An O-succinylmercaptolyase mutant, encoding gene and application thereof

Abstract

The present invention belongs to the field of biotechnology, and specifically relates to an O-succinylhomoserine sulfhydrolase mutant, its encoding gene, and its application in the biocatalytic preparation of L-methionine. The mutant is obtained by single-point mutation or multi-point mutation at the 59th position and / or the 125th position of the amino acid sequence shown in SEQ ID NO.2. The beneficial effects of the present invention are mainly reflected in: the present invention provides an OSHS mutant with high conversion rate. Using the crude enzyme solution obtained by disrupting the recombinant bacteria containing the OSHS mutant as a catalyst, using O-succinyl-L-homoserine as a substrate, and sodium methanethiolate as a sulfhydryl donor to synthesize L-methionine, the industrial cost is relatively low; under the optimal system, the conversion rate for substrates of 400-800 mM reaches 80%-85%. Compared with the conversion rate of 71% of the wild-type substrate, it is increased by 13%-20% respectively, and it has the potential for large-scale application, and has extremely high application value in the industrial production of L-methionine.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an O-succinylthiol lyase mutant and a coding gene thereof, and application of the mutant in biocatalytic preparation of L-methionine. Background Art

[0002] L-methionine is one of the sulfur-containing "essential amino acids" for the human body. It participates in the transfer of methyl groups and the metabolism of phosphorus in animals, as well as the synthesis of adrenaline, choline and creatine, and the synthesis of protein and cystine. It is widely used in medicine, health products, food, cosmetics and feed industries. China's demand for methionine was about 270,000 tons in 2019 and about 300,000 tons in 2020, which cannot meet market demand.

[0003] Among the reported L-methionine synthesis technology routes, the chemical method has harsh production conditions and strong substrate toxicity; the biological fermentation method has advantages such as green economy. However, the metabolic synthesis pathway of L-methionine in microorganisms is complex and is affected by multi-level regulation. Therefore, it is difficult to realize the industrial application of biological fermentation to produce L-methionine.

[0004] Fermentation-enzyme coupling can efficiently produce L-methionine. The fermentation production of O-succinyl-L-homoserine is combined with the enzyme-catalyzed production of L-methionine by O-succinyl-L-homoserine, and a two-step production model of L-methionine is proposed. Based on this model, an L-methionine synthesis route is constructed. Using the fermentation broth of the constructed strain and sodium methyl mercaptan as substrates, O-succinyl-L-homoserine sulfhydryl lyase crude enzyme liquid is used to catalyze the production of L-methionine, and high-yield L-methionine can be obtained. Compared with chemical and biological fermentation methods, it has the advantages of being green, efficient, cheap and easy to obtain.

[0005] In order to maximize the cost-effectiveness of industrial production, enzymes need to meet several requirements. First, the enzyme should have the characteristics of high activity and high conversion rate. Secondly, the enzyme should have thermal stability to maintain its activity during the reaction period of 1 to 5 hours. Finally, the enzyme needs to maintain a high reaction rate with a high concentration of thiol donors, so it is necessary to screen suitable thiol donors, which should have low inhibition on enzyme activity while ensuring the substrate conversion rate. Using molecular biological methods to transform the genes of enzymes and thus change the structure of enzyme molecules, the stability of the enzyme can be enhanced and the enzyme activity can be improved. Screening out suitable thiol donors can reduce the inhibition of thiol toxicity on cells and even the entire catalytic reaction system, reduce the impact on the product, and achieve the requirements of maximizing the cost-effectiveness of industrial production. In 2013, CJ CheilJedang Co., Ltd. in South Korea discovered the Mycelium monocytogenes (Hyphomonas neptunium)O-acetylhomoserine sulfhydrylase is applied to the biocatalytic reaction for synthesizing L-methionine from O-acetylhomoserine, and the substrate conversion rate is increased to 80% (US 9029105 B2). Li Renxiang et al. published a novel O-acetylhomoserine sulfhydrylase variant and a method for producing L-methionine, and the maximum conversion rate of O-acetylhomoserine to L-methionine is 86% (CN108138205A).

[0006] At present, the research on mercapto lyase at home and abroad has problems such as fewer enzyme sources, unsatisfactory catalytic activity, and poor stability, which to a certain extent limit the application of mercapto lyase in the production of L-methionine. Summary of the Invention

[0007] The object of the present invention is to provide an O-succinyl mercapto lyase mutant, its coding gene, and its application in the biocatalytic preparation of L-methionine in view of the above deficiencies. The mutant has high enzyme activity, good stability, and strong organic solvent tolerance, and uses O-succinyl-L-homoserine and sodium methanethiolate as substrates and pyridoxal 5'-phosphate as a cofactor to achieve the efficient preparation of L-methionine.

[0008] The technical solution adopted by the present invention is as follows:

[0009] An O-succinyl mercapto lyase mutant is obtained by single-point mutation at the 58th or 124th position of the amino acid sequence shown in SEQ ID NO.2.

[0010] Preferably, the amino acid sequence of the mutant is as shown in SEQ ID NO.3 or SEQ ID NO.4.

[0011] The O-succinyl mercapto lyase mutant of the present invention is directed to the original OSHS sequence, and the nucleotide sequence of OSHS is mutated using the saturation mutation method. After the obtained amplification product is purified, it is induced to express in Escherichia coli, and mutants with improved activity are obtained by screening to improve its catalytic activity towards O-succinyl-L-homoserine.

[0012] Due to the particularity of the amino acid sequence, any fragment or variant of the peptide protein containing the amino acid sequence shown in the present invention, such as its conservative variant, bioactive fragment or derivative, as long as the homology of the fragment or peptide protein variant of the peptide protein to the aforementioned amino acid sequence is more than 90%, belongs to the scope of protection of the present invention. Specifically, the changes include deletion, insertion or substitution of amino acids in the amino acid sequence; among them, for conservative changes of the variant, the substituted amino acid has a structure or chemical property similar to the original amino acid, such as substituting serine with alanine.

[0013] The present invention also relates to a gene encoding the O-succinylhomoserine sulfhydrylase mutant, as well as a recombinant vector and a genetically engineered bacterium containing the encoding gene.

[0014] The vector can be various conventional vectors in the art, such as various plasmids, phages or viral vectors, etc., and preferably pET-28b is better. There is no limitation on the host cell into which the DNA encoding the mutant OSHS of the present invention is introduced, as long as a recombinant expression system has been established for it, and it satisfies that the recombinant expression vector can stably self-replicate and the OSHS mutant gene carried by it can be effectively expressed. For example, Escherichia coli, Bacillus subtilis, yeast, actinomycetes, Aspergillus, as well as animal cells and higher plant cells. The present invention preferably uses Escherichia coli, and more preferably Escherichia coli E. coli BL21 (DE3). The recombinant plasmid pET28b-OSHS m is transformed into E. coli BL21 (DE3) to obtain the engineered bacterium E. coli BL21 (DE3) / pET28b-OSHS m . m

[0015] The present invention induces and cultures the recombinant Escherichia coli containing the OSHS mutant sequence to achieve the expression of the OSHS mutant. The medium can be a medium in the art that can enable the transformant to grow and produce the OSHS of the present invention, and preferably LB medium: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, the solvent is deionized water, and the pH is 7.2. There are no special limitations on the culture method and culture conditions, as long as the transformant can grow and produce bacterial cells. The following method is preferred: The recombinant E. coli BL21 (DE3) / pET28b-OSHS involved in the present invention m is seeded into LB medium containing 50 μg / ml kanamycin and cultured at 37 °C until the OD 600 reaches 0.6-1 (preferably 0.6), and under the induction of isopropyl-β-D-thiogalactopyranoside (IPTG) with a final concentration of 0.1-1.0 mM (preferably 0.1 mM), the OSHS mutant protein of the present invention can be highly expressed.

[0016] The present invention also relates to the application of the O-succinylhomoserine sulfhydrylase mutant in the biocatalytic preparation of L-methionine.

[0017] Specifically, the application is as follows: using the wet bacterial cells obtained by fermenting and culturing a recombinant genetic engineering bacterium containing the O-succinylhomoserine sulfhydrylase mutant gene or the crude enzyme solution extracted from the wet bacterial cells as a catalyst, using O-succinyl-L-homoserine as a substrate, using sodium methanethiolate as a sulfhydryl donor, and using a buffer solution with a pH of 5.0 to 9.0 as a reaction medium to form a reaction system, and performing a conversion reaction at a temperature of 20 to 45 °C (preferably 30 °C). The reaction solution is separated and purified to obtain the L-methionine.

[0018] In the reaction system, the final concentration of the substrate added is 50 to 500 mM, and the final concentration of the sulfhydryl donor added is 100 mM to 1 M; the final concentration of the catalyst added to the reaction system based on the weight of the wet bacterial cells is 20 to 100 g / L.

[0019] The crude enzyme solution extracted from the wet bacterial cells is obtained by resuspending the wet bacterial cells with a buffer solution with a pH of 6.0 to 9.0, followed by ultrasonic disruption, centrifugation, and collecting the supernatant as the crude enzyme solution; the addition amount of the sulfhydryl donor sodium methanethiolate is 100 mM to 200 mM (preferably 100 mM); the reaction time is 2 h to 6 h (preferably 2 h); the buffer solution with a pH of 5.0 to 9.0 is preferably 100 mM PBS buffer solution with a pH of 7.0.

[0020] The wet bacterial cells can be prepared by the following method:

[0021] Inoculate a recombinant genetic engineering bacterium containing the O-succinylhomoserine sulfhydrylase mutant gene into an LB liquid medium containing kanamycin resistance with a final concentration of 50 μg / mL, culture at 37 °C and 200 rpm for 8 h, and then inoculate into a fresh fermentation medium containing kanamycin resistance with a final concentration of 50 μg / mL at an inoculation amount of 10% by volume, and culture at 37 °C and 500 rpm until the OD of the bacterial cells 600 reaches 0.6 to 0.8, add IPTG with a final concentration of 0.1 mM, induce and culture at 28 °C for 11 h, centrifuge the fermentation broth at 4 °C and 10000 rpm for 10 min, discard the supernatant, collect the wet bacterial cells, and store at -20 °C.

[0022] Disruption of wet bacterial cells: Weigh 2 g of resting cells, resuspend them in 20 mL of Tris-HCl (50 mM, pH 7.0) buffer solution, and use ultrasonic waves to disrupt and release intracellular proteins. The disruption program is as follows: disruption time 2 s, interval time 2 s, power 40 W, and total disruption time 20 min. Maintain an ice bath condition during the disruption process. Subsequently, centrifuge the cell disruption solution at 12000 rpm and 4 °C for 20 min to remove solid substances such as cell debris, and collect the supernatant crude enzyme solution. The volume of the buffer solution with a pH of 5.0 to 9.0 used is 10 mL / g based on the wet weight of the wet bacterial cells.

[0023] Definition of enzyme activity: At 30 °C, the amount of enzyme required to catalyze the formation of 1 μmol of product per minute is defined as 1 enzyme activity unit, denoted by U.

[0024] Detection method of the product in the present invention: Ultra-high-speed fully automatic amino acid analyzer: LA 8080 (HITACHI), mobile phase is ninhydrin solution (1 L), filtered through an organic membrane. Flow rate: 0.35 mL / min, column temperature: 135 °C, retention time: product: 14.2 min; substrate O-succinyl-L-homoserine: 5.2 min.

[0025] The beneficial effects of the present invention are mainly reflected in: The present invention provides an OSHS mutant with high conversion rate. Using the crude enzyme solution obtained by disrupting the recombinant bacteria containing the OSHS mutant as a catalyst, O-succinyl-L-homoserine as a substrate, and sodium methanethiolate as a mercapto donor to synthesize L-methionine, the industrial cost is relatively low; under the optimal system, the conversion rate of the substrate of 400-800 mM reaches 80%-85%. Compared with the conversion rate of the wild-type substrate of 71%, it is increased by 13%-20% respectively, and has the potential for large-scale application, and has extremely high application value in the industrial production of L-methionine. Detailed implementation mode

[0026] The following further describes the present invention in detail with specific examples, but the present invention is not limited to the following examples:

[0027] The final concentration composition of the LB medium described in the present invention is: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, agar 15 g / L, and the solvent is deionized water with pH 7.0. The preparation method of the PBS buffer solution (100 mM, pH 7.0) is: weigh 43.7 g of Na2HPO4•12H2O, add 1 L of ultrapure water to completely dissolve it; then weigh 12.17 g of NaH2PO4・2H2O in 1 L of ultrapure water, and mix the two.

[0028] Example 1: Construction of a genetic engineering bacterium of mercapto-lyase

[0029] The full gene was synthesized according to the sequence SEQ ID NO.1 of the genetically engineered bacterium and ligated to the plasmid pET-28b to construct a heterologous expression recombinant plasmid containing the mercaptide lyase encoding gene. The expression recombinant plasmid was transformed into the host bacterium E. coli BL21(DE3) to obtain a recombinant genetically engineered bacterium containing the recombinant plasmid. Using the recombinant bacterium (E. coli BL21(DE3) / pET28b-OSHS) containing the expression vector pET28b-OSHS as the starting strain, a mutant library was constructed by semi-rational design, and through site-directed saturation mutagenesis technology, the catalytic activity of mercaptide lyase towards the substrate O-succinyl-L-homoserine was further enhanced.

[0030] The bacterial liquid of the starting strain of the present invention can be obtained by the following method: The recombinant expression plasmid pET28b-OSHS of the present invention m was transferred into E. coli DH5α competent cells, spread on an LB plate containing kanamycin at a final concentration of 50 μg / ml, cultured overnight at 37 °C, and positive transformants were picked and identified by sequencing. The verified positive monoclonal was inoculated into 5 mL of LB liquid medium containing kanamycin at a final concentration of 50 μg / mL, cultured overnight at 37 °C, and after the plasmid was extracted and verified, the recombinant expression vector was transferred into E. coli BL21(DE3) strain to obtain the recombinant bacterium E. coli BL21(DE3) / pET28b-OSHS (i.e., the parental strain), which was cultured overnight at 37 °C on an LB plate containing kanamycin at a final concentration of 50 μg / ml. A monoclonal was picked and inoculated into 5 mL of LB liquid medium containing kanamycin at a final concentration of 50 μg / mL, cultured overnight at 37 °C, and then stored at -80 °C in glycerol for standby.

[0031] Example 2: Establishment of a recombinant mercaptide lyase mutant library

[0032] Using the bacterial liquid of the recombinant plasmid pET28b-OSHS obtained in Example 1 as a template, a mutant sequence was obtained by PCR amplification.

[0033] Site-directed saturation mutagenesis:

[0034] Gly(G)58:

[0035] Upstream primer 1: 5’-GGTGGCCAGGAGCCG NNK AATATTTATAGCAGA-3’

[0036] Downstream primer 2: 5’-TCCGCAGCACAAGCAGCAGCACGTTTTGGT-3’

[0037] Asn(N)124:

[0038] Forward primer 3: 5’-ACAACAAGCCTGTTT NNK ACCTATCTGACACGG-3’

[0039] Reverse primer 4: 5’-AAACAGGCTTGTTGTGGTACCAAAAACTGC-3’

[0040] The saturation mutagenesis primers are as described above, and the mutation sites are underlined. Using the plasmid DNA containing the SCR gene as a template, mutations were introduced by PCR.

[0041] The amplification system was: 20 μl reaction system: 2×Phanta Max Buffer: 10 μl; 10 mM dNTP mixture (2.5 mM each of dATP, dCTP, dGTP and dTTP) 0.5 μl; 1 μl each of the forward primer and the reverse primer, template (E. coli broth): 1 μl; Phanta Max Super-Fidelity DNA Polymerase: 1 μl; The system was made up with double-distilled water.

[0042] The PCR reaction conditions were: pre-denaturation at 95 °C for 5 min, then enter the temperature cycle of 95 °C for 30 s, 68 °C for 30 s, 72 °C for 4 min, for a total of 30 cycles, and finally extension at 72 °C for 5 min, and the termination temperature was 4 °C.

[0043] The PCR product was treated with DpnI at 37 °C for 2 h, inactivated and then transformed into the E. coli BL21 (DE3) recipient bacteria, and spread on an LB solid plate containing kanamycin resistance at a final concentration of 50 μg / ml. After culturing at 37 °C for 12 h, all the transformants on the plate were picked into a 96-well deep-well plate containing 1 mL of LB medium / well at a final concentration of 50 μg / ml, and a cloned mutant library was obtained.

[0044] Example 3: High-throughput screening of recombinant mercaptolysin mutants

[0045] To identify positive mutants with substrate specificity, O-succinyl-L-homoserine was dissolved in 0.1 M PBS buffer (pH 7.0) to a final concentration of 400 mM, and 5 used as a coenzyme was added ，Pyridoxal - phosphate was added to the reaction mixture to a final concentration of 10 mM, and another substrate sodium methanethiolate was added to the reaction mixture to a final concentration of 2 mM. 1 ml of the reaction mixture was placed at 30 °C, and then 10 μl of the crude enzyme solution of each mutant was added to make the final protein concentration 10 mg / ml. By collecting 100 μl of the reaction mixture after the start of the reaction and adding it to 900 μl of 4 mg / ml DTNB solution. The absorbance at 415 nm was measured to confirm the progress of the reaction.

[0046] DTNB reacts with the -SH group of sodium methanethiolate remaining in the reaction mixture to generate a yellow substance. Therefore, the progress of the reaction can be monitored by observing the conversion of sodium methanethiolate in the reaction mixture to methionine, that is, the disappearance of yellow in the reaction mixture. Further, if the difference in the absorbance of DTNB before and after the reaction (OD 415 ) is higher, it indicates stronger enzyme activity.

[0047] Effective mutations were screened among the candidate mutants. The mutant sequences were confirmed by sequencing analysis. Then enzyme activity assay analysis was carried out to establish the optimal mutation sites.

[0048] Example 4: Preparation of recombinant mercaptide lyase mutant wet cells

[0049] (1) Seed culture: Recombinant Escherichia coli containing the gene expressing recombinant mercaptide lyase mutant (recombinant Escherichia coli BL21(DE3) / pET28b - mut - Gly58Ala, recombinant Escherichia coli BL21(DE3) / pET28b - mut - Asn124Gly) was inoculated into LB liquid medium containing kanamycin at a final concentration of 50 μg / ml and cultured at 37 °C and 220 rpm for 8 h - 10 h to obtain seed liquid.

[0050] (2) Fermentation culture: The seed liquid was inoculated into LB liquid medium containing kanamycin resistance at a final concentration of 50 μg / ml at an inoculum volume concentration of 1%, cultured at 37 °C and 220 rpm until the OD 600 value reached 0.6, IPTG with a final concentration of 0.1 mM was added, and the culture was continued at 28 °C for 12 h. After centrifugation at 12000 rpm with an ultra - low - temperature high - speed centrifuge, the wet cells were collected and stored at -20 °C for standby.

[0051] (4) Preparation of crude enzyme solution: Weigh 2 g of resting cells, resuspend them in 20 mL of Tris-HCl (50 mM, pH 7.0) buffer, and use ultrasonic disruption to release intracellular proteins. The disruption program is as follows: disruption time 2 s, interval time 2 s, power 40 W, and total disruption time 20 min. The disruption process is maintained under ice bath conditions. Subsequently, centrifuge the cell disruption solution at 12000 rpm and 4 °C for 20 min to remove solid substances such as cell debris, and collect the supernatant crude enzyme solution. This crude enzyme solution can be directly used as a biocatalyst or for protein purification.

[0052] Prepare the crude enzyme solution of recombinant Escherichia coli BL21(DE3) / pET28b-OSHS containing the gene expressing the recombinant mutant mercaptide lyase by the same method.

[0053] Example 5: Isolation and purification of recombinant mutant mercaptide lyase

[0054] When the mercaptide lyase gene was synthesized, it was already ligated to the pET-28b(+) expression vector, and the N-terminus of the mercaptide lyase carried a His-tag. Since the histidine peptide segment can chelate with divalent nickel ions, the mercaptide lyase can be purified by metal affinity chromatography. In this example, the Ni-NTA column was used to purify the supernatant crude enzyme solution, and the specific steps are as follows:

[0055] (1) Prepare buffers: Prepare 300 mM NaCl, 50 mM sodium phosphate buffer, pH 8.0; add 10 mM, 50 mM, and 300 mM imidazole to the standard solution respectively to prepare the equilibration buffer (buffer A), binding buffer (buffer B), and elution buffer (buffer C);

[0056] (2) Wash the pipeline: Connect all the pipelines on the protein purifier, and wash the pipeline with buffer A, setting the flow rate to 1.0 mL / min;

[0057] (3) Loading: Correctly install the Ni-NTA column on the protein purifier, set the flow rate to 0.5 mL / min, and run until the voltage and conductivity are balanced. Load the crude enzyme solution onto the column, change the flow rate to 0.3 mL / min, and the loading volume is 10 mL;

[0058] (4) Elution: After loading, change the flow rate to 0.5 mL / min, and use buffer B as the mobile phase to remove the unbound target protein and miscellaneous proteins in the column;

[0059] (5)Collect the target protein: Change the flow rate to 0.3 mL / min, use buffer C as the mobile phase, elute the target protein from the chromatography column, collect it and store it on ice. Analyze and verify the collected eluate by SDS-PAGE, and determine the target protein eluate according to the electrophoresis results.

[0060] The entire purification process was carried out at 4 °C. After dialysis and purification of the target protein eluate, the retentate was taken to determine the protein content by the BCA kit method and stored frozen in an -80 °C refrigerator to obtain pure enzymes of mercapto-lyase OSHS and its mutants mut-Gly58Ala and mut-Asn124Gly.

[0061] Example 6: Determination of the activity of mercapto-lyase

[0062] In a 10 mL reaction system, add substrate O-succinyl-L-homoserine with a final concentration of 400 mM, sodium methanethiolate with a final concentration of 100 mM, and sodium methanethiolate with a final concentration of 10 mM in sequence. Add the pure enzyme solution prepared by the method of Example 5, and adjust the reaction solution to pH 6.5 with a pH 7.0, 100 mM PBS buffer. React the reaction system at 30 °C for 30 min, add 5 mL of 6M hydrochloric acid to terminate the reaction, and take samples to detect the enzyme activity. The results are shown in Table 1.

[0063] Table 1: Results of the determination of the activity of OSHS and its mutants

[0064] enzyme Enzyme specificity (U / mg) wild type 71.2 mutant - Gly58Ala 80.9 mutant - Asn124Gly 85.3

[0065] The definition of enzyme activity unit (U) is: The amount of enzyme required to produce 1 µmol of product L-methionine within 1 min under the conditions of 30 °C and pH 6.5 is defined as 1 U. The production amount of the product is determined by detection with an ultra-high-speed fully automatic amino acid analyzer.

[0066] Example 7: Application of recombinant mercapto-lyase OSHS in the preparation of L-methionine

[0067] (1) Use the recombinant Escherichia coli BL21(DE3) / pET28b-OSHS wet cells containing the expression recombinant plasmid obtained in Example 4 as a biocatalyst, and use O-succinyl-L-homoserine as a substrate to carry out a biotransformation reaction to prepare L-methionine.

[0068] The composition of the catalytic system and the catalytic conditions are as follows: In a 10 mL reaction system, recombinant mercapto lyase OSHS wet cells (with a dosage of 50 g / L buffer) were added, sodium methanethiolate with a final concentration of 100 mM and sodium methanethiolate with a final concentration of 10 mM, the initial substrate final concentration was 400 mM, and the reaction solution was adjusted to pH 6.5 with pH 7.0, 100 mM PBS buffer. A 30 °C water bath was used, with a magnetic stirring device at 600 rpm. Samples were taken at regular intervals during the reaction, with a sampling volume of 100 μL, diluted 2-fold with 6 M hydrochloric acid, and the conversion rate was determined by an ultra-high-speed fully automatic amino acid analyzer. The results showed that the yield reached 71% after 2 h of catalysis.

[0069] (2)Detection method of L-methionine by amino acid analyzer

[0070] The detection method of the product in the present invention: Ultra-high-speed fully automatic amino acid analyzer: LA 8080 (HITACHI), the mobile phase is ninhydrin solution (1 L), passing through an organic filter membrane. Flow rate: 0.35 mL / min, column temperature: 135 °C.

[0071] Retention time: Product L-methionine: 14.2 min; Substrate O-succinyl-L-homoserine: 5.2 min.

[0072] Example 8: Application of recombinant mercapto lyase mutant Gly58Ala in the preparation of L-methionine

[0073] Using the recombinant Escherichia coli BL21(DE3) / pET28b-Gly58Ala wet cells containing the expression recombinant plasmid obtained in Example 2 as a biocatalyst, and using O-succinyl-L-homoserine as a substrate, a biotransformation reaction was carried out to prepare L-methionine.

[0074] In a 10 mL reaction system, recombinant mercapto lyase mutant wet cells (with a dosage of 50 g / L buffer) were added, sodium methanethiolate with a final concentration of 100 mM and sodium methanethiolate with a final concentration of 10 mM, the initial substrate final concentration was 400 mM, and the reaction solution was adjusted to pH 6.5 with pH 7.0, 100 mM PBS buffer. A 30 °C water bath was used, with a magnetic stirring device at 600 rpm. Samples were taken at regular intervals during the reaction, with a sampling volume of 100 μL, diluted 2-fold with 6 M hydrochloric acid, and the conversion rate was determined by an ultra-high-speed fully automatic amino acid analyzer. The results showed that the yield reached 81% after 2 h of catalysis.

[0075] Example 9: Application of recombinant mercapto lyase mutant Asn124Gly in the preparation of L-methionine

[0076] Using the recombinant Escherichia coli BL21(DE3) / pET28b-Asn124Gly wet cells containing the expression recombinant plasmid obtained in Example 2 as the biocatalyst, and O-succinyl-L-homoserine as the substrate, a biotransformation reaction was carried out to prepare L-methionine.

[0077] In a 10 mL reaction system, recombinant mercaptolase mutant wet cells (dosage: 50 g / L buffer), sodium methanethiol at a final concentration of 100 mM and sodium methanethiol at a final concentration of 10 mM were added. The initial substrate final concentration was 400 mM, and the reaction solution was adjusted to pH 6.5 with a pH 7.0, 100 mM PBS buffer. The reaction was carried out in a 30 °C water bath with a magnetic stirring device at 600 rpm. Samples were taken at regular intervals during the reaction, with a sampling volume of 100 μL, diluted 2-fold with 6 M hydrochloric acid, and the conversion rate was measured by an ultra-high speed fully automatic amino acid analyzer. The results showed that the yield reached 85% after 2 h of catalysis.

[0078] The technical features of the above-described embodiments can be combined arbitrarily. For the sake of brevity of description, 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, it should be considered to be within the scope described in this specification.

Claims

1. An O-succinyl mercaptolase mutant, the amino acid sequence of the mutant is shown in SEQ ID NO.3 or SEQ ID NO.

4.

2. A gene encoding the O-succinyl mercaptolase mutant according to claim 1.

3. A recombinant vector containing the encoding gene according to claim 2.

4. A recombinant genetically engineered bacterium containing the encoding gene according to claim 2.

5. Use of the O-succinyl mercaptolase mutant according to claim 1 in the biocatalytic preparation of L-methionine.

6. The application according to claim 5, characterized in that The use is as follows: using the wet cells obtained by fermentation culture of a recombinant genetically engineered bacterium containing the O-succinyl mercaptolase mutant gene or the crude enzyme solution extracted from the wet cells as a catalyst, using O-succinyl-L-homoserine as a substrate, using sodium methanethiolate as a mercapto donor, using a buffer solution with a pH of 5.0 to 9.0 as a reaction medium to form a reaction system, carrying out a conversion reaction at a temperature of 20 to 45 °C, separating and purifying the reaction solution to obtain the L-methionine.

7. The application according to claim 6, characterized in that In the reaction system, the final concentration of the substrate added is 50 to 500 mM, and the final concentration of the mercapto donor added is 100 mM to 1 M; the final concentration of the catalyst added in the reaction system based on the weight of the wet cells is 20 to 100 g / L.