CN116064445A - Leucine dehydrogenase mutant and application thereof in production of L-2-aminobutyric acid - Google Patents
Leucine dehydrogenase mutant and application thereof in production of L-2-aminobutyric acid Download PDFInfo
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Abstract
The invention discloses a leucine dehydrogenase mutant and application thereof in producing L-2-aminobutyric acid, and belongs to the technical fields of enzyme engineering and microbial engineering. The leucine dehydrogenase mutant is obtained by mutating valine at position 125 of leucine dehydrogenase with an amino acid sequence shown as SEQ ID NO.1 into methionine, wherein the amino acid sequence of the mutant is shown as SEQ ID NO.3, and the nucleotide sequence of the mutant is shown as SEQ ID NO. 4. The catalytic activity of the leucine dehydrogenase mutant is improved, and compared with a wild type, the enzymatic activity of the mutant enzyme is improved by 55%. Under the condition of 300mM concentration of 2-carbonyl sodium butyrate, the conversion rate can reach more than 94% after 6 hours of reaction.
Description
Technical Field
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
Leucine dehydrogenase (Leucine dehydrogenase, leuDH, EC1.4.1.9) is a coenzyme NADH dependent oxidoreductase, which can hydrogenate and reduce chiral keto acids to generate corresponding chiral unnatural amino acids, and has the advantages of mild reaction conditions, optically pure products, high conversion efficiency and the like. In 1961, sanwal et al first cloned and expressed leucine dehydrogenase in Bacillus cereus, and subsequently cloned and heterologously expressed leucine dehydrogenase genes derived from different strains, which could provide more ways for biosynthesis of some chiral unnatural amino acids. Leucine dehydrogenase is a common oxidoreductase and is widely available. The two-shore acid dehydrogenases with different sources have different catalytic properties and functions, and the reaction can be improved by constructing a strain with high enzyme activity through a genetic engineering means.
The L-2-aminobutyric acid catalyzed by the sodium 2-carbonyl butyrate is an important drug intermediate and is widely applied to the fields of medicine, agriculture, chemical industry and the like. The (S) -2 aminobutanamide prepared by amidation is a key precondition of antiepileptic drugs levetiracetam and brivaracetam, and has great use value and application scene in the market. In addition, (S) -2-aminobutanol produced by L-2-aminobutyric acid is an important precursor substance of anti-binding drug butanol. It is seen that there is a great demand in the market in the future, and thus there is an urgent need for a more efficient and cost-effective synthesis method to meet the market demand. There is no report on mutation of leucine dehydrogenase (LeuDH) derived from microorganism Exiguobacterium sibiricum, which is an amino acid starting from the present application.
Disclosure of Invention
In order to solve the above problems, the present invention provides a leucine dehydrogenase mutant with improved catalytic activity, which mutates valine Val at position 125 of a starting amino acid into methionine Met, and has an activity improved by 55% relative to that of a wild-type leucine dehydrogenase, specifically, the leucine dehydrogenase mutant V125M has an enzyme activity reaching 33.33 U.mg -1 Whereas the enzyme activity of the wild leucine dehydrogenase is only 21.50U.mg -1 Thus, the leucine dehydrogenase mutant of the present invention is catalyticThe method has good performance in the process and has industrial application value.
The first object of the invention is to provide a leucine dehydrogenase mutant, which is obtained by mutating valine at position 125 of leucine dehydrogenase with an amino acid sequence shown as SEQ ID NO.1 into methionine. Specifically, the amino acid sequence of the mutant V125M is shown as SEQ ID NO. 3.
A second object of the present invention is to provide a gene encoding the leucine dehydrogenase mutant.
Further, the nucleotide sequence of the gene for encoding the leucine dehydrogenase mutant is shown as SEQ ID NO. 4.
A third object of the present invention is to provide a recombinant plasmid carrying the gene.
Further, the recombinant plasmid vector is pET-28a (+) plasmid, pET-28b (+) plasmid or pET-20b (+) plasmid.
It is a fourth object of the present invention to provide host cells expressing the leucine dehydrogenase mutants.
Further, the host cell is a bacterial, fungal, plant cell or animal cell.
Further, the bacterium is E.coli, preferably E.coli BL21 (DE 3).
Further, the invention also provides a preparation method of the leucine dehydrogenase mutant, which comprises the following steps:
(1) Designing a mutation primer according to a mutation site, carrying out site-directed mutagenesis by taking a carrier carrying a wild leucine dehydrogenase encoding gene with an amino acid sequence shown as SEQ ID NO.1 as a template, and constructing an expression carrier containing the leucine dehydrogenase encoding mutant gene;
(2) Transforming the expression vector containing the gene for encoding the leucine dehydrogenase mutant obtained in the step (1) into a host;
(3) Inoculating the host expressing the leucine dehydrogenase mutant into a fermentation medium for fermentation to obtain fermentation liquor; centrifuging the fermentation liquor, and collecting thalli; crushing thalli, and centrifuging to obtain cell crushing supernatant; and extracting cell disruption supernatant to obtain the leucine dehydrogenase mutant.
Further, in step (1), the forward and reverse mutation primers are:
Val125-F：5’-TATTACCGCAGAAGACWYRAATACCACCGTTGCAG-3’；
Val125-R：5’-CTGCAACGGTGGTATTYRWGTCTTCTGCGGTAATA-3’。
further, in step (1), the mutation site is determined on the basis of a three-dimensional structural model of the starting amino acid LeuDH and conservation.
Further, in the step (2), a recombinant plasmid pET28a (+) -LeuDH is prepared by taking pET28a (+) as a vector, and the recombinant plasmid pET28a (+) -LeuDH is used as a template for mutation.
Further, in step (2), leucine dehydrogenase LeuDH was mutated using a whole plasmid PCR method.
Further, in the step (2), the constructed mutant plasmid is introduced into an expression host E.coli BL21 (DE 3), and positive monoclonal clones after verification are selected from a 96-well plate for induction expression culture.
Further, in the step (3), the leucine dehydrogenase mutant with the best activity is centrifuged, the bacterial solution is resuspended by using a buffer solution and then subjected to ultrasonic disruption, and the leucine dehydrogenase mutant V125M is obtained by purifying by nickel ion affinity chromatography.
A fifth object of the present invention is to provide the use of the leucine dehydrogenase mutant, gene, recombinant plasmid or host cell described above for the production of L-2-aminobutyric acid.
Furthermore, the application is to produce L-2-aminobutyric acid by taking 2-carbonyl butyrate as a substrate and taking the leucine dehydrogenase mutant as a catalyst for catalytic conversion.
Further, specifically, the leucine dehydrogenase mutant is added into a reaction system containing sodium 2-carbonyl butyrate for reaction to obtain a reaction solution; extracting the reaction liquid to obtain the L-2-aminobutyric acid.
Further, the reaction system also contains coenzyme.
Further, the coenzyme is NADP + 、NADPH、NAD + And NADH.
Further, the addition amount of the leucine dehydrogenase mutant in a reaction system is 1-10 kU/L.
Further, the concentration of 2-carbonyl butyrate in the reaction system is 20 to 1000mM.
Further, the concentration of the coenzyme in the reaction system is 0.01 to 1M.
Further, the reaction system is a buffer solution containing sodium 2-carbonyl butyrate, coenzyme and the like.
Further, the buffer solution is an ammonium chloride ammonia water buffer solution.
Further, the concentration of the Tris-HCl buffer solution is 1-3 mol/L.
Further, the reaction temperature is 20-40 ℃ and the pH is 8.0-10.0.
The invention has the beneficial effects that:
the invention modifies the molecular structure of leucine dehydrogenase by semi-rational design and site-directed mutagenesis technology on the basis of leucine dehydrogenase from microorganism Exiguobacterium sibiricum, and finally obtains leucine dehydrogenase mutant V125M with improved catalytic activity. Incubation at 25℃and the leucine dehydrogenase mutant V125M had an enzyme activity of 33.33U mg -1 The enzyme activity of the wild type is 21.50U mg -1 The activity of leucine dehydrogenase mutant V125M is improved by 55% compared with the wild type. The leucine dehydrogenase mutant has high catalytic efficiency in catalyzing sodium 2-carbonyl butyrate to produce L-2-aminobutyric acid, so that the leucine dehydrogenase mutant has extremely high application prospect in producing L-2-aminobutyric acid.
Drawings
FIG. 1 is a conservative analysis of the amino acids of the present invention;
FIG. 2 shows SDS-PAGE of the leucine dehydrogenase of the present invention (44 kDa) after purification by a nickel column.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.
The invention is based on a high-enzyme activity LeuDH recombinant escherichia coli, wherein the recombinant escherichia coli expresses leucine dehydrogenase with an amino acid sequence shown as SEQ ID NO.1, and the nucleotide sequence of the leucine dehydrogenase shown as SEQ ID NO.1 is shown as SEQ ID NO. 2. According to the uniprotKB database, the invention selects a sequence (number: 13051; date: 28/07/2020) with a length of 300-400 and no redundant protein from all sequences in Glu/Leu/Phe/Val dehydrogenases family, compares 13051 sequences in a MAFFT server by using a 'MAFFT-add' module based on structure, uses software to count the occurrence frequency of all amino acids in 13051 sequences at all sites (the result is shown in figure 1), uses the result as the basis to guide mutation of sites near a substrate pocket, and screens from 96 pore plates to obtain a leucine dehydrogenase mutant with enhanced catalytic activity, thereby laying a foundation for widening industrial application of leucine dehydrogenase.
The invention provides a leucine dehydrogenase mutant with improved catalytic activity, which takes leucine dehydrogenase (LeuDH) from microorganism Exiguobacterium sibiricum as a parent, and the 125 th mutation, in particular to the mutation of Val125 th leucine in the parent into methionine Met, so as to obtain mutant enzyme, which is named as V125M.
The raw materials used in the invention are all conventional commercial products unless specified; the methods used in the present invention are conventional in the art unless otherwise specified.
The leucine dehydrogenase activity determination method comprises the following steps:
activity is measured on NH containing 20mM substrate and 0.2mM NADH 4 CL/NH 4 The reaction was carried out in an OH buffer (2M, pH 9.5) and the activity parameter was calculated from the consumption of NADH.
Under the above reaction conditions, the amount of enzyme required to produce 1. Mu. Mol of NADH per minute is defined as 1 enzyme activity unit, i.e., 1U.
The enzyme activity calculation formula: enzyme activity(U)＝EW×V×10 3 /(6220×L)；
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).
The media involved in the examples are as follows:
LB liquid medium: 5g/L of yeast extract, 10g/L of tryptone and 10g/L of NaCl; sterilizing at 121deg.C for 20min at pH 7.0; the LB solid culture medium is agar powder with the mass concentration of 20g/L added on the basis of the liquid culture medium.
TB liquid medium: 24g/L of yeast extract, 12g/L of tryptone and 4mL/L of glycerol; k (K) 2 HPO 4 72mmol/L，KH 2 PO 4 17mmol/L, and sterilizing at 121deg.C for 20min.
EXAMPLE 196 well plate screening
Based on the conservation of the site near the pocket, degenerate codon primers containing other possible amino acids were designed, transformants on selected plates were grown in 96-well plates with 200. Mu.L LB medium (kan concentration 50. Mu.g/mL) per well and then placed on a constant temperature shaker at 37℃and 250rpm with shaking overnight. After that, 50. Mu.L of the seed solution was aspirated from each well and transferred to a 96-well plate corresponding to another position, and each well contained 400. Mu.L of LB medium (kan concentration: 50. Mu.g/mL). Shake culturing at 37deg.C and 250rpm on a constant temperature shaker for 3-4 hr. Next, 50. Mu.L of 2mM IPTG was added to induce the expression of LeuDH, and the mixture was shake-cultured on a thermostatic shaker at 25℃and 250rpm for 18 hours. After induction, 100. Mu.L of the bacterial liquid was aspirated to measure OD. After centrifugation in a high throughput deep well plate centrifuge (4 ℃,190 Xg, 5 min), 200. Mu.L of Tris-HCl buffer containing lysozyme and DNAseI was added to each well and incubated for 15min at room temperature. Finally 2600 and x g are centrifugated for 10min, and the clarified supernatant is crude enzyme liquid. Firstly, a strain with improved enzyme activity of crude enzyme is selected from a large number of mutant strains through measurement of crude enzyme liquid, and then an accurate conclusion is obtained through measurement of pure enzyme.
EXAMPLE 2 expression and purification of mutant enzyme V125M
Recombinant E.coli was overexpressed in shake flasks, induced by addition of IPTG at a final concentration of 0.3mM when OD reached 0.6, and then shake-cultured at constant temperature for 16h (25 ℃,180 rpm). Crushing on ice using an ultrasonic crusher. After that, the mixture was centrifuged at 12000rpm at 4℃for 20 minutes, and the supernatant was used for protein purification or crude enzyme extraction. For protein purification, the supernatant after ultrafiltration through a 0.22 μm membrane was loaded onto a Ni-NTA gel column, eluted with a 20-500mM concentration gradient of imidazole in Tris-HCl (500 mM) buffer containing 500mM NaCl at a washing rate of 1mL/min, and the majority of the protein was collected in the eluate. The eluate was analyzed by SDS gel electrophoresis. The imidazole was ultrafiltered out using a filter column of MWCO 30000 da. 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. As a result, as can be seen from FIG. 2, when pure enzyme with correct band and high purity is obtained, the calculation of enzyme activity is obtained by dividing the enzyme activity unit by the protein content, and the sample preparation before enzyme activity detection is performed for standby.
Example 3 enzyme Activity assay for mutant enzyme V125M
At a temperature of 25 ℃,200 μl of the reaction system comprises: an amount of pure enzyme, 20mM substrate, 0.2mM NADH,2M ammonium chloride ammonia buffer (pH 9.5). After 2min incubation, the reaction was started by adding an appropriate enzyme solution for 1min and the change in absorbance at 340nm was measured. The enzyme activity was calculated from the decrease in NADH.
Experimental results:
TABLE 1 enzyme Activity measurement of esi leucine dehydrogenase on substrate sodium 2-carbonyl butyrate
Conclusion of experiment: the activity of V125M is improved by 55% compared with the wild type.
Example 4 transformation experiment
And respectively adding 0.02M NADH and 0.02-0.3M sodium 2-carbonyl butyrate (pH is adjusted to 8.5 by NaOH) into 20mL of a catalytic system, adding a proper amount of enzyme solution, and finally supplementing the reaction volume to 20mL by using 0.1M PBS buffer solution with pH of 8.5, wherein the reaction temperature is 30 ℃, the rotating speed is 180rpm, taking the reaction solution in different time periods, and detecting substrate conversion and product generation conditions. And the effect of substrate concentration on the synthesis of L-2-aminobutyric acid was investigated.
And detecting the content of L-2-aminobutyric acid and the content of sodium 2-carbonyl butyrate in the reaction liquid by using a high performance liquid chromatograph. The method for measuring L-2-aminobutyric acid comprises the following steps: the amino acid pre-column on-line derivatization HPLC method employs Agilent high performance liquid chromatograph 1260, with a column of Diamonsil C18 (5 μm,4.6 mm. Times.250 mm). The mobile phase A is a buffer solution containing 10 mmol.L-1 Na2HPO4 and 10 mmol.L-1 Na2B4O7, the pH value of the buffer solution is adjusted to 8.2 by HCl, and the mobile phase B is an organic mixed phase (methanol: acetonitrile: water=45:45:10). The column temperature box is 40 ℃, the flow rate is 1mL min < -1 >, the detection wavelength of VWD is 338nm, and the sample injection amount is 10 mu L. The derivative positions 1-4 are boric acid, water, OPA and water in sequence. The method for measuring the sodium 2-carbonyl butyrate comprises the following steps: the column was Bio-rad (5 μm,4.6 mm. Times.250 mm) using an Agilent high performance liquid chromatograph 1260. The mobile phase was 0.05mM dilute sulfuric acid. The temperature of the column is 25 ℃, the flow rate is 0.8mL min < -1 >, the detection wavelength of VWD is 254nm, and the sample injection amount is 10 mu L.
Experimental results:
TABLE 2 catalytic reactions at different sodium 2-carbonyl butyrate concentrations
Conclusion of experiment: under the condition of 300mM concentration of 2-carbonyl sodium butyrate, the conversion rate can reach more than 94% after 6 hours of reaction.
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.
Claims (10)
1. A leucine dehydrogenase mutant, characterized in that: the amino acid sequence of the leucine dehydrogenase mutant is shown as SEQ ID NO. 3.
2. A gene encoding the leucine dehydrogenase mutant of claim 1.
3. The gene according to claim 2, characterized in that: the nucleotide sequence of the gene is shown as SEQ ID NO. 4.
4. A recombinant plasmid carrying the gene of claim 2 or 3.
5. A host cell expressing the leucine dehydrogenase mutant of claim 1.
6. The host cell of claim 5, wherein: the host cell is a bacterial, fungal, plant cell or animal cell.
7. Use of the leucine dehydrogenase mutant of claim 1, the gene of claim 2 or 3, the recombinant plasmid of claim 4 or the host cell of claim 5 or 6 for the production of L-2-aminobutyric acid.
8. The use according to claim 7, characterized in that: the application is to produce L-2-aminobutyric acid by using 2-carbonyl butyrate as a substrate and using the leucine dehydrogenase mutant as a catalyst for catalytic conversion.
9. The use according to claim 8, characterized in that: the concentration of the substrate is 20-1000 mM.
10. The use according to claim 7, characterized in that: the reaction temperature is 20-40 ℃ and the pH is 8.0-10.0.
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