvate, and the L-amino acid is L-tert-leucine.

[0013] In a sixth aspect, embodiments of this application provide a method for synthesizing L-tertiary leucine, comprising: contacting the above-mentioned leucine dehydrogenase mutant, a transformed host cell containing the above-mentioned leucine dehydrogenase mutant, or a culture of a transformed host cell containing the above-mentioned leucine dehydrogenase mutant with trimethylpyruvate to convert trimethylpyruvate into L-tertiary leucine.

[0014] Optionally, the pH of the reaction system is 8.5–10.

[0015] The leucine dehydrogenase mutants and their applications provided in this application involve screening wild-type leucine dehydrogenase for mutations to obtain leucine dehydrogenase mutants with amino acid sequences as shown in SEQ ID NO: 1 or SEQ ID NO: 2. These mutants exhibit improved catalytic efficiency and thermal stability for asymmetric reductive amination of prochiral α-keto acid compounds, which is beneficial for improving the synthesis efficiency of L-amino acids.

[0016] These or other aspects of this application will become more apparent in the following description of the embodiments. Attached Figure Description

[0017] Figure 1 The schematic diagram of the reaction principle for the asymmetric reductive amination to prepare L-amino acids in this application is shown. Detailed Implementation

[0018] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of the invention. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0020] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0021] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0022] In this article, the term "leucine dehydrogenase" refers to an enzyme that exhibits asymmetric reductive ammoniation activity of prochiral α-keto acids, which can asymmetricly reductively ammoniate prochiral α-keto acids to convert them into L-amino acids.

[0023] The reaction principle for converting prochiral α-keto acids into L-amino acids using the aforementioned leucine dehydrogenase is shown below:

[0024]

[0025] The description herein refers to "a polypeptide, protein, mutant, or enzyme having the amino acid sequence shown in SEQ ID NO:". Obviously, polypeptides, proteins, mutants, or enzymes having the amino acid sequence shown in SEQ ID NO:, even with some sequence deletions, modifications, substitutions, conserved substitutions, or additions, can also be used in this application, as long as they exhibit the same or corresponding activity as the polypeptide, protein, mutant, or enzyme with the amino acid sequence shown in SEQ ID NO:. For example, it is not excluded to add sequences that do not alter protein function, naturally occurring mutations, their silent mutations, or conserved substitutions before or after "the polypeptide, protein, mutant, or enzyme with the amino acid sequence shown in SEQ ID NO:". Furthermore, polypeptides, proteins, mutants, or enzymes having the amino acid sequence shown in SEQ ID NO:, when subjected to the addition of the aforementioned sequences that do not alter protein function, naturally occurring mutations, their silent mutations, or conserved substitutions, also fall within the scope of this application, as long as they exhibit the same or corresponding activity as the amino acid sequence shown in SEQ ID NO: after the addition of the aforementioned sequences.

[0026] This application provides a leucine dehydrogenase mutant, which is generated by at least one mutation in the amino acid sequence shown in SEQ ID NO: 3.

[0027] The amino acid sequence shown in SEQ ID NO: 3 can be obtained from NCBI's GenBank. Specifically, the amino acid sequence shown in SEQ ID NO: 3 is derived from Roseibium aggregatum, with the GenBank accession number WP_006932274.1. In this specification, the enzyme formed by the amino acid sequence shown in SEQ ID NO: 3 can be called wild-type leucine dehydrogenase, and the enzyme formed by the mutated amino acid sequences can be called mutant leucine dehydrogenase or leucine dehydrogenase mutant.

[0028] In one leucine dehydrogenase mutant of this embodiment, the following mutations occur in the amino acid sequence corresponding to SEQ ID NO: 3: aspartic acid D at position 24 is mutated to leucine L, glutamic acid E at position 124 is mutated to valine V, S at position 129 is mutated to threonine T, and valine V at position 243 is mutated to alanine A (SEQ ID NO: 1). In the corresponding mutations, the amino acid defects at the corresponding positions in the amino acid sequence shown in SEQ ID NO: 3 are replaced by other amino acids. The above-mentioned leucine dehydrogenase mutant has the amino acid sequence shown in SEQ ID NO: 1.

[0029] In one leucine dehydrogenase mutant of this embodiment, the following mutations occur in the amino acid sequence corresponding to SEQ ID NO: 3: aspartic acid D at position 24 is mutated to alanine A, glutamic acid E at position 124 is mutated to valine V, S at position 129 is mutated to threonine T, and valine V at position 243 is mutated to alanine A (SEQ ID NO: 2). In the corresponding mutations, the amino acid defect at the corresponding position in the amino acid sequence corresponding to SEQ ID NO: 3 is replaced by other amino acids. The above-mentioned leucine dehydrogenase mutant has the amino acid sequence shown in SEQ ID NO: 2.

[0030] In this embodiment, the term "other amino acids" is not limited, as long as it differs from the amino acids corresponding to each position. Specifically, the other amino acids in this embodiment may be one or more amino acids selected from the following: nonpolar amino acids glycine (G), alanine (A), valine (V), leucine (L), isoleucine (I), methionine (M), phenylalanine (F), tryptophan (W), and proline (P); polar amino acids serine (S), threonine (T), cysteine ​​(C), tyrosine (Y), asparagine (N), and glutamine (Q); acidic amino acids aspartic acid (D) and glutamic acid (E); and basic amino acids lysine (K), arginine (R), and histidine (H), but are not limited thereto.

[0031] In this embodiment, by screening wild-type leucine dehydrogenase for mutations, leucine dehydrogenase mutants with amino acid sequences as shown in SEQ ID NO: 1 or SEQ ID NO: 2 were obtained. These mutants have improved catalytic efficiency for asymmetric reductive amination of prochiral α-keto acid compounds and improved thermal stability, which is beneficial for improving the synthesis efficiency of L-amino acids.

[0032] This application also provides a polynucleotide encoding a leucine dehydrogenase mutant, the polynucleotide comprising the nucleotide sequence corresponding to the amino acid sequence of the aforementioned leucine dehydrogenase mutant.

[0033] The polynucleotide is a DNA or RNA chain formed by the polymerization of several nucleotides.

[0034] The polynucleotide only needs to encode the leucine dehydrogenase mutant mentioned above, and any nucleotide in the polynucleotide can be chemically modified.

[0035] This application also provides a recombinant vector comprising the polynucleotide encoding a leucine dehydrogenase mutant as described above.

[0036] In this context, a recombinant vector refers to a DNA preparation containing a polynucleotide sequence encoding a leucine dehydrogenase mutant. It may also contain control sequences. In the recombinant vector, the polynucleotide sequence encoding the leucine dehydrogenase mutant is operatively linked to a suitable control sequence, allowing the leucine dehydrogenase mutant to be expressed in a suitable host. Specifically, the control sequence may include, but is not limited to, promoters capable of initiating transcription, arbitrary operon sequences for regulating transcription, suitable mRNA ribosome binding sites, and sequences for controlling transcription and translation termination. After transformation into a suitable host cell, the recombinant vector can replicate or function independently of the host genome, or it can integrate into the genome itself for replication or function.

[0037] There are no particular restrictions on the type of recombinant vector; any vector known in the art can be used as long as it can replicate in the host cell. Exemplarily, commonly used vectors in the art can include plasmids, granules, viruses, and bacteriophages in their natural or recombinant states. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, and Charon21A can be used as phage vectors or granule vectors, and the pBR system, pUC system, pBluescript II system, pGEM system, pTZ system, pCL system, and pET system can be used as plasmid vectors. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, and pCC1BAC vectors can be used.

[0038] This application also provides a host cell, including the recombinant vector described above.

[0039] In this embodiment, the recombinant vector is transformed into a host cell, enabling the synthesis of the leucine dehydrogenase mutant within the host cell. The recombinant vector, when introduced into the host cell, allows the polynucleotide encoding the leucine dehydrogenase mutant to be expressed in the host cell, thus enabling the host cell to synthesize the leucine dehydrogenase mutant. This polynucleotide can be inserted into the host cell's chromosome, located outside the host cell's chromosome, or simultaneously inserted into the host cell's chromosome and located outside the chromosome. The polynucleotide can be DNA or RNA, as long as it can be expressed in the host cell. Exemplarily, the recombinant vector can be an expression cassette, including a promoter, transcription termination element, ribosomal domain, and translation termination element operatively linked to the polynucleotide.

[0040] In one implementation, the host cell may be a species of Escherichia, Erwinia, Serratia, Providencia, Corynebacterium, or Brevibacterium; exemplary, the host cell may be Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum, or Aspergillus oryzae.

[0041] This application also provides a method for preparing a leucine dehydrogenase mutant, comprising the following steps: culturing and transforming host cells with a recombinant vector, so that the leucine dehydrogenase mutant accumulates in the bacterial cells of the transformed host cells, wherein the recombinant vector contains a nucleotide sequence encoding the above-mentioned leucine dehydrogenase mutant.

[0042] The process involves growing transformed host cells containing the recombinant vector under appropriate environmental conditions. The methods of culturing host cells can include, but are not limited to, batch culture, continuous culture, or fed-batch culture. To achieve appropriate environmental conditions, the pH can be adjusted using alkaline or acidic compounds to a suitable value, such as 5–9, 6–8, or 6.5–7.0. For example, the alkaline compound can be sodium hydroxide, potassium hydroxide, or ammonia, and the acidic compound can be hydrochloric acid, phosphoric acid, or sulfuric acid. To achieve appropriate environmental conditions, the temperature can be controlled at 25–60°C, or at 30–45°C. Alternatively, oxygen or an oxygen-containing gas mixture can be injected to maintain an aerobic environment.

[0043] The carbon source in the host cell culture medium may include, but is not limited to, at least one of carbohydrates, oils or fats, fatty acids, alcohols, or organic acids. Carbohydrates include, for example, glucose, sucrose, lactose, fructose, maltose, molasses, starch, and cellulose; oils or fats include, for example, soybean oil, sunflower oil, peanut oil, and coconut oil; fatty acids include, for example, palmitic acid, stearic acid, and linoleic acid; alcohols include, for example, glycerol and ethanol; and organic acids include, for example, acetic acid. The phosphorus source in the host cell culture medium may include, but is not limited to, at least one of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, sodium dihydrogen phosphate, and disodium hydrogen phosphate.

[0044] The culture medium for the host cells may further include metal salts, such as magnesium sulfate or ferric sulfate, and / or the culture medium for the host cells may further include other substances required for growth, such as amino acids or vitamins.

[0045] As one implementation, the recombinant vector may include a nucleotide sequence corresponding to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0046] One embodiment of this application provides the application of the above-mentioned leucine dehydrogenase mutant in the asymmetric reductive amination of prochiral α-keto acid compounds.

[0047] In this method, prochiral α-keto acid compounds are used as substrates, and leucine dehydrogenase mutants are used as catalysts. The substrates are converted into L-amino acids by utilizing the asymmetric reductive amination catalytic activity of leucine dehydrogenase mutants.

[0048] Specifically, because the leucine dehydrogenase mutant is NAD + NADH-dependent oxidoreductases, the catalytic reaction of asymmetric reductive amination requires the coenzyme NAD+. + It is carried out in the presence of the coenzyme regeneration system.

[0049] NAD + It is the oxidized state of nicotinamide adenine dinucleotide, which does not have an additional hydride and therefore presents a positive charge. NAD + In cells, it primarily functions as an electron acceptor, participating in various redox reactions. NADH is the reduced state of nicotinamide adenine dinucleotide, which structurally contains an additional hydride (i.e., a negatively charged hydrogen atom).

[0050] like Figure 1 As shown, during the asymmetric reductive amination of pre-chiral α-keto acids catalyzed by the leucine dehydrogenase mutant to generate L-amino acids, NADH is converted to NAD. +In the presence of the coenzyme regeneration system, NAD... + It is converted into NADH.

[0051] The coenzyme regeneration system includes a cosubstrate and an oxidoreductase. During the process of the cosubstrate being oxidized to form byproducts under the catalysis of the oxidoreductase, NAD+... + It is converted into NADH. Specifically, the cosubstrate can be isopropanol, and the oxidoreductase can be isopropanol dehydrogenase, which oxidizes isopropanol to isoacetone. Alternatively, the cosubstrate can be formate, and the oxidoreductase can be formate dehydrogenase. Another possible cosubstrate is glucose, and the oxidoreductase can be glucose dehydrogenase.

[0052] Specifically, transformant host cells containing the recombinant vector are cultured and transformed to allow the leucine dehydrogenase mutant to accumulate in the bacterial cells of the transformed host cells. The resulting bacterial cells or the crude enzyme solution formed by the disruption of the resulting bacterial cells, prochiral α-keto acid compounds, and coenzyme NAD are then processed. + A coenzyme regeneration system, ammonia or ammonium chloride are added to the buffer solution to form a reaction system, and a reductive amination reaction is carried out at 30℃~55℃.

[0053] For example, the prochiral α-keto acid compound can be trimethylpyruvate, and the L-amino acid can be L-tert-leucine. Trimethylpyruvate is converted to L-tert-leucine via the catalytic activity of asymmetric reductive amination of a leucine dehydrogenase mutant.

[0054] An embodiment of this application also provides a method for synthesizing L-tert-leucine, comprising the following steps:

[0055] The above-mentioned leucine dehydrogenase mutant, the transformed host cell containing the above-mentioned leucine dehydrogenase mutant, or the culture of the transformed host cell containing the above-mentioned leucine dehydrogenase mutant are brought into contact with trimethylpyruvate to convert trimethylpyruvate into L-tert-leucine.

[0056] The pH of the reaction system is 8.5–10.

[0057] In some embodiments, transformed host cells containing the recombinant vector are cultured to allow the leucine dehydrogenase mutant to accumulate in the bacterial cells of the transformed host cells. The resulting bacterial cells or the crude enzyme solution formed by the disruption of the resulting bacterial cells, trimethylpyruvate, and coenzyme NAD are then processed. + Isopropanol, isopropanol dehydrogenase preparation, ammonia or ammonium chloride are added to a buffer solution to form a reaction system, and a reductive amination reaction is carried out at 30℃~55℃. The isopropanol dehydrogenase preparation can be wet cells obtained by inducing and culturing recombinant genetically engineered bacteria containing the isopropanol dehydrogenase encoding gene, or crude enzyme solution obtained by ultrasonic disruption of wet cells, or immobilized cells prepared from wet cells.

[0058] Example

[0059] The encoding genes of the leucine dehydrogenase mutants in Examples 1 and 2 of this application and the leucine dehydrogenase in Comparative Example 1 can be called LaLeuDH genes. The LaLeuDH gene contains the nucleotide sequence corresponding to the amino acid sequence of the above-mentioned leucine dehydrogenase mutant or leucine mutant enzyme. For example, the LaLeuDH gene in Examples 1 and 2 contains the nucleotide sequence corresponding to the amino acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2. The LaLeuDH gene is constructed in the pET-28a plasmid to obtain a recombinant vector.

[0060] The recombinant vector used in the various embodiments and Comparative Example 1 of this application is the pET-28a plasmid containing the LaLeuDH gene, hereinafter referred to as pET-28a-LaLeuDH; the host cell used in the various embodiments and Comparative Example 1 of this application is Escherichia coli.

[0061] The steps in the embodiments and Comparative Example 1 of this application are as follows:

[0062] protein expression

[0063] a. Transform pET-28a-LaLeuDH into Escherichia coli; pick a single clone of pET-28a-LaLeuDH or a strain preserved at -80℃ and inoculate it into a small test tube containing 5 mL of LB liquid medium (Kan+, 100 μg / mL), and incubate overnight at 37℃ and 220 rpm to obtain the seed culture.

[0064] b. Transfer the seed culture to 50 mL of LB liquid medium (Kan+, 100 μg / mL) and incubate at 37 °C and 220 rpm on a shaker for reactivation.

[0065] c. Transfer the reactivated bacterial culture to 800 mL of 2YT liquid medium (Kan+, 100 μg / mL) at an inoculation rate of 1%, and incubate at 37 °C and 220 rpm on a shaker until the OD 600 is approximately 0.6-0.8.

[0066] d. Lower the temperature of the shaker to 16℃-18℃. After the temperature of the cultured bacterial solution has decreased, add isopropylthio-β-D-galactoside (IPTG) to a final concentration of 0.5mM and induce expression for 14-16h.

[0067] e. After expression is complete, collect the above culture solution into a bottle, pre-cool the centrifuge to 4°C, and centrifuge at 5500 rpm for 10 min.

[0068] f. Remove the supernatant, add 30 mL of protein purification buffer, and resuspend the bacterial cells using a vortex mixer.

[0069] g. Centrifuge the resuspended bacterial cells again at 5500 rpm for 10 min. Discard the supernatant, add 30 mL of protein purification buffer, and vortex to resuspend the bacterial cells (there should be no solid particles). Transfer the resuspended cells to a 50 mL centrifuge tube and store at -80°C.

[0070] Protein purification

[0071] a. Preparation of crude enzyme solution: 1.0 g of collected wet bacterial cells were added to 20 mL of equilibration buffer for resuspending. The resuspended cells were then disrupted using a cell disruptor set to 300 W to prevent excessive temperature from affecting enzyme activity. The disruption program was set to run for 1 second and pause for 3 seconds. The disruption solution was continuously cooled with an ice-water mixture until the suspension became clear and transparent. The disruption solution was then centrifuged at 12000 rpm for 10 min at 4 °C. The supernatant was collected and filtered through a 0.22 μm filter to obtain the crude enzyme solution. All proteins used in this study were unlabeled, and the predicted isoelectric point (PI) was 6.35; therefore, weakly basic anionic groups were selected for purification.

[0072] b. Regeneration and equilibration of ion exchange chromatography column: Protein purification was performed using a DEAE Sepharose Fast Flow anion exchange column. The column was washed with a high-salt buffer (containing 1-2 M NaCl) at a flow rate of 1 mL / min for 3-5 column volumes, then washed with 0.1 M NaOH for 3-5 column volumes, then washed with elution buffer for 3-5 column volumes, and finally washed with equilibration buffer until the detector parameters such as OD280, conductivity, and pH value stabilized.

[0073] c. Loading and elution of crude enzyme solution: Load the prepared crude enzyme solution at a loading rate of 0.5 mL / min, with a loading volume of 20 mL. After loading, wash with equilibration buffer for 3–5 column volumes, then elute using an increasing salt concentration gradient with elution buffer. Collect each fraction and confirm by protein electrophoresis. If the purification effect is unsatisfactory, this step can be repeated, or purification can be performed again using agarose gel G75 FF.

[0074] d. Protein concentration: The collected target protein was concentrated using ultrafiltration membrane concentration method. The protein was concentrated using a 10kDa protein concentration tube and centrifuged at 5000rpm for 30min at 4℃.

[0075] e. Protein desalting: Dilute the concentrated protein with an appropriate amount of PBS buffer (20mM, pH 7.0) and place it in a dialysis bag (molecular weight cutoff 8-14kDa). Use 20mM, pH 7.0 PBS dialysate and let it stand overnight at 4°C. The dialysate needs to be changed once during the process.

[0076] f. Preservation of ion exchange chromatography columns: After use, rinse the ion exchange chromatography column with 1M NaOH for 3-5 column volumes, then rinse with 20% ethanol, and store in a refrigerator at 4°C.

[0077] Electrophoretic analysis of proteins

[0078] a. Protein sample preparation: Add the purified protein solution and 5× loading buffer at a ratio of 1:4 (v / v), heat in boiling water for 10 min, and set aside for later use.

[0079] b. Sample loading and electrophoresis: Place the precast protein gel (Genscript, SurePAGE, 4%–20%) in the electrophoresis tank, and add the protein sample and marker to the sample wells of the protein gel using a pipette.

[0080] c. Staining and destaining: Remove the outer shell of the pre-cast gel after electrophoresis, and automatically destain and stain using a protein staining and destaining instrument for 15 minutes.

[0081] d. Gel image analysis: The stained and destained protein gels were photographed and saved using a gel imaging system.

[0082] Isopropanol dehydrogenase preparation

[0083] The gene encoding isopropanol dehydrogenase (amino acid sequence as shown in SEQ ID NO: 4) was constructed into the pET-28a plasmid to obtain the recombinant plasmid pET-28a-IPADH.

[0084] pET-28a-IPADH was transformed into Escherichia coli; single clones of pET-28a-IPADH or strains preserved at -80℃ were picked and inoculated into small test tubes containing 5 mL of LB liquid medium (Kan+, 100 μg / mL) and cultured overnight at 37℃ and 220 rpm to obtain seed culture.

[0085] The seed culture was transferred to 50 mL of LB liquid medium (Kan+, 100 μg / mL) and cultured on a shaker at 37 °C and 220 rpm for reactivation.

[0086] The reactivated bacterial culture was transferred to 800 mL of 2YT liquid medium (Kan+, 100 μg / mL) at an inoculation rate of 1%, and cultured at 37 °C and 220 rpm on a shaker until the OD 600 was approximately 0.6-0.8.

[0087] Lower the temperature of the shaker to 16℃-18℃. After the temperature of the cultured bacterial solution has decreased, add isopropylthio-β-D-galactoside (IPTG) to a final concentration of 0.5mM and induce expression for 14-16h.

[0088] After expression, the above culture solution was collected into a bottle, and the centrifuge was pre-cooled to 4°C and centrifuged at 5500 rpm for 10 min.

[0089] Remove the supernatant, add 30 mL of protein purification buffer, and resuspend the bacterial cells using a vortex mixer.

[0090] Centrifuge the resuspended bacterial cells again at 5500 rpm for 10 min. Discard the supernatant, add 30 mL of buffer, and vortex to resuspend the bacterial cells (there should be no solid particles). Transfer the resuspended cells to a 50 mL centrifuge tube and store at -80°C.

[0091] Enzyme-catalyzed reactions

[0092] In vitro enzyme catalytic reaction conditions:

[0093] Reaction buffer: Diammonium phosphate buffer at concentrations of 100mM, 200mM, and 300mM;

[0094] Reaction pH: pH 8, pH 8.5, pH 9;

[0095] The concentrations of trimethylpyruvic acid are 50 g / L and 100 g / L;

[0096] The ammonia concentrations are 0.6 mL / 10 mL, 0.8 mL / 10 mL, 1.0 mL / 10 mL, 1.1 mL / 10 mL, and 1.2 mL / 10 mL.

[0097] The volume concentrations of isopropanol were 0.6 mL / 10 mL, 0.8 mL / 10 mL, and 1.0 mL / 10 mL.

[0098] NAD + The concentrations are 1 g / L, 2 g / L, 3 g / L, 4 g / L, and 5 g / L;

[0099] The addition amounts of leucine dehydrogenase / leucine dehydrogenase mutant were 10 g / L, 20 g / L, 30 g / L, and 40 g / L;

[0100] The dosage of isopropanol dehydrogenase added was 10 g / L and 20 g / L;

[0101] Reaction temperatures: 30℃, 35℃, 40℃, 45℃, 50℃, 55℃;

[0102] Reaction time: 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h or 24h;

[0103] The total reaction volume is 10 mL.

[0104] The embodiments and comparative examples can adopt the following conditions: the reaction system contains trimethylpyruvic acid at a concentration of 100 g / L, ammonia at a volume concentration of 0.6 mL / 10 mL, isopropanol at a volume concentration of 0.6 mL / 10 mL, and NAD+. + The concentration of the enzyme was 2 g / L, the amount of leucine dehydrogenase / leucine dehydrogenase mutant added was 10 g / L, the amount of isopropanol dehydrogenase added was 20 g / L, the pH was 8.5, the reaction was carried out at 40°C for 10 hours, and the substrate conversion rate was measured every 1 hour to measure the conversion activity of each example and comparative example.

[0105] Experimental results:

[0106] The leucine dehydrogenase mutant in Example 1 completely converted the substrate trimethylpyruvate within 5 hours;

[0107] The leucine dehydrogenase mutant in Example 2 completely converted the substrate trimethylpyruvate within 5 hours;

[0108] Comparative Example 1 showed that the leucine dehydrogenase converted 30% of the substrate trimethylpyruvate within 7 hours.

[0109] Compared with the wild-type leucine dehydrogenase of Comparative Example 1, the catalytic activities of the leucine dehydrogenase mutants of Example 1 and Example 2 were improved.

[0110] Thermal stability comparison

[0111] Before the catalytic reaction, the leucine dehydrogenase mutant of Example 1, the leucine dehydrogenase mutant of Example 2, and the leucine dehydrogenase of Comparative Example 1 were incubated at 55°C for 30 minutes and then cooled on ice for 20 minutes.

[0112] The embodiments and comparative examples can adopt the following conditions: the reaction system contains trimethylpyruvic acid at a concentration of 100 g / L, ammonia at a volume concentration of 0.6 mL / 10 mL, isopropanol at a volume concentration of 0.6 mL / 10 mL, and NAD+. +The concentration of the enzyme was 2 g / L, the amount of leucine dehydrogenase / leucine dehydrogenase mutant added was 10 g / L, the amount of isopropanol dehydrogenase added was 20 g / L, the pH was 8.5, the reaction was carried out at 40°C for 10 hours, and the substrate conversion rate was measured every 1 hour to measure the conversion activity of each example and comparative example.

[0113] Experimental results:

[0114] The leucine dehydrogenase mutant in Example 1 completely converted the substrate trimethylpyruvate within 5 hours;

[0115] The leucine dehydrogenase mutant in Example 2 completely converted the substrate trimethylpyruvate within 5 hours;

[0116] The leucine dehydrogenase in Comparative Example 1 achieved a conversion rate of less than 15% for the substrate trimethylpyruvate within 7 hours.

[0117] The relative activity of the leucine dehydrogenase mutant in Example 1 was 1 within 3 hours, and the relative activity of the leucine dehydrogenase mutant in Example 2 was 1.48 within 3 hours.

[0118] The leucine dehydrogenase mutants of Example 1 and Example 2 still exhibited high catalytic activity after incubation at 55°C; furthermore, the thermal stability of the leucine dehydrogenase mutant of Example 2 was superior to that of the leucine dehydrogenase mutant of Example 1.

[0119] The above description is merely an embodiment of this application. It should be noted that those skilled in the art can make improvements without departing from the inventive concept of this application, but these improvements all fall within the protection scope of this application.

Claims

1. A leucine dehydrogenase mutant, characterized in that, The amino acid sequence of the leucine dehydrogenase mutant is shown in SEQ ID NO: 1 or SEQ ID NO:

2.

2. A polynucleotide, characterized in that, The polynucleotide encodes the leucine dehydrogenase mutant as described in claim 1.

3. A recombinant vector, characterized in that, The recombinant vector comprises the polynucleotide as described in claim 2.

4. A host cell, characterized in that, The host cell includes the recombinant vector as described in claim 3.

5. The application of the leucine dehydrogenase mutant according to claim 1 in the asymmetric reductive amination of prochiral α-keto acid compounds, in the coenzyme NAD+. + In the presence of a coenzyme regeneration system, the leucine dehydrogenase mutant of claim 1 catalyzes the asymmetric reductive amination of the prochiral α-keto acid compound to generate L-amino acids, wherein, The prochiral α-keto acid compound is trimethylpyruvate, and the L-amino acid is L-tert-leucine.

6. The application according to claim 5, characterized in that, The coenzyme regeneration system includes a cosubstrate and an oxidoreductase, wherein the cosubstrate is isopropanol, formate, or glucose, and the oxidoreductase is isopropanol dehydrogenase, formate dehydrogenase, or glucose dehydrogenase.

7. A method for synthesizing L-tert-leucine, characterized in that, include: The leucine dehydrogenase mutant of claim 1 or a transformed host cell containing the leucine dehydrogenase mutant of claim 1 is brought into contact with trimethylpyruvate to convert trimethylpyruvate into L-tert-leucine.

8. The method for synthesizing L-tert-leucine according to claim 7, characterized in that, The pH of the reaction system is 8.5–10.

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