Coenzyme Cycle System and its Applications

Abstract

This application relates to the field of biotechnology, and in particular to a coenzyme cycling system and its applications. The coenzyme cycling system and its applications provided in the embodiments of this application relate to the coenzyme NAD+. + In the presence of formate dehydrogenase, ammonium formate is catalyzed to produce NH3 and NAD3. + It is reduced to the coenzyme NADH; in the presence of coenzyme NADH, the leucine dehydrogenase mutant uses the generated NH3 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 a coenzyme cycle 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 and amination prochiral α-keto acid compounds. The asymmetric reducing and 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 a coenzyme cycling 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 a coenzyme cycling system, including NAD+. + Ammonium formate, formate dehydrogenase, and leucine dehydrogenase mutant, wherein the leucine dehydrogenase mutant has an amino acid sequence as shown in any one of SEQ ID NO: 1 to SEQ ID NO: 5.

[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 formate dehydrogenase is derived from a second recombinant engineered bacterium containing a formate dehydrogenase encoding gene.

[0009] Optionally, the formate 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] Optionally, the formate dehydrogenase has the amino acid sequence shown in SEQ ID NO: 7, and the mass ratio of the leucine dehydrogenase mutant to the formate dehydrogenase is 1:1.8 to 2.2.

[0011] Optionally, the formate dehydrogenase has the amino acid sequence shown in SEQ ID NO: 8, and the mass ratio of the leucine dehydrogenase mutant to the formate dehydrogenase is 1:4.5 to 5.5.

[0012] Optionally, the coenzyme cycle system further includes NADH and / or pyridoxal phosphate.

[0013] Secondly, embodiments of this application provide a method for preparing L-tert-leucine using the above-described coenzyme cycle system, comprising: contacting the coenzyme cycle system with trimethylpyruvate to convert the trimethylpyruvate into L-tert-leucine.

[0014] Optionally, the molar ratio of ammonium formate to trimethylpyruvic acid is 5 to 6:1.

[0015] The coenzyme cycling system and its application provided in this application's embodiments are related to coenzyme NAD. + In the presence of formate dehydrogenase, ammonium formate is catalyzed to produce NH3 and NAD3. + It is reduced to the coenzyme NADH; in the presence of coenzyme NADH, the leucine dehydrogenase mutant uses the generated NH3 to asymmetricly reduce and amination the prochiral α-keto acid compound, 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 coenzyme cycle system in an embodiment of this application.

[0017] Figure 2 This is a schematic diagram of the reaction principle of the coenzyme cycle system in an embodiment of this application.

[0018] Figure 3 The results of the substrate addition ratio experiments for Examples 1-5 are shown in the figure.

[0019] Figure 4The results of the substrate addition ratio experiments for Examples 6-9 are shown in the figure.

[0020] Figure 5 The figure shows the experimental results of substrate addition ratio in Example 10.

[0021] Figure 6 The figure shows the experimental results of the addition ratio of recombinant engineered bacteria in Examples 11-15.

[0022] Figure 7 The figure shows the experimental results of the addition ratio of recombinant engineered bacteria in Examples 16-20. Detailed Implementation

[0023] 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.

[0024] 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.

[0025] 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.

[0026] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0027] 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.

[0028] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0029] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0030] 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.

[0031] The reaction principle for converting prochiral α-keto acids into L-amino acids using the aforementioned leucine dehydrogenase is shown below:

[0032] .

[0033] The description herein refers to "a polypeptide, protein, mutant, or enzyme having the amino acid sequence shown in SEQ ID NO: ". Obviously, a polypeptide, protein, mutant, or enzyme having the amino acid sequence shown in SEQ ID NO: , with some of its sequences deleted, modified, substituted, conservatively substituted, or added, can also be used in this application, as long as it exhibits the same or corresponding activity as the polypeptide, protein, mutant, or enzyme having the amino acid sequence shown in SEQ ID NO: . For example, it is not excluded to add a sequence that does not change the protein function, a naturally occurring mutation, its silent mutation, or a conservative substitution before or after "the polypeptide, protein, mutant, or enzyme having the amino acid sequence shown in SEQ ID NO: ". Furthermore, a polypeptide, protein, mutant, or enzyme having the amino acid sequence shown in SEQ ID NO: , when it is supplemented with the aforementioned sequence that does not change the protein function, a naturally occurring mutation, its silent mutation, or a conservative substitution, also falls within the scope of this application, as long as it exhibits the same or corresponding activity as the amino acid sequence shown in SEQ ID NO: .

[0034] One embodiment of this application provides a coenzyme cycling system, which includes NAD+. + Ammonium formate, formate dehydrogenase, and leucine dehydrogenase mutants, wherein the leucine dehydrogenase mutants have any of the amino acid sequences shown in SEQ ID NO: 1 to SEQ ID NO: 5.

[0035] 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. + In cells, it mainly acts as an electron acceptor, participating in a variety of redox reactions. NADH is the reduced state of nicotinamide adenine dinucleotide, which structurally contains an additional hydride (i.e., a negatively charged hydrogen atom).

[0036] Please see Figure 1 As shown, in the reaction of converting oxidized coenzyme to reduced coenzyme, ammonium formate acts as the substrate, and formate dehydrogenase acts as the catalyst. Utilizing the oxidative catalytic activity of formate dehydrogenase, ammonium formate is converted to NH3. During the oxidation of ammonium formate to form product NH3 under the catalysis of formate dehydrogenase, NAD... + It is converted into NADH.

[0037] 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 the NH3 product formed by the oxidation of ammonium formate 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. + .

[0038] This leucine dehydrogenase mutant produces at least one mutation in the amino acid sequence shown in SEQ ID NO: 6, which enhances the enzyme's catalytic activity 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 formate to NH3 catalyzed by formate dehydrogenase, as well as the conversion of the oxidized coenzyme to the reduced coenzyme, thus increasing the rates of NADH and NAD+ conversion. + The circulation speed increases.

[0039] 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.

[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 (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.

[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, 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.

[0042] 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.

[0043] 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.

[0044] 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 corresponding 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.

[0045] In one implementation, the leucine dehydrogenase mutant is derived from a first recombinant engineered bacterium containing the leucine dehydrogenase mutant encoding gene.

[0046] 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.

[0047] 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.

[0048] The first recombinant vector was transformed into a host cell to obtain the first recombinant engineered bacterium, enabling the synthesis of the leucine dehydrogenase mutant within this bacterium. The first recombinant engineered bacterium could be *Escherichia coli* (Escherichia coli). Escherichia ) genus, Erwinia ( Erwinia ) genus, Serratia ( Serratia ) genus, Providencia ( Providencia ) genus, Corynebacterium ( Corynebacterium ) genus or short bacilli ( Brevibacterium ) genus; for example, the host cell can be *Escherichia coli* (E. coli). Escherichia coli Bacillus subtilis ( Bacillus subtilis ), Corynebacterium glutamicum ( Corynebacterium glutamicum ) or Aspergillus oryzae ( Aspergillus oryzae ).

[0049] 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.

[0050] As one implementation method, formate dehydrogenase is derived from a second recombinant engineered bacterium containing a formate dehydrogenase encoding gene.

[0051] Specifically, a second recombinant vector capable of expressing formate dehydrogenase is constructed. This second recombinant vector comprises a polynucleotide encoding formate dehydrogenase, wherein the polynucleotide comprises the nucleotide sequence corresponding to the amino acid sequence of the aforementioned formate dehydrogenase. The polynucleotide is a DNA or RNA chain formed by the polymerization of several nucleotides. The polynucleotide only needs to encode the aforementioned formate dehydrogenase, and any nucleotide in the polynucleotide can be chemically modified.

[0052] The second recombinant vector is a DNA preparation containing a nucleic acid sequence of a polynucleotide encoding formate dehydrogenase, and may also contain a control sequence. In the second recombinant vector, the nucleic acid sequence of the polynucleotide encoding formate dehydrogenase is operatively linked to a suitable control sequence, so that formate dehydrogenase can be expressed in a suitable host.

[0053] The second recombinant vector was transformed into the host cell to obtain the second recombinant engineered bacteria, which enabled formate dehydrogenase to be synthesized in the second recombinant engineered bacteria.

[0054] In some embodiments, formate 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 above-mentioned second wet cell, or an immobilized cell prepared from the above-mentioned second wet cell.

[0055] In this embodiment, ammonium formate is added in batches to maintain the concentration ratio of formate dehydrogenase to ammonium formate in the reaction system within the above-mentioned range, which can improve the conversion rate of ammonium formate.

[0056] In one embodiment, the formate dehydrogenase has the amino acid sequence shown in SEQ ID NO: 7, and the mass ratio of the leucine dehydrogenase mutant to the formate dehydrogenase is 1:1.8 to 2.2.

[0057] In this embodiment, controlling the mass ratio of the leucine dehydrogenase mutant and the formate dehydrogenase having the amino acid sequence shown in SEQ ID NO: 7 within the above range is beneficial to improving the conversion efficiency of the prochiral α-keto acid compound substrate.

[0058] In one embodiment, the formate dehydrogenase has the amino acid sequence shown in SEQ ID NO: 8, and the mass ratio of the leucine dehydrogenase mutant to the formate dehydrogenase is 1:4.5 to 5.5.

[0059] In this embodiment, controlling the mass ratio of the leucine dehydrogenase mutant and the formate dehydrogenase having the amino acid sequence shown in SEQ ID NO: 8 within the above range is beneficial to improving the conversion efficiency of the prochiral α-keto acid compound substrate.

[0060] In one implementation, the coenzyme cycle system also includes NADH and / or pyridoxal phosphate.

[0061] Pyridoxal phosphate is a coenzyme for the amination of leucine dehydrogenase mutants, and can promote the asymmetric reductive amination of leucine dehydrogenase mutants.

[0062] One embodiment of this application provides a method for preparing L-tert-leucine using the above-described coenzyme cycling system, comprising the following steps:

[0063] The coenzyme cycle described above is brought into contact with trimethylpyruvate to convert trimethylpyruvate into L-tert-leucine.

[0064] Please see Figure 2 As shown, in the reaction of converting oxidized coenzyme to reduced coenzyme, ammonium formate acts as the substrate, and formate dehydrogenase acts as the catalyst. Utilizing the oxidative catalytic activity of formate dehydrogenase, ammonium formate is converted to NH3. During the oxidation of ammonium formate to form product NH3 under the catalysis of formate dehydrogenase, NAD... + It is converted into NADH.

[0065] 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 oxidation of ammonium formate (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. + .

[0066] In some embodiments, the total mass concentration of formate dehydrogenase and leucine dehydrogenase mutant in the reaction system can be 25 g / L to 35 g / L. For example, the total mass concentration of formate dehydrogenase and leucine dehydrogenase mutant can be 25 g / L, 27 g / L, 30 g / L, 33 g / L or 35 g / L.

[0067] In some embodiments, the molar ratio of ammonium formate to trimethylpyruvic acid in the reaction system is 5 to 6:1.

[0068] Preparation of leucine dehydrogenase mutants

[0069] 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.

[0070] 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.

[0071] Expression of leucine dehydrogenase mutant

[0072] 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.

[0073] b. Transfer the seed culture to 50 mL LB liquid medium (Kan+, 100 μg / mL) and incubate at 37 ℃ and 220 rpm on a shaker for reactivation.

[0074] 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 ℃ and 220 rpm on a shaker until the OD 600 is approximately 0.6-0.8.

[0075] d. Lower the temperature of the shaker to 16 ℃-18 ℃. After the temperature of the cultured bacterial solution has decreased, add isopropylthio-β-D-galactoside (IPTG) to a final concentration of 0.5 mM and induce expression for 14-16 h.

[0076] 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.

[0077] f. Remove the supernatant, add 30 mL of protein purification buffer, and resuspend the bacterial cells using a vortex mixer.

[0078] 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 (ensuring no solid particles remain). Transfer the resuspended cells to a 50 mL centrifuge tube and store at -80 °C.

[0079] Purification of leucine dehydrogenase mutant protein

[0080] 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 centrifuged at 12000 rpm for 10 min at 4 ℃. 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.

[0081] 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, followed by washing with 0.1 M NaOH for 3-5 column volumes, then washing with elution buffer for 3-5 column volumes, and finally washing with equilibration buffer until the detector parameters such as OD280, conductivity, and pH value stabilized.

[0082] 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.

[0083] d. Protein concentration: The collected target protein was concentrated using ultrafiltration membrane concentration method. The concentration was carried out using a 10 kDa protein concentration tube and centrifuged at 4 ℃ and 5000 rpm for 30 min.

[0084] e. Protein desalting: Dilute the concentrated protein with an appropriate amount of PBS buffer (20 mM, pH 7.0) and place it in a dialysis bag (molecular weight cutoff 8~14 kDa). Use 20 mM, pH 7.0 PBS dialysate and let it stand overnight at 4 ℃. The dialysate needs to be changed once during the process.

[0085] f. Storage of ion exchange chromatography columns: After use, the ion exchange chromatography column should be rinsed with 1 M NaOH for 3-5 column volumes, then rinsed with 20% ethanol, and stored in a refrigerator at 4 ℃.

[0086] Electrophoretic analysis of leucine dehydrogenase mutant protein

[0087] a. Protein sample processing: 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.

[0088] 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.

[0089] 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.

[0090] d. Gel image analysis: The stained and destained protein gels were photographed and saved using a gel imaging system.

[0091] Preparation of formate dehydrogenase

[0092] The gene encoding formate dehydrogenase can be called the FDH gene. The FDH gene contains the nucleotide sequence corresponding to the amino acid sequence of formate dehydrogenase. For example, the FDH gene contains the nucleotide sequence corresponding to the amino acid sequence shown in SEQ ID NO: 7 to SEQ ID NO: 8. The FDH gene is constructed in pET-28a plasmid to obtain the second recombinant vector.

[0093] The second recombinant vector is a pET-28a plasmid containing the FDH gene, hereinafter referred to as pET-28a-FDH; the host cell used in the various embodiments and comparative examples in this application is Escherichia coli.

[0094] Formate dehydrogenase expression

[0095] a. Transform pET-28a-FDH into Escherichia coli (e.g., competent E.coli-BL21 cells); pick a single clone of pET-28a-FDH 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.

[0096] b. Transfer the seed culture to 50 mL LB liquid medium (Kan+, 100 μg / mL) and incubate at 37 ℃ and 220 rpm on a shaker for reactivation.

[0097] 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 ℃ and 220 rpm on a shaker until the OD 600 is approximately 0.6-0.8.

[0098] d. Lower the temperature of the shaker to 16 ℃-18 ℃. After the temperature of the cultured bacterial solution has decreased, add isopropylthio-β-D-galactoside (IPTG) to a final concentration of 0.5 mM and induce expression for 14-16 h.

[0099] 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.

[0100] f. Remove the supernatant, add 30 mL of protein purification buffer, and resuspend the bacterial cells using a vortex mixer.

[0101] 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 (ensuring no solid particles remain). Transfer the resuspended cells to a 50 mL centrifuge tube and store at -80 °C.

[0102] Purification of formate dehydrogenase protein

[0103] 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 centrifuged at 12000 rpm for 10 min at 4 ℃. 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.

[0104] 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, followed by washing with 0.1 M NaOH for 3-5 column volumes, then washing with elution buffer for 3-5 column volumes, and finally washing with equilibration buffer until the detector parameters such as OD280, conductivity, and pH value stabilized.

[0105] 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.

[0106] d. Protein concentration: The collected target protein was concentrated using ultrafiltration membrane concentration method. The concentration was carried out using a 10 kDa protein concentration tube and centrifuged at 4 ℃ and 5000 rpm for 30 min.

[0107] e. Protein desalting: Dilute the concentrated protein with an appropriate amount of PBS buffer (20 mM, pH 7.0) and place it in a dialysis bag (molecular weight cutoff 8~14 kDa). Use 20 mM, pH 7.0 PBS dialysate and let it stand overnight at 4 ℃. The dialysate needs to be changed once during the process.

[0108] f. Storage of ion exchange chromatography columns: After use, the ion exchange chromatography column should be rinsed with 1 M NaOH for 3-5 column volumes, then rinsed with 20% ethanol, and stored in a refrigerator at 4 ℃.

[0109] Electrophoretic analysis of formate dehydrogenase protein

[0110] a. Protein sample processing: 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.

[0111] 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.

[0112] 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.

[0113] d. Gel image analysis: The stained and destained protein gels were photographed and saved using a gel imaging system.

[0114] Enzyme-catalyzed reaction system

[0115] In vitro enzyme catalytic reaction conditions:

[0116] Reaction buffer: Diammonium phosphate buffer at concentrations of 100mM, 200mM, and 300mM;

[0117] Reaction pH: pH 8, pH 8.5, pH 9;

[0118] The total concentration of ammonium formate and trimethylpyruvic acid is 100 g / L;

[0119] NAD + The concentrations were 1 g / L, 2 g / L, 3 g / L, 4 g / L, and 5 g / L;

[0120] The total amount of leucine dehydrogenase mutant and formate dehydrogenase added was 30 g / L;

[0121] Reaction temperatures: 30℃, 35℃, 40℃, 45℃;

[0122] Reaction time: 2h, 4h, 6h, 8h, 10h, 12h, 14h, 16h, 18h, 20h, 22h, or 24h;

[0123] The total reaction volume is 10 mL.

[0124] Substrate addition ratio experiment

[0125] The two substrates are ammonium formate and trimethylpyruvic acid.

[0126] Leucine dehydrogenase mutant and formate 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.

[0127] Examples 1-9 can be performed with the following method: the total concentration of ammonium formate and trimethylpyruvic acid in the reaction system is 100 g / L (added in batches: 35 g / L for 0 hours of reaction, 35 g / L for 2 hours of reaction, and 30 g / L for 6 hours of reaction), NAD + The concentration of the substrate was 2 g / L, and the total amount of leucine dehydrogenase mutant and formate dehydrogenase added was 30 g / L (10 g / L of leucine dehydrogenase mutant and 20 g / L of formate dehydrogenase). The leucine dehydrogenase mutant had the amino acid sequence shown in SEQ ID NO: 2, and the formate dehydrogenase had the amino acid sequence shown in SEQ ID NO: 7. The reaction was carried out at pH 8.5 and temperature 40℃ for 10 hours, and the total concentration of the two substrates was measured every 1 hour.

[0128] The molar ratios of ammonium formate and trimethylpyruvic acid in each embodiment are shown in Table 1:

[0129] Table 1. Molar ratio of ammonium formate to trimethylpyruvic acid

[0130]

[0131] Please see Figure 3 and Figure 4 As shown, the conversion of both substrates accelerated with increasing molar ratio of ammonium formate to trimethylpyruvic acid.

[0132] Example 10 can be performed with the following configuration: the total concentration of ammonium formate and trimethylpyruvate in the reaction system is 100 g / L (added in batches: 33.3 g / L for 0 hours of reaction, 33.3 g / L for 2 hours of reaction, and 33.3 g / L for 4.5 hours of reaction), and the molar ratio of ammonium formate to trimethylpyruvate is 6:1. NAD + The concentration of the substrate was 2 g / L, and the total amount of leucine dehydrogenase mutant and formate dehydrogenase added was 30 g / L (10 g / L of leucine dehydrogenase mutant and 20 g / L of formate dehydrogenase). The leucine dehydrogenase mutant had the amino acid sequence shown in SEQ ID NO: 2, and the formate dehydrogenase had the amino acid sequence shown in SEQ ID NO: 7. The reaction was carried out at pH 8.5 and temperature 40℃ for 10 hours, and the total concentration of the two substrates was measured every 1 hour.

[0133] Please see Figure 5 As shown, when the molar ratio of ammonium formate to trimethylpyruvic acid is 6:1, both substrates are consumed within 8 hours.

[0134] Recombinant engineered bacteria addition ratio experiment

[0135] Leucine dehydrogenase mutant and formate 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.

[0136] Examples 11-20 can be performed with the following configuration: the total concentration of ammonium formate and trimethylpyruvic acid in the reaction system is 100 g / L (added in batches: 35 g / L for 0 hours of reaction, 35 g / L for 2 hours of reaction, and 30 g / L for 6 hours of reaction), the molar ratio of ammonium formate to trimethylpyruvic acid is 5:1, and NAD... + The concentration of the substrate was 2 g / L, and the total amount of leucine dehydrogenase mutant and formate dehydrogenase added was 30 g / L. The leucine dehydrogenase mutant had the amino acid sequence shown in SEQ ID NO: 2, and the formate dehydrogenase had the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 8. The pH was 8.5, and the reaction was carried out at 40 °C for 10 hours. The total concentration of the two substrates was measured every 1 hour.

[0137] The mass fractions of the leucine dehydrogenase mutant and formate dehydrogenase in each embodiment are shown in Table 2:

[0138] Table 2. Mass ratio of leucine dehydrogenase mutant to formate dehydrogenase

[0139]

[0140] Please see Figure 6 As shown, the conversion rate of the two substrates was highest when the mass ratio of leucine dehydrogenase mutant to formate dehydrogenase (SEQ ID NO: 7) was 1:2.

[0141] Please see Figure 7 As shown, the conversion rate of the two substrates was highest when the mass ratio of leucine dehydrogenase mutant to formate dehydrogenase (SEQ ID NO: 8) was 1:5.

[0142] Comparative experiment on the activity of leucine dehydrogenase mutants

[0143] Leucine dehydrogenase mutant and formate 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.

[0144] Examples 21-25 and Comparative Example 1 can be performed with the following configuration: the total concentration of ammonium formate and trimethylpyruvate in the reaction system is 100 g / L (added in batches: 33.3 g / L for 0 hours of reaction, 33.3 g / L for 2 hours of reaction, and 33.3 g / L for 4.5 hours of reaction), and the molar ratio of ammonium formate to trimethylpyruvate is 6:1. NAD + The concentration of the substrate was 2 g / L, and the total amount of leucine dehydrogenase mutant and formate dehydrogenase added was 30 g / L (the amount of leucine dehydrogenase mutant added was 10 g / L, and the amount of formate dehydrogenase added was 20 g / L). The formate dehydrogenase had the amino acid sequence shown in SEQ ID NO: 7. The pH was 8.5, the reaction was carried out at 40 °C for 10 hours, and the total concentration of the two substrates was measured every 1 hour.

[0145] The leucine dehydrogenase mutants or leucine dehydrogenases in each embodiment and comparative example are shown in Table 3:

[0146] Table 3. Amino acid sequences of leucine dehydrogenase mutants or leucine dehydrogenases

[0147]

[0148] In Examples 21 to 25, the leucine dehydrogenase mutant completely transformed both substrates within 8 hours; in Comparative Example 1, the leucine dehydrogenase achieved a transformation rate of less than 40% for both substrates within 12 hours.

[0149] Compared with the wild-type leucine dehydrogenase used in Comparative Example 1, the catalytic activity of the leucine dehydrogenase mutants used in Examples 21 to 25 was improved.

[0150] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0151] 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. A coenzyme cycling system, characterized in that, Including NAD + Ammonium formate, formate dehydrogenase, and leucine dehydrogenase mutants, wherein the amino acid sequence of the leucine dehydrogenase mutant is shown in any one of SEQ ID NO: 1 to SEQ ID NO:

5.

2. The coenzyme cycling 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 coenzyme cycling 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 coenzyme cycling system according to claim 1, characterized in that, The formate dehydrogenase is derived from a second recombinant engineered bacterium containing a formate dehydrogenase encoding gene.

5. The coenzyme cycling system according to claim 4, characterized in that, The formate 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. The coenzyme cycling system according to claim 1, characterized in that, The amino acid sequence of the formate dehydrogenase is shown in SEQ ID NO: 7, and the mass ratio of the leucine dehydrogenase mutant to the formate dehydrogenase is 1:

2.

7. The coenzyme cycling system according to claim 1, characterized in that, The coenzyme cycle system also includes NADH and / or pyridoxal phosphate.

8. A method for preparing L-tert-leucine using the coenzyme cycling system as described in any one of claims 1 to 7, characterized in that, include: The coenzyme cycle system is contacted with trimethylpyruvate to convert the trimethylpyruvate into L-tert-leucine.

9. The method for preparing L-tert-leucine using a coenzyme cycle system according to claim 8, characterized in that, The molar ratio of ammonium formate to trimethylpyruvic acid is 5-6:1.

Images