CN117551630A - S-adenosylmethionine synthetase mutant based on 121 locus and application thereof - Google Patents
S-adenosylmethionine synthetase mutant based on 121 locus and application thereof Download PDFInfo
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- C12Y205/01006—Methionine adenosyltransferase (2.5.1.6), i.e. adenosylmethionine synthetase
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Abstract
The invention belongs to the technical field of bioengineering, and particularly relates to an S-adenosylmethionine synthetase mutant based on 121 loci and application thereof. Wild-type bacteria of the S-adenosylmethionine synthetase mutants of the invention are derived fromGuyparkeria spXI15, obtaining mutants with mutation of core amino acid related to enzyme catalytic activity through site-directed mutagenesis; only one of the following mutations is present in positions 1 to 390 of the amino acid sequence corresponding to SEQ ID NO. 1: D22S; G121D;D244A; K251I; G266S; A307S; the amino acid sequence is shown as one of SEQ ID NO 3-8; the S-adenosylmethionine synthetase mutant realizes the application in the biosynthesis of S-adenosylmethionine, so that the yield of S-adenosylmethionine can reach 25.7g/L, and the purity of a process product can reach 89.7%.
Description
This application is a divisional application of patent application No. 202211409009.9; the application date of the original application is 11/2022, the application number is 202211409009.9, and the S-adenosylmethionine synthetase mutant and the application thereof are named.
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to an S-adenosylmethionine synthetase mutant based on 121 loci and application of the mutant.
Background
S-adenosylmethionine (SAM), which is named S-adenosyl-methionine, is an important metabolic intermediate in life such as animals, plants, and the like, is also an important physiologically active substance in human body, and participates in various biochemical reactions, such as intracellular methyl donor, and SAM plays an important role in methylation modification of molecules such as nucleic acid, protein, lipid, and the like. Meanwhile, SAM also participates in important biochemical reactions such as intracellular sulfur transfer reaction and polyamine synthesis. SAM is also a very valuable pharmaceutical molecule, playing an important role in the treatment of liver disease, depression and rheumatoid arthritis.
SAM is enzymatically synthesized from the substrates L-methionine and ATP via S-adenosylmethionine synthetase, and thus S-adenosylmethionine synthetase is a key enzyme for SAM synthesis. The existing industrial production process of the adenomethionine mainly comprises a yeast fermentation method and an enzymatic conversion method. SAM is prepared by intracellular expression using a specially cultivated SAM-expressing Saccharomyces cerevisiae strain. The genetically engineered Saccharomyces cerevisiae is researched in China, SAM is expressed by fermentation, but the highest expression is only within 10g/L of fermentation liquor at present, and the purity of the adenomethionine in the fermentation liquor is lower, so that the overall yield is lower, and the production cost is still higher. The enzyme method is mainly used for extracting and purifying adenomethionine synthetase from beer yeast or other engineering bacteria, and SAM is prepared by converting by optimizing the enzymatic reaction conditions, the method has high raw material conversion rate, and SAM is easy to extract and purify, but the method still has the problems of low catalytic efficiency of enzyme, high overall production cost and the like.
Disclosure of Invention
In order to solve the technical problems, the invention provides an S-adenosylmethionine synthetase mutant. The invention obtains the high-efficiency S-adenosylmethionine synthetase by excavation, and modifies the S-adenosylmethionine synthetase by a genetic engineering means to obtain the S-adenosylmethionine synthetase mutant, and the activity and the yield of synthesizing the S-adenosylmethionine by the S-adenosylmethionine synthetase mutant are obviously improved.
The wild strain of the S-adenosylmethionine synthetase mutant is derived from Guyparkleia sp.XI15, and the mutant with the mutation of core amino acid related to the catalytic activity of the enzyme is obtained through site-directed mutagenesis.
The S-adenosylmethionine synthetase mutant provided by the invention has more than 80% of homology (preferably more than or equal to 90%, more preferably more than or equal to 95%) with an amino acid sequence shown in SEQ ID NO. 1, has the capability of synthesizing S-adenosylmethionine, and remarkably improves the catalytic activity.
The present invention provides an S-adenosylmethionine synthetase mutant, wherein only the 22 nd, 123, 244, 251, 266, 307 th site mutation exists in the 1 st to 390 th sites of the amino acid sequence corresponding to SEQ ID NO. 1.
Preferably, the above mutants have only one of the following mutations in positions 1 to 390 of the amino acid sequence corresponding to SEQ ID NO. 1: D22S; G121D; D244A; K251I; G266S; A307S; the amino acid sequence is shown in one of SEQ ID NO 3-8.
The coding gene of the amino acid sequence shown in SEQ ID NO. 1 is the nucleotide sequence shown in SEQ ID NO. 2.
The invention also provides the coding gene of the mutant, and the gene sequence of the coding gene is shown as NO 9-14; further provided are expression vectors and recombinant cells containing the genes.
The preparation method of the S-adenosylmethionine synthetase mutant comprises the following steps:
(1) Synthesizing a coding gene of an amino acid sequence shown in SEQ ID NO. 1 to obtain a nucleotide sequence shown in SEQ ID NO. 2;
(2) Carrying out site-directed mutagenesis on the nucleotide sequence shown in SEQ ID NO. 2 to obtain coding genes of SEQ ID NO. 3-8;
(3) Adding a soluble tag into the coding gene of SEQ ID NO 3-8, wherein the protein sequence is as follows:
MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK, then carrying out gene cloning to obtain a plasmid containing a fusion soluble label S-adenosylmethionine synthetase mutant coding gene;
(4) Introducing a plasmid containing the S-adenosylmethionine synthetase mutant coding gene with increased soluble label into competent cells to obtain an expression strain of the S-adenosylmethionine synthetase mutant;
(5) And (3) performing induced fermentation on the expression strain of the S-adenosylmethionine synthetase mutant to obtain the S-adenosylmethionine synthetase mutant.
The invention also provides S-adenosylmethionine prepared by using the S-adenosylmethionine synthetase mutant.
The S-adenosylmethionine is obtained by fermenting and culturing engineering bacteria expressing the coding gene of the S-adenosylmethionine synthetase mutant to a certain extent, then performing cell wall breaking to obtain crude enzyme liquid, adding methionine and ATP precursor substances into the crude enzyme liquid, and performing catalytic reaction at the pH of 6.5-7.2 and the temperature of 25-30 ℃.
Preferably, the concentration of the crude enzyme solution is 20-30g/L.
Preferably, the precursor ATP is added at a concentration of 80-110g/L, methionine is added at a concentration of 30-35g/L, and the total reaction time is 5-10h.
The invention develops an S-adenosylmethionine synthetase mutant capable of catalyzing S-adenosylmethionine with high efficiency. The multi-angle multi-layer system from the primary structure to the higher structure compares the difference between S-adenosylmethionine synthetase with the ID of WP_058573953.1 in GenBank (the amino acid sequence is shown as SEQ ID NO: 1) and homologous enzyme protein, determines that key amino acid sites influencing the enzymatic properties of the S-adenosylmethionine synthetase are amino acids 22, 123, 244, 251, 266 and 307, and then mutates the sites by codon substitution as follows: D22S; G121D; D244A; K251I; G266S; A307S to obtain S-adenosylmethionine synthetase mutant; the mutant enzyme protein is obtained by constructing a 6 XHis fusion expression vector of the mutant gene and introducing the expression vector into a genetically engineered bacterium E.coli BL21 (DE 3) for induced expression.
The invention provides an S-adenosylmethionine synthetase mutant, the protein of which is non-natural protein and has the characteristic of efficiently catalyzing the synthesis of S-adenosylmethionine. The S-adenosylmethionine synthetase mutant is obtained by mutating amino acids 22, 123, 244, 251, 266 and 307 of S-adenosylmethionine synthetase of GenBank ID WP_ 058573953.1.
The invention has the beneficial effects that:
1. compared with the wild type S-adenosylmethionine synthetase mutant, the S-adenosylmethionine synthetase mutant prepared by the invention has the characteristic of high-efficiency catalysis of S-adenosylmethionine synthesis, the yield can reach 25.7g/L, and the purity of a process product can reach 89.7%;
2. the S-adenosylmethionine synthetase of the invention is added with a soluble label, so that the soluble expression of the enzyme is obviously improved, and compared with the case that the soluble label is not added, the mutant gene with obviously improved soluble expression is obtained, and according to figure 2, the soluble expression is improved by more than 70%.
3. And compared with the method for preparing S-adenosyl methionine in the background art, the cost is greatly reduced by more than 30 percent.
Drawings
FIG. 1 is a SDS-PAGE electrophoresis of the expression in S-adenosylmethionine synthetase mutant G266S main; BL21-CodonPlus (DE 3), BL21 (DE 3), rosetta (DE 3) and Overexpress C43 (DE 3) are four E.coli protein expression hosts, and Marker is a protein molecular weight standard;
FIG. 2 shows the expression of the protein after the addition of the solubility tag, and the GST-S-adenosylmethionine synthetase mutant G266S shows the mutant after the addition of the solubility tag;
FIG. 3 is a HPLC detection chart of a product synthesized by S-adenosylmethionine synthetase mutant G266S;
FIG. 4 is an HPLC detection chart of S-adenosylmethionine standard.
Detailed Description
The present invention will now be further described in connection with specific embodiments in order to enable those skilled in the art to better understand the invention. The following examples are only illustrative of the present invention and are not intended to limit the scope of the invention.
Example 1
1. S-adenosylmethionine synthetase mutant design
The S-adenosylmethionine synthetase gene with Genbank ID of WP_058573953.1 is obtained by screening from NCBI database through gene mining, the full length of an open reading frame of the gene is 1170bp, the coded S-adenosylmethionine synthetase consists of 390 amino acids, the amino acid sequence (390 aa) is SEQ ID NO:1, and the nucleotide sequence of the coded S-adenosylmethionine synthetase is SEQ ID NO:2.
By comparing the identity of S-adenosylmethionine synthetase (amino acid sequence shown as SEQ ID NO: 1) having an ID of WP_058573953.1 in Genbank with the homologous enzyme protein from the primary structure to the higher structure in multiple layers, it was confirmed that the key amino acid positions affecting the enzymatic properties of S-adenosylmethionine synthetase were amino acids 22, 123, 244, 251, 266, 307, and then these positions were subjected to the following mutations by codon substitution: D22S; G121D; D244A; K251I; G266S; A307S, the amino acid sequence of which is shown in SEQ ID NO 3-8.
2. Acquisition of S-adenosylmethionine synthetase mutant Gene
The S-adenosylmethionine synthetase mutant gene can be obtained by a total gene synthesis method or a molecular cloning method. The experiment adopts a total gene synthesis method to obtain an S-adenosylmethionine synthetase gene with the Genbank ID of WP_058573953.1, and adopts a PCR method to obtain an S-adenosylmethionine synthetase mutant gene.
1. Complete gene synthesis of WP_ 058573953.1S-adenosylmethionine synthetase
The S-adenosylmethionine synthetase with Genbank ID WP_058573953.1 was subjected to total gene synthesis, and the synthesized gene fragment was ligated into pUC57 plasmid, and the above gene was synthesized by the company of Shanghai, inc.
2. Site-directed mutagenesis of S-adenosylmethionine synthetase gene
(1) Site-directed mutagenesis primer design
And designing a primer for the gene sequence of the S-adenosylmethionine synthetase mutant, and introducing site-directed mutation. The nucleotide sequences of the primers are shown in Table 1. The primers were synthesized by the division of the Committee bioengineering (Shanghai).
TABLE 1 nucleotide sequences of primers
(2) Site-directed mutagenesis
The S-adenosylmethionine synthetase mutant gene was amplified by the following PCR system and procedure using the pUC57 plasmid containing the S-adenosylmethionine synthetase gene of PRQ77350.1 as a template and the upstream primer and the downstream primer obtained in the step (1). PCR system: KOD-Plus-0.5. Mu.L, plasmid template 0.8. Mu.L, upstream primer F5 '(10. Mu.M) 1. Mu.L, downstream primer R5' (10. Mu.M) 1. Mu.L, 10 XPCR Buffer 2.5. Mu.L, dNTPs (2 mM) 3. Mu.L, mgSO 4 (25mM)1.5μL，ddH 2 O14.7. Mu.L. The PCR procedure was as follows: pre-denaturing at 94℃for 4min; denaturation at 94℃for 40sec, annealing at 68℃and extension for 7.5min;18 cycles; c.68℃for 20min. A template plasmid containing the S-adenosylmethionine synthase gene of WP_058573953.1 was digested with DpnI enzyme at 37℃for 30min.
(3) DH5 alpha competent cell transformation
The pUC57 plasmid containing the S-adenosylmethionine synthetase mutant gene obtained in the step (2) was transformed into DH 5. Alpha. Competent cells. DH5 alpha competent cells were placed on ice, 10. Mu.L of plasmid solution was added after thawing, and placed on ice for 30min; heat shock is carried out for 50s at 42 ℃, and then the mixture is kept stand on ice for 3min; adding 600 mu L of sterile LB liquid medium, and culturing for 1h at 37 ℃ in a shaking table at 200rpm; 200. Mu.L of the cultured bacterial liquid was aspirated, and the resulting mixture was spread on LB plate medium containing Amp resistance (100. Mu.g/mL) and cultured at 37℃overnight in an inverted state.
(4) Positive clone screening
Single colonies on LB plates were picked, inoculated into LB liquid medium containing Amp resistance (100. Mu.g/mL), and shake cultured overnight at 37℃and 220rpm for plasmid extraction. Plasmids were extracted according to the instructions using OMEGA Plasmid Mini Kit I (cat# D6943) and the extracted plasmids were sent to Jin Weizhi (China, suzhou) company for sequencing to identify whether the S-adenosylmethionine synthetase of WP_058573953.1 was mutated successfully.
3. S-adenosylmethionine synthetase mutant gene cloning
(1) Gene cloning
The primer is designed for the gene sequence of the S-adenosylmethionine synthetase mutant, and is constructed into a pET28 (a) plasmid, and the nucleotide sequence of the primer is shown in Table 2. Principal for lifeThe primers were synthesized by engineering (Shanghai) Co., ltd. The S-adenosylmethionine synthetase mutant gene was amplified by using pUC57 plasmid containing the S-adenosylmethionine synthetase mutant gene as a template, and the obtained upstream primer and downstream primer according to the following PCR system and procedure. PCR system: primeSTAR Max Premix (2X) 25. Mu.L, plasmid template 0.5. Mu.L, upstream primer NcoI-F (10. Mu.M) 2. Mu.L, downstream primer XhoI-R (10. Mu.M) 2. Mu.L, ddH 2 O was added to a total volume of 50. Mu.L. The PCR procedure was as follows: a.98 ℃ pre-denaturation for 2min; denaturation at 98℃for 10sec, annealing at 65℃for 10sec, extension at 72℃for 30sec;30 cycles; c.72℃for 3min. The PCR amplification products were detected by 1.0% agarose gel electrophoresis to give a band of interest of approximately 1600bp in size. The target band was excised under an ultraviolet lamp, and the S-adenosylmethionine synthetase mutant gene fragment was recovered using Omega Gel Extraction Kit (cat# D2500) according to the kit instructions.
TABLE 2 nucleotide sequences of primers
(2) Expression vector construction
Double digestion was performed on the S-adenosylmethionine synthetase mutant gene and pET28 (a) vector using NcoI and Xho I restriction enzymes, respectively. Enzyme cleavage system (gene): s-adenosylmethionine synthetase mutant gene 25. Mu.L, ncoI enzyme 2. Mu.L, xhoI enzyme 2. Mu.L, 10 XBuffer 5. Mu.L, and sterile double distilled water was added to the system to 50. Mu.L. Enzyme cutting system (carrier): pET28 (a) vector 2. Mu.L, ncoI enzyme 0.5. Mu.L, xhoI enzyme 0.5. Mu.L, 10 XBuffer 1. Mu.L, and sterile double distilled water was added to the system to 10. Mu.L. Enzyme cutting conditions: and enzyme cutting at 37 ℃ for 30min. Then, the double digested mutant gene of S-adenosylmethionine and the linear vector of pET28 (a) are connected by using T4 DNA ligase, and the plasmid pET28 (a) containing the mutant gene of S-adenosylmethionine is obtained after overnight connection at 16 ℃.
(3) Construction of expression vector fused with soluble tag containing S-adenosylmethionine synthetase mutant
The soluble tag sequence was designed and constructed into pET28 (a) plasmid containing S-adenosylmethionine synthetase mutant gene, the nucleotide sequence of the primer is shown in Table 3. The primers were synthesized by the division of the Committee bioengineering (Shanghai). The soluble tag fragment was amplified using the pET-GST plasmid as a template and the obtained upstream primer and downstream primer according to the following PCR system and procedure. PCR system: primeSTAR Max Premix (2X) 25. Mu.L, pET-GST plasmid template 0.5. Mu.L, upstream primer XhoI-Tag-F (10. Mu.M) 2. Mu.L, downstream primer XhoI-Tag-R (10. Mu.M) 2. Mu.L, ddH 2 O was added to a total volume of 50. Mu.L. The PCR procedure was as follows: a.98 ℃ pre-denaturation for 2min; denaturation at 98℃for 10sec, annealing at 65℃for 10sec, extension at 72℃for 30sec;30 cycles; c.72℃for 3min. The PCR amplification product was detected by 1.0% agarose gel electrophoresis to give a target band of approximately 650bp in size. The target band was cut under a UV lamp, and the soluble tag fragment was recovered using Omega Gel Extraction Kit (cat# D2500) according to the kit instructions.
The soluble tag fragment and pET28 (a) plasmid containing the S-adenosylmethionine synthetase mutant gene were subjected to single cleavage using Xho I restriction enzyme, respectively. Enzyme cleavage system (soluble tag): 25. Mu.L of the soluble tag fragment, 2. Mu.L of Xho I enzyme, 10 XBuffer, 5. Mu.L, and 50. Mu.L of the system were supplemented with sterile double distilled water. Enzyme cutting system (carrier): pET28 (a) plasmid containing S-adenosylmethionine synthetase mutant gene 2. Mu.L, xhoI enzyme 0.5. Mu.L, 10 XBuffer 1. Mu.L, and sterile double distilled water was added to the system to 10. Mu.L. Enzyme cutting conditions: and enzyme cutting at 37 ℃ for 30min. Adding phosphate groups into the soluble tag fragments after single enzyme digestion, and reacting: 4. Mu.L of the soluble tag fragment after single cleavage, 0.5. Mu.L of T4 polynucleotide kinase, 2. Mu.L of 10 xT 4 ligase Buffer, and 20. Mu.L of sterile double distilled water were added to the system, and the mixture was heated at 37℃for 45min and at 70℃for 15min. Removing phosphate groups from a single digested pET28 (a) plasmid fragment containing an S-adenosylmethionine synthetase mutant gene, and reacting: 10 mu L of pET28 (a) plasmid fragment containing S-adenosylmethionine synthetase mutant gene after single enzyme digestion, 1 mu L of alkaline phosphatase, 2 mu L of 10 Xalkaline phosphatase Buffer, and 20 mu L of sterile double distilled water are added, and the mixture is heated for 1h at 37 ℃ and 15min at 70 ℃. Then, a T4 DNA ligase is used for connecting a soluble tag fragment added with phosphate groups after single enzyme digestion and a pET28 (a) plasmid fragment containing S-adenosyl methionine synthetase mutant genes with phosphate groups removed after single enzyme digestion, and the plasmid pET28 (a) containing S-adenosyl methionine synthetase mutant genes fused with the soluble tag is obtained after overnight connection at 16 ℃.
The protein sequence of the soluble tag is as follows:
MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYGVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK
the soluble tag sequence is shown as SEQ ID NO. 15.
TABLE 3 nucleotide sequences of primers
| Primer name | Primer sequences |
| XhoI-Tag-F | CCGCTCGAGTCCCCTATACTAGGTTATTGGAAA |
| XhoI-Tag-R | CCGCTCGAGCAACCCGGCGTCTGCGCGCAGAG |
(4) DH5 alpha competent cell transformation
Transforming pET28 (a) plasmid containing S-adenosylmethionine synthetase mutant gene obtained in step (2) and (3) into DH5 alpha competent cells. DH5 alpha competent cells were placed on ice, 10. Mu.L of plasmid solution was added after thawing, and placed on ice for 30min; heat shock is carried out for 50s at 42 ℃, and then the mixture is kept stand on ice for 3min; adding 600 mu L of sterile LB liquid medium, and culturing for 1h at 37 ℃ in a shaking table at 200rpm; 200. Mu.L of the cultured broth was aspirated, plated on LB plate medium containing Kana resistance (50. Mu.g/mL), and cultured upside down at 37℃overnight.
(5) Positive clone screening
Single colonies on LB plates were picked, inoculated into LB liquid medium containing Kana resistance (50. Mu.g/mL), and shake cultured overnight at 37℃and 220rpm for plasmid extraction. The plasmid was extracted according to the instructions using OMEGA Plasmid Mini Kit I and the extracted plasmid was sent to Jin Weizhi (China, suzhou) company for sequencing to identify whether the S-adenosylmethionine synthetase mutant gene expression vector was constructed successfully.
4. Heterologous expression of S-adenosylmethionine synthetase mutant 6 XHis fusion proteins
(1) Protein expression competent cell transformation
E.coli BL21 (DE 3), E.coli Rosetta (DE 3) and Overexpress C43 (DE 3) competent cells were taken out of the-80℃ultra-low temperature refrigerator, respectively, and placed on ice. After the cells were thawed, 1. Mu.L of pET28- (a) plasmid containing the S-adenosylmethionine synthetase mutant gene was added to each competent cell, and the mixture was left on ice for 30min; heat shock is carried out for 50s at 42 ℃, and then the mixture is placed on ice for 3min; 600 μl of sterile LB liquid medium was added, and shake culture was performed at 37deg.C and 200rpm for 1h; 200. Mu.L of the cultured broth was aspirated, plated on LB plate medium containing Kana resistance (50. Mu.g/mL), and cultured upside down at 37℃overnight.
(2) Preservation of S-adenosylmethionine synthetase mutant expression strain
Single colonies on LB plates were picked, inoculated into LB liquid medium containing Kana resistance (50. Mu.g/mL), and shake cultured overnight at 37℃and 220rpm, and used as expression strains for S-adenosylmethionine synthetase mutants. Adding sterile glycerol with final volume concentration of 15% into the bacterial liquid, and storing in ultralow temperature refrigerator at-80deg.C for a long time.
(3) Protein expression
a. Culturing seeds in a shaking bottle: 100 mu L S-adenosineThe expression strain of the methionine synthetase mutant is inoculated into 50mL sterile TB liquid culture medium, the final concentration of kanamycin is 50 mug/mL, and the strain is cultured for 8 hours at 37 ℃ and 200rpm, so as to obtain the expression seed bottle bacterial liquid of the S-adenosyl methionine synthetase mutant. b. Fermenting and shaking culture: 10mL of seed bottle bacterial liquid for expressing the S-adenosyl methionine synthetase mutant is inoculated into 350mL of sterile TB liquid culture medium, and the final concentration of kanamycin is 50 mug/mL, 37 ℃ and 200rpm; waiting for OD 600 When the enzyme is=0.6-0.8, sterile IPTG with the final concentration of 0.3mM is added, the temperature is 28 ℃, the induction culture is carried out for 12 hours at 200rpm, the enzyme-producing thalli of the S-adenosylmethionine synthetase are obtained, and then the high-pressure homogenization and the crushing are carried out to obtain the crude enzyme liquid of the S-adenosylmethionine synthetase.
(4) Protein detection
a. Protein sample preparation: the crude enzyme solution of S-adenosylmethionine synthetase which was not centrifuged was used as a whole-cell sample (whole cell). 1mL of the crude S-adenosylmethionine synthetase enzyme solution was placed in a 1.5mL centrifuge tube, and centrifuged at 12000rpm for 5min, and the supernatant was taken as a soluble S-adenosylmethionine synthetase sample (supernatant). After the supernatant was completely aspirated, the pellet was resuspended with 1mL of distilled water as an insoluble S-adenosylmethionine synthetase sample (pellet). A whole cell sample, a supernatant sample and a precipitate sample of 40. Mu. L S-adenosylmethionine synthetase were taken, 10. Mu.L of 5X Protein Loading Buffer were added thereto, and the mixture was heated in boiling water for 10 minutes to completely denature the protein. SDS-PAGE gel preparation: SDS-PAGE gel preparation glass plates were mounted on a gel preparation rack. Into a 50mL centrifuge tube were added 2.8mL of distilled water, 3.2mL of 30% acrylamide solution, 2mL of seperate Buffer solution (pH=8.8), 80. Mu.L of 10% ammonium persulfate solution, and 4. Mu.L of TEMED, respectively. Immediately after mixing, 7.5mL of the gum-separating solution was added to the gel formulation glass plate well and the liquid surface was flattened with 0.5mL of isopropyl alcohol. After the separation gel is solidified, the upper isopropanol layer is discarded, and the separation gel is washed by distilled water and then is sucked by absorbent paper. Into a 50mL centrifuge tube were added 2.28mL of distilled water, 0.68mL of 30% acrylamide solution, 1mL of concentrated gum Buffer solution (pH=6.8), 40. Mu.L of 10% ammonium persulfate solution, and 4. Mu.L of TEMED, respectively. Immediately after mixing, the concentrated gum solution was added to the gel formulation glass plate well to fill the entire well. The gel preparation comb was slowly inserted vertically into the concentrated gel. SDS-PAGE electrophoresis: SDS-PAGE gels were mounted on a vertical electrophoresis rack with gel formulation glass and the other side was closed with a special plastic plate. Filling the middle groove of the vertical electrophoresis frame with 1 XSDS-PAGE electrophoresis buffer; a1 XSDS-PAGE running buffer was poured into the vertical electrophoresis tank so as to be about 5cm beyond the platinum wire electrode. The gel preparation comb was slowly and vertically removed, 10 μl of each of the different Protein electrophoresis samples was added to the concentrated gel, and 5 μl of Protein Marker reagent was added to the sample adjacent wells. 90V constant voltage electrophoresis for 40min and 150V constant voltage electrophoresis for 55min. SDS-PAGE gel staining and decolorizing: SDS-PAGE gel was removed from the glass plate interlayer, stained with Coomassie blue R-250 stain for 45min, and then destained overnight with Coomassie blue destaining solution, with Massa blue destaining solution changed 3-4 times. SDS-PAGE gel imaging: SDS-PAGE gels were photographed in a gel imager.
Example 2
S-adenosylmethionine synthesis was performed using S-adenosylmethionine synthetase mutant G266S. The precursor substances are added into the S-adenosylmethionine synthetase crude enzyme solution, the concentration of the crude enzyme solution is 25g/L, the addition concentration of precursor ATP is 100g/L, the addition concentration of methionine is 33g/L, the fermentation pH is about 7.0, the reaction temperature is maintained at 28 ℃, the reaction solution is filtered after the reaction is carried out for 6 hours, the content and the purity of the product are detected by HPLC, and the detection results are shown in Table 4 and figure 3.
Example 3
S-adenosylmethionine synthesis was performed using other mutants of S-adenosylmethionine synthetase. The precursor substances are added into the S-adenosylmethionine synthetase crude enzyme solution, the concentration of the crude enzyme solution is 25g/L, the addition concentration of precursor ATP is 100g/L, the addition concentration of methionine is 33g/L, the fermentation pH is maintained at about 7.0, the reaction temperature is maintained at 28 ℃, the reaction solution is filtered after the reaction is carried out for 6 hours, the content and the purity of the product are detected by HPLC, and the detection result is shown in Table 4.
Comparative example 1
The test uses S-adenosylmethionine synthetase initial enzyme to synthesize S-adenosylmethionine.
The precursor substances are added into the S-adenosylmethionine synthetase crude enzyme solution, the concentration of the crude enzyme solution is 25g/L, the addition concentration of precursor ATP is 100g/L, the addition concentration of methionine is 33g/L, the fermentation pH is maintained at about 7.0, the reaction temperature is maintained at 28 ℃, the reaction solution is filtered after the reaction is carried out for 6 hours, the content and the purity of the product are detected by HPLC, and the detection result is shown in Table 4.
Comparative example 2
In the test, S-adenosylmethionine is synthesized by adopting S-adenosylmethionine synthetase initial enzyme and changing a soluble label into MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNI DQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLA.
The precursor substances are added into the S-adenosylmethionine synthetase crude enzyme solution, the concentration of the crude enzyme solution is 25g/L, the addition concentration of precursor ATP is 100g/L, the addition concentration of methionine is 33g/L, the fermentation pH is maintained at about 7.0, the reaction temperature is maintained at 28 ℃, the reaction solution is filtered after the reaction is carried out for 6 hours, the content and the purity of the product are detected by HPLC, and the detection result is shown in Table 4.
Comparative example 3
In the test, S-adenosylmethionine is synthesized by adopting S-adenosylmethionine synthetase initial enzyme and changing a soluble label into MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMD SLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG.
The precursor substances are added into the S-adenosylmethionine synthetase crude enzyme solution, the concentration of the crude enzyme solution is 25g/L, the addition concentration of precursor ATP is 100g/L, the addition concentration of methionine is 33g/L, the fermentation pH is maintained at about 7.0, the reaction temperature is maintained at 28 ℃, the reaction solution is filtered after the reaction is carried out for 6 hours, the content and the purity of the product are detected by HPLC, and the detection result is shown in Table 4.
TABLE 4 comparison of catalytic effects of different mutants
Claims (7)
1. A mutant S-adenosylmethionine synthetase based on position 121, characterized in that said mutant has only the following mutations in positions 1 to 390 of the amino acid sequence corresponding to SEQ ID No. 1: G121D; the amino acid sequence is shown as SEQ ID NO. 4.
2. The coding gene of the S-adenosylmethionine synthetase mutant according to claim 1, wherein the coding gene has a sequence shown in SEQ ID NO. 10.
3. An expression vector comprising the gene encoding the mutant S-adenosylmethionine synthetase of claim 2.
4. A recombinant cell comprising the gene encoding the mutant S-adenosylmethionine synthetase of claim 2.
5. Use of the mutant S-adenosylmethionine synthetase according to claim 1 for preparing S-adenosylmethionine.
6. The application according to claim 5, wherein the specific application method is as follows: the S-adenosylmethionine is obtained by adding methionine and ATP precursor into crude enzyme solution obtained by breaking wall of engineering bacteria expressing coding genes of the S-adenosylmethionine synthetase mutant.
7. The use according to claim 6, wherein the concentration of the crude enzyme solution is 20-30g/L, the concentration of the precursor ATP is 80-110g/L, the concentration of methionine is 30-35g/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 hours.
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