Leucine dehydrogenase mutant and application thereof

Abstract

The invention relates to the technical field of biology, in particular to a leucine dehydrogenase mutant and application thereof. According to the leucine dehydrogenase mutant and the application thereof provided by the embodiment of the invention, wild leucine dehydrogenase is subjected to mutation screening to obtain the leucine dehydrogenase mutant with an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, the catalytic efficiency of asymmetric reductive amination of a prochiral alpha-keto acid compound is improved, and the thermal stability of the leucine dehydrogenase mutant is improved; the synthesis efficiency of the L-amino acid is favorably improved.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a leucine dehydrogenase mutant and an application thereof. Background Art

[0002] Chiral non-natural amino acids, such as L-tert-leucine, are becoming increasingly prominent in various fields, and the exploration of green and efficient synthetic routes has gradually become the focus of researchers. The biocatalytic method of chiral non-natural amino acids is to use α-keto acid compounds as substrates and inorganic ammonia to prepare them through reductive amination under the catalysis of amino acid dehydrogenase.

[0003] Leucine dehydrogenase (LeuDH) belongs to the amino acid dehydrogenase superfamily and is a type of NAD + / NADH-dependent oxidoreductase, which is an important biological pathway for the synthesis of L-amino acids. In the background art, the thermal stability and conversion efficiency of leucine dehydrogenase are poor, which is not conducive to improving the efficiency of L-amino acid synthesis. Summary of the invention

[0004] In view of the above problems, the embodiments of the present application provide a leucine dehydrogenase mutant and its application to solve the technical problem that the thermal stability and conversion efficiency of leucine dehydrogenase are not good for improving the efficiency of L-amino acid synthesis.

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

[0006] In a second aspect, an embodiment of the present application provides a polynucleotide encoding the above-mentioned leucine dehydrogenase mutant.

[0007] In a third aspect, an embodiment of the present application provides a recombinant vector, which includes the above-mentioned polynucleotide.

[0008] In a fourth aspect, an embodiment of the present application provides a host cell, wherein the host cell comprises the above-mentioned recombinant vector.

[0009] In a fifth aspect, the embodiments of the present application provide the use of the above-mentioned 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 acid.

[0011] Optionally, the coenzyme regeneration system comprises a cosubstrate and an oxidoreductase, 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 trimethylpyruvic acid, and the L-amino acid is L-tert-leucine.

[0013] In a sixth aspect, an embodiment of the present application provides a method for synthesizing L-tert-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 the trimethylpyruvate into L-tert-leucine.

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

[0015] The leucine dehydrogenase mutants and applications thereof provided in the embodiments of the present application are obtained by performing mutation screening on the wild-type leucine dehydrogenase to obtain a leucine dehydrogenase mutant with an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, which has improved catalytic efficiency for asymmetric reductive amination of prochiral α-keto acid compounds and improved thermal stability, thereby facilitating improved L-amino acid synthesis efficiency.

[0016] These and other aspects of the present application will become more clearly understood in the description of the following embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The schematic diagram of the reaction for preparing L-amino acids by asymmetric reductive amination in the present application is shown. DETAILED DESCRIPTION

[0018] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

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

[0020] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0021] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0022] The term "leucine dehydrogenase" herein refers to an enzyme that exhibits asymmetric reductive amination activity of prochiral α-keto acid compounds, and is capable of asymmetric reductive amination of prochiral α-keto acid compounds to convert the prochiral α-keto acid compounds into L-amino acids.

[0023] The reaction principle formula for converting prochiral α-keto acid compounds into L-amino acids using the above-mentioned leucine dehydrogenase is as follows:

[0024]

[0025] Described herein as "a polypeptide, protein, mutant or enzyme having an amino acid sequence as shown in SEQ ID NO:", it is obvious that a polypeptide, protein, mutant or enzyme having an amino acid sequence as shown in SEQ ID NO:, in which some sequences are deleted, modified, substituted, conservatively substituted or added, can also be used in the present application, as long as it can show the same or corresponding activity as the polypeptide, protein, mutant or enzyme having an amino acid sequence as shown in SEQ ID NO:. For example, it is not excluded to add a sequence that does not change the function of the protein, a naturally occurring mutation, a silent mutation or a conservative substitution before or after "a polypeptide, protein, mutant or enzyme having an amino acid sequence as shown in SEQ ID NO:"; and a polypeptide, protein, mutant or enzyme having an amino acid sequence as shown in SEQ ID NO:, when it is added with the above-mentioned sequence that does not change the function of the protein, a naturally occurring mutation, a silent mutation or a conservative substitution, also falls within the scope of the present application, as long as it shows the same or corresponding activity as the amino acid sequence as shown in SEQ ID NO: after the above-mentioned sequence is added.

[0026] The present embodiment provides a leucine dehydrogenase mutant, wherein the leucine dehydrogenase mutant has 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 GenBank of NCBI. Specifically, the amino acid sequence shown in SEQ ID NO: 3 is derived from Roseibium aggregatum, and the number in GenBank is WP_006932274.1. In this specification, the enzyme formed by the amino acid sequence shown in SEQ ID NO: 3 can be referred to as wild-type leucine dehydrogenase, and the enzyme formed by each mutated amino acid sequence can be referred to as mutant leucine dehydrogenase or leucine dehydrogenase mutant.

[0028] In a leucine dehydrogenase mutant of this embodiment, the following mutations occur in the amino acid sequence shown in SEQ ID NO: 3: aspartic acid D at position 24 mutates to leucine L, glutamic acid E at position 124 mutates to valine V, S at position 129 mutates to threonine T, and valine V at position 243 mutates to alanine A (SEQ ID NO: 1). In the corresponding mutations, the amino acid residues 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 a leucine dehydrogenase mutant of this embodiment, the following mutations occur in the amino acid sequence shown in SEQ ID NO: 3: aspartic acid D at position 24 mutates to alanine A, glutamic acid E at position 124 mutates to valine V, S at position 129 mutates to threonine T, and valine V at position 243 mutates to alanine A (SEQ ID NO: 2). In the corresponding mutations, the amino acid residues 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: 2.

[0030] In this embodiment, the above-mentioned "other amino acids" are not limited as long as they are different from the amino acids corresponding to each position. Specifically, the other amino acids in the embodiments of the present application can be one or more amino acids selected from the following: non-polar 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 example, by performing mutation screening on the wild-type leucine dehydrogenase, a leucine dehydrogenase mutant having an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2 is obtained, which has improved catalytic efficiency for asymmetric reductive amination of prochiral α-keto acid compounds and improved thermal stability, which is beneficial to improving the synthesis efficiency of L-amino acids.

[0032] The present application also provides a polynucleotide encoding a leucine dehydrogenase mutant, wherein the polynucleotide comprises a nucleotide sequence corresponding to the amino acid sequence of the leucine dehydrogenase mutant.

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

[0034] As long as the polynucleotide can encode the above-mentioned leucine dehydrogenase mutant, any nucleotide in the polynucleotide may be chemically modified.

[0035] The present application also provides a recombinant vector, comprising the polynucleotide encoding the leucine dehydrogenase mutant.

[0036] Wherein, a recombinant vector refers to a DNA preparation containing a nucleic acid sequence of a polynucleotide encoding a leucine dehydrogenase mutant, and it may also contain a control sequence, in which the nucleic acid sequence of a polynucleotide encoding a leucine dehydrogenase mutant is operably linked to a suitable control sequence so that the leucine dehydrogenase mutant can be expressed in a suitable host. Specifically, the control sequence may include, but is not limited to, a promoter capable of initiating transcription, any operator sequence for regulating transcription, a suitable mRNA ribosome binding site, and a sequence for controlling transcription and translation termination. After transformation into a suitable host cell, the recombinant vector may replicate or function independently of the host genome, or may be integrated into the genome itself for replication or function.

[0037] Among them, there is no particular restriction on the type of recombinant vector, as long as it can replicate in the host cell, any vector known in the art can be used. Exemplarily, commonly used vectors in the art may include plasmids, cosmids, viruses and phages in natural or recombinant states. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used as phage vectors or cosmid vectors, and pBR system, pUC system, pBluescript II system, pGEM system, pTZ system, pCL system, pET system, etc. can be used as plasmid vectors. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vectors, etc. can be used.

[0038] The present application also provides a host cell, comprising the above-mentioned recombinant vector.

[0039] Wherein, the above-mentioned recombinant vector is transformed into a host cell so that the leucine dehydrogenase mutant can be synthesized in the host cell of the present embodiment. The recombinant vector is introduced into a host cell, and the polynucleotide encoding the leucine dehydrogenase mutant in the recombinant vector can be expressed in the host cell so that the host cell can synthesize the above-mentioned leucine dehydrogenase mutant. The polynucleotide can be inserted into the chromosome of the host cell or be located outside the chromosome of the host cell or simultaneously inserted into the chromosome of the host cell and be located outside the chromosome, and 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, a transcription termination element, a ribosomal domain and a translation termination element operably connected to the polynucleotide.

[0040] As an embodiment, the host cell can be Escherichia, Erwinia, Serratia, Providencia, Corynebacterium or Brevibacterium; exemplarily, the host cell can be Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum or Aspergillus oryzae.

[0041] The present application also provides a method for preparing a leucine dehydrogenase mutant, comprising the following steps: culturing a transformed host cell transformed with a recombinant vector to allow the leucine dehydrogenase mutant to accumulate in the cells of the transformed host cell, wherein the recombinant vector contains a nucleotide sequence encoding the leucine dehydrogenase mutant.

[0042] Among them, the transformed host cells transformed with the recombinant vector are grown under appropriate environmental conditions, and the host cell culture method may include but is not limited to batch culture, continuous culture or fed-batch culture. In order to achieve appropriate environmental conditions, the pH can be adjusted using alkaline compounds or acidic compounds to adjust the pH to an appropriate value, for example, the pH can be adjusted to 5 to 9, or the pH can be adjusted to 6 to 8, or the pH can be adjusted to 6.5 to 7.0. Exemplarily, the alkaline compound can be sodium hydroxide, potassium hydroxide or ammonia, and the acidic compound can be hydrochloric acid, phosphoric acid or sulfuric acid. In order to achieve appropriate environmental conditions, the temperature can be controlled at 25 to 60°C, or the temperature can be controlled at 30 to 45°C, or oxygen or an oxygen-containing gas mixture can be injected to keep the culture environment in an aerobic state.

[0043] The carbon source contained 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 such as glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils or fats such as soybean oil, sunflower oil, peanut oil and coconut oil, fatty acids such as palmitic acid, stearic acid and linoleic acid, alcohols such as glycerol and ethanol, and organic acids such as acetic acid. The phosphorus source contained 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 host cell culture medium may further include a metal salt, such as magnesium sulfate or iron sulfate, and / or the host cell culture medium may further include other substances required for growth, such as amino acids or vitamins.

[0045] As an embodiment, 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 the present application provides a use of the above-mentioned leucine dehydrogenase mutant in asymmetric reductive amination of prochiral α-keto acid compounds.

[0047] Among them, 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 catalytic activity of asymmetric reductive amination of the leucine dehydrogenase mutants.

[0048] Specifically, since the leucine dehydrogenase mutant is NAD + / NADH-dependent oxidoreductase, catalysis of asymmetric reductive amination requires the coenzyme NAD + and coenzyme regeneration system.

[0049] NAD + It is the oxidized state of nicotinamide adenine dinucleotide, which does not carry an additional hydride and therefore has a positive charge. NAD + It mainly acts as an electron acceptor in cells and participates in various redox reactions. NADH is the reduced state of nicotinamide adenine dinucleotide, which contains an additional hydride (i.e., a negatively charged hydrogen atom) in its structure.

[0050] like Figure 1 As shown, in the process of asymmetric reductive amination of prochiral α-ketoacid compounds catalyzed by leucine dehydrogenase mutants to produce L-amino acids, NADH is converted to NAD +In the presence of a coenzyme regeneration system, NAD + is converted into NADH.

[0051] Among them, the coenzyme regeneration system includes cosubstrate and oxidoreductase. In the process of cosubstrate being oxidized to form by-products under the catalysis of oxidoreductase, NAD + is converted into NADH. Specifically, the cosubstrate can be isopropanol, the oxidoreductase can be isopropanol dehydrogenase, and the isopropanol is oxidized by the isopropanol dehydrogenase to form isopropanone. The cosubstrate can be formate, and the oxidoreductase can be formate dehydrogenase. The cosubstrate can also be glucose, and the oxidoreductase can be glucose dehydrogenase.

[0052] Specifically, the transformed host cells transformed with the recombinant vector are cultured to allow the leucine dehydrogenase mutant to accumulate in the bacterial cells of the transformed host cells, and the obtained bacterial cells or the crude enzyme solution, prochiral α-keto acid compounds, coenzyme NAD + , a coenzyme regeneration system, ammonia water or ammonium chloride are added to the buffer to form a reaction system, and the reductive amination reaction is carried out at 30°C to 55°C.

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

[0054] An embodiment of the present 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 is contacted with trimethylpyruvate to convert the trimethylpyruvate into L-tert-leucine.

[0056] Wherein, the pH of the reaction system is 8.5-10.

[0057] In some embodiments, the transformed host cell transformed with the recombinant vector is cultured to allow the leucine dehydrogenase mutant to accumulate in the bacterial cells of the transformed host cell, and the obtained bacterial cells or the crude enzyme solution, trimethylpyruvate, coenzyme NAD + , isopropanol, isopropanol dehydrogenase preparation, ammonia water or ammonium chloride are added to a buffer to form a reaction system, and a reductive amination reaction is carried out at 30° C. to 55° C. The isopropanol dehydrogenase preparation can be wet cells obtained by inducing and culturing recombinant genetic engineering bacteria containing an isopropanol dehydrogenase encoding gene, or a crude enzyme solution obtained by ultrasonically crushing wet cells, or immobilized cells prepared from wet cells.

[0058] Example

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

[0060] The recombinant vector used in each embodiment and comparative example 1 of the present application is a pET-28a plasmid containing the LaLeuDH gene, hereinafter referred to as pET-28a-LaLeuDH; the host cell used in each embodiment and comparative example 1 of the present application is Escherichia coli.

[0061] The steps of each embodiment and comparative example 1 in this application are as follows:

[0062] Protein expression

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

[0064] b. Transfer the seed solution into 50 mL LB liquid medium (Kan+, 100 μg / mL) and culture in a shaking incubator at 37°C and 220 rpm for reactivation.

[0065] c. The reactivated bacterial solution was transferred into 800 mL 2YT liquid culture medium (Kan+, 100 μg / mL) at a 1% inoculation volume and cultured in a shaking incubator at 37°C and 220 rpm until OD 600 was approximately 0.6-0.8.

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

[0067] e. After the expression is completed, the above culture solution is collected into a bottle, and the centrifuge is precooled to 4°C, 5500rpm, and centrifuged for 10 minutes.

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

[0069] g. Centrifuge the resuspended cells again at 5500rpm for 10min. Pour off the supernatant, add 30mL of protein purification buffer, resuspend the cells with a vortex oscillator (no solid particles), pour into a 50mL centrifuge tube, and store in a -80℃ refrigerator.

[0070] Protein purification

[0071] a. Preparation of crude enzyme solution: Add 1.0g of collected wet bacteria to 20mL of equilibrium buffer for resuspending, and use a cell disruptor to disrupt the resuspended cells. The cell disruptor parameters are set to 300W to prevent the high temperature from affecting the enzyme activity. The disruption program is set to work for 1s and pause for 3s. At the same time, the disruption liquid needs to be cooled with an ice-water mixture. The disruption can be stopped when the suspension is clear and transparent. Centrifuge the disruption liquid at 4°C and 12000rpm for 10min, collect the supernatant, and filter through a 0.22μm filter membrane to obtain a crude enzyme solution. The proteins used in this study are all unlabeled, and the protein isoelectric point is predicted to be PI 6.35, so a weakly alkaline anion group is selected for purification.

[0072] b. Regeneration and equilibration of ion exchange chromatography column: Use DEAE Sepharose Fast Flow anion exchange column for protein purification, flush with high salt concentration buffer (containing 1-2M NaCl) at a flow rate of 1mL / min for 3-5 column volumes, then flush with 0.1M NaOH for 3-5 column volumes, then flush with elution buffer for 3-5 column volumes, and then wash with equilibration solution to equilibrium for 3-5 column volumes until the detector parameters such as OD280, conductivity, pH value, etc. are stable.

[0073] c. Loading and elution of crude enzyme solution: Load the prepared crude enzyme solution at a loading speed of 0.5 mL / min, with a loading volume of 20 mL. After loading, use the equilibrium buffer to wash 3 to 5 column volumes, then use the method of increasing the salt concentration gradient elution, use the elution buffer for elution, collect each component, and confirm it by protein electrophoresis. If the purification effect is not good, repeat this step, or use agarose gel G75 FF for secondary purification.

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

[0075] e. Protein desalting: Add an appropriate amount of PBS buffer (20mM, pH 7.0) to dilute the concentrated protein, and place it in a dialysis bag (molecular weight cutoff 8-14kDa). Use 20mM, pH 7.0 PBS dialysis fluid and place it in a 4°C refrigerator overnight. The dialysis fluid needs to be replaced once in the middle.

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

[0077] Electrophoresis analysis of proteins

[0078] a. Protein sample treatment: 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 after heating.

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

[0080] c. Staining and decolorization: Remove the outer shell of the precast gel after electrophoresis, and use a protein staining and decolorizing instrument to automatically decolorize and stain it. The time is set to 15 minutes.

[0081] d. Protein gel image analysis: The stained and destained protein gel is photographed and saved using a gel imager.

[0082] Isopropanol dehydrogenase preparation

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

[0084] Transform pET-28a-IPADH into Escherichia coli; pick a single clone of pET-28a-IPADH or a strain stored at -80°C and inoculate it into a small test tube containing 5 mL LB liquid culture medium (Kan+, 100 μg / mL), and culture it overnight at 37°C and 220 rpm as the seed solution.

[0085] The seed solution was transferred into 50 mL LB liquid medium (Kan+, 100 μg / mL) and cultured in a shaking incubator at 37°C and 220 rpm for reactivation.

[0086] The reactivated bacterial liquid was transferred into 800 mL 2YT liquid culture medium (Kan+, 100 μg / mL) at a 1% inoculation volume and cultured in a shaking incubator at 37° C. and 220 rpm until OD 600 reached about 0.6-0.8.

[0087] Lower the shaking temperature to 16°C-18°C. After the temperature of the cultured bacterial solution drops, add isopropylthio-β-D-galactoside (IPTG) to a final concentration of 0.5 mM and induce expression for 14-16 hours.

[0088] After the expression was completed, the culture solution was collected into a bottle, and the centrifuge was precooled to 4°C, 5500 rpm, and centrifuged for 10 minutes.

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

[0090] Centrifuge the resuspended cells again at 5500 rpm for 10 min. Pour off the supernatant, add 30 mL of buffer, resuspend the cells with a vortex oscillator (no solid particles), pour into a 50 mL centrifuge tube, and store in a -80°C refrigerator.

[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 concentration of trimethylpyruvic acid was 50 g / L and 100 g / L;

[0096] The volume concentration of ammonia water is 0.6mL / 10mL, 0.8mL / 10mL, 1.0mL / 10mL, 1.1mL / 10mL, 1.2mL / 10mL;

[0097] The volume concentration of isopropanol is 0.6mL / 10mL, 0.8mL / 10mL, and 1.0mL / 10mL;

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

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

[0100] The addition amount of isopropanol dehydrogenase was 10 g / L and 20 g / L;

[0101] Reaction temperature: 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 was 10 mL.

[0104] Each embodiment and comparative example can be used: the reaction system has a concentration of 100 g / L of trimethylpyruvic acid, a volume concentration of 0.6 mL / 10 mL of ammonia water, a volume concentration of 0.6 mL / 10 mL of isopropanol, and NAD + The concentration of is 2 g / L, the addition amount of leucine dehydrogenase / leucine dehydrogenase mutant is 10 g / L, the addition amount of isopropanol dehydrogenase is 20 g / L, the pH is 8.5, the temperature is 40°C, and the reaction is carried out for 10 hours. The substrate conversion rate is measured every 1 hour to measure the conversion activity of each embodiment and comparative example.

[0105] Experimental results:

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

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

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

[0109] Compared with the wild-type leucine dehydrogenase of Comparative Example 1, the catalytic activities of the leucine dehydrogenase mutant of Example 1 and the leucine dehydrogenase mutant of Example 2 were respectively 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] Each embodiment and comparative example can be used: the reaction system has a concentration of 100 g / L of trimethylpyruvic acid, a volume concentration of 0.6 mL / 10 mL of ammonia water, a volume concentration of 0.6 mL / 10 mL of isopropanol, and NAD +The concentration of is 2 g / L, the addition amount of leucine dehydrogenase / leucine dehydrogenase mutant is 10 g / L, the addition amount of isopropanol dehydrogenase is 20 g / L, the pH is 8.5, the temperature is 40°C, and the reaction is carried out for 10 hours. The substrate conversion rate is measured every 1 hour to measure the conversion activity of each embodiment and comparative example.

[0113] Experimental results:

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

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

[0116] The conversion rate of the leucine dehydrogenase of Comparative Example 1 to the substrate trimethylpyruvate within 7 hours was less than 15%.

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

[0118] After being incubated at 55° C., the leucine dehydrogenase mutant of Example 1 and the leucine dehydrogenase mutant of Example 2 still have high catalytic activity; and the thermal stability of the leucine dehydrogenase mutant of Example 2 is better than that of the leucine dehydrogenase mutant of Example 1.

[0119] The above is only an implementation method of the present application. It should be pointed out that a person skilled in the art can make improvements without departing from the inventive concept of the present application, but these improvements are within the scope of protection of the present application.

Claims

1. A leucine dehydrogenase mutant, characterized in that: The leucine dehydrogenase mutant has an amino acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO:

2.

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

3. A recombinant vector, characterized in that: The recombinant vector comprises the polynucleotide according to claim 2.

4. A host cell, characterized in that The host cell comprises the recombinant vector according to claim 3.

5. Use of the leucine dehydrogenase mutant according to claim 1 in asymmetric reductive amination of prochiral α-keto acid compounds.

6. The use according to claim 5, characterized in that: In the coenzyme NAD + In the presence of a coenzyme regeneration system, the leucine dehydrogenase mutant according to claim 1 is used to catalyze the asymmetric reductive amination of the prochiral α-keto acid compound to generate L-amino acid.

7. The use according to claim 6, characterized in that: The coenzyme regeneration system comprises a cosubstrate and an oxidoreductase, wherein the cosubstrate is isopropanol, formate or glucose, and the oxidoreductase is isopropanol dehydrogenase, formate dehydrogenase or glucose dehydrogenase.

8. The use according to claim 7, characterized in that: The prochiral α-keto acid compound is trimethylpyruvic acid, and the L-amino acid is L-tert-leucine.

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

10. The method for synthesizing L-tert-leucine according to claim 9, characterized in that: The pH of the reaction system is 8.5-10.

Images