266 site-based s-adenosylmethionine synthetase mutant and application thereof

Abstract

This invention belongs to the field of bioengineering technology, specifically relating to a mutant S-adenosylmethionine synthase based on the 266-site and its applications. The wild-type strain of the S-adenosylmethionine synthase mutant of this invention is derived from... Guyparkeria sp XI15, a mutant obtained by site-directed mutagenesis, has a mutation in the core amino acid related to enzyme catalytic activity; only one of the following mutations exists in positions 1 to 390 of the amino acid sequence corresponding to SEQ ID NO:1: D22S; ​​G123D; D244A; K251I; G266S; A307S; its amino acid sequence is shown in one of SEQ ID NO:3-8; the S-adenosylmethionine synthase mutant of the present invention has been applied in the biosynthesis of S-adenosylmethionine, so that the yield of S-adenosylmethionine can reach 25.7 g / L and the purity of the process product can reach 89.7%.

Description

[0001] This application is a divisional application of patent application No. 202211409009.9; the original application was filed on November 11, 2022, with application number 202211409009.9, and entitled "An S-adenosylmethionine synthase mutant and its application". Technical Field

[0002] This invention belongs to the field of bioengineering technology, and specifically relates to an S-adenosylmethionine synthase mutant based on the 266 site, as well as the application of the above mutant. Background Technology

[0003] S-adenosylmethionine (SAM) is an important metabolic intermediate in living organisms such as animals and plants, and also a vital physiologically active substance in the human body. It participates in various biochemical reactions, such as acting as an intracellular methyl donor, playing a crucial role in the methylation modification of molecules like nucleic acids, proteins, and lipids. SAM also participates in important intracellular transsulfurization reactions and the synthesis of polyamines. Furthermore, SAM is a valuable pharmaceutical molecule, playing a significant role in the treatment of liver disease, depression, and rheumatoid arthritis.

[0004] SAM is synthesized from the substrate L-methionine and ATP via enzymatic conversion using S-adenosylmethionine synthase. Therefore, S-adenosylmethionine synthase is the key enzyme in SAM synthesis. Currently, the main industrial production processes for SAM include yeast fermentation and enzymatic conversion. Internationally, specially cultivated Saccharomyces cerevisiae strains expressing SAM are often used for intracellular expression to prepare SAM. Domestically, some researchers have developed genetically engineered Saccharomyces cerevisiae for fermentation expression of SAM, but the highest expression level achieved so far is only within 10 g / L of fermentation broth, and the purity of SAM in the fermentation broth is low, resulting in a low overall yield and still high production costs. Enzymatic conversion often involves extracting and purifying S-adenosylmethionine synthase from Saccharomyces cerevisiae or other engineered strains, and then converting it into SAM by optimizing the enzymatic reaction conditions. This method has a high raw material conversion rate and SAM is easy to extract and purify; however, it still suffers from low enzyme catalytic efficiency and high overall production costs. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides an S-adenosylmethionine synthase mutant. This invention obtains a highly efficient S-adenosylmethionine synthase through discovery and then modifies it using genetic engineering techniques to obtain an S-adenosylmethionine synthase mutant. This mutant exhibits significantly improved activity and yield in synthesizing S-adenosylmethionine.

[0006] The wild-type S-adenosylmethionine synthase mutant of this invention is derived from Guyparkeria sp. XI15. Through site-directed mutagenesis, a mutant with mutations in the core amino acids related to enzyme catalytic activity was obtained.

[0007] The S-adenosylmethionine synthase mutant provided by the present invention has more than 80% homology (preferably ≥90%, more preferably ≥95%) with the amino acid sequence shown in SEQ ID NO:1, and the mutant has the ability to synthesize S-adenosylmethionine and its catalytic activity is significantly improved.

[0008] The present invention provides an S-adenosylmethionine synthase mutant with a single site mutation at positions 22, 123, 244, 251, 266, and 307 in the amino acid sequence corresponding to positions 1 to 390 of SEQ ID NO:1.

[0009] Preferably, the mutant has only one of the following mutations in positions 1 to 390 of the amino acid sequence corresponding to SEQ ID NO:1: D22S; ​​G123D; D244A; K251I; G266S; A307S; and its amino acid sequence is shown in one of SEQ ID NO:3-8.

[0010] The gene encoding the amino acid sequence shown in SEQ ID NO:1 is the nucleotide sequence shown in SEQ ID NO:2.

[0011] The present invention also provides the coding gene of the above mutant, the gene sequence of which is shown in NO:9-14; and further provides an expression vector containing the gene and recombinant cells.

[0012] The S-adenosylmethionine synthase mutant of the present invention is prepared by the following steps:

[0013] (1) Synthesize the gene encoding the amino acid sequence shown in SEQ ID NO:1 to obtain the nucleotide sequence shown in SEQ ID NO:2;

[0014] (2) The nucleotide sequence shown in SEQ ID NO:2 was subjected to site-directed mutagenesis to obtain the coding gene of SEQ ID NO:3-8;

[0015] (3) A soluble tag was added to the coding gene of SEQ ID NO:3-8, and its protein sequence is as follows:

[0016] MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK, and then gene cloning was performed to obtain a plasmid containing the gene encoding a mutant of the fused soluble tag S-adenosylmethionine synthase;

[0017] (4) The plasmid containing the S-adenosylmethionine synthase mutant encoding gene with added soluble tag was introduced into competent cells to obtain an expression strain of S-adenosylmethionine synthase mutant.

[0018] (5) The expression strain of S-adenosylmethionine synthase mutant was induced to ferment and obtain S-adenosylmethionine synthase mutant.

[0019] The present invention also provides S-adenosylmethionine prepared using an S-adenosylmethionine synthase mutant.

[0020] The aforementioned S-adenosylmethionine is obtained by fermenting engineered bacteria expressing the coding gene of the S-adenosylmethionine synthase mutant to a certain extent, followed by cell wall disruption to obtain a crude enzyme solution, adding methionine and ATP precursor substances to the crude enzyme solution, and carrying out a catalytic reaction under the conditions of pH 6.5-7.2 and temperature 25-30℃.

[0021] Preferably, the concentration of the crude enzyme solution is 20-30 g / L.

[0022] Preferably, the concentration of the precursor ATP is 80-110 g / L, the concentration of methionine is 30-35 g / L, and the total reaction time is 5-10 h.

[0023] This invention develops a mutant S-adenosylmethionine synthase capable of efficiently catalyzing S-adenosylmethionine. The invention involved multi-layered comparisons of the S-adenosylmethionine synthase (amino acid sequence shown in SEQ ID NO:1) in GenBank (ID WP_058573953.1) with its homologous enzyme protein from primary to higher-order structures. The key amino acid sites affecting the enzymatic properties of the S-adenosylmethionine synthase were identified as amino acids at positions 22, 123, 244, 251, 266, and 307. These sites were then mutated using codon substitutions as follows: D22S; ​​G123D; D244A; K251I; G266S; A307S, resulting in the S-adenosylmethionine synthase mutant. The mutant enzyme protein was obtained by constructing a 6×His fusion expression vector and inducing expression in the genetically engineered bacterium *E. coli* BL21(DE3).

[0024] This invention provides a mutant S-adenosylmethionine synthase, the protein of which is a non-natural protein and exhibits highly efficient catalysis of S-adenosylmethionine synthesis. The S-adenosylmethionine synthase mutant of this invention is obtained by mutating amino acids at positions 22, 123, 244, 251, 266, and 307 of the S-adenosylmethionine synthase in GenBank ID: WP_058573953.1.

[0025] The beneficial effects of this invention are:

[0026] 1. The S-adenosylmethionine synthase mutant prepared by this invention has the characteristic of highly efficient catalysis of S-adenosylmethionine synthesis compared with the wild type, with a yield of up to 25.7 g / L and a product purity of 89.7%.

[0027] 2. The S-adenosylmethionine synthase of the present invention has been tagged with a soluble tag, which significantly improves the soluble expression of the enzyme. Compared with the untagged mutant gene, a mutant gene with significantly enhanced soluble expression is obtained. Figure 2 Soluble expression increased by more than 70%.

[0028] 3. Compared with the method for preparing S-adenosylmethionine mentioned in the background art, the cost is significantly reduced, by more than 30%. Attached Figure Description

[0029] Figure 1 This is an SDS-PAGE electrophoresis image of the S-adenosylmethionine synthase mutant G266S expressed in the host cell; among them, BL21-CodonPlus (DE3), BL21 (DE3), Rosetta (DE3) and OverexpressC43 (DE3) are four E. coli protein expression hosts, and the marker is the protein molecular weight standard;

[0030] Figure 2 This shows the soluble expression of the protein after adding the soluble tag. The GST-S-adenosylmethionine synthase mutant G266S is a mutant after adding the soluble tag.

[0031] Figure 3 This is an HPLC chromatogram of the product synthesized using the S-adenosylmethionine synthase mutant G266S.

[0032] Figure 4 This is an HPLC chromatogram of S-adenosylmethionine standard. Detailed Implementation

[0033] To enable those skilled in the art to better understand the present invention, it will now be further described in conjunction with specific embodiments. The following examples are for illustrative purposes only and are not intended to limit the scope of the invention.

[0034] Example 1

[0035] I. Design of S-adenosylmethionine synthase mutants

[0036] Through gene mining, the S-adenosylmethionine synthase gene with Genbank ID WP_058573953.1 was obtained from the NCBI database. The open reading frame of this gene is 1170 bp in length, and the S-adenosylmethionine synthase it encodes consists of 390 amino acids. Its amino acid sequence (390 aa) is SEQ ID NO:1, and the nucleotide sequence encoding this S-adenosylmethionine synthase is SEQ ID NO:2.

[0037] By comparing the S-adenosylmethionine synthase (amino acid sequence shown in SEQ ID NO:1) with homologous enzyme proteins from GenBank using a multi-layered, multi-dimensional approach from primary to higher-order structures, the key amino acid sites affecting the enzymatic properties of S-adenosylmethionine synthase were identified as amino acids at positions 22, 123, 244, 251, 266, and 307. These sites were then mutated using codon substitutions as follows: D22S; ​​G123D; D244A; K251I; G266S; A307S, resulting in S-adenosylmethionine synthase mutants, whose amino acid sequences are shown in SEQ ID NO:3-8.

[0038] II. Obtaining the S-adenosylmethionine synthase mutant gene

[0039] The S-adenosylmethionine synthase mutant gene can be obtained through whole-gene synthesis or molecular cloning. In this experiment, the S-adenosylmethionine synthase gene with GenBank ID WP_058573953.1 was obtained using whole-gene synthesis, and the S-adenosylmethionine synthase mutant gene was obtained using PCR.

[0040] 1. Synthesis of the complete S-adenosylmethionine synthase gene of WP_058573953.1

[0041] The S-adenosylmethionine synthase with Genbank ID WP_058573953.1 was synthesized in its entirety. The synthesized gene fragment was ligated into the pUC57 plasmid, and the gene was then synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0042] 2. Site-directed mutation of S-adenosylmethionine synthase gene

[0043] (1) Site-directed mutagenesis primer design

[0044] Primers were designed based on the gene sequence of the S-adenosylmethionine synthase mutant to introduce site-directed mutagenesis. The nucleotide sequences of the primers are shown in Table 1. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0045] Table 1. Nucleotide sequences of primers

[0046]

[0047]

[0048] (2) Site-directed mutation

[0049] Using the pUC57 plasmid containing the S-adenosylmethionine synthase gene PRQ77350.1 as a template, the S-adenosylmethionine synthase mutant gene was amplified using the upstream and downstream primers obtained in step (1) according to the following PCR system and program. PCR system: KOD-Plus-0.5μL, plasmid template 0.8μL, upstream primer F5' (10μM) 1μL, downstream primer R5' (10μM) 1μL, 10×PCR Buffer 2.5μL, dNTPs (2mM) 3μL, MgSO4 (25mM) 1.5μL, ddH2O 14.7μL. PCR program as follows: a. 94℃ pre-denaturation for 4min; b. 94℃ denaturation for 40sec, 68℃ annealing and extension for 7.5min; 18 cycles; c. 68℃ extension for 20min. The template plasmid containing the S-adenosylmethionine synthase gene WP_058573953.1 was digested with DpnI enzyme at 37°C for 30 min.

[0050] (3) DH5α competent cell transformation

[0051] The pUC57 plasmid containing the S-adenosylmethionine synthase mutant gene obtained in step (2) was transformed into DH5α competent cells. DH5α competent cells were placed on ice, and after the cells thawed, 10 μL of plasmid solution was added and the cells were placed on ice for 30 min; heat-shocked at 42℃ for 50 s, and then placed on ice for 3 min; 600 μL of sterile LB liquid medium was added, and the cells were cultured at 37℃ and 200 rpm for 1 h in a shaker; 200 μL of the cultured bacterial solution was taken and spread on LB agar plates containing Amp resistance (100 μg / mL), and incubated upside down at 37℃ overnight.

[0052] (4) Screening for positive clones

[0053] Single colonies were picked from LB agar plates and inoculated into LB liquid medium containing Amp resistance (100 μg / mL). The culture was incubated overnight at 37°C and 220 rpm for plasmid extraction. Plasmids were extracted using the OMEGA Plasmid Mini Kit I (catalog number: D6943) according to the manufacturer's instructions. The extracted plasmids were then sent to Genewiz (Suzhou, China) for sequencing to determine whether the S-adenosylmethionine synthase in WP_058573953.1 had been successfully mutated.

[0054] 3. Cloning of the S-adenosylmethionine synthase mutant gene

[0055] (1) Gene cloning

[0056] Primers were designed based on the S-adenosylmethionine synthase mutant gene sequence and constructed into the pET28(a) plasmid. The nucleotide sequences of the primers are shown in Table 2. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. Using the pUC57 plasmid containing the S-adenosylmethionine synthase mutant gene as a template, the obtained upstream and downstream primers were used to amplify the S-adenosylmethionine synthase mutant gene according to the following PCR system and program. PCR system: PrimeSTAR Max Premix (2×) 25 μL, plasmid template 0.5 μL, upstream primer NcoI-F (10 μM) 2 μL, downstream primer XhoI-R (10 μM) 2 μL, ddH2O added to a total volume of 50 μL. PCR program: a. 98℃ pre-denaturation for 2 min; b. 98℃ denaturation for 10 sec, 65℃ annealing for 10 sec, 72℃ extension for 30 sec; 30 cycles; c. 72℃ extension for 3 min. The PCR amplification products were detected by 1.0% agarose gel electrophoresis, yielding a target band of approximately 1600 bp. The target band was excised under UV light, and the S-adenosylmethionine synthase mutant gene fragment was recovered using the Omega Gel Extraction Kit (catalog number: D2500) according to the kit instructions.

[0057] Table 2. Nucleotide sequences of primers

[0058]

[0059]

[0060] (2) Construction of expression vector

[0061] The S-adenosylmethionine synthase mutant gene and the pET28(a) vector were double-digested using NcoI and XhoI restriction endonucleases, respectively. Digestion system (gene): 25 μL S-adenosylmethionine synthase mutant gene, 2 μL NcoI enzyme, 2 μL XhoI enzyme, 5 μL 10× Buffer, and sterile double-distilled water to a final volume of 50 μL. Digestion system (vector): 2 μL pET28(a) vector, 0.5 μL NcoI enzyme, 0.5 μL XhoI enzyme, 1 μL 10× Buffer, and sterile double-distilled water to a final volume of 10 μL. Digestion conditions: 37℃ for 30 min. Then, the double-digested S-adenosylmethionine synthase mutant gene and the pET28(a) linear vector were ligated using T4 DNA ligase and incubated overnight at 16°C to obtain the pET28(a) plasmid containing the S-adenosylmethionine synthase mutant gene.

[0062] (3) Construction of an expression vector containing an S-adenosylmethionine synthase mutant with a soluble tag

[0063] Primers were designed for the soluble tag sequence and constructed into the pET28(a) plasmid containing the S-adenosylmethionine synthase mutant gene. The nucleotide sequences of the primers are shown in Table 3. The primers were synthesized by Sangon Biotech (Shanghai) Co., Ltd. Using the pET-GST plasmid as a template, the soluble tag fragment was amplified using the obtained upstream and downstream primers according to the following PCR system and program. PCR system: PrimeSTAR Max Premix (2×) 25 μL, pET-GST plasmid template 0.5 μL, upstream primer XhoI-Tag-F (10 μM) 2 μL, downstream primer XhoI-Tag-R (10 μM) 2 μL, ddH2O added to a total volume of 50 μL. PCR program: a. 98℃ pre-denaturation for 2 min; b. 98℃ denaturation for 10 sec, 65℃ annealing for 10 sec, 72℃ extension for 30 sec; 30 cycles; c. 72℃ extension for 3 min. The PCR amplification products were detected by 1.0% agarose gel electrophoresis, yielding a target band of approximately 650 bp. The target band was excised under UV light, and the soluble tag fragment was recovered using the Omega Gel Extraction Kit (catalog number: D2500) according to the kit instructions.

[0064] Single digestion of the soluble tag fragment and the pET28(a) plasmid containing the S-adenosylmethionine synthase mutant gene was performed using Xho I restriction endonuclease. Digestion system (soluble tag): 25 μL soluble tag fragment, 2 μL Xho I enzyme, 5 μL 10× Buffer, and sterile double-distilled water to a final volume of 50 μL. Digestion system (vector): 2 μL pET28(a) plasmid containing the S-adenosylmethionine synthase mutant gene, 0.5 μL Xho I enzyme, 1 μL 10× Buffer, and sterile double-distilled water to a final volume of 10 μL. Digestion conditions: 37℃ for 30 min. Phosphate groups were added to the soluble tag fragment after single-enzyme digestion. The reaction system consisted of 4 μL of the soluble tag fragment after single-enzyme digestion, 0.5 μL of T4 polynucleotide kinase, 2 μL of 10×T4 ligase buffer, and sterile double-distilled water to a final volume of 20 μL. The mixture was heated at 37°C for 45 min and then at 70°C for 15 min. Phosphate groups were removed from the pET28(a) plasmid fragment containing the S-adenosylmethionine synthase mutant gene after single-enzyme digestion. The reaction system consisted of 10 μL of the pET28(a) plasmid fragment containing the S-adenosylmethionine synthase mutant gene after single-enzyme digestion, 1 μL of alkaline phosphatase, 2 μL of 10× alkaline phosphatase buffer, and sterile double-distilled water to a final volume of 20 μL. The mixture was heated at 37°C for 1 h and then at 70°C for 15 min. Then, the soluble tag fragment with added phosphate group after single enzyme digestion and the pET28(a) plasmid fragment containing the S-adenosylmethionine synthase mutant gene after single enzyme digestion were ligated using T4 DNA ligase. The ligation was carried out overnight at 16°C to obtain the pET28(a) plasmid with soluble tag containing the S-adenosylmethionine synthase mutant gene.

[0065] The protein sequence of the soluble tag is as follows:

[0066] MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRI AYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK

[0067] The soluble tag sequence is shown in SEQ ID NO:15.

[0068] Table 3. Nucleotide sequences of primers

[0069] Primer name Primer sequence XhoI-Tag-F <![CDATA[CCG CTCGAG TCCCCTATACTAGGTTATTGGAAA]]> XhoI-Tag-R <![CDATA[CCG CTCGAG CAACCCGGCGTCTGCGCGCAGAG]]>

[0070] (4) DH5α competent cell transformation

[0071] The pET28(a) plasmid containing the S-adenosylmethionine synthase mutant gene obtained in steps (2) and (3) was transformed into DH5α competent cells. DH5α competent cells were placed on ice, and after the cells thawed, 10 μL of plasmid solution was added and the cells were placed on ice for 30 min; heat-shocked at 42°C for 50 s, and then placed on ice for 3 min; 600 μL of sterile LB liquid medium was added and cultured at 37°C and 200 rpm for 1 h in a shaker; 200 μL of the cultured bacterial solution was taken and spread on LB agar plates containing Kana resistance (50 μg / mL) and incubated upside down at 37°C overnight.

[0072] (5) Screening for positive clones

[0073] Single colonies were picked from LB agar plates and inoculated into LB liquid medium containing Kana resistance (50 μg / mL). The culture was incubated overnight at 37°C and 220 rpm for plasmid extraction. Plasmids were extracted using the OMEGA Plasmid Mini Kit I according to the manufacturer's instructions, and the extracted plasmids were sent to Genewiz (Suzhou, China) for sequencing to determine whether the S-adenosylmethionine synthase mutant gene expression vector was successfully constructed.

[0074] 4. Heterologous expression of the S-adenosylmethionine synthase mutant 6×His fusion protein

[0075] (1) Transformation of protein-expressing competent cells

[0076] E. coli BL21(DE3), E. coli Rosetta(DE3), and OverexpressC43(DE3) competent cells were taken from an ultra-low temperature freezer at -80℃ and placed on ice. After the cells thawed, 1 μL of pET28-(a) plasmid containing the S-adenosylmethionine synthase mutant gene was added to each type of competent cell, and the cells were placed on ice for 30 min; heat-shocked at 42℃ for 50 s, then placed on ice for 3 min; 600 μL of sterile LB liquid medium was added, and the cells were cultured at 37℃ and 200 rpm for 1 h on a shaker; 200 μL of the cultured bacterial solution was aspirated and spread onto LB agar plates containing Kana resistance (50 μg / mL), and incubated upside down at 37℃ overnight.

[0077] (2) Preservation of S-adenosylmethionine synthase mutant expression strains

[0078] Single colonies were picked from LB agar plates and inoculated into LB liquid medium containing Kana resistance (50 μg / mL). The cultures were incubated overnight at 37°C and 220 rpm on a shaker to produce an expression strain for the S-adenosylmethionine synthase mutant. Sterile glycerol was added to the bacterial culture to a final volume concentration of 15%, and the culture was stored at -80°C for long-term preservation.

[0079] (3) Protein expression

[0080] a. Seed culture in shake flasks: 100 μL of the expression strain of S-adenosylmethionine synthase mutant was inoculated into 50 mL of sterile TB liquid medium, with a final kanamycin concentration of 50 μg / mL. The culture was incubated at 37℃ and 200 rpm for 8 h to obtain the expression seed culture of the S-adenosylmethionine synthase mutant. b. Fermentation culture in shake flasks: 10 mL of the expression seed culture of the S-adenosylmethionine synthase mutant was inoculated into 350 mL of sterile TB liquid medium, with a final kanamycin concentration of 50 μg / mL. The culture was incubated at 37℃ and 200 rpm until OD500 was achieved. 600 When the concentration of S-adenosylmethionine synthase is 0.6-0.8, sterile IPTG with a final concentration of 0.3 mM is added, and the cells are induced and cultured at 28°C and 200 rpm for 12 h to obtain enzyme-producing cells of S-adenosylmethionine synthase. Then, the cells are homogenized under high pressure to obtain crude enzyme solution of S-adenosylmethionine synthase.

[0081] (4) Protein detection

[0082] a. Protein Sample Preparation: The uncentrifuged crude S-adenosylmethionine synthase solution was used as the whole bacterial sample (whole bacteria). 1 mL of the crude S-adenosylmethionine synthase solution was placed in a 1.5 mL centrifuge tube and centrifuged at 12000 rpm for 5 min. The supernatant was used as the soluble S-adenosylmethionine synthase sample (supernatant). After completely removing the supernatant, the precipitate was resuspended in 1 mL of distilled water as the insoluble S-adenosylmethionine synthase sample (precipitate). 40 μL of the whole S-adenosylmethionine synthase sample, supernatant, and precipitate were added to each, and then heated in boiling water for 10 min to completely denature the protein. b. SDS-PAGE Gel Preparation: The SDS-PAGE gel preparation glass plate was mounted on the gel preparation rack. Add 2.8 mL of distilled water, 3.2 mL of 30% acrylamide solution, 2 mL of separating gel buffer (pH = 8.8), 80 μL of 10% ammonium persulfate solution, and 4 μL of TEMED to a 50 mL centrifuge tube. After mixing thoroughly, immediately add 7.5 mL of the separating gel solution to the well of the gel preparation glass plate and press the liquid surface flat with 0.5 mL of isopropanol. After the separating gel solidifies, discard the upper layer of isopropanol, wash with distilled water, and blot dry with absorbent paper. Add 2.28 mL of distilled water, 0.68 mL of 30% acrylamide solution, 1 mL of stacking gel buffer (pH = 6.8), 40 μL of 10% ammonium persulfate solution, and 4 μL of TEMED to a 50 mL centrifuge tube. After mixing thoroughly, immediately add the stacking gel solution to the well of the gel preparation glass plate, filling the entire well. Slowly and vertically insert the gel preparation comb into the stacking gel. c. SDS-PAGE electrophoresis: Mount the SDS-PAGE gel and gel preparation glass together onto the vertical electrophoresis stand, sealing the other side with a dedicated plastic plate. Pour 1×SDS-PAGE electrophoresis buffer into the central groove of the vertical electrophoresis stand; pour 1×SDS-PAGE electrophoresis buffer into the vertical electrophoresis tank, ensuring it covers the platinum wire electrode by approximately 5 cm. Slowly and vertically remove the gel preparation comb, and add 10 μL of different protein electrophoresis samples to the stacking gel, respectively. Add 5 μL of protein marker reagent to the wells adjacent to the samples. Electrophoresis at 90V for 40 min, then at 150V for 55 min. d. SDS-PAGE gel staining and destaining: Remove the SDS-PAGE gel from the glass plate interlayer, stain with Coomassie Brilliant Blue R-250 staining solution for 45 min, then destain overnight with Coomassie Brilliant Blue destaining solution, changing the destaining solution 3–4 times during this period. e. SDS-PAGE gel imaging: Taking pictures of SDS-PAGE gels using a gel imaging system.

[0083] Example 2

[0084] S-Adenosylmethionine synthase mutant G266S was used for S-adenosylmethionine synthesis. Precursor substances were added to the crude S-adenosylmethionine synthase solution at a concentration of 25 g / L, the ATP precursor concentration was 100 g / L, and the methionine concentration was 33 g / L. The fermentation pH was approximately 7.0, and the reaction temperature was maintained at 28℃. After 6 hours of reaction, the reaction solution was filtered, and the product content and purity were determined by HPLC. The results are shown in Table 4. Figure 3 .

[0085] Example 3

[0086] S-Adenosylmethionine synthase mutants were used to synthesize S-adenosylmethionine. Precursor substances were added to the crude S-adenosylmethionine synthase solution at a concentration of 25 g / L, the ATP precursor concentration was 100 g / L, and the methionine concentration was 33 g / L. The fermentation pH was maintained at approximately 7.0, and the reaction temperature was maintained at 28℃. After 6 hours of reaction, the reaction solution was filtered, and the product content and purity were determined by HPLC. The results are shown in Table 4.

[0087] Comparative Example 1

[0088] This experiment used S-adenosylmethionine synthase as the initial enzyme for the synthesis of S-adenosylmethionine.

[0089] Precursor substances were added to the crude enzyme solution of S-adenosylmethionine synthase. The concentration of the crude enzyme solution was 25 g / L, the concentration of the precursor ATP was 100 g / L, and the concentration of methionine was 33 g / L. The fermentation pH was maintained at around 7.0, the reaction temperature was maintained at 28℃, and the reaction solution was filtered after 6 h. The content and purity of the product were detected by HPLC. The test results are shown in Table 4.

[0090] Comparative Example 2

[0091] This experiment used S-adenosylmethionine synthase as the initial enzyme, with the soluble label changed to MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNI DQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLA for the synthesis of S-adenosylmethionine.

[0092] Precursor substances were added to the crude enzyme solution of S-adenosylmethionine synthase. The concentration of the crude enzyme solution was 25 g / L, the concentration of the precursor ATP was 100 g / L, and the concentration of methionine was 33 g / L. The fermentation pH was maintained at around 7.0, the reaction temperature was maintained at 28℃, and the reaction solution was filtered after 6 h. The content and purity of the product were detected by HPLC. The test results are shown in Table 4.

[0093] Comparative Example 3

[0094] This experiment used S-adenosylmethionine synthase as the initial enzyme, with the soluble label changed to MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMD SLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG for the synthesis of S-adenosylmethionine.

[0095] Precursor substances were added to the crude enzyme solution of S-adenosylmethionine synthase. The concentration of the crude enzyme solution was 25 g / L, the concentration of the precursor ATP was 100 g / L, and the concentration of methionine was 33 g / L. The fermentation pH was maintained at around 7.0, the reaction temperature was maintained at 28℃, and the reaction solution was filtered after 6 h. The content and purity of the product were detected by HPLC. The test results are shown in Table 4.

[0096] Table 4 Comparison of catalytic effects of different mutants

[0097]

Claims

1. A 266-site based mutant of S-adenosylmethionine synthetase, characterized in that, The mutant contains only the following mutation in positions 1 to 390 of the amino acid sequence corresponding to SEQ ID NO:1: G266S; its amino acid sequence is shown in SEQ ID NO:

7.

2. The gene encoding the mutant S-adenosylmethionine synthetase according to claim 1, characterized in that, The sequence of the encoding gene is shown in SEQ ID NO:

13.

3. An expression vector comprising the gene encoding the S-adenosylmethionine synthase mutant as described in claim 2.

4. Recombinant cells comprising the gene encoding the S-adenosylmethionine synthase mutant as described in claim 2.

5. The application of the S-adenosylmethionine synthase mutant according to claim 1 in the preparation of S-adenosylmethionine.

6. Use according to claim 5, wherein The specific application method is as follows: methionine and ATP precursor are added to the crude enzyme solution of engineered bacteria expressing the coding gene of the S-adenosylmethionine synthase mutant after cell wall disruption to obtain S-adenosylmethionine.

7. The application as described in claim 6, characterized in that, The concentration of the crude enzyme solution is 20-30 g / L, the concentration of the precursor ATP is 80-110 g / L, the concentration of methionine is 30-35 g / L, the pH of the reaction system is 6.5-7.2, the reaction temperature is 25-30℃, and the total reaction time is 5-10 h.

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