L-amino acid synthesis system and application

Abstract

The invention relates to the technical field of biology, in particular to an L-amino acid synthesis system and application. According to the L-amino acid synthesis system and application provided by the embodiment of the invention, in the presence of coenzyme NAD < + >, isopropanol is catalyzed by isopropanol dehydrogenase to generate acetone, and NAD < + > is reduced into coenzyme NADH; in the presence of coenzyme NADH, the leucine dehydrogenase mutant utilizes NH3 generated by ammonia water volatilization to perform asymmetric reductive amination on a prochiral alpha-keto acid compound, the NADH is oxidized into NAD +, the catalytic efficiency of asymmetric reductive amination of the leucine dehydrogenase mutant on the prochiral alpha-keto acid compound is improved, the circulating speed of the coenzyme NADH and NAD + is increased, and the catalytic activity of the leucine dehydrogenase mutant on the prochiral alpha-keto acid compound is improved. The catalytic efficiency of asymmetric reductive amination of the leucine dehydrogenase is favorably improved.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to an L-amino acid synthesis system and application. Background Art

[0002] Leucine dehydrogenase (LeuDH) belongs to the amino acid dehydrogenase superfamily and is a type of NADH (Nicotinamide adenine dinucleotide)-dependent oxidoreductase that can asymmetrically reductively aminize prochiral α-keto acid compounds. The asymmetric reductive amination of leucine dehydrogenase is an important biological pathway for the synthesis of L-amino acids.

[0003] In the process of asymmetric reductive amination by leucine dehydrogenase, the coenzyme NADH is needed for electron transfer. The coenzyme NADH is continuously consumed. After the reaction is completed, the coenzyme NADH is converted to NAD + The coenzyme NADH exists in the form of an oxidized state. Due to its high cost and poor stability, it cannot be added in large quantities during the reaction, which is not conducive to improving the catalytic efficiency of the asymmetric reductive amination of leucine dehydrogenase. Summary of the invention

[0004] Based on this, the present application provides an L-amino acid synthesis system and application to solve the above-mentioned technical problem that the catalytic efficiency of asymmetric reductive amination of leucine dehydrogenase cannot be improved due to the inability to add a large amount of coenzyme NADH during the reaction.

[0005] In a first aspect, the present invention provides an L-amino acid synthesis system, comprising NAD + , isopropanol, isopropanol dehydrogenase and a leucine dehydrogenase mutant, the leucine dehydrogenase mutant having an amino acid sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 5, the isopropanol dehydrogenase having an amino acid sequence as shown in SEQ ID NO: 7, and the mass ratio of the leucine dehydrogenase mutant to the isopropanol dehydrogenase is 1: 1 to 4.

[0006] Optionally, the leucine dehydrogenase mutant is derived from a first recombinant engineered bacterium containing a gene encoding the leucine dehydrogenase mutant.

[0007] Optionally, the leucine dehydrogenase mutant is a first wet cell obtained by inducing culture of the first recombinant engineered bacteria, a crude enzyme solution obtained by crushing the first wet cell, or an immobilized cell prepared from the first wet cell.

[0008] Optionally, the isopropanol dehydrogenase is derived from a second recombinant engineered bacterium containing a gene encoding isopropanol dehydrogenase.

[0009] Optionally, the isopropanol dehydrogenase is a second wet cell obtained by inducing culture of the second recombinant engineered bacteria, a crude enzyme solution obtained by crushing the second wet cell, or immobilized cells prepared from the second wet cell.

[0010] In a second aspect, an embodiment of the present application provides a method for synthesizing L-tert-leucine, wherein the above-mentioned L-amino acid synthesis system is contacted with trimethylpyruvate in the presence of aqueous ammonia to convert the trimethylpyruvate into L-tert-leucine.

[0011] Optionally, the NAD + The concentration of isopropanol dehydrogenase is 10 g / L to 20 g / L.

[0012] Optionally, the volume ratio of the isopropanol to the buffer solution in the reaction system is 1 to 1.3:10, and the volume ratio of the ammonia water to the buffer solution in the reaction system is 1 to 1.3:10.

[0013] Optionally, the total added concentration of the trimethylpyruvic acid is 100 g / L to 110 g / L, and the pH of the reaction system is 8.5 to 10.

[0014] Optionally, the mass ratio of the leucine dehydrogenase mutant to the isopropanol dehydrogenase is 1:1-2.

[0015] The L-amino acid synthesis system and application provided in the embodiments of the present application are based on the coenzyme NAD + In the presence of NAD + Reduced to coenzyme NADH; in the presence of coenzyme NADH, the leucine dehydrogenase mutant uses NH3 generated by the volatilization of ammonia to asymmetrically reduce the prochiral α-keto acid compounds and NADH is oxidized to NAD + Since the leucine dehydrogenase mutant has an improved catalytic efficiency in the asymmetric reductive amination of prochiral α-ketoacid compounds, the coenzymes NADH and NAD + The cycle rate is accelerated, which is beneficial to improving the catalytic efficiency of the asymmetric reductive amination of leucine dehydrogenase. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Schematic diagram of the reaction principle of the L-amino acid synthesis system of the present application embodiment.

[0017] Figure 2 Schematic diagram of the reaction principle of synthesizing L-tert-leucine by the L-amino acid synthesis system of the embodiment of the present application.

[0018] Figure 3 This is a graph showing the experimental results of the amount of isopropanol added in Examples 1 to 3.

[0019] Figure 4 This is a graph showing the experimental results of the amount of ammonia water added in Examples 5 to 8.

[0020] Figure 5 This is a graph showing the experimental results of adding proportions of the recombinant engineered bacteria in Examples 9 to 11.

[0021] Figure 6 This is a graph showing the experimental results of adding proportions of the recombinant engineered bacteria of Examples 12 to 14. DETAILED DESCRIPTION

[0022] In order to facilitate the understanding of the present application, the present application will be described more fully below with reference to the relevant drawings. The preferred embodiments of the present application are given in the drawings. However, the present application can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the disclosure of the present application more thorough and comprehensive.

[0023] It should be noted that when an element is referred to as being "fixed to" another element, it may be directly on the other element or there may be an intermediate element. When an element is considered to be "connected to" another element, it may be directly connected to the other element or there may be an intermediate element at the same time.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. The terms used herein in the specification of this application are only for the purpose of describing specific embodiments and are not intended to limit this application.

[0025] 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.

[0026] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0027] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0028] The terms "leucine dehydrogenase" and "leucine dehydrogenase mutant" herein are both enzymes that exhibit asymmetric reductive amination activity of prochiral α-keto acid compounds, which can asymmetric reductive amination of prochiral α-keto acid compounds to convert prochiral α-keto acid compounds into L-amino acids.

[0029] The reaction principle formula for converting prochiral α-keto acid compounds into L-amino acids using the above-mentioned leucine dehydrogenase is as follows:

[0030]

[0031] 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.

[0032] One embodiment of the present application provides an L-amino acid synthesis system, wherein the L-amino acid synthesis system comprises NAD + , isopropanol, isopropanol dehydrogenase and leucine dehydrogenase mutant, the leucine dehydrogenase mutant has an amino acid sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 5, the isopropanol dehydrogenase has an amino acid sequence as shown in SEQ ID NO: 7, and the mass ratio of the leucine dehydrogenase mutant to the isopropanol dehydrogenase is 1: 1 to 4.

[0033] Among them, NAD + It is the oxidized state of nicotinamide adenine dinucleotide, which has no additional hydride (i.e., a negatively charged hydrogen atom) 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.

[0034] 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.

[0035] Please continue reading Figure 1 As shown, in the reaction of converting the reduced coenzyme into the oxidized coenzyme, the prochiral α-keto acid compound is used as the substrate, the leucine dehydrogenase mutant is used as the catalyst, and the catalytic activity of the asymmetric reductive amination of the leucine dehydrogenase mutant is utilized, and at the same time, the product NH3 formed by the volatilization of ammonia water in the reaction system is utilized to convert the substrate into L-amino acid; in the process of the asymmetric reductive amination reaction of the prochiral α-keto acid compound catalyzed by the leucine dehydrogenase mutant to generate L-amino acid, NADH is converted into NAD + .

[0036] The leucine dehydrogenase mutant has at least one mutation in the amino acid sequence shown in SEQ ID NO: 6, and the at least one mutation can improve the catalytic activity of the enzyme in asymmetric reductive amination. The catalytic activity of the leucine dehydrogenase mutant is improved, and the speed of asymmetric reductive amination of prochiral α-ketoacid compounds to generate L-amino acids is accelerated, thereby accelerating the speed of the reduced coenzyme to convert to the oxidized coenzyme, thereby accelerating the speed of isopropanol dehydrogenase catalyzing the conversion of isopropanol to acetone and the speed of the oxidized coenzyme to convert to the reduced coenzyme, and NADH and NAD + The cycle speed is accelerated.

[0037] In the present specification, the enzyme formed by the amino acid sequence shown in SEQ ID NO: 6 may be referred to as wild-type leucine dehydrogenase, and the enzyme formed by each mutated amino acid sequence may be referred to as mutant leucine dehydrogenase or leucine dehydrogenase mutant.

[0038] In a leucine dehydrogenase mutant in this embodiment, the following mutations occur in the amino acid sequence shown in SEQ ID NO: 6: 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: 6 are replaced by other amino acids. The above-mentioned leucine dehydrogenase mutant has the amino acid sequence shown in SEQ ID NO: 1.

[0039] In a leucine dehydrogenase mutant in this embodiment, the following mutations occur in the amino acid sequence shown in SEQ ID NO: 6: 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: 6 are replaced by other amino acids. The above-mentioned leucine dehydrogenase mutant has the amino acid sequence shown in SEQ ID NO: 2.

[0040] In a leucine dehydrogenase mutant in this embodiment, the following mutations occur in the amino acid sequence shown in SEQ ID NO: 6: aspartic acid D at position 24 mutates to leucine A, glutamic acid E at position 124 mutates to valine L, S at position 129 mutates to threonine T, and valine V at position 243 mutates to alanine A (SEQ ID NO: 3). In the corresponding mutations, the amino acid residues at the corresponding positions in the amino acid sequence shown in SEQ ID NO: 6 are replaced by other amino acids. The above-mentioned leucine dehydrogenase mutant has the amino acid sequence shown in SEQ ID NO: 3.

[0041] In a leucine dehydrogenase mutant in this embodiment, the following mutations occur in the amino acid sequence shown in SEQ ID NO: 6: 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, proline P at position 130 mutates to tyrosine Y, and valine V at position 243 mutates to alanine A (SEQ ID NO: 4). In the corresponding mutations, the amino acid residues at the corresponding positions in the amino acid sequence shown in SEQ ID NO: 6 are replaced by other amino acids. The above-mentioned leucine dehydrogenase mutant has the amino acid sequence shown in SEQ ID NO: 4.

[0042] In a leucine dehydrogenase mutant in this embodiment, the following mutations occur in the amino acid sequence shown in SEQ ID NO: 6: cysteine ​​C at position 53 mutates to methionine M, glutamic acid E at position 124 mutates to valine V, and S at position 129 mutates to threonine T (SEQ ID NO: 5). In the corresponding mutations, the amino acid residues at the corresponding positions in the amino acid sequence shown in SEQ ID NO: 6 are replaced by other amino acids. The above-mentioned leucine dehydrogenase mutant has the amino acid sequence shown in SEQ ID NO: 5.

[0043] As an embodiment, the leucine dehydrogenase mutant is derived from a first recombinant engineered bacterium containing a gene encoding the leucine dehydrogenase mutant.

[0044] Specifically, a first recombinant vector capable of expressing a leucine dehydrogenase mutant is constructed, wherein the first recombinant vector comprises a polynucleotide encoding the leucine dehydrogenase mutant, and the polynucleotide comprises a nucleotide sequence corresponding to the amino acid sequence of the leucine dehydrogenase mutant. The polynucleotide is a DNA chain or RNA chain formed by polymerization of a plurality of nucleotides. As long as the polynucleotide can encode the leucine dehydrogenase mutant, any nucleotide in the polynucleotide may be chemically modified.

[0045] The first recombinant vector refers to a DNA preparation containing a nucleic acid sequence of a polynucleotide encoding a leucine dehydrogenase mutant, which may also contain a control sequence, in which the nucleic acid sequence of the polynucleotide encoding the leucine dehydrogenase mutant is operably linked to a suitable control sequence so that the leucine dehydrogenase mutant can be expressed in a suitable host. Exemplarily, 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 first recombinant vector may replicate or function independently of the host genome, or may be integrated into the genome of the host cell itself for replication or function.

[0046] The first recombinant vector is transformed into a host cell to obtain a first recombinant engineered bacterium, so that the leucine dehydrogenase mutant can be synthesized in the first recombinant engineered bacterium. The first recombinant engineered bacterium can be Escherichia, Erwinia, Serratia, Providencia, Corynebacterium or Brevibacterium; illustratively, the host cell can be Escherichia coli, Bacillus subtilis, Corynebacterium glutamicum or Aspergillus oryzae.

[0047] In some embodiments, the leucine dehydrogenase mutant can be a first wet cell obtained by inducing culture of a first recombinant engineered bacterium, or a crude enzyme solution obtained by crushing the first wet cell, or an immobilized cell prepared from the first wet cell.

[0048] As an embodiment, the isopropanol dehydrogenase is derived from a second recombinant engineered bacterium containing a gene encoding isopropanol dehydrogenase.

[0049] Specifically, a second recombinant vector capable of expressing isopropanol dehydrogenase is constructed, wherein the second recombinant vector comprises a polynucleotide encoding isopropanol dehydrogenase, and the polynucleotide comprises a nucleotide sequence corresponding to the amino acid sequence of the above-mentioned isopropanol dehydrogenase. The polynucleotide is a DNA chain or RNA chain formed by polymerization of a plurality of nucleotides. As long as the polynucleotide can encode the above-mentioned isopropanol dehydrogenase, any nucleotide in the polynucleotide can be chemically modified.

[0050] The second recombinant vector refers to a DNA preparation, which contains a nucleic acid sequence of a polynucleotide encoding isopropanol dehydrogenase, and it may also contain a control sequence. In the second recombinant vector, the nucleic acid sequence of the polynucleotide encoding isopropanol dehydrogenase is operably linked to a suitable control sequence so that the isopropanol dehydrogenase can be expressed in a suitable host.

[0051] The second recombinant vector is transformed into a host cell to obtain a second recombinant engineered bacterium, so that isopropanol dehydrogenase can be synthesized in the second recombinant engineered bacterium.

[0052] In some embodiments, the isopropanol dehydrogenase is a second wet cell obtained by inducing culture of a second recombinant engineered bacterium, or a crude enzyme solution obtained by crushing the second wet cell, or an immobilized cell prepared from the second wet cell.

[0053] In this embodiment, the coenzyme NAD + In the presence of NAD + Reduced to coenzyme NADH; in the presence of coenzyme NADH, the leucine dehydrogenase mutant uses NH3 generated by the volatilization of ammonia to asymmetrically reduce the prochiral α-keto acid compounds and NADH is oxidized to NAD + Since the leucine dehydrogenase mutant has an improved catalytic efficiency in the asymmetric reductive amination of prochiral α-ketoacid compounds, the coenzymes NADH and NAD + The cycle rate is accelerated, which is beneficial to improving the catalytic efficiency of the asymmetric reductive amination of leucine dehydrogenase.

[0054] An embodiment of the present application provides a method for synthesizing L-tert-leucine, which utilizes the above-mentioned L-amino acid synthesis system to contact with trimethylpyruvate to convert the trimethylpyruvate into L-tert-leucine.

[0055] See also Figure 2 As shown in the figure, in the reaction of converting the oxidized coenzyme into the reduced coenzyme, isopropanol is used as a substrate and isopropanol dehydrogenase is used as a catalyst. The oxidation catalytic activity of isopropanol dehydrogenase is used to convert isopropanol into acetone. In the process of isopropanol being oxidized to form the product acetone under the catalysis of isopropanol dehydrogenase, NAD + is converted into NADH.

[0056] Please continue reading Figure 2 As shown, in the reaction of reducing coenzyme to oxidizing coenzyme, trimethylpyruvate is used as substrate, leucine dehydrogenase mutant is used as catalyst, and the catalytic activity of asymmetric reductive amination of leucine dehydrogenase mutant is utilized, and the product NH3 (NH3 reacts with H2O to form ammonia water, and ammonia water precipitates NH4 + ) converts the substrate into L-tert-leucine; in the process of the leucine dehydrogenase mutant catalyzing the asymmetric reductive amination reaction of trimethylpyruvate to produce L-tert-leucine, NADH is converted into NAD + .

[0057] In some embodiments, in the reaction system, NAD + The concentration of isopropanol dehydrogenase is 10 g / L to 20 g / L, the concentration of leucine dehydrogenase mutant is 0.6 g / L to 1.0 g / L, the concentration of leucine dehydrogenase mutant is 5 g / L to 20 g / L, and the concentration of isopropanol dehydrogenase is 10 g / L to 20 g / L.

[0058] In some embodiments, in the reaction system, the volume ratio of isopropanol to the buffer solution in the reaction system is 1 to 1.3:10, and the volume ratio of ammonia water to the buffer solution in the reaction system is 1 to 1.3:10.

[0059] In some embodiments, in the reaction system, before the reaction starts, the concentration of trimethylpyruvic acid is 100 g / L to 110 g / L, and the pH of the reaction system is 8.5 to 10.

[0060] In some embodiments, in the reaction system, the mass ratio of the leucine dehydrogenase mutant to the isopropanol dehydrogenase is 1:1-2.

[0061] Preparation of leucine dehydrogenase mutants

[0062] The leucine dehydrogenase mutant and the gene encoding leucine dehydrogenase 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 contains a nucleotide sequence corresponding to the amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 6, and the LaLeuDH gene is constructed into the pET-28a plasmid to obtain the first recombinant vector.

[0063] The first recombinant vector 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 of the present application is Escherichia coli.

[0064] Expression of leucine dehydrogenase mutants

[0065] a. Transform pET-28a-LaLeuDH into Escherichia coli (e.g., competent cells E. coli DH5α); 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 a seed solution.

[0066] 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.

[0067] 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.

[0068] d. Lower the shaking temperature to 16-18°C. After the temperature of the cultured bacterial solution has dropped, add isopropyl-β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM and induce expression for 14-16 hours.

[0069] 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.

[0070] f. Remove the supernatant, add 30 mL of protein purification buffer, and resuspend the cells using a vortex oscillator.

[0071] 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.

[0072] Purification of leucine dehydrogenase mutant protein

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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.

[0077] 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.

[0078] 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.

[0079] Electrophoretic Analysis of Leucine Dehydrogenase Mutant Proteins

[0080] 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.

[0081] 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.

[0082] 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.

[0083] d. Protein gel image analysis: The stained and destained protein gel is photographed and saved using a gel imager.

[0084] Preparation of isopropanol dehydrogenase

[0085] The gene encoding isopropanol dehydrogenase can be called the IPADH gene, which contains a nucleotide sequence corresponding to the amino acid sequence of the above-mentioned isopropanol dehydrogenase. For example, the IPADH gene contains a nucleotide sequence corresponding to the amino acid sequence shown in SEQ ID NO: 7. The IPADH gene is constructed into the pET-28a plasmid to obtain a second recombinant vector.

[0086] The second recombinant vector is a pET-28a plasmid containing the IPADH gene, hereinafter referred to as pET-28a-IPADH; the host cell used in each embodiment and comparative example of the present application is Escherichia coli.

[0087] Expression of isopropanol dehydrogenase

[0088] 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.

[0089] 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.

[0090] 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.

[0091] Lower the shaking temperature to 16-18°C. After the temperature of the cultured bacterial solution drops, add isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.5 mM and induce expression for 14-16 hours.

[0092] 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.

[0093] Remove the supernatant, add 30 mL of protein purification buffer, and resuspend the bacteria using a vortex oscillator.

[0094] Centrifuge the resuspended cells again at 5500 rpm for 10 min. Pour off the supernatant, add 30 mL of protein purification 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.

[0095] Enzyme catalyzed reactions

[0096] In vitro enzyme catalytic reaction conditions:

[0097] Reaction buffer: diammonium phosphate buffer at concentrations of 100mM, 200mM, and 300mM;

[0098] Reaction pH: pH8, pH 8.5, pH 9, pH10;

[0099] The concentrations of trimethylpyruvic acid were 100 g / L and 110 g / L;

[0100] The volume concentration of ammonia water is 0.6mL / 10mL, 0.8mL / 10mL, 1.0mL / 10mL, 1.1mL / 10mL, 1.2mL / 10mL, 1.3mL / 10mL;

[0101] The volume concentration of isopropanol is 0.6mL / 10mL, 0.8mL / 10mL, 1.0mL / 10mL, 1.1mL / 10mL, 1.2mL / 10mL, 1.3mL / 10mL;

[0102] NAD + The concentrations are 0.6g / L, 0.7g / L, 0.8g / L, 0.9g / L, and 1.0g / L;

[0103] The addition amount of leucine dehydrogenase / leucine dehydrogenase mutant was 10 g / L, 20 g / L, 30 g / L, and 40 g / L;

[0104] The addition amount of isopropanol dehydrogenase was 10 g / L, 20 g / L, 30 g / L, and 40 g / L;

[0105] Reaction temperature: 30°C, 35°C, 40°C, 45°C;

[0106] Reaction time: 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, or 24h;

[0107] The total reaction volume was 10 mL.

[0108] The amino acid sequence of the leucine dehydrogenase mutant in Examples 1 to 14 is shown in SEQ ID NO:2.

[0109] Isopropyl alcohol addition experiment

[0110] The leucine dehydrogenase mutant and the isopropanol dehydrogenase are crude enzymes obtained by crushing the wet bacterial cells of the induced culture of the first recombinant engineering bacteria and the crude enzymes obtained by crushing the wet bacterial cells of the induced culture of the second recombinant engineering bacteria, respectively.

[0111] Examples 1 to 4 can be used: the reaction system has a trimethylpyruvic acid concentration of 110 g / L (adopting a batch addition method: the amount of trimethylpyruvic acid added after 0 hours of reaction is 36.7 g / L, the amount of trimethylpyruvic acid added after 4 hours of reaction is 36.7 g / L, and the amount of trimethylpyruvic acid added after 7 hours of reaction is 36.7 g / L), the volume concentration of ammonia water is 1.1 mL / 10 mL, and NAD + The concentration is 0.6 g / L, the addition amount of leucine dehydrogenase / leucine dehydrogenase mutant is 15 g / L, the addition amount of isopropanol dehydrogenase is 15 g / L, the buffer is 100 mM diammonium phosphate, the volume of the buffer is 10 mL, the temperature is 40°C for 12 hours, and the substrate conversion rate is measured every 1 hour to measure the conversion activity of each embodiment and comparative example.

[0112] The volume of isopropanol in each embodiment is shown in Table 1:

[0113] Table 1 Addition amount of isopropanol

[0114] Volume of isopropyl alcohol Example 1 1.0mL Example 2 1.1mL Example 3 1.2mL Example 4 1.3mL

[0115] The results are as follows Figure 3 As shown, Examples 1 to 4 can consume a large amount of the substrate trimethylpyruvate within 12 hours, among which Example 1 has the fastest consumption rate.

[0116] Ammonia addition experiment

[0117] The leucine dehydrogenase mutant and the isopropanol dehydrogenase are crude enzymes obtained by crushing the wet bacterial cells of the induced culture of the first recombinant engineering bacteria and the crude enzymes obtained by crushing the wet bacterial cells of the induced culture of the second recombinant engineering bacteria, respectively.

[0118] Examples 5 to 8 may be used: the reaction system has a trimethylpyruvic acid concentration of 110 g / L (adopting a batch addition method: the amount of trimethylpyruvic acid added after 0 hours of reaction is 36.7 g / L, the amount of trimethylpyruvic acid added after 2 hours of reaction is 36.7 g / L, and the amount of trimethylpyruvic acid added after 6 hours of reaction is 36.7 g / L), the volume concentration of isopropanol is 1.1 mL / 10 mL, and the NAD + The concentration is 0.6 g / L, the addition amount of leucine dehydrogenase / leucine dehydrogenase mutant is 15 g / L, the addition amount of isopropanol dehydrogenase is 15 g / L, the buffer is 100 mM diammonium phosphate, the volume of the buffer is 10 mL, the temperature is 40°C for 12 hours, and the substrate conversion rate is measured every 1 hour to measure the conversion activity of each embodiment and comparative example.

[0119] The volume of isopropanol in each embodiment is shown in Table 2:

[0120] Table 2 Amount of ammonia added

[0121] Volume of ammonia Example 5 1.0mL Example 6 1.1mL Example 7 1.2mL Example 8 1.3mL

[0122] The results are as follows Figure 4 As shown, Examples 5 to 8 can consume a large amount of the substrate trimethylpyruvate within 12 hours, among which Example 5 has the fastest consumption rate.

[0123] Experiment on two enzyme addition amounts

[0124] The leucine dehydrogenase mutant and the isopropanol dehydrogenase are crude enzymes obtained by crushing the wet bacterial cells of the induced culture of the first recombinant engineering bacteria and the crude enzymes obtained by crushing the wet bacterial cells of the induced culture of the second recombinant engineering bacteria, respectively.

[0125] Examples 9 to 14 can be used: the reaction system has a trimethylpyruvic acid concentration of 110 g / L (adopting a batch addition method: the amount of trimethylpyruvic acid added after 0 hours of reaction is 36.7 g / L, the amount of trimethylpyruvic acid added after 2 hours of reaction is 36.7 g / L, and the amount of trimethylpyruvic acid added after 6 hours of reaction is 36.7 g / L), the volume concentration of isopropanol is 1.0 mL / 10 mL, the volume concentration of ammonia water is 1.0 mL / 10 mL, and NAD + The concentration is 0.6 g / L, the buffer is 100 mM diammonium phosphate, the volume of the buffer is 10 mL, the temperature is 40° C. for 12 hours, and the substrate conversion rate is measured every 1 hour to measure the conversion activity of each embodiment and comparative example.

[0126] The addition amounts of the leucine dehydrogenase mutant and isopropanol dehydrogenase in each embodiment are shown in Table 3:

[0127] Table 3 Addition amount of leucine dehydrogenase mutant and isopropanol dehydrogenase

[0128]

[0129]

[0130] The results are as follows Figure 5 and Figure 6 As shown, Examples 9 to 14 can consume a large amount of the substrate trimethylpyruvate within 12 hours, among which, Examples 11 and 14 have the fastest consumption speed.

[0131] Activity comparison experiment of leucine dehydrogenase mutants

[0132] The leucine dehydrogenase mutant and the isopropanol dehydrogenase are respectively the induced culture wet cells of the first recombinant engineering bacteria and the induced culture wet cells of the second recombinant engineering bacteria.

[0133] Examples 15 to 19 and Comparative Example 1 can be used: the reaction system has a trimethylpyruvic acid concentration of 110 g / L (adopting a batch addition method: the amount of trimethylpyruvic acid added after 0 hours of reaction is 36.7 g / L, the amount of trimethylpyruvic acid added after 2 hours of reaction is 36.7 g / L, and the amount of trimethylpyruvic acid added after 6 hours of reaction is 36.7 g / L), the volume concentration of isopropanol is 1.0 mL / 10 mL, the volume concentration of ammonia water is 1.0 mL / 10 mL, and NAD + The concentration is 0.6 g / L, the buffer is 100 mM diammonium phosphate, the volume of the buffer is 10 mL, the temperature is 40° C. for 12 hours, and the substrate conversion rate is measured every 1 hour to measure the conversion activity of each embodiment and comparative example.

[0134] The leucine dehydrogenase mutants or leucine dehydrogenases in the examples and comparative examples are shown in Table 4:

[0135] Table 4 Leucine dehydrogenase mutants or leucine dehydrogenase amino acid sequences

[0136] Leucine dehydrogenase Embodiment 15 SEQ ID NO: 1 Example 16 SEQ ID NO: 2 Embodiment 17 SEQ ID NO: 3 Embodiment 18 SEQ ID NO: 4 Embodiment 19 SEQ ID NO: 5 Comparative Example 1 SEQ ID NO: 6

[0137] In Examples 15 to 19, the leucine dehydrogenase mutants were used, and the two substrates were consumed in large quantities within 12 hours; the conversion rate of the leucine dehydrogenase in Comparative Example 1 for the two substrates was less than 30% within 12 hours.

[0138] Compared with the wild-type leucine dehydrogenase used in Comparative Example 1, the catalytic activities of the leucine dehydrogenase mutants used in Examples 15 to 19 were respectively improved.

[0139] The above embodiments only express the preferred implementation methods of the present application, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the patent application. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present application, and these all belong to the protection scope of the present application. Therefore, the protection scope of the patent application shall be subject to the attached claims.

Claims

1. An L-amino acid synthesis system, characterized in that: NAD included + , isopropanol, isopropanol dehydrogenase and a leucine dehydrogenase mutant, the leucine dehydrogenase mutant having an amino acid sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 5, the isopropanol dehydrogenase having an amino acid sequence as shown in SEQ ID NO: 7, and the mass ratio of the leucine dehydrogenase mutant to the isopropanol dehydrogenase is 1: 1 to 4.

2. The L-amino acid synthesis system according to claim 1, characterized in that: The leucine dehydrogenase mutant is derived from a first recombinant engineering bacterium containing a gene encoding the leucine dehydrogenase mutant.

3. The L-amino acid synthesis system according to claim 2, characterized in that: The leucine dehydrogenase mutant is the first wet bacterial body obtained by inducing culture of the first recombinant engineered bacteria, or the crude enzyme solution obtained by crushing the first wet bacterial body, or the immobilized cells prepared from the first wet bacterial body.

4. The L-amino acid synthesis system according to claim 1, characterized in that: The isopropanol dehydrogenase is derived from a second recombinant engineering bacterium containing an isopropanol dehydrogenase encoding gene.

5. The L-amino acid synthesis system according to claim 4, characterized in that: The isopropanol dehydrogenase is a second wet cell obtained by inducing and culturing the second recombinant engineered bacteria, a crude enzyme solution obtained by crushing the second wet cell, or immobilized cells prepared from the second wet cell.

6. A method for synthesizing L-tert-leucine, characterized in that: The L-amino acid synthesis system according to any one of claims 1 to 5 is contacted with trimethylpyruvate in the presence of aqueous ammonia to convert the trimethylpyruvate into L-tert-leucine.

7. The method for synthesizing L-tert-leucine according to claim 6, characterized in that: The NAD + The concentration of isopropanol dehydrogenase is 10 g / L to 20 g / L.

8. The method for synthesizing L-tert-leucine according to claim 7, characterized in that: The volume ratio of the isopropanol to the buffer solution in the reaction system is 1 to 1.3:10, and the volume ratio of the ammonia water to the buffer solution in the reaction system is 1 to 1.3:

10.

9. The method for synthesizing L-tert-leucine according to claim 7, characterized in that: The total added concentration of the trimethylpyruvic acid is 100 g / L to 110 g / L, and the pH of the reaction system is 8.5 to 10.

10. The method for synthesizing L-tert-leucine according to claim 7, characterized in that: The mass ratio of the leucine dehydrogenase mutant to the isopropanol dehydrogenase is 1:1-2.

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