A leucine dehydrogenase mutant and its application in producing L-2-aminobutyric acid

Abstract

The present invention discloses a leucine dehydrogenase mutant and its application in producing L-2-aminobutyric acid, belonging to the technical fields of enzyme engineering and microbial engineering. The leucine dehydrogenase mutant of the present invention is obtained by mutating the 125th valine of the leucine dehydrogenase shown in the amino acid sequence of SEQ ID NO.1 to methionine, 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 leucine dehydrogenase mutant of the present invention is improved, and the enzyme activity of the mutant enzyme is increased by 55% relative to the wild type. Under the condition of 300mM concentration of sodium 2-carbonylbutyrate, the conversion rate can reach more than 94% after 6h of reaction.

Description

Technical Field

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

[0002] Leucine dehydrogenase (LeuDH, EC1.4.1.9) is a coenzyme NADH-dependent oxidoreductase that can hydrogenate and reduce chiral keto acids to generate corresponding chiral non-natural amino acids, and has the advantages of mild reaction conditions, optically pure products and high conversion efficiency. In 1961, Sanwal et al. first cloned and expressed leucine dehydrogenase in Bacillus cereus. Subsequently, leucine dehydrogenase genes from different strains were cloned and heterologously expressed, which can provide more pathways for the biosynthesis of some chiral non-natural amino acids. Leucine dehydrogenase is a common oxidoreductase with a wide range of sources. Leucine dehydrogenases from different sources have different catalytic properties and functions. The reaction can be improved by constructing strains with high enzyme activity through genetic engineering.

[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. The (S)-2-aminobutyramide prepared by amidation is the 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 substance for the anti-binding drug butanol. It can be seen that its demand in the market in the future is huge, so a more efficient and low-cost synthesis method is urgently needed to meet market demand. There is no relevant report on the mutation of the starting amino acid of the present invention application-leucine dehydrogenase (LeuDH) derived from the microorganism Exiguobacterium sibiricum. Summary of the invention

[0004] To solve the above problems, the present invention provides a leucine dehydrogenase mutant with improved catalytic activity, wherein the 125th amino acid valine Val is mutated to methionine Met, and the activity of the wild-type leucine dehydrogenase is increased by 55%. Specifically, the enzyme activity of the leucine dehydrogenase mutant V125M reaches 33.33 U·mg -1 , while the enzyme activity of wild-type leucine dehydrogenase was only 21.50 U·mg -1 Therefore, the leucine dehydrogenase 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 a leucine dehydrogenase mutant, wherein the valine at position 125 of the leucine dehydrogenase with an amino acid sequence as shown in SEQ ID NO.1 is mutated to methionine to obtain a leucine dehydrogenase mutant V125M. Specifically, the amino acid sequence of the mutant V125M is shown in SEQ ID NO.3.

[0006] The second object of the present invention is to provide a gene encoding the leucine dehydrogenase mutant.

[0007] Furthermore, the nucleotide sequence of the gene encoding the leucine dehydrogenase 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 leucine dehydrogenase 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 a leucine dehydrogenase 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 leucine dehydrogenase 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 leucine dehydrogenase mutant;

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

[0016] (3) inoculating the host expressing the leucine dehydrogenase 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 leucine dehydrogenase mutant.

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

[0018] Val125-F:5'-TATTACCGCAGAAGAC Wlq AATACCACCGTTGCAG-3′;

[0019] Val125-R: 5'-CTGCAACGGTGGTATT Yj Y GTCTTCTGCGGTAATA-3'.

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

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

[0022] Furthermore, in step (2), the leucine dehydrogenase LeuDH 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 leucine dehydrogenase 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 leucine dehydrogenase mutant V125M.

[0025] A fifth object of the present invention is to provide the use of the above-mentioned leucine dehydrogenase 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 leucine dehydrogenase mutant as a catalyst to catalyze the conversion and produce L-2-aminobutyric acid.

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

[0028] Furthermore, the reaction system also contains coenzyme.

[0029] Furthermore, the coenzyme is NADP + , NADPH, NAD + and NADH.

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

[0031] Furthermore, in the reaction system, the concentration of 2-carbonylbutyrate is 20 to 1000 mM.

[0032] Furthermore, in the reaction system, the concentration of the coenzyme is 0.01-1M.

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

[0034] Furthermore, the buffer solution is an ammonium chloride aqueous buffer solution.

[0035] Furthermore, the concentration of the Tris-HCl buffer is 1 to 3 mol / L.

[0036] Furthermore, the reaction temperature is 20-40° C. and the pH is 8.0-10.0.

[0037] Beneficial effects of the present invention:

[0038] Based on the leucine dehydrogenase from the microorganism Exiguobacterium sibiricum, the present invention transforms the molecular structure of leucine dehydrogenase through semi-rational design and site-directed mutagenesis technology, and finally obtains a leucine dehydrogenase mutant V125M with improved catalytic activity. Incubated at 25°C, the enzyme activity of the leucine dehydrogenase mutant V125M is 33.33U mg -1 The wild-type enzyme activity is 21.50 U mg -1 The activity of the leucine dehydrogenase mutant V125M is 55% higher than that of the wild type. The leucine dehydrogenase mutant of the present invention has high catalytic efficiency in catalyzing sodium 2-carbonylbutyrate to produce L-2-aminobutyric acid. Therefore, the leucine dehydrogenase 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 conservative analysis of the starting amino acids of the present invention;

[0040] Figure 2 The leucine dehydrogenase (44 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 a highly enzymatically active LeuDH recombinant Escherichia coli, which expresses a leucine dehydrogenase having an amino acid sequence as shown in SEQ ID NO.1, and the nucleotide sequence of the leucine dehydrogenase shown in SEQ ID NO.1 is shown in SEQ ID NO.2. According to the uniprotKB database, the present invention selects non-redundant protein sequences with a length of 300-400 (quantity: 13051; date: 28 / 07 / 2020) from all sequences in the Glu / Leu / Phe / Val dehydrogenases family, and uses the "mafft-add" module on the MAFFT server to perform a structure-based alignment of these 13051 sequences, and uses the software to count the occurrence frequencies of all amino acids in the 13051 sequences (see the results). Figure 1 ), and based on this, the sites near the substrate pocket were mutated, and leucine dehydrogenase mutants with enhanced catalytic activity were obtained through screening from 96-well plates, laying a foundation for expanding the industrial application of leucine dehydrogenase.

[0043] The present invention provides a leucine dehydrogenase mutant with improved catalytic activity. The mutant takes the leucine dehydrogenase (LeuDH) from the microorganism Exiguobacterium sibiricum as a parent, and the 125th position is mutated, specifically, the leucine at the Val125 position in the parent is mutated to methionine Met, thereby obtaining a mutant enzyme named V125M.

[0044] 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.

[0045] The method for determining the activity of leucine dehydrogenase of the present invention is as follows:

[0046] The activity was measured in NH4CL / NH4OH buffer (2 M, pH 9.5) containing 20 mM substrate and 0.2 mM NADH, and the activity parameters were calculated from the consumption of NADH.

[0047] Under the above reaction conditions, the amount of enzyme required to produce 1 μmol NADH per minute was defined as 1 enzyme activity unit, i.e. 1 U.

[0048] Enzyme activity calculation formula: Enzyme activity (U) = EW × V × 10 3 / (6220×L);

[0049] EW is the change in absorbance within 1 min; V is the reaction volume (mL); 6220 is the molar extinction coefficient of NADH; L is the optical path distance (cm).

[0050] The culture medium involved in the embodiment is as follows:

[0051] 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.

[0052] TB liquid culture medium: yeast extract 24 g / L, trypsin 12 g / L, glycerol 4 mL / L; K2HPO4 72 mmol / L, KH2PO4 17 mmol / L, sterilization at 121°C for 20 min.

[0053] Example 1 96-well plate screening

[0054] According to the conservation of sites near the pocket, degenerate codon primers containing other possible amino acids were designed. Transformants on the plate were selected and cultured in 96 deep-well plates, each well containing 200 μL LB medium (kan concentration was 50 μg / mL), and then placed on a constant temperature shaker at 37°C and 250 rpm for shaking overnight. After that, 50 μL of seed solution was transferred from each well to another 96 deep-well plate corresponding to the position, each well containing 400 μL LB medium (kan concentration was 50 μg / mL). Shake and culture on a constant temperature shaker at 37°C and 250 rpm for 3-4 hours. Then, 50 μL 2mM IPTG was added to induce the expression of LeuDH, and the culture was shaken on a constant temperature shaker at 25°C and 250 rpm for 18 hours. 100 μL of the induced bacterial solution was taken to measure OD. Afterwards, the bacteria were collected by centrifugation in a high-throughput deep-well plate centrifuge (4°C, 1900x g, 5 min), and 200 μL of Tris-HCl buffer containing lysozyme and DNAseI was added to each well, and incubated at room temperature for 15 min. Finally, centrifugation was performed at 2600x g for 10 min, and the clarified supernatant was the crude enzyme solution. First, the crude enzyme solution was measured to select strains with improved crude enzyme activity from a large number of mutant strains, and then the pure enzyme was measured to reach an accurate conclusion.

[0055] Example 2 Expression and purification of mutant enzyme V125M

[0056] The recombinant E. coli was overexpressed in a shake flask. When the OD reached 0.6, IPTG with a final concentration of 0.3mM was added to induce expression, and then cultured in a constant temperature shaker for 16h (25°C, 180rpm). The supernatant was crushed on ice using an ultrasonic disruptor. After centrifugation at 4°C, 12000rpm for 20min, the supernatant was used for protein purification or extraction of crude enzyme solution. For protein purification, the supernatant after ultrafiltration through a 0.22μm filter membrane was loaded onto a Ni-NTA gel column, and eluted with a 20-500mM concentration gradient of imidazole in a Tris-HCl (500mM) buffer containing 500mM NaCl at a washing rate of 1mL / 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 filter column with MWCO 30000da. The purified enzyme was collected in a 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. Results 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.

[0057] Example 3 Determination of the activity of mutant enzyme V125M

[0058] At 25°C, 200 μL of the reaction system contains: a certain amount of pure enzyme, 20 mM substrate, 0.2 mM NADH, 2 M ammonium chloride buffer (pH 9.5). After 2 minutes of incubation, add appropriate enzyme solution to start the reaction for 1 minute and measure the change in absorbance at 340 nm. The enzyme activity is calculated based on the reduction in NADH.

[0059] Experimental results:

[0060] Table 1 Enzyme activity measurement of esi leucine dehydrogenase for substrate sodium 2-oxobutyrate

[0061]

[0062] Experimental conclusion: The activity of V125M is 55% higher than that of the wild type.

[0063] Example 4 Transformation Experiment

[0064] In a 20mL catalytic system, 0.02M NADH and 0.02-0.3M sodium 2-carbonylbutyrate (pre-adjusted to pH 8.5 with NaOH) were added, along with an appropriate amount of enzyme solution, and finally the reaction volume was supplemented to 20mL with 0.1M pH8.5 PBS buffer. 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.

[0065] 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.

[0066] Experimental results:

[0067] Table 2 Catalytic reaction under different concentrations of sodium 2-carbonylbutyrate

[0068]

[0069] Experimental conclusion: Under the condition of 300mM concentration of sodium 2-carbonylbutyrate, the conversion rate can reach more than 94% after 6h of reaction.

[0070] 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. A leucine dehydrogenase mutant, characterized in that: The amino acid sequence of the leucine dehydrogenase mutant is shown in SEQ ID NO.

3.

2. A gene encoding the leucine dehydrogenase mutant according to claim 1.

3. A recombinant plasmid carrying the gene according to claim 2. A host cell expressing the leucine dehydrogenase mutant according to claim 1.

5. The host cell according to claim 4, characterized in that: The host cell is a bacterium, a fungus, a plant cell or an animal cell.

6. Use of the leucine dehydrogenase mutant according to claim 1, the gene according to claim 2, the recombinant plasmid according to claim 3 or the host cell according to claim 4 or 5 in producing L-2-aminobutyric acid.

7. The use according to claim 6, characterized in that: The application is to use 2-carbonylbutyrate as a substrate and the leucine dehydrogenase mutant as a catalyst to catalyze the conversion and produce L-2-aminobutyric acid.

8. The use according to claim 7, characterized in that: The concentration of the substrate is 20-1000 mM.

9. The use according to claim 6, characterized in that: The reaction temperature is 20~40°C and the pH is 8.0~10.0.

Images