CN113249365B - L-threonine aldolase mutant and method for preparing L-syn-p-methylsulfonylbenzserine - Google Patents
L-threonine aldolase mutant and method for preparing L-syn-p-methylsulfonylbenzserine Download PDFInfo
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- 238000006243 chemical reaction Methods 0.000 claims abstract description 29
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- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 claims abstract description 14
- PSVPUHBSBYJSMQ-UHFFFAOYSA-N 4-methylsulfonylbenzaldehyde Chemical compound CS(=O)(=O)C1=CC=C(C=O)C=C1 PSVPUHBSBYJSMQ-UHFFFAOYSA-N 0.000 claims abstract description 11
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- 229960000318 kanamycin Drugs 0.000 description 1
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- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
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- 229910021645 metal ion Inorganic materials 0.000 description 1
- PGXWDLGWMQIXDT-UHFFFAOYSA-N methylsulfinylmethane;hydrate Chemical compound O.CS(C)=O PGXWDLGWMQIXDT-UHFFFAOYSA-N 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- WHQSYGRFZMUQGQ-UHFFFAOYSA-N n,n-dimethylformamide;hydrate Chemical compound O.CN(C)C=O WHQSYGRFZMUQGQ-UHFFFAOYSA-N 0.000 description 1
- -1 p-methylsulfonylbenzylserine Chemical compound 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/04—Alpha- or beta- amino acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y401/00—Carbon-carbon lyases (4.1)
- C12Y401/02—Aldehyde-lyases (4.1.2)
- C12Y401/02005—L-Threonine aldolase (4.1.2.5)
Abstract
The application discloses an L-threonine aldolase mutant and a method for preparing L-syn-p-methylsulfonylphenyl serine. The L-threonine aldolase mutant is obtained by mutating wild L-threonine aldolase, and can catalyze condensation reaction to generate L-syn-p-methylsulfonyl phenylserine by taking glycine and p-methylsulfonyl benzaldehyde as substrates and pyridoxal phosphate as coenzyme. The L-syn-p-methylsulfonylphenyl serine produced by using the L-threonine aldolase mutant has the following advantages: 1. the production process is simple, and the reaction condition is mild; 2. the L-threonine aldolase has high selectivity and high optical purity, and does not need resolution; 3. the atom utilization rate is high, and the theory can reach 100%; 4. the production process is environment-friendly, has less pollution, and accords with the green chemical idea; 5. the product is simple to separate and purify.
Description
The application relates to a division application of an L-threonine aldolase mutant, a gene and a method for preparing L-syn-p-methylsulfonylbenzserine, wherein the application date of the original application is 2020, 1 month and 17 days, and the application number is 2020100508531.
Technical Field
The application relates to the technical field of enzyme engineering, in particular to an L-threonine aldolase mutant, a gene and a method for preparing L-syn-p-methylsulfonylbenzenesulfone.
Background
L-syn-p-methylsulfonylbenzylserine is an important medical intermediate and is applied to the synthesis of various antibiotics. The p-methylsulfonylbenzylserine has two chiral centers and four isomers, which are respectively: l-syn-p-methylsulfonylphenyl serine, L-anti-p-methylsulfonylphenyl serine, D-syn-p-methylsulfonylphenyl serine, D-anti-p-methylsulfonylphenyl serine (FIG. 1).
At present, two main production methods exist for L-syn-p-methylsulfonylbenzylserine. One is chemical synthesis, copper sulfate is used as a catalyst, and by utilizing the complexation of metal ions, two cis products are generated by the selective reaction of methylsulfonyl benzaldehyde and glycine: the L-syn-p-methylsulfonylphenyl serine copper and the D-syn-p-methylsulfonylphenyl serine copper cannot be directly split, and can only be continuously subjected to esterification reaction with ethanol to generate racemic D, L-syn-p-methylsulfonylphenyl serine ethyl ester, and then chiral resolution is performed to realize separation of D-isomer and L-isomer, so that the optically pure L-syn-p-methylsulfonylphenyl serine ethyl ester is prepared. The process mainly has the following defects: the theoretical yield is only 50%, the ineffective enantiomer is difficult to apply and the process is complex. Meanwhile, the equimolar chiral resolving agent is required to be used, the single resolution yield is low, and a large amount of waste water and waste salt can be generated by recycling the resolving agent and copper salt in the reaction. The other is to combine the chemical method and the biological method, the main process is to firstly utilize the p-methylsulfonyl benzaldehyde and glycine to generate racemized p-methylsulfonyl phenylserine copper salt under the catalysis of copper sulfate, and the main products are L-syn-p-methylsulfonyl phenylserine copper and D-syn-p-methylsulfonyl phenylserine copper (figure 2). Removing copper ions in the product to generate a mixture of L-syn-p-methylsulfonylphenyl serine and D-syn-p-methylsulfonylphenyl serine. And then D-threonine aldolase is used for decomposing D-syn-p-methylsulfonylphenyl serine to achieve the aim of resolution, so as to obtain the optically pure L-syn-p-methylsulfonylphenyl serine. This process has the following disadvantages: the production process is complex, the atom utilization rate is not high, the theoretical yield can only reach 50%, and the problem of copper salt environmental pollution can also be generated.
In contrast, the use of the L-threonine aldolase mutant to catalyze the direct production of L-syn-p-methylsulfonylbenzaldehyde and glycine has the following advantages:
1. the production process is simple, and the reaction condition is mild;
2. the L-threonine aldolase has high selectivity and high optical purity;
3. the method does not need resolution, has high atom utilization rate and can reach 100% in theory;
4. the production process is environment-friendly, has less pollution, and accords with the green chemical idea;
5. the downstream separation and purification process is simple.
The key point of catalyzing and synthesizing the L-syn-p-methylsulfonyl phenylserine by using the L-threonine aldolase is to obtain the enzyme capable of synthesizing the L-syn-p-methylsulfonyl phenylserine with high selectivity. The L-threonine aldolase reported at present has higher selectivity to alpha-carbon, and ee (%) >99%. The selectivity to beta-carbon is not high, and de (%) is between 20 and 50%, so that a mixture of L-syn-p-methylsulfonylphenyl serine and L-anti-p-methylsulfonylphenyl serine is produced. Therefore, no research report on directly using L-threonine aldolase to synthesize L-syn-p-methylsulfonylphenyl serine with high chiral purity exists at present.
Disclosure of Invention
Aiming at the defects of the existing L-syn-p-methylsulfonylbenzylserine synthesis process, the application provides a method for preparing L-syn-p-methylsulfonylbenzylserine by using an L-threonine aldolase mutant, which has high raw material utilization rate and can be recycled. The product is easy to separate and purify, and has high conversion rate, yield and chiral purity; compared with chemical catalysis technology, the technology is simple and pollution is small.
An L-threonine aldolase mutant obtained by mutating a wild-type L-threonine aldolase having accession numbers wp_015261381.1, SIS82838.1, wp_016204489.1, wp_023973138.1, wp_077361571.1 or wp_069998496.1, respectively, specifically one of the following mutations:
(1) The mutant is obtained by mutating wild type L-threonine aldolase with the accession number WP_015261381.1, and the mutation site is one of the following: 5F, 5W, 8H, 8F, 31H, 31M, 125K, 125V, 143K, 143R, 227R, 252F, 252H, 305R, 305K, 307H, 307R, 308H, 308K, 5F/8H/31H/125V, 5F/8H/31H/305R, 31H/125V/227R/305R, 5F/8H/31H/125V, 5F/125V/227R/E305R, 8H/31H/125V/227R, 8H/31H/143R/305R, 5F/8H/31H/125V/227R/E305K 5F/8H/Y31M/125V/227R/305R, 5W/8H/31H/125V/227R/305R, 5F/8F/31H/125K/227R/E305R, 8H/31H/125V/143R/252F/305R, 5F/8H/31H/125V/143K/305R/307R, 8F/31H/125V/143R/227R/305R/307H, 8F/31H/125V/143R/252F/305R/308H, 8F/31M/125V/143R/252F/305R/308K;
(2) The mutant is obtained by mutating wild type L-threonine aldolase with accession number SIS82838.1, and the mutation site is one of the following: 18F, 18W, 21H, 21F, 44H, 44M, 138K, 138V, 157K, 157R, 241F, 266H, 319R, 319K, 321H, 321R, 322H, 322K, 18F/21H/44H/138V, 18F/21H/44/319R, 44H/138V/241R/319R, 18F/21H/44H/138V, 18F/138V/241R/319R, 21H/44H/138V/241R, 21H/44H/157R/319R, 13F/21H/44H/138V/241R/319K 13F/21H/44M/138V/241R/319R, 13W/21H/44H/138V/241R/319R, 13F/21F/44H/138K/241R/319R, 21H/31H/138V/157R/266F/319R, 13F/21H/44H/138V/143K/319R/322R, 21F/44H/138V/157R/241R/319/322H, 21F/44H/138V/157R/266F/319H, 21F/44M/138V/157R/266F/319R/323K;
(3) The mutant is obtained by mutating wild type L-threonine aldolase with the accession number WP_016204489.1, and the mutation site is one of the following: 5F, 5W, 8H, 8F, 31H, 31M, 125K, 125V, 143K, 143R, 227R, 252F, 252H, 305R, 305K, 307H, 307R, 308H, 308K, 5F/8H/31H/125V, 5F/8H/31H/305R, 31H/125V/227R/305R, 5F/8H/31H/125V, 5F/125V/227R/E305R, 8H/31H/125V/227R, 8H/31H/143R/305R, 5F/8H/31H/125V/227R/E305K 5F/8H/Y31M/125V/227R/305R, 5W/8H/31H/125V/227R/305R, 5F/8F/31H/125K/227R/E305R, 8H/31H/125V/143R/252F/305R, 5F/8H/31H/125V/143K/305R/307R, 8F/31H/125V/143R/227R/305R/307H, 8F/31H/125V/143R/252F/305R/308H, 8F/31M/125V/143R/252F/305R/308K;
(4) The mutant is obtained by mutating wild type L-threonine aldolase with the accession number WP_023973138.1, and the mutation site is one of the following: 5F, 5W, 8H, 8F, 31H, 31M, 125K, 125V, 143K, 143R, 227R, 252F, 252H, 305R, 305K, 307H, 307R, 308H, 308K, 5F/8H/31H/125V, 5F/8H/31H/305R, 31H/125V/227R/305R, 5F/8H/31H/125V, 5F/125V/227R/E305R, 8H/31H/125V/227R, 8H/31H/143R/305R, 5F/8H/31H/125V/227R/E305K 5F/8H/Y31M/125V/227R/305R, 5W/8H/31H/125V/227R/305R, 5F/8F/31H/125K/227R/E305R, 8H/31H/125V/143R/252F/305R, 5F/8H/31H/125V/143K/305R/307R, 8F/31H/125V/143R/227R/305R/307H, 8F/31H/125V/143R/252F/305R/308H, 8F/31M/125V/143R/252F/305R/308K;
(5) The mutant is obtained by mutating wild type L-threonine aldolase with the accession number WP_077361571.1, and the mutation site is one of the following: 5F, 5W, 8H, 8F, 31H, 31M, 125K, 125V, 143K, 143R, 227R, 252F, 252H, 305R, 305K, 307H, 307R, 308H, 308K, 5F/8H/31H/125V, 5F/8H/31H/305R, 31H/125V/227R/305R, 5F/8H/31H/125V, 5F/125V/227R/E305R, 8H/31H/125V/227R, 8H/31H/143R/305R, 5F/8H/31H/125V/227R/E305K 5F/8H/Y31M/125V/227R/305R, 5W/8H/31H/125V/227R/305R, 5F/8F/31H/125K/227R/E305R, 8H/31H/125V/143R/252F/305R, 5F/8H/31H/125V/143K/305R/307R, 8F/31H/125V/143R/227R/305R/307H, 8F/31H/125V/143R/252F/305R/308H, 8F/31M/125V/143R/252F/305R/308K;
(6) The mutant is obtained by mutating wild type L-threonine aldolase with the accession number WP_069998496.1, and the mutation site is one of the following: 5F, 5W, 8H, 8F, 31H, 31M, 125K, 125V, 143K, 143R, 227R, 252F, 252H, 305R, 305K, 307H, 307R, 308H, 308K, 5F/8H/31H/125V, 5F/8H/31H/305R, 31H/125V/227R/305R, 5F/8H/31H/125V, 5F/125V/227R, 8H/31H/143R/305R, 5F/8H/31H/125V/305R, 305K, 5F/8H/31M/125V/305R, 5F/8H/125V/227R, 5W/8H/31H/125V/305R, 5F/8F/125H/125F/305R, 8F/305R, 143H/31H/305R, 8F/305R, 125H/305R, 143H/305R, 125H/305R, 252F/305R, and 252F/305R, 35H/305R, 5F/8H/31H/305R, and 305R, 5F/305R, 35H/305R, and 305R, 5F/35H/35R, and 305R, 5F/8H/305R, and/143R, and/305R.
The application also provides a gene encoding the L-threonine aldolase mutant.
The application also provides an expression vector comprising the gene. Preferably, the expression vector is a pET28a plasmid into which the gene is inserted.
The application also provides a genetic engineering bacterium containing the gene.
The application also provides application of the L-threonine aldolase mutant, the gene or one of the genetically engineered bacteria in preparation of L-syn-p-methylsulfonylbenzenesis.
The application also provides a method for preparing L-syn-p-methylsulfonylbenzylserine, which takes glycine and p-methylsulfonylbenzaldehyde as substrates, pyridoxal phosphate as coenzyme, and utilizes the L-threonine aldolase mutant or cells expressing the L-threonine aldolase mutant as catalysts to generate the L-syn-p-methylsulfonylbenzylserine through condensation reaction. The cell expressing the L-threonine aldolase mutant may be the above genetically engineered bacterium containing the gene.
Preferably, the reaction is carried out in an organic solvent-water mixed solution or an aqueous solution. More preferably, the organic solvent is DMF or DMSO, and the addition amount of the organic solvent is less than or equal to 20 percent of the total volume. The reaction can be carried out in an aqueous phase, but after adding some organic solvent, the conversion rate and the de value of the reaction can be improved to a certain extent.
In the reaction system, the catalyst is used in the form of crude enzyme liquid after cell disruption, engineering bacteria resting cells expressing recombinant enzyme, purified pure enzyme or immobilized enzyme.
Compared with the prior art, the application has the following beneficial effects:
1. the production process is simple, and the reaction condition is mild;
2. the L-threonine aldolase has high selectivity and high optical purity, and does not need resolution;
3. the atom utilization rate is high, and the theory can reach 100%;
4. the production process is environment-friendly, has less pollution, and accords with the green chemical idea;
5. the product is simple to separate and purify.
Drawings
FIG. 1 is a schematic diagram of four configuration molecular structures of p-methylsulfonylphenyl serine.
FIG. 2 is an equation for the chemical synthesis of L-syn-p-methylsulfonylbenzylserine.
FIG. 3 is an equation for synthesizing L-syn-p-methylsulfonylbenzylserine by a biological synthesis method.
FIG. 4 is a diagram showing the sequence alignment and the corresponding mutation sites of threonine aldolase from different sources.
FIG. 5 is a high performance liquid chromatography (achiral analysis) of a substrate p-methylsulfonyl benzaldehyde standard sample, and the retention time of the p-methylsulfonyl benzaldehyde is 7.439min.
FIG. 6 is a high performance liquid chromatography (achiral analysis) of the product p-methylsulfonylbenzeneserine standard, with a p-methylsulfonylbenzeneserine retention time of 2.620min.
FIG. 7 is a liquid phase diagram (chiral analysis) of L-anti-p-methylsulfonylbenzeneserine standard; wherein the retention time is 6.333min,
FIG. 8 is a liquid phase diagram (chiral analysis) of L-syn-p-methylsulfonylbenzeneserine standard; wherein the retention time is 7.891min.
FIG. 9 is a high performance liquid chromatography (chiral analysis) of a reaction solution (after completion of the reaction) of wild-type L-threonine aldolase DdLTA in example 4: wherein, retention time: l-anti-p-methylsulfonylphenyl serine is 6.284 min and L-syn-p-methylsulfonylphenyl serine is 7.436min.
Detailed Description
The experimental methods in the application are all conventional methods unless otherwise specified, and the gene cloning operation can be specifically found in the "molecular cloning Experimental guidelines" by J.Sam Broker et al.
Reagents for upstream genetic engineering operations: restriction enzymes, primer STAR DNA polymerase, DNA ligase, and recombinase used in the examples of the application are all purchased from TaKaRa; genome extraction kit, plasmid extraction kit, DNA recovery purification kit were purchased from Axygen; coli BL21 (DE 3), plasmids, etc. are available from Novagen; DNA marker, low molecular weight standard protein, agarose electrophoresis reagent were purchased from Beijing full gold biotechnology Co., ltd; primer synthesis and gene sequencing work are completed by the catalpa sinensis biological technology limited company in Hangzhou. The above methods of reagent use are referred to in the commercial specifications.
Reagents for downstream catalytic processes: glycine, p-methylsulfonylbenzaldehyde, L-syn-p-methylsulfonylbenzylserine, pyridoxal phosphate are all analytically pure.
The structural formula of the p-methylsulfonyl benzaldehyde is shown as a formula (1); the structural formula of the L-syn-p-methylsulfonylbenzylserine is shown as a formula (2); the method comprises the following steps:
the application analyzes the concentration of the substrate and the product in the reaction liquid by High Performance Liquid Chromatography (HPLC) and monitors the reaction. The HPLC analysis method is as follows:
chromatographic column model: QS-C18, 5 μm, 4.6X1250 mm. Mobile phase: KH (KH) 2 PO 4 (50 mM): acetonitrile=21:79, ph=8.0; detection wavelength: 225nm, flow rate: 1.0mL/min, column temperature: 40 ℃. The peak appearance of the substrate (standard) and the product (standard) is shown in FIGS. 5 and 6. Chiral analysis and concentration analysis of the product (standard) require pre-column derivatization of the peak-out conditions of L-anti-p-methylsulfonylphenyl serine and L-syn-p-methylsulfonylphenyl serine, as shown in FIG. 7 and FIG. 8.
Example 1: construction of wild enzyme engineering bacteria
The amino acid sequence wp_015261381.1 (derived from Desulfitobacterium dichloroeliminans (DdLTA)), SIS82838.1 (derived from Chryseobacterium chaponense (CcLTA)), wp_016204489.1 (derived from Bacillus nealsonii (BnLTA)), wp_023973138.1 (derived from Clostridium beijerinckii (CbLTA)), wp_077361571.1 (derived from Clostridium saccharoperbutylacetonicum (CsLTA)), wp_069998496.1 (derived from cellulilyticum sp.15 g10i2 (CpLTA)) encoding the L-threonine aldolase was searched and selected by inputting a keyword such as threonine aldolase into National Coalition Building Institute (NCBI) database. Six amino acid sequences were converted to nucleotide sequences (the nucleotide sequences are shown in SEQ ID Nos. 1 to 6) according to E.coli codon preference. Six nucleotide sequences were synthesized entirely by chemical methods (Anhui general organism) and integrated between the multiple cloning sites BamHI and HindIII of the expression vector pET-28 a. And finally, introducing the constructed plasmid into escherichia coli BL21 (DE 3) to construct wild L-threonine aldolase engineering bacteria.
Example 2: construction of mutant enzymes
1. Activation of engineering bacteria and plasmid extraction
All engineering bacteria (constructed and obtained in example 1) were activated and cultured using LB medium, and the formulation was: 10g/L peptone, 5g/L yeast powder and 10g/L NaCl, and is dissolved in deionized water, then the volume is fixed, and the solution is sterilized at 115 ℃ for 30min for later use. The solid medium was LB medium supplemented with 2% agar.
The stored engineering bacteria glycerol tube was inoculated into a test tube containing 10mL of LB medium, and cultured at 37℃for 12 hours at 200 rpm. After obtaining the cultured cells, plasmid extraction was performed according to the instructions of the Axygen plasmid extraction kit. The obtained plasmid can be directly used for point mutation or can be stored at-20deg.C for a long time.
2. Site-directed mutagenesis of genes
The mutation gene is obtained by adopting a whole plasmid PCR method. FIG. 4 is a diagram showing the sequence alignment and the corresponding mutation sites of threonine aldolase from different sources.
PCR amplification system:
PCR amplification conditions:
1) Pre-denaturation: 98 ℃ for 5min;
2) Denaturation: 98 ℃ for 30s; annealing: 30s at 60 ℃; extension: 90s at 72 ℃; cycling for 30 times;
3) Rear extension: 72 ℃ for 10min;
4) Preserving at 4 ℃.
After the completion of PCR amplification, the amplified products were detected by 0.9% agarose gel electrophoresis, and the results showed that the amplified products were single bands, each having a size of about 7000 bp. The amplified products are purified and recovered by using a DNA recovery and purification kit, and specific steps are referred to the instruction of the purification kit.
The primer required for mutating lysine at 5 th position of L-threonine aldolase DdLTA into phenylalanine:
an upstream primer: ATGATCAGTTTCTTTAACGATTACAGCGA
A downstream primer: TCGCTGTAATCGTTAAAGAAACTGATCAT
Other mutation site primers were designed according to this principle.
3. Construction of mutant engineering bacteria
The purified gene fragment was digested with dpnl to remove the template and then recombined with recombinase. The recombinant product is transformed into E.coli BL21 (DE 3) competent cells, plated, picked single colony to LB liquid culture, and the PCR method is used for identifying the positive transformant which is successfully constructed, and the correctness of the mutation site is verified by sequencing. After verification, sterile glycerol with a final concentration of 25% is added, numbered and placed at-80 ℃ for preservation.
Example 3: culture of thallus and preparation of crude enzyme solution
1. Culture of bacterial cells
LB liquid medium composition: 10g/L peptone, 5g/L yeast powder and 10g/L NaCl, and is dissolved in deionized water, then the volume is fixed, and the solution is sterilized at 115 ℃ for 30min for later use.
After streaking and activating the engineering bacteria containing the L-threonine aldolase gene by a plate, single colonies are selected and inoculated into 5mL of LB liquid medium containing 50 mug/mL of kanamycin, and shake culture is carried out for 12 hours at 37 ℃. Transferring into 50mL fresh LB liquid medium containing 50 mug/mL Kan according to 2% inoculum size, shake culturing at 37deg.C until OD600 reaches about 0.6, adding IPTG to final concentration of 0.5mM, and inducing culturing at 28deg.C for 10 hr. After the culture is finished, the culture solution is centrifuged at 10000rpm for 10min, the supernatant is discarded, and the thalli are collected and stored in an ultralow temperature refrigerator at-80 ℃ for standby.
2. Preparation of crude enzyme solution
The cells collected after the completion of the culture were washed twice with 50mM phosphate buffer solution pH 8.0. The cells were then resuspended in phosphate buffer pH 8.0 and sonicated 30 times at 400W power for 3s each and for 7s each. The cell disruption solution was centrifuged at 12000rpm for 3min at 4℃to remove the precipitate, and the obtained supernatant was a crude enzyme solution containing recombinant L-threonine aldolase.
Example 4: preparation of L-syn-p-methylsulfonylphenyl serine by L-threonine aldolase and mutant thereof in aqueous solution
Engineering bacteria capable of expressing L-threonine aldolase and mutants thereof were cultured as in example 3 to obtain a crude enzyme solution. 0.1M p-methylsulfonyl benzaldehyde and 1M glycine are quantitatively weighed into a 1L reactor, the volume is fixed to 1L by 100mM pH 8.0NaOH-Gly buffer solution, the final concentration of pyridoxal phosphate is 1 mu M, and the concentration of wet thalli is 10g/L. The reaction temperature is controlled to be 30 ℃ through water bath, magnetic stirring is carried out, and after the reaction is carried out for 10min, the concentration of the substrate and the concentration of the product and the L-syn-p-methylsulfonylbenzeneserine de value are detected.
The reaction end data are shown in Table 1. FIG. 9 is a high performance liquid chromatography (chiral analysis) of a wild-type L-threonine aldolase DdLTA reaction solution (after completion of the reaction): wherein, retention time: l-anti-p-methylsulfonylphenyl serine is 6.284 min and L-syn-p-methylsulfonylphenyl serine is 7.436min.
Table 1 conversion rate and de (%) value of L-threonine aldolase and mutant thereof in various systems
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Example 5: preparation of L-syn-p-methylsulfonylbenzylserine by L-threonine aldolase and mutant thereof in water-DMF solution
Engineering bacteria capable of expressing L-threonine aldolase and mutants thereof were cultured as in example 3 to obtain a crude enzyme solution. Quantitatively weighing 0.1M p-methylsulfonyl benzaldehyde and 1M glycine into a 1L reactor, wherein the volume fraction of DMF in the reaction system is 20%, the volume of the reaction system is fixed to 1L by using 100mM pH 8.0NaOH-Gly buffer solution, the final concentration of pyridoxal phosphate is 1 mu M, and the concentration of wet thalli is 10g/L. Controlling the reaction temperature to be 30 ℃ through water bath, magnetically stirring, detecting the concentration of a substrate and a product by using achiral liquid chromatography after reacting for 2 hours, and detecting the L-syn-p-methylsulfonylbenzylserine de value by using a chiral column.
The reaction end data are shown in Table 1.
Example 6: preparation of L-syn-p-methylsulfonylbenzylserine by L-threonine aldolase and mutant thereof in water-DMSO solution
Engineering bacteria capable of expressing L-threonine aldolase and mutants thereof were cultured as in example 3 to obtain a crude enzyme solution. And quantitatively weighing 0.1M p-methylsulfonyl benzaldehyde and 1M glycine into a 1L reactor, wherein the volume fraction of DMSO in the reaction system is 20%, the volume of the reaction system is fixed to 1L by using 100mM pH 8.0NaOH-Gly buffer solution, the final concentration of pyridoxal phosphate is 1mM, and the concentration of wet thalli is 10g/L. Controlling the reaction temperature to be 30 ℃ through water bath, magnetically stirring, detecting the concentration of a substrate and a product by using achiral liquid chromatography after reacting for 2 hours, and detecting the L-syn-p-methylsulfonylbenzylserine de value by using a chiral column.
The reaction end data are shown in Table 1.
Sequence listing
<110> university of Zhejiang
<120> L-threonine aldolase mutant and method for preparing L-syn-p-methylsulfonylbenzserine
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1023
<212> DNA
<213> desulphurized dichloro bacillus (Desulfitobacterium dichloroeliminans)
<400> 1
atgatcagtt tcaagaacga ttacagcgaa ggtgcccatc cgcgtattct ggaagcactg 60
ctgaaaagta atctgattca ggaacagggt tatggtgaag atagcttttg cctgggtgcc 120
agcgaactgc tgcgcgaacg tctgggcaat aaggatctga ccattcatta tctgaccggc 180
ggtacacagg caaatctggt ggccattagc gcctttctgc gtccgcatga agccgccatt 240
gccgcacaga ccggccatat ttttgtgcat gaaaccggcg ccattgaagc aaccggccat 300
aaagtgctga ccagcgaaag caaagatggt aaactgaccc cggcacagat tcaggaagtt 360
ctgaccgcac ataccgatga acacatggtg aaaccgaaac tggtttatat tagcaatacc 420
accgaagttg gtacagttta tagtaaaagc gaactgcagg cactgagtca gttttgccgt 480
gaaaagaatc tgtatctgtt tatggatggc gcacgcctgg gtagtgccct gtgtagcgaa 540
ggcaatgatc tggatctggc agatctgccg aaactggtgg atgcctttta tattggcggt 600
acaaaaaatg gcgcactgct gggcgaagca ctggtgctgt gtaatgaagc actgaaaccg 660
gattttcgtt atcacatgaa acagaaaggt gcactgctgg ccaaaggccg tgttattggc 720
ctgcagtttc tggaactgtt tcgtgataat ctgtattttg atctggcaat tcatgcaaat 780
accatggcat ataaactgcg tgatgaactg aaagaagcag gtgttaaatt tctggccgaa 840
agtagtagta atcaggtgtt tccgattttt agcgatgcca ttgttgaaca gctgaaagtg 900
aattatcatt ttgaaatctg gggtaaagtg ggcacccaga ccgccattcg cctggttacc 960
agttgggcca cccgtgaaga agccgtggat agctttatgg cagatctgaa aaagattctg 1020
taa 1023
<210> 2
<211> 1074
<212> DNA
<213> Zhang Shi golden yellow bacillus (Chryseobacterium chaponense)
<400> 2
atggtgttta gtccgtttct gtttattttt atcaacatga agttcagctt caagaatgat 60
tatgcagaag gttgtcatcc gcgtattctg gaagccctga gcgccaccaa tctggttcag 120
cagaatggct atggcctgga tgattattgt ctgaatgcag aacatctgat tcagaaaaag 180
attaataacc cgcgcgcaaa agtgcatttt gttagcggcg gcacccaggc caatctgatt 240
gtgattagtg catttctgcg tccgcatgaa agcgttgtga gcccggccac cggccatatt 300
tttaccaatg aaagcggcgc cattgaagca accggccata aagtgcatgc agttgaaacc 360
gaagatggca aaattcgtcc ggaagatatt cgcaaagtga ttgatgtgca tcagaataag 420
ccgcatcagg tgaaacagaa actggtttat attagcaata gcaccgaagt tggcaccatc 480
tatagcaaaa aagaactgat tgatctgtac cagttttgcc aggaaaataa tctgtttctg 540
tttgttgatg gtgcccgtct gggccatgca ctgaaagcag aaaccaatga tctgaccctg 600
gaagattttg gcaaatatac cgatgccttt tattggggtg gcaccaaaaa tggcgccctg 660
attggtgaag caattgtgat taataatgag gatctgcagg gcgaatttgg ctttcatctg 720
aaacagaaag gcgccatgct ggcaaaaggc cgcctgctgg gtattcagtt tcaggaactg 780
ctgaaagatg atctgtattt tgatctggcc aatctggcca atcagaaagc catgaaaatt 840
aagaaaagtt ttcaggaaat cggctgccat tttctgagtg aaacctttac caatcagatt 900
tttccgattc tgaataatag ccagattgaa aaactgagtg aaaattttga cttctacgtg 960
tggaaaaaga ttgatgaaga taaaagtgcc gttcgcatta ttaccagttg ggccaccagc 1020
gatgaaattg tggaaaaact gattgcagaa attagtagcc tgcgtaatag ttaa 1074
<210> 3
<211> 1011
<212> DNA
<213> Nielsen bacillus (Bacillus nealsonii)
<400> 3
atgtacagtt tcaacaacga ttacagtgaa ggcgcacatc cgcgtattct gcaggcactg 60
gtggaaagca atctgcagca ggaaattggt tatggtcagg atagttttac caataaggcc 120
gccgaagttc tgaaaaccaa aatgaatagc gatgaagttg atgtgcatct gctggttggc 180
ggtacccaga ccaatctgat tgcaattagt gcctttctgc gcccgcatga agcagcaatt 240
gcagccagta ccggtcatat ttttgttcat gaaaccggtg caattgaagc aaccggtcat 300
aaagtgatta ccgttgatgc caaatatggt aaactgaccc cgagtctggt tcagagcgtg 360
ctggatgaac ataccgatga acatatggtg aaaccgaaac tggtttatat tagcaatagt 420
accgaaattg gcaccatcta tagtaaaagc gaactggaac agctgagtca gttttgccag 480
attaataatc tgattttcta catggacggc gcccgcctgg gtagtgccct gtgtgcaaaa 540
gataatgatc tggttctgag tgattttccg aaactgctgg atgcctttta tattggcggc 600
accaaaaatg gtgcactgat gggcgaagcc ctggttatta agaatgatag tctgaaaacc 660
gatttccgtt atcatattaa gcagaaaggt gccatgctgg caaaaggccg cctgctgggt 720
attcagtttt atgaactgtt taaagacgac ctgtttttcg aactggcaga atatgccaat 780
aagatggcag aacgtctgaa tattgccctg gccgaaaaag attatcgttt tctgaccccg 840
tcaagcacca atcaggtgtt tccgattttt agtaatgaaa aaatcaccat gctgcagaaa 900
aattatcagt ttaatatctg ggagaagatc gataaagatc atagtgccat tcgtctggtg 960
accagctggg caaccaaaga agcagaagtt gaagccttta ttaatgaaat t 1011
<210> 4
<211> 1017
<212> DNA
<213> Clostridium beijerinckii (Clostridium beijerinckii)
<400> 4
atgtacagct ttaagaacga ttacagtgaa ggtgcccata gccgcattct gaatgcactg 60
gttgaaacca atctggaaca gaccgatggt tatggcaccg atcagtatac cgaacgcagt 120
gttaatctgc tgaaaaagaa aattgaccgc gaagatgttg atattcatct gctggttggc 180
ggtacccagg tgaatctgac cgcaattagc gcatttctgc gcccgcatca ggcaaccatt 240
ggcgcagata ccagtcatat taattgtcat gaaaccggcg ccattgaagc aaccggtcat 300
aaagttatta ccatgaaaac caatgacggt aaactgaccc cgaatctgat tcagaatgtg 360
gttgatagtc atagtgatga acatatggtt cagccgaaac tggtttatat tagcaatagc 420
accgaactgg gcaccctgta taccaaagca gaactgattg atctgcgcga ttgttgtaaa 480
cgtaataagc tgctgctgta tctggatggt gcccgtctgg gtagcgcact ggttgccgaa 540
gaaaatgatc tgaccctggc cgatattgca aaactggttg atgcctttta tattggtggt 600
accaaaaatg gtgcactgtt tggcgaagca ctggttattt gcaatgatga actgaaagaa 660
gatttcatct atttcatcaa gcagaaaggt ggtctgctgg caaaaggtcg tctgctgggt 720
attcagtttg aagaactgtt taaagatgac ctgtattttg aactggcaaa acatgcaaat 780
aagatggcac tgatgctgaa aggtgcaatt gtggatgaag aatataaatt tctgaccgaa 840
agttttacca accagcagtt tccgattttt ccgaataatc tgattgaaaa actgagtgaa 900
aagtacagct ttaatatcga acgcgtgatt gatagtaatt ataccgccat tcgcctggtt 960
accagctggg caaccaaaga agaaattgtt ctggaattca ttgaggatct gcatctg 1017
<210> 5
<211> 1020
<212> DNA
<213> Clostridium sucrose butoxide (Clostridium saccharoperbutylacetonicum)
<400> 5
atgtacagct ttaagaacga ttacagtgaa ggcgcccatg aacgtattct gaatgccctg 60
gttgaaagta attttgaaca gaccgatggc tatggtgaag attatcatac cgaacgcgca 120
gtgcagattc tgaaagataa aattgataac cagaacgttg acattcatct gctggttggc 180
ggcacccagg ttaatctgac cgccattagt gcatttctgc gtccgcatca ggcagcaatt 240
ggtgcagata ccagtcatat taattgccat gaaaccggcg ccattgaagc caccggccat 300
aaagttatta ccgttaaaac cgatgatggt aaactgaccc cgagtctgat tcagaaagtt 360
gtggataccc atcaggatga acacatggtt cagccgaaac tggtgtatat tagcgatagc 420
accgaactgg gcaccctgta taccaaagca gaactgaccg atctgcataa ttgctgcaaa 480
aagaataagc tgctgctgta tctggatggc gcccgcctgg gcgcagcatt aaccgcagaa 540
aagaatgatc tgaccctggc cgatattgca aaactggtgg atgcatttta tattggtggt 600
acaaaaaacg gcgccctgtt tggtgaagca ctggttattt gcaaagatga actgaaagaa 660
gatttccgtt attttatcaa gcagaaaggc ggcctgctgg ccaaaggccg tctgctgggt 720
attcagtttg aagaactgtt taaagatgac ctgtattttg aactggccaa atatgcaaat 780
aacctggcaa ttattctgaa aaatgccctg attgaaaagg gttatgaatt tctgtgcgaa 840
agttatacca atcagcagtt tccgattctg ccgaatgaag ttgttaataa gattagcgaa 900
aagtacagtt tcaacgtgga acgtgttatt gatgaaaata ataccgttat ccgtctggtg 960
accagctggg ccaccagtaa agaaaaagtt ctggaatttg ttgaggaact gaatctgtaa 1020
<210> 6
<211> 1029
<212> DNA
<213> cellulolytic bacteria (Cellulosilyticum sp.)
<400> 6
atgtacagtt tcaagaacga ttacagtgaa ggtgcacatc cgaaaattct ggaagcactg 60
attgccagca atctggaaca gaccgaaggc tatggcgaag atcattatag tcagaaagca 120
gcatggctgc tgaaagaaat gattggtcgt gatgatattg cagttcattt ctttgtgggc 180
ggtacacaga ccaatctgac cgcaattagc gcatttctgc gcccgcatca ggccgttatt 240
gcagccgcaa ccggccatat tgcaacccat gaaaccggcg caattgaagc caccggccat 300
aaagtgatta ccgttgaaac cagcgatggc aaactgcgta tggatcatat tcagagtgtt 360
ctggatggcc ataccgatga acacatggtt agtccgaaaa tggtttatat tagcaatagc 420
accgaagttg gtagtatcta taaaaaagca gaactggaag gtctgagtca gttttgtaaa 480
gccaataatc tgctgctgta tctggatggc gcacgcctgg gcagtgcact gaccagcaaa 540
gaaaatgata tgaccctgct ggatctgggt cgtctgaccg atgtgtttta tattggcggt 600
acaaaaaatg gtgccctgat gggtgaagca ctgatcattt gtaatgattt tctgaaagag 660
gacttccgct ttcatattaa gcagaaaggt gcactgctgg caaaaggccg tctgctgggt 720
attcagtttg aagcactgtt taaagataac ctgtattttg aactggccga acatgccaat 780
cagatggcag tgcgcctgca ggatgaaatt aagaaactgg gctttagctt tctgattagc 840
agcccgagca atcaggtgtt tccgattttt ccgaatagtg tgattgaaaa actgcaggaa 900
aaatatgcct ttcatatttg ggaaaaggtg gatgatagct atagtgccat tcgcctggtt 960
accagctggg ccaccaaaga agaagccgtg agtaattttg ttaaagatct gaataacatc 1020
gtgttctaa 1029
<210> 7
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 7
atgatcagtt tctttaacga ttacagcga 29
<210> 8
<211> 29
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 8
tcgctgtaat cgttaaagaa actgatcat 29
Claims (9)
1. An L-threonine aldolase mutant characterized by a mutation of the wild-type L-threonine aldolase of accession number wp_077361571.1, specifically one of the following mutations:
31H/125V/227R/305R；8H/31H/143R/305R；5F/8H/Y31M/125V/227R/305R；8F/31H/125V/143R/252F/305R/308H。
2. a gene encoding the L-threonine aldolase mutant according to claim 1.
3. An expression vector comprising the gene of claim 2.
4. The expression vector of claim 3, which is a pET28a plasmid into which the gene of claim 2 has been inserted.
5. A genetically engineered bacterium comprising the gene of claim 2.
6. The use of one of the L-threonine aldolase mutants according to claim 1, the genes according to claim 2 or the genetically engineered bacteria according to claim 5 for the preparation of L-syn-p-methylsulfonylbenzeneserine.
7. A method for preparing L-syn-p-methylsulfonylbenzylserine, characterized in that glycine and p-methylsulfonylbenzaldehyde are used as substrates, pyridoxal phosphate is used as a coenzyme, and the L-syn-p-methylsulfonylbenzylserine is produced by condensation reaction using the L-threonine aldolase mutant according to claim 1 or a cell expressing the L-threonine aldolase mutant according to claim 1 as a catalyst.
8. The method of claim 7, wherein the reaction is carried out in an organic solvent-water mixed solution or an aqueous solution.
9. The method of claim 8, wherein the organic solvent is DMF or DMSO and the organic solvent is added in an amount of 20% or less of the total volume.
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| CN110272856B (en) * | 2019-05-08 | 2022-05-03 | 江南大学 | Recombinant bacterium for expressing D-threonine aldolase and construction method and application thereof |
| CN112175892B (en) * | 2020-09-03 | 2022-08-30 | 浙江大学 | Engineering bacterium for co-expressing L-threonine aldolase and PLP synthase and application thereof |
| CN115433727B (en) * | 2021-06-02 | 2023-11-17 | 弈柯莱生物科技(集团)股份有限公司 | L-threonine aldolase and preparation method and application thereof |
| CN114134134B (en) * | 2021-10-29 | 2023-07-14 | 宁波泓森生物科技有限公司 | L-threonine aldolase mutant and application thereof in synthesis of L-syn-p-methylsulfonyl phenylserine |
| CN114736939B (en) * | 2022-06-13 | 2022-09-02 | 山东国邦药业有限公司 | Method for promoting enzymatic preparation of (2R, 3S) -p-methylsulfonylphenylserine |
| CN115747194B (en) * | 2022-10-28 | 2023-11-14 | 浙江大学 | L-threonine aldolase mutant, gene and method for preparing L-anti-p-methylsulfonylphenyl serine |
| CN115611785B (en) * | 2022-12-19 | 2023-03-14 | 天津工微生物科技有限公司 | Purification method of (2R, 3S) -2-amino-3-hydroxy-3- (4- (methylsulfonyl) phenyl) propionic acid |
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| JP2007054012A (en) * | 2005-08-26 | 2007-03-08 | Research Institute Of Innovative Technology For The Earth | L-threo type highly stereoselective l-threonine aldolase and gene encoding the same |
| EP2004836A1 (en) * | 2006-04-13 | 2008-12-24 | DSMIP Assets B.V. | Process for the preparation of enantiomeri cally enriched beta-amino alcohols starting from glycine and an aldehyde in the presence of a threonine aldolase and a decarboxylase |
| CN104774825B (en) * | 2015-03-23 | 2017-11-07 | 浙江工业大学 | Nitrilase mutants and its application |
| CN107743520A (en) * | 2015-05-06 | 2018-02-27 | 特雷里斯公司 | Composition and method for the biological production of methionine |
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