A ω-transaminase mutant and its application in producing L-2-aminobutyric acid

Abstract

The present invention discloses an ω-transaminase mutant and its application in producing L-2-aminobutyric acid, belonging to the technical fields of enzyme engineering and microbial engineering. The ω-transaminase mutant of the present invention is obtained by mutating the 153rd valine of the ω-transaminase shown in the amino acid sequence of SEQ ID NO.1 to alanine, the amino acid sequence of the mutant is shown in SEQ ID NO.3, and the nucleotide sequence is shown in SEQ ID NO.4. The catalytic activity of the ω-transaminase mutant of the present invention is improved, and the enzyme activity of the mutant enzyme is increased by 578% relative to the wild type. Under the condition of 300mM concentration of sodium 2-carbonylbutyrate, the conversion rate can reach more than 76% after 12h of reaction.

Description

Technical Field

[0001] The invention relates to an ω-aminotransferase mutant and application thereof in producing L-2-aminobutyric acid, belonging to the technical fields of enzyme engineering and microbial engineering. Background Art

[0002] Transaminases (TA) (EC 2.6.1.X) are a class of 5′-pyridoxal phosphate (PLP)-dependent enzymes. Scientists discovered transaminases as early as the middle of the last century, but it was not until the end of the last century that research on transaminases made significant progress based on the need for chiral synthesis, such as GW starting to use transaminases to prepare chiral amines with enantioselectivity. Transaminases can currently be divided into five major categories: D-transaminases (D-TA), ω-transaminases (ω-TA), aspartate transaminases (Asp-TA), aromatic transaminases (Aro-TA) and branched-chain transaminases (BC-TA). ω-transaminases (ω-TA) are naturally occurring enzymes that can mediate asymmetric reductive amination reactions of ketones. Compared with other amine oxidative dehydrogenases that can catalyze reductive amination, they have the advantages of not requiring cofactors and having high product selection specificity.

[0003] L-2-aminobutyric acid catalyzed by sodium 2-carbonylbutyrate is an important drug intermediate and is widely used in the fields of medicine, agriculture, and chemical industry. (S)-2-aminobutyramide prepared by amidation is a key prerequisite for the anti-epileptic drugs levetiracetam and brivaracetam, and has great use value and application scenarios in the market. In addition, (S)-2-aminobutanol produced by L-2-aminobutyric acid is an important precursor of the anti-binding drug butanol. It can be seen that there will be huge demand for it in the market in the future, so a more efficient and low-cost synthesis method is urgently needed to meet market demand. Summary of the invention

[0004] In order to solve the above problems, the present invention provides a ω-aminotransferase mutant with improved catalytic activity, wherein the 153rd amino acid valine Val is mutated to alanine Ala, and the activity of the mutant is increased by 578% compared with the wild-type ω-aminotransferase. Specifically, the enzyme activity of the ω-aminotransferase mutant V153A reaches 3.53 U·mg -1 , while the enzyme activity of wild-type ω-aminotransferase was only 0.52 U·mg -1 Therefore, the ω-aminotransferase mutant of the present invention performs well in the catalytic process and has industrial application value.

[0005] The first object of the present invention is to provide an ω-aminotransferase mutant, wherein the valine at position 153 of the ω-aminotransferase with an amino acid sequence as shown in SEQ ID NO.1 is mutated to alanine to obtain the ω-aminotransferase mutant V153A. Specifically, the amino acid sequence of the mutant V153A is shown in SEQ ID NO.3.

[0006] The second object of the present invention is to provide a gene encoding the ω-aminotransferase mutant.

[0007] Furthermore, the nucleotide sequence of the gene encoding the ω-aminotransferase mutant is shown in SEQ ID NO.4.

[0008] The third object of the present invention is to provide a recombinant plasmid carrying the gene.

[0009] Furthermore, the vector of the recombinant plasmid is pET-28a(+) plasmid, pET-28b(+) plasmid or pET-20b(+) plasmid.

[0010] The fourth object of the present invention is to provide a host cell expressing the ω-aminotransferase mutant.

[0011] Furthermore, the host cell is a bacterium, a fungus, a plant cell or an animal cell.

[0012] Furthermore, the bacteria is Escherichia coli, preferably Escherichia coli BL21 (DE3).

[0013] Furthermore, the present invention also provides a method for preparing an ω-aminotransferase mutant, comprising the following steps:

[0014] (1) designing mutation primers according to the mutation site, performing site-directed mutagenesis using a vector carrying a wild-type ω-transaminase encoding gene with an amino acid sequence as shown in SEQ ID NO.1 as a template, and constructing an expression vector containing a gene encoding a mutant ω-transaminase;

[0015] (2) transforming the expression vector containing the gene encoding the ω-transaminase mutant obtained in step (1) into a host;

[0016] (3) inoculating the host expressing the ω-aminotransferase mutant into a fermentation medium for fermentation to obtain a fermentation broth; centrifuging the fermentation broth to collect the bacterial cells; crushing the bacterial cells and centrifuging to obtain a cell crushing supernatant; extracting the cell crushing supernatant to obtain the ω-aminotransferase mutant.

[0017] Further, in step (1), the forward and reverse mutation primers are:

[0018] Val153-F: 5'-GAACGCGTATCATGGCGCGACCGCGGTGAGCGCG

[0019] Val153-R: 5'-CGCGCTCACCGCGGTCGCGCCATGATACGCGTTCC.

[0020] Furthermore, in step (1), the mutation site is determined based on the three-dimensional structural model and conservation of the starting amino acid ω-TA.

[0021] Furthermore, in step (2), pET28a(+) is used as a vector to prepare a recombinant plasmid pET28a(+)-ω-TA, and the recombinant plasmid pET28a(+)-ω-TA is used as a template for mutation.

[0022] Furthermore, in step (2), the ω-transaminase ω-TA is mutated using a whole plasmid PCR method.

[0023] Furthermore, in step (2), the successfully constructed mutant plasmid is introduced into the expression host E. coli BL21 (DE3), and the positive single clones after verification are selected in a 96-well plate for induced expression culture.

[0024] Furthermore, in step (3), the bacterial solution of the ω-aminotransferase mutant with the best activity is centrifuged, resuspended in a buffer solution, and then ultrasonically disrupted, and purified by nickel ion affinity chromatography to obtain the ω-aminotransferase mutant V153A.

[0025] The fifth object of the present invention is to provide the use of the above-mentioned ω-aminotransferase mutant, gene, recombinant plasmid or host cell in the production of L-2-aminobutyric acid.

[0026] Furthermore, the application is to use 2-carbonylbutyrate as a substrate and the ω-aminotransaminase mutant as a catalyst to catalyze the conversion and produce L-2-aminobutyric acid.

[0027] Further, specifically, the above-mentioned ω-transaminase mutant is added to a reaction system containing sodium 2-carbonylbutyrate to react to obtain a reaction solution; the reaction solution is extracted to obtain L-2-aminobutyric acid.

[0028] Furthermore, the reaction system also contains a cofactor.

[0029] Furthermore, the cofactor is PLP (pyridoxal phosphate).

[0030] Furthermore, the amount of the ω-aminotransferase mutant added to the reaction system is 1-10 kU / L.

[0031] Furthermore, in the reaction system, the concentration of sodium 2-carbonylbutyrate is 20 to 500 mM.

[0032] Furthermore, in the reaction system, the concentration of PLP is 5-15 mM.

[0033] Furthermore, the reaction system is a buffer solution containing sodium 2-carbonylbutyrate, cofactors, etc.

[0034] Furthermore, the buffer is PBS buffer.

[0035] Furthermore, the concentration of the PBS buffer is 1 mol / L.

[0036] Furthermore, the reaction temperature is 20-40°C and the pH is 7.8-8.2.

[0037] Beneficial effects of the present invention:

[0038] Based on the ω-aminotransferase from the microorganism Paracoccus pantotrophus, the present invention transforms the molecular structure of the ω-aminotransferase through semi-rational design and site-directed mutagenesis technology, and finally obtains the ω-aminotransferase mutant V153A with improved catalytic activity. Incubated at 30°C, the enzyme activity of the ω-aminotransferase mutant V153A is 3.53U mg -1 The wild-type enzyme activity is 0.52 U mg -1 The activity of the ω-aminotransferase mutant V153A is 578% higher than that of the wild type. The ω-aminotransferase mutant of the present invention has high catalytic efficiency in catalyzing sodium 2-carbonylbutyrate to produce L-2-aminobutyric acid. Therefore, the ω-aminotransferase mutant of the present invention has a very high application prospect in the production of L-2-aminobutyric acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 It is the active pocket and active amino acid residue of the starting amino acid of the present invention;

[0040] Figure 2 The ω-aminotransferase (50 kDa) of the present invention was purified by nickel column and then verified by SDS-PAGE. DETAILED DESCRIPTION

[0041] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0042] The present invention is based on an ω-TA recombinant Escherichia coli engineering bacterium with high enzyme activity, and the Escherichia coli expresses an ω-transaminase with an amino acid sequence as shown in SEQ ID NO.1, and the nucleotide sequence of the ω-transaminase shown in SEQ ID NO.1 is shown in SEQ ID NO.2. The present invention downloads the PDTA (PDB: 4GRX) three-dimensional protein model from the PDB protein database (https: / / www.rcsb.org / ), downloads the substrate and ligand three-dimensional structure (sodium 2-carbonylbutyrate, PLP) from PubChem (https: / / pubchem.ncbi.nlm.nih.gov / ), and uses chem3D to combine PLP with the ligand through a Schiff base to form a resonance structure and perform energy minimization. Pymol ligands are hydrogenated and protein receptors are dehydrated. AutoDock Tools are used to simulate the docking of protein receptors and ligands, and the results are imported into Pymol for modification. The distribution of the S and L pockets of ppTA is consistent with the basic structure of ω-transaminase. The S pocket of ppTA is composed of three key residues: V153, F321, and T322. The mutation optimization of these three amino acid residues is conducive to the expansion of the S pocket of ppTA to accommodate larger groups of substrates. Based on this, the mutation of sites near the substrate pocket was guided, and ω-transaminase mutants with enhanced catalytic activity were obtained from the 96-well plate screening, laying a foundation for broadening the industrial application of ω-transaminase.

[0043] The present invention provides an ω-aminase mutant with improved catalytic activity. The mutant uses an ω-aminase (ω-TA) derived from the microorganism Paracoccus pantotrophus as a parent, and the 153rd position is mutated, specifically, the leucine at the Val153 position in the parent is mutated to alanine Ala to obtain a mutant enzyme named V153A.

[0044] For the mutation at position 153, specifically, the valine at position Val153 in the parent was mutated to alanine Ala to obtain a mutant enzyme, which was named V153A.

[0045] The raw materials used in the present invention, unless otherwise specified, are conventional commercially available products; the methods used in the present invention, unless otherwise specified, are conventional methods in the art.

[0046] The method for determining the activity of ω-aminotransferase of the present invention is as follows:

[0047] The activity was measured in PBS buffer (pH 7.8) containing 20 mM substrate and 1 mM PLP, and the activity parameter was calculated from the amount of L-2-aminobutyric acid produced.

[0048] Under the above reaction conditions, the amount of enzyme required to produce 1 μmol of L-2-aminobutyric acid per minute was defined as 1 enzyme activity unit, i.e. 1U.

[0049] Enzyme activity calculation formula: Enzyme activity (U) = C × V1 × 10 3 / (T×V2×M);

[0050] C is the concentration of L-2-aminobutyric acid produced; V1 is the reaction volume (mL); T is the reaction time; V2 is the amount of protein added (mL).

[0051] The culture medium involved in the embodiment is as follows:

[0052] LB liquid medium: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl; pH 7.0, sterilization at 121°C for 20 min; LB solid medium is liquid medium with agar powder added at a mass concentration of 20 g / L.

[0053] TB medium: tryptone 20 g·L -1 , yeast extract 24g·L -1 , KH2PO4 0.17mol·L-1, K2HPO4 0.72mol·L -1 , 0.4% glycerol, sterilized at 121°C for 20 min, wherein phosphate was prepared into a high-concentration mother solution and sterilized separately, and TB was added to the required concentration in an ultra-clean workbench.

[0054] Example 1: Construction of V153A mutant strain

[0055] The method for preparing a site-directed mutant comprises the following steps: according to the sequence of the ω-aminase of Paracoccus pantotrophus (the amino acid sequence is shown in SEQ ID NO.1), respectively designing and synthesizing primers for introducing site-directed mutations, performing site-directed mutation on the V153A position of the ω-aminase, determining the DNA coding sequence, and respectively sequencing to confirm whether the coding gene of the ω-aminase mutant is correct; connecting the mutant gene to an expression vector pET28a and introducing it into Escherichia coli for expression, thereby obtaining the corresponding ω-aminase site-directed mutant.

[0056] PCR amplification of site-directed mutant encoding gene: PCR technology was used with expression vector pET28a as template. The mutation primers for introducing V153A site-directed mutation were:

[0057] Val153-F: 5'-GAACGCGTATCATGGCGCGACCGCGGTGAGCGCG-3'

[0058] Val153-R: 5'-CGCGCTCACCGCGGTCGCGCCATGATACGCGTTCC-3'

[0059] The PCR amplification program was set as follows: first, pre-denaturation at 98°C for 5 min; then 30 cycles: denaturation at 98°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 5 min; finally extension at 72°C for 10 min, and insulation at 4°C. PCR products were detected by 1% agarose gel electrophoresis.

[0060] After the PCR product was purified, DPn I was added, and the template was degraded by water bath at 37°C for 1 hour, and then E. coli JM109 was transformed, and positive clones were picked, cultured in LB liquid medium for 8-10 hours, and the glycerol tube was preserved and sent for sequencing. The mutant with correct sequencing (amino acid sequence as shown in SEQ ID NO.3, nucleotide sequence as shown in SEQ ID NO.4) was inoculated from the glycerol tube into LB medium, cultured overnight, and the plasmid was extracted. The plasmid was transformed into the expression host Escherichia coli BL21 (DE3) competent cells to obtain a recombinant strain capable of expressing the mutant V153A.

[0061] Example 2: Expression and purification of mutant enzyme V153

[0062] The recombinant Escherichia coli was overexpressed in a shake flask. When the OD reached 0.6, IPTG was added at a final concentration of 0.2 mM to induce expression, and then cultured in a constant temperature shaker for 12 hours (16°C, 180 rpm). The cells were broken using an ultrasonic disruptor on ice. After centrifugation at 4°C, 12000 rpm for 20 minutes, the supernatant was used for protein purification or extraction of crude enzyme solution. For protein purification, the supernatant after ultrafiltration through a 0.22 μm filter membrane was loaded onto a Ni-NTA gel column and eluted in a PBS (500 mM) buffer containing 500 mM NaCl using a 20-500 mM concentration gradient of imidazole at a flushing rate of 1 mL / min. Most of the protein was collected in the eluate. The eluate was analyzed by SDS gel electrophoresis. Imidazole was removed by ultrafiltration using a MWCO 30000da filter column. The purified enzyme was collected in phosphate buffer. The protein concentration of the purified enzyme solution was determined according to the Bradford method, and the protein purity was detected by SDS-PAGE. The results are shown in Figure 2 As can be seen from the figure, after obtaining pure enzyme with correct band and high purity, the enzyme activity is calculated by dividing the enzyme activity by the protein content. This is the sample preparation work before enzyme activity detection and is for future use.

[0063] Example 3: Determination of enzyme activity of mutant enzyme V153A

[0064] At 37°C, 1 mL of the reaction system contains: a certain amount of pure enzyme, 20 mM substrate, 1 mM PLP, and PBS buffer (pH 7.8). The reaction is started by adding the pure enzyme, and 1 / 10 volume of 1M hydrochloric acid is added after 30 minutes to terminate the reaction. The concentration of L-2-aminobutyric acid in the reaction solution is detected by high performance liquid chromatography, and the enzyme activity is calculated.

[0065] Experimental results:

[0066] Table 1 Enzyme activity measurement of ppTA for substrate sodium 2-oxobutyrate

[0067]

[0068] Experimental conclusion: The activity of V153A is 578% higher than that of the wild type.

[0069] Example 4: Transformation Experiment

[0070] In a 20mL catalytic system, 1mM PLP and 0.02-0.3M sodium 2-carbonylbutyrate (pre-adjusted to pH 7.8 with NaOH), an appropriate amount of enzyme solution, and finally 0.1M pH8.5 PBS buffer was used to make up the reaction volume to 20mL. The reaction temperature was 30°C and the speed was 180rpm. The reaction solution was taken at different time periods to detect substrate conversion and product generation. The effect of substrate concentration on the synthesis of L-2-aminobutyric acid was also explored.

[0071] The content of L-2-aminobutyric acid and sodium 2-carbonylbutyrate in the reaction solution was detected by high performance liquid chromatography. The determination method of L-2-aminobutyric acid was as follows: amino acid pre-column online derivatization HPLC method, using Agilent high performance liquid chromatograph 1260, and the chromatographic column was Diamonsil C18 (5μm, 4.6mm×250mm). The mobile phase A was a buffer containing 10mmol·L-1Na2HPO4 and 10mmol·L-1Na2B4O7, HCl adjusted pH 8.2, and the mobile phase B was an organic mixed phase (methanol: acetonitrile: water = 45:45:10). The column oven was 40℃, the flow rate was 1mL·min-1, the VWD detection wavelength was 338nm, and the injection volume was 10μL. The derivatization positions 1-4 were boric acid, water, OPA and water, respectively. The determination method of sodium 2-carbonylbutyrate is as follows: Agilent high performance liquid chromatograph 1260 is used, the chromatographic column is Bio-rad (5 μm, 4.6 mm×250 mm), the mobile phase is 0.05 mM dilute sulfuric acid, the column temperature is 25°C, the flow rate is 0.8 mL·min-1, the VWD detection wavelength is 254 nm, and the injection volume is 10 μL.

[0072] Experimental results:

[0073] Table 2 Catalytic reaction under different concentrations of sodium 2-carbonylbutyrate

[0074]

[0075] Experimental conclusion: Under the condition of 300mM concentration of sodium 2-carbonylbutyrate, the conversion rate can reach more than 76% after 12h of reaction.

[0076] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. An application of an ω-aminotransferase mutant in the production of L-2-aminobutyric acid, characterized in that: The amino acid sequence of the ω-aminotransferase mutant is shown in SEQ ID NO.3, and the application is to use 2-carbonylbutyrate as a substrate and the ω-aminotransferase mutant as a catalyst to catalyze the conversion and produce L-2-aminobutyric acid.

2. The use according to claim 1, characterized in that: The nucleotide sequence of the gene encoding the ω-aminotransferase mutant is shown in SEQ ID NO.

4.

3. The use according to claim 1, characterized in that: The concentration of the substrate is 20-500 mmol / L.

4. The use according to claim 1, characterized in that: The reaction temperature is 20-40°C and the pH is 7.8-8.2.

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