L-amino acid synthesis systems and applications

Abstract

This application relates to the field of biotechnology, and in particular to an L-amino acid synthesis system and its application. The L-amino acid synthesis system and its application provided in the embodiments of this application are relevant to the coenzyme NAD+. + In the presence of isopropanol dehydrogenase, isopropanol is catalyzed to produce acetone and NAD+. + It is reduced to the coenzyme NADH; in the presence of coenzyme NADH, the leucine dehydrogenase mutant uses NH3 generated from the volatilization of ammonia to asymmetricly reduce and amination the prochiral α-keto acid compound, and NADH is oxidized to NAD. + Because the leucine dehydrogenase mutant has increased catalytic efficiency in the asymmetric reductive amination of prochiral α-keto acids, the coenzymes NADH and NAD... + The increased cycle speed is beneficial to improving the catalytic efficiency of asymmetric reductive amination of leucine dehydrogenase.

Description

Technical Field

[0001] This application relates to the field of biotechnology, and in particular to an L-amino acid synthesis system and its applications. Background Technology

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

[0003] During the asymmetric reductive amination of leucine dehydrogenase, electron transfer is dependent on the coenzyme NADH. NADH is continuously consumed, and after the reaction, it is converted into NAD+. + It exists in its oxidized form. Due to the high cost and poor stability of the coenzyme NADH, it cannot be added in large quantities during the reaction, which is detrimental to improving the catalytic efficiency of asymmetric reductive amination of leucine dehydrogenase. Summary of the Invention

[0004] Based on this, this application provides an L-amino acid synthesis system and its application to solve the aforementioned technical problem that the inability to add large amounts of coenzyme NADH during the reaction process is detrimental to improving the catalytic efficiency of asymmetric reductive amination of leucine dehydrogenase.

[0005] In a first aspect, embodiments of this application provide an L-amino acid synthesis system, including NAD+. + The mixture contains isopropanol, isopropanol dehydrogenase, and a leucine dehydrogenase mutant, wherein the leucine dehydrogenase mutant has an amino acid sequence as shown in any 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.

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

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

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

[0009] Optionally, the isopropanol dehydrogenase is a second wet cell obtained by inducing and culturing the second recombinant engineered bacteria, or a crude enzyme solution obtained by breaking the second wet cell, or an immobilized cell prepared from the second wet cell.

[0010] Secondly, embodiments of this application provide a method for synthesizing L-tert-leucine, wherein the above-mentioned L-amino acid synthesis system is contacted with trimethylpyruvate in the presence of ammonia to convert the trimethylpyruvate into L-tert-leucine.

[0011] Optionally, the NAD + The concentration of the enzyme is 0.6 g / L to 1.0 g / L, the concentration of the leucine dehydrogenase mutant is 5 g / L to 20 g / L, and the concentration of the isopropanol dehydrogenase is 10 g / L to 20 g / L.

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

[0013] Optionally, the total 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 to 2.

[0015] The L-amino acid synthesis system and its application provided in this application's embodiments are relevant to the coenzyme NAD+ synthesis. + In the presence of isopropanol dehydrogenase, isopropanol is catalyzed to produce acetone and NAD+. + It is reduced to coenzyme NADH; in the presence of coenzyme NADH, the leucine dehydrogenase mutant uses NH3 generated from the volatilization of ammonia to asymmetricly reduce and amination prochiral α-keto acid compounds, and NADH is oxidized to NAD. + Because the leucine dehydrogenase mutant has improved catalytic efficiency in the asymmetric reductive amination of prochiral α-keto acids, the coenzymes NADH and NAD... + The increased cycle speed is beneficial to improving the catalytic efficiency of asymmetric reductive amination of leucine dehydrogenase. Attached Figure Description

[0016] Figure 1 This is a schematic diagram of the reaction principle of the L-amino acid synthesis system according to an embodiment of this application.

[0017] Figure 2 This is a schematic diagram illustrating the reaction principle of the L-amino acid synthesis system for synthesizing L-tert-leucine according to an embodiment of this application.

[0018] Figure 3 The graph shows the experimental results of isopropanol addition in Examples 1-3.

[0019] Figure 4 The graph shows the experimental results of ammonia addition in Examples 5-8.

[0020] Figure 5 The figure shows the experimental results of the addition ratio of recombinant engineered bacteria in Examples 9-11.

[0021] Figure 6 The figures show the experimental results of the addition ratio of recombinant engineered bacteria in Examples 12-14. Detailed Implementation

[0022] To facilitate understanding of this application, a more complete description will be provided below with reference to the accompanying drawings. Preferred embodiments of this application are shown in the drawings. However, this application can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of this application.

[0023] It should be noted that when a component is said to be "fixed to" another component, it can be directly attached to the other component or there may be an intervening component. When a component is said to be "connected to" another component, it can be directly connected to the other component or there may be an intervening component.

[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application.

[0025] 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.

[0026] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0027] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0028] In this article, the terms "leucine dehydrogenase" and "leucine dehydrogenase mutant" refer to enzymes that exhibit 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.

[0029] The reaction principle for converting prochiral α-keto acids into L-amino acids using the aforementioned leucine dehydrogenase is shown below:

[0030]

[0031] 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.

[0032] One embodiment of this application provides an L-amino acid synthesis system, which includes NAD+. + Isopropanol, isopropanol dehydrogenase, and leucine dehydrogenase mutant, wherein the leucine dehydrogenase mutant has an amino acid sequence as shown in any of SEQ ID NO: 1 to SEQ ID NO: 5, and 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 does not have an additional hydride (i.e., a negatively charged hydrogen atom), 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).

[0034] 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.

[0035] Please continue reading. Figure 1 As shown, in the reaction of reducing coenzyme to oxidizing coenzyme, prochiral α-keto acid compounds serve as substrates, and leucine dehydrogenase mutants act as catalysts. The asymmetric reductive amination catalytic activity of the leucine dehydrogenase mutant is utilized, while NH3, a product formed by the volatilization of ammonia in the reaction system, converts the substrate into L-amino acids. During the asymmetric reductive amination of prochiral α-keto acid compounds catalyzed by the leucine dehydrogenase mutant to generate L-amino acids, NADH is converted to NAD. + .

[0036] This leucine dehydrogenase mutant produces at least one mutation in the amino acid sequence shown in SEQ ID NO: 6, which enhances the catalytic activity of the enzyme in asymmetric reductive amination. The increased catalytic activity of this leucine dehydrogenase mutant accelerates the asymmetric reductive amination of prochiral α-keto acids to L-amino acids, thereby accelerating the conversion of the reduced coenzyme to the oxidized coenzyme. This, in turn, accelerates the conversion of isopropanol to acetone catalyzed by isopropanol dehydrogenase, as well as the conversion of the oxidized coenzyme to the reduced coenzyme, thus increasing the rates of NADH and NAD. + The circulation speed increases.

[0037] In this specification, the enzyme formed by the amino acid sequence shown in SEQ ID NO: 6 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.

[0038] In this embodiment, a leucine dehydrogenase mutant exhibits the following mutations in the amino acid sequence corresponding to SEQ ID NO: 6: 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 these mutations, the amino acid residues at the corresponding sites in the amino acid sequence shown in SEQ ID NO: 6 are replaced by other amino acids. The aforementioned leucine dehydrogenase mutant has the amino acid sequence shown in SEQ ID NO: 1.

[0039] In this embodiment, a leucine dehydrogenase mutant exhibits the following mutations in the amino acid sequence corresponding to SEQ ID NO: 6: 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 these mutations, the amino acid residues at the corresponding sites in the amino acid sequence shown in SEQ ID NO: 6 are replaced by other amino acids. The aforementioned leucine dehydrogenase mutant has the amino acid sequence shown in SEQ ID NO: 2.

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

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

[0042] In this embodiment, a leucine dehydrogenase mutant exhibits the following mutations in the amino acid sequence corresponding to SEQ ID NO: 6: cysteine ​​(C) at position 53 is mutated to methionine (M), glutamate (E) at position 124 is mutated to valine (V), and S at position 129 is mutated to threonine (T) (SEQ ID NO: 5). In these mutations, the amino acid residues at the corresponding sites in the amino acid sequence shown in SEQ ID NO: 6 are replaced by other amino acids. The aforementioned leucine dehydrogenase mutant has the amino acid sequence shown in SEQ ID NO: 5.

[0043] In one implementation, the leucine dehydrogenase mutant is derived from a first recombinant engineered bacterium containing the leucine dehydrogenase mutant encoding gene.

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

[0045] The first recombinant vector is a DNA preparation containing a nucleic acid sequence encoding a polynucleotide of a leucine dehydrogenase mutant. It may also contain a control sequence in which the nucleic acid 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. Exemplarily, the control sequence may include, but is not limited to, a promoter capable of initiating transcription, any operon sequence for regulating transcription, a suitable mRNA ribosome binding site, and sequences 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 it may integrate into the host cell's genome itself for replication or function.

[0046] The first recombinant vector is transformed into a host cell to obtain a first recombinant engineered bacterium, enabling the synthesis of the leucine dehydrogenase mutant within the first recombinant engineered bacterium. The first recombinant engineered bacterium can be of the genera *Escherichia*, *Erwinia*, *Serratia*, *Providencia*, *Corynebacterium*, or *Brevibacterium*; exemplary, 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 and culturing a first recombinant engineered bacterium, or a crude enzyme solution obtained by breaking down the first wet cell, or an immobilized cell prepared from the first wet cell.

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

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

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

[0051] The second recombinant vector was transformed into the host cell to obtain the second recombinant engineered bacteria, which enabled isopropanol dehydrogenase to be synthesized in the second recombinant engineered bacteria.

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

[0053] In this embodiment, in coenzyme NAD + In the presence of isopropanol dehydrogenase, isopropanol is catalyzed to produce acetone and NAD+. + It is reduced to coenzyme NADH; in the presence of coenzyme NADH, the leucine dehydrogenase mutant uses NH3 generated from the volatilization of ammonia to asymmetricly reduce and amination prochiral α-keto acid compounds, and NADH is oxidized to NAD. + Because the leucine dehydrogenase mutant has improved catalytic efficiency in the asymmetric reductive amination of prochiral α-keto acids, the coenzymes NADH and NAD... + The increased cycle speed is beneficial to improving the catalytic efficiency of asymmetric reductive amination of leucine dehydrogenase.

[0054] One embodiment of this application provides a method for synthesizing L-tert-leucine, which involves contacting trimethylpyruvate with the aforementioned L-amino acid synthesis system to convert trimethylpyruvate into L-tert-leucine.

[0055] Please see Figure 2 As shown, in the reaction of converting oxidized coenzyme to reduced coenzyme, isopropanol serves as the substrate, and isopropanol dehydrogenase acts as the catalyst. Utilizing the oxidative catalytic activity of isopropanol dehydrogenase, isopropanol is converted to acetone. During the oxidation of isopropanol to acetone under the catalysis of isopropanol dehydrogenase, NAD+... + It 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 the substrate and leucine dehydrogenase mutant is used as the catalyst. The asymmetric reductive amination catalytic activity of leucine dehydrogenase mutant is utilized, while the product NH3 formed by the volatilization of ammonia water (NH3 reacts with H2O to form ammonia water, and NH4 is precipitated from the ammonia water) is utilized. + The substrate is converted to L-tert-leucine; during the asymmetric reductive amination of trimethylpyruvate to L-tert-leucine catalyzed by the leucine dehydrogenase mutant, NADH is converted to NAD. + .

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

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

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

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

[0061] Preparation of leucine dehydrogenase mutants

[0062] The leucine dehydrogenase mutant and the gene encoding leucine dehydrogenase can be called the LaLeuDH gene. The LaLeuDH gene contains the nucleotide sequence corresponding to the amino acid sequence of the aforementioned leucine dehydrogenase mutant or leucine mutant enzyme. For example, the LaLeuDH gene contains the nucleotide sequence corresponding to the amino acid sequence shown in SEQ ID NO: 1 to SEQ ID NO: 6. The first recombinant vector is obtained by constructing the LaLeuDH gene in the pET-28a plasmid.

[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 the various embodiments and comparative examples in this application is Escherichia coli.

[0064] Expression of leucine dehydrogenase mutant

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

[0066] 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.

[0067] 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.

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

[0069] 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.

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

[0071] 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.

[0072] Purification of leucine dehydrogenase mutant protein

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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℃.

[0077] 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.

[0078] 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.

[0079] Electrophoretic analysis of leucine dehydrogenase mutant protein

[0080] 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.

[0081] 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.

[0082] 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.

[0083] d. Gel image analysis: The stained and destained protein gels were photographed and saved using a gel imaging system.

[0084] Preparation of isopropanol dehydrogenase

[0085] The gene encoding isopropanol dehydrogenase can be called the IPADH gene. The IPADH gene contains the nucleotide sequence corresponding to the amino acid sequence of the isopropanol dehydrogenase mentioned above. For example, the IPADH gene contains the 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 the 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 the various embodiments and comparative examples in this application is Escherichia coli.

[0087] Expression of isopropanol dehydrogenase

[0088] 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.

[0089] 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.

[0090] 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.

[0091] Lower the temperature of the shaker to 16℃-18℃. After the temperature of the cultured bacterial solution has decreased, add isopropyl β-D-1-thiogalactopyranoside (IPTG) to a final concentration of 0.5mM and induce expression for 14-16h.

[0092] 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.

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

[0094] 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.

[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: pH 8, pH 8.5, pH 9, pH 10;

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

[0100] The ammonia concentrations are 0.6 mL / 10 mL, 0.8 mL / 10 mL, 1.0 mL / 10 mL, 1.1 mL / 10 mL, 1.2 mL / 10 mL, and 1.3 mL / 10 mL.

[0101] The volume concentrations of isopropanol are 0.6 mL / 10 mL, 0.8 mL / 10 mL, 1.0 mL / 10 mL, 1.1 mL / 10 mL, 1.2 mL / 10 mL, and 1.3 mL / 10 mL.

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

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

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

[0105] Reaction temperatures: 30℃, 35℃, 40℃, 45℃;

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

[0107] The total reaction volume is 10 mL.

[0108] The amino acid sequences of the leucine dehydrogenase mutants in Examples 1 to 14 are shown in SEQ ID NO: 2.

[0109] Isopropanol addition experiment

[0110] Leucine dehydrogenase mutant and isopropanol dehydrogenase are crude enzymes obtained by breaking the induced culture wet cells of the first recombinant engineered bacteria and the induced culture wet cells of the second recombinant engineered bacteria, respectively.

[0111] Examples 1-4 can be performed with the following reaction system: trimethylpyruvate concentration of 110 g / L (added in batches: 36.7 g / L at 0 hours of reaction, 36.7 g / L at 4 hours of reaction, and 36.7 g / L at 7 hours of reaction), ammonia volume concentration of 1.1 mL / 10 mL, and NAD... + The concentration of the enzyme was 0.6 g / L, the amount of leucine dehydrogenase / leucine dehydrogenase mutant added was 15 g / L, the amount of isopropanol dehydrogenase added was 15 g / L, the buffer was 100 mM diammonium phosphate, the buffer volume was 10 mL, the reaction was carried out at 40 °C for 12 hours, and the substrate conversion rate was measured every 1 hour to measure the conversion activity of each example and comparative example.

[0112] The volumes of isopropanol in each embodiment are shown in Table 1:

[0113] Table 1. Amount of Isopropanol Added

[0114] Volume of isopropanol 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 all consume a large amount of the substrate trimethylpyruvate within 12 hours, with Example 1 showing the fastest consumption rate.

[0116] Ammonia addition experiment

[0117] Leucine dehydrogenase mutant and isopropanol dehydrogenase are crude enzymes obtained by breaking the induced culture wet cells of the first recombinant engineered bacteria and the induced culture wet cells of the second recombinant engineered bacteria, respectively.

[0118] Examples 5-8 can be performed with the following reaction system: trimethylpyruvate concentration of 110 g / L (added in batches: 36.7 g / L at 0 hours of reaction, 36.7 g / L at 2 hours of reaction, and 36.7 g / L at 6 hours of reaction), isopropanol volume concentration of 1.1 mL / 10 mL, and NAD... + The concentration of the enzyme was 0.6 g / L, the amount of leucine dehydrogenase / leucine dehydrogenase mutant added was 15 g / L, the amount of isopropanol dehydrogenase added was 15 g / L, the buffer was 100 mM diammonium phosphate, the buffer volume was 10 mL, the reaction was carried out at 40 °C for 12 hours, and the substrate conversion rate was measured every 1 hour to measure the conversion activity of each example and comparative example.

[0119] The volumes of isopropanol in each embodiment are shown in Table 2:

[0120] Table 2 Ammonia water addition amount

[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 all consume a large amount of the substrate trimethylpyruvate within 12 hours, with Example 5 showing the fastest consumption rate.

[0123] Experiment on the addition of two enzymes

[0124] Leucine dehydrogenase mutant and isopropanol dehydrogenase are crude enzymes obtained by breaking the induced culture wet cells of the first recombinant engineered bacteria and the induced culture wet cells of the second recombinant engineered bacteria, respectively.

[0125] Examples 9-14 can be performed with the following reaction system: trimethylpyruvate concentration of 110 g / L (added in batches: 36.7 g / L at 0 hours, 36.7 g / L at 2 hours, and 36.7 g / L at 6 hours), isopropanol volume concentration of 1.0 mL / 10 mL, ammonia volume concentration of 1.0 mL / 10 mL, and NAD... + The concentration of the active ingredient was 0.6 g / L, the buffer solution was 100 mM diammonium phosphate, the buffer volume was 10 mL, and the reaction was carried out at 40 °C for 12 hours. The substrate conversion rate was measured every hour to measure the conversion activity of each example and comparative example.

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

[0127] Table 3. Dosage 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, with Examples 11 and 14 showing the fastest consumption rate.

[0131] Comparative experiment on the activity of leucine dehydrogenase mutants

[0132] Leucine dehydrogenase mutant and isopropanol dehydrogenase were the induced culture wet cells of the first recombinant engineered bacteria and the induced culture wet cells of the second recombinant engineered bacteria, respectively.

[0133] Examples 15-19 and Comparative Example 1 can be performed with the following reaction system: trimethylpyruvate concentration of 110 g / L (added in batches: 36.7 g / L at 0 hours, 36.7 g / L at 2 hours, and 36.7 g / L at 6 hours), isopropanol volume concentration of 1.0 mL / 10 mL, ammonia volume concentration of 1.0 mL / 10 mL, and NAD... + The concentration of the active ingredient was 0.6 g / L, the buffer solution was 100 mM diammonium phosphate, the buffer volume was 10 mL, and the reaction was carried out at 40 °C for 12 hours. The substrate conversion rate was measured every hour to measure the conversion activity of each example and comparative example.

[0134] The leucine dehydrogenase mutants or leucine dehydrogenases in each embodiment and comparative example are shown in Table 4:

[0135] Table 4. Amino acid sequences of leucine dehydrogenase mutants or leucine dehydrogenases

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

[0137] In Examples 15-19, the leucine dehydrogenase mutant consumed a large amount of both substrates within 12 hours; in Comparative Example 1, the leucine dehydrogenase showed a conversion rate of less than 30% for both substrates within 12 hours.

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

[0139] The above embodiments merely illustrate preferred implementations of this application, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Therefore, the scope of protection of this patent application should be determined by the appended claims.

Claims

1. An L-amino acid synthesis system, characterized in that, including NAD + isopropanol, isopropanol dehydrogenase, and a leucine dehydrogenase mutant, an amino acid sequence of the leucine dehydrogenase mutant is shown as any one of SEQ ID NO: 1 to SEQ ID NO: 5, an amino acid sequence of the isopropanol dehydrogenase is shown as SEQ ID NO: 7, and a 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 was derived from a first recombinant engineered bacterium containing the leucine dehydrogenase mutant encoding gene.

3. The L-amino acid synthesis system according to claim 2, characterized in that, The leucine dehydrogenase mutant is the first wet cell obtained by inducing and culturing the first recombinant engineered bacteria, or the crude enzyme solution obtained by breaking the first wet cell, or the immobilized cell prepared from the first wet cell.

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

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

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

7. The method for synthesizing L-tert-leucine according to claim 6, characterized in that, The concentration of the NAD + is 0.6 g / L to 1.0 g / L, the concentration of the leucine dehydrogenase mutant is 5 g / L to 20 g / L, and the concentration of the 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 isopropanol to the buffer solution in the reaction system is 1 to 1.3:10, and the volume ratio of ammonia 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 concentration of trimethylpyruvic acid added 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 to 2.

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