CN111139230A - A kind of L-threonine aldolase mutant, gene and method for preparing L-syn-p-methylsulfonyl phenylserine - Google Patents
A kind of L-threonine aldolase mutant, gene and method for preparing L-syn-p-methylsulfonyl phenylserine Download PDFInfo
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Abstract
本发明公开了一种L‑苏氨酸醛缩酶突变体、基因及制备L‑syn‑对甲砜基苯丝氨酸的方法。所述L‑苏氨酸醛缩酶突变体由野生型L‑苏氨酸醛缩酶突变所得，能够以甘氨酸和对甲砜基苯甲醛为底物，以磷酸吡哆醛为辅酶，催化缩合反应生成L‑syn‑对甲砜基苯丝氨酸。使用本发明L‑苏氨酸醛缩酶突变体生产L‑syn‑对甲砜基苯丝氨酸具有以下优点：1、生产工艺简单，反应条件温和；2、L‑苏氨酸醛缩酶的选择性高，产品光学纯度高，无需拆分；3、原子利用率高，理论可达100％；4、生产过程环保，污染较小，符合绿色化学理念；5、产品分离纯化简单。
The invention discloses an L-threonine aldolase mutant, a gene and a method for preparing L-syn-p-methylsulfonylphenylserine. The L-threonine aldolase mutant is obtained by mutation of wild-type L-threonine aldolase, and can use glycine and p-methylsulfonylbenzaldehyde as substrates and pyridoxal phosphate as coenzyme to catalyze condensation The reaction produces L‑syn‑p-methylsulfonylphenylserine. Using the L-threonine aldolase mutant of the present invention to produce L-syn-p-methylsulfonylphenylserine has the following advantages: 1. the production process is simple and the reaction conditions are mild; 2. the selection of L-threonine aldolase High performance, high optical purity of the product, no need for splitting; 3. High atom utilization rate, theoretically up to 100%; 4. The production process is environmentally friendly, with less pollution, in line with the concept of green chemistry; 5. The product is simple to separate and purify.
Description
Technical Field
The invention 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-methylsulfonylphenylserine.
Background
L-syn-p-methylsulfonylphenylserine is an important medical intermediate and is applied to the synthesis of various antibiotics. P-methylsulfonylphenylserine has two chiral centers and has four isomers, which are respectively: l-syn-p-methylsulfonylphenylserine, L-anti-p-methylsulfonylphenylserine, D-syn-p-methylsulfonylphenylserine, and D-anti-p-methylsulfonylphenylserine (FIG. 1).
Currently, there are two main production methods for L-syn-p-methylsulfonylphenylserine. One is synthesized by a chemical method, copper sulfate is used as a catalyst, and the methyl sulfone benzaldehyde and glycine are selectively reacted to generate two cis-form products by utilizing the complexation of metal ions: the method comprises the steps of carrying out chiral resolution on L-syn-p-methylsulfonylphenylserine copper and D-syn-p-methylsulfonylphenylserine copper to obtain optically pure L-syn-p-methylsulfonylphenylserine ethyl ester, wherein acid cannot be directly resolved but can only be continuously subjected to esterification reaction with ethanol to generate racemic D, L-syn-p-methylsulfonylphenylserine ethyl ester, and separating D-type and L-type isomers. The process mainly has the following defects: the theoretical yield is only 50 percent, the invalid enantiomer is difficult to apply and the process is complex. Meanwhile, an equimolar amount of chiral resolution reagent is required, the single resolution yield is low, the recycling of the resolution reagent and copper salt in the reaction generate a large amount of wastewater and waste salt. The other method combines a chemical method and a biological method, and the main process is to firstly utilize p-methylsulfonylbenzaldehyde and glycine to generate racemic p-methylsulfonylphenylserine copper salt under the catalysis of copper sulfate, wherein the main products are L-syn-p-methylsulfonylphenylserine copper salt and D-syn-p-methylsulfonylphenylserine copper salt (figure 2). Removing copper ions in the product to generate a mixture of L-syn-p-methylsulfonylphenylserine and D-syn-p-methylsulfonylphenylserine. And D-threonine aldolase is used for decomposing D-syn-p-methylsulfonylphenylserine to achieve the aim of resolution, so that the optically pure L-syn-p-methylsulfonylphenylserine is obtained. 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 percent, and the problem of environmental pollution caused by copper salt can also be generated.
In contrast, the use of the L-threonine aldolase mutant for catalyzing p-methylsulfonylbenzaldehyde and glycine to directly produce L-syn-p-methylsulfonylphenylserine (FIG. 3) has the following advantages:
1. the production process is simple, and the reaction conditions are mild;
2. the selectivity of the L-threonine aldolase is high, and the optical purity of the product is high;
3. the method does not need to be split, has high atom utilization rate and can reach 100 percent theoretically;
4. the production process is environment-friendly, has less pollution and accords with the green chemical concept;
5. the downstream separation and purification process is simple.
The key point of utilizing L-threonine aldolase to catalyze and synthesize L-syn-p-methylsulfonylphenylserine is to obtain an enzyme capable of synthesizing the L-syn-p-methylsulfonylphenylserine with high selectivity, the currently reported L-threonine aldolase has high selectivity on α -carbon, ee (%) > 99%, the selectivity on β -carbon is not high, and de (%) is between 20 and 50%, so that a mixture of the L-syn-p-methylsulfonylphenylserine and the L-anti-p-methylsulfonylphenylserine is generated.
Disclosure of Invention
The invention provides a method for preparing L-syn-p-methylsulfonylphenylserine by using an L-threonine aldolase mutant aiming at the defects of the existing L-syn-p-methylsulfonylphenylserine synthesis process, and the method has high utilization rate of raw materials and can be recycled. The product is easy to separate and purify, and the conversion rate, yield and chiral purity are high; compared with chemical catalysis process, the process is simple and has less pollution.
An L-threonine aldolase mutant is obtained by mutation of wild-type L-threonine aldolase with the accession numbers of WP _015261381.1, SIS82838.1, WP _016204489.1, WP _023973138.1, WP _077361571.1 or WP _069998496.1 respectively, and specifically comprises one of the following mutations:
(1) resulting from a mutation in the wild-type L-threonine aldolase with accession number WP _015261381.1 at 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/8H/31H/227R/305R, and/227R, 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) resulting from a mutation in the wild-type L-threonine aldolase with accession number SIS82838.1 at one of the following mutation sites: 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/138H/R/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/319/323H, 21F/44M/138V/157R/266F/319R/323K;
(3) resulting from a mutation in the wild-type L-threonine aldolase with accession number WP _016204489.1 at 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/8H/31H/227R/305R, and/227R, 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) resulting from a mutation in the wild-type L-threonine aldolase with accession number WP _023973138.1 at 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/8H/31H/227R/305R, and/227R, 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) resulting from a mutation in the wild-type L-threonine aldolase with accession number WP _077361571.1 at 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/8H/31H/227R/305R, and/227R, 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) resulting from a mutation in the wild-type L-threonine aldolase with accession number WP _069998496.1 at 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/8H/31H/227R/305R, and/227R, 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.
The invention also provides a gene for coding the L-threonine aldolase mutant.
The invention also provides an expression vector containing the gene. Preferably, the expression vector is pET28a plasmid inserted with the gene.
The invention also provides a genetically engineered bacterium containing the gene.
The invention also provides the application of the L-threonine aldolase mutant, the gene or one of the genetic engineering bacteria in the preparation of L-syn-p-methylsulfonyl phenylserine.
The invention also provides a method for preparing L-syn-p-methylsulfonyl phenylserine, which takes glycine and p-methylsulfonyl benzaldehyde as substrates, pyridoxal phosphate as a coenzyme, and takes the L-threonine aldolase mutant or a cell expressing the L-threonine aldolase mutant as a catalyst to carry out condensation reaction to generate the L-syn-p-methylsulfonyl phenylserine. The cell expressing the L-threonine aldolase mutant may be the above-mentioned 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 the conversion rate and de value of the reaction can be improved to a certain extent after some organic solvent is added.
In the reaction system, the catalyst is used in the form of crude enzyme solution after cell disruption, engineering bacteria resting cells for expressing recombinase, purified pure enzyme or immobilized enzyme.
Compared with the prior art, the invention has the following beneficial effects:
1. the production process is simple, and the reaction conditions are mild;
2. the selectivity of the L-threonine aldolase is high, the optical purity of the product is high, and the product does not need to be split;
3. the atom utilization rate is high and can reach 100% theoretically;
4. the production process is environment-friendly, has less pollution and accords with the green chemical concept;
5. the product is simple to separate and purify.
Drawings
FIG. 1 is a schematic diagram of the molecular structure of four configurations of p-methylsulfonylphenylserine.
FIG. 2 shows the equation for synthesizing L-syn-p-methylsulfonylphenylserine by the chemical synthesis method.
FIG. 3 is a formula for synthesizing L-syn-p-methylsulfonylphenylserine by the biosynthesis method.
FIG. 4 is a diagram showing the alignment of threonine aldolase sequences from different sources and the corresponding mutation sites.
FIG. 5 is a high performance liquid detection spectrum (achiral analysis) of a standard sample of p-methylsulfonylbenzaldehyde as a substrate, and the retention time of the p-methylsulfonylbenzaldehyde is 7.439 min.
FIG. 6 is a high performance liquid chromatography detection spectrum (achiral analysis) of a product p-methylsulfonylphenylserine standard sample, and the retention time of the p-methylsulfonylphenylserine is 2.620 min.
FIG. 7 is a liquid phase spectrum (chiral analysis) of L-anti-p-methylsulfonylphenylserine; wherein the retention time is 6.333min,
FIG. 8 is a liquid phase spectrum (chiral analysis) of L-syn-p-methylsulfonylphenylserine standard; wherein the retention time is 7.891 min.
FIG. 9 is a high performance liquid chromatography detection profile (chiral analysis) of the reaction solution (after the reaction) of wild-type L-threonine aldolase DdLTA in example 4: wherein, the retention time: the content of L-anti-p-methylsulfonylphenylserine is 6.286min, and the content of L-syn-p-methylsulfonylphenylserine is 7.436 min.
Detailed Description
The experimental methods in the present invention are conventional methods unless otherwise specified, and the gene cloning procedures can be specifically described in molecular cloning protocols, compiled by J. Sambruka et al.
Reagents used for upstream genetic engineering: restriction enzymes, Primer STARDNA polymerase, DNA ligase, and recombinase used in the examples of the invention were all purchased from TaKaRa; the genome extraction kit, the plasmid extraction kit and the DNA recovery and purification kit are purchased from Axygen; coli BL21(DE3), plasmids, etc. were purchased from Novagen corporation; DNA marker, low molecular weight standard protein and agarose electrophoresis reagent are purchased from Beijing all-style gold biotechnology limited; primer synthesis and gene sequencing work are completed by Hangzhou Zhikexi biotechnology limited. The method of using the above reagent is referred to the commercial specification.
Reagents used in the downstream catalytic process: glycine, p-methylsulfonylbenzaldehyde, L-syn-p-methylsulfonylphenylserine, and pyridoxal phosphate were all commercially available as analytical reagents.
The structural formula of the p-methylsulfonylbenzaldehyde is shown as a formula (1); the structural formula of the L-syn-p-methylsulfonylphenylserine is shown as a formula (2); the method comprises the following specific steps:
the invention analyzes the concentration of the substrate and the product in the reaction solution by High Performance Liquid Chromatography (HPLC) and monitors the reaction progress.
The HPLC analysis method is as follows:
the type of the chromatographic column: QS-C18, 5 μm, 4.6X 250 mm. Mobile phase: KH (Perkin Elmer)2PO4(50 mM): acetonitrile 21: 79, pH 8.0; detection wavelength: 225nm, flow rate: 1.0mL/min, column temperature: at 40 ℃. Substrate (standards) and product (standards) peaked as shown in FIGS. 5 and 6. Chiral analysis and concentration analysis of the product (standard) require pre-column derivatization of the peak appearance of L-anti-p-methylsulfonylphenylserine and L-syn-p-methylsulfonylphenylserine, as shown in FIG. 7 and FIG. 8.
Example 1: construction of wild enzyme engineering bacteria
Inputting a keyword such as threoninealdolase into a National coordination Institute (NCBI) database to search and select an amino acid sequence WP _015261381.1 (derived from Desulfiobacterium dichlorelininans (DdLTA)), SIS82838.1 (derived from Chryseobacterium chaponensis (CcLTA)), WP _016204489.1 (derived from Bacillus neolisonii (BnLTA)), WP _023973138.1 (derived from Clostridium beijirinckii (CbLTA)), WP _077361571.1 (derived from Clostridium saccharolyticum (CbLTA)), WP _069998496.1 (derived from Cellulosilyticum sp.1I5G0I2 (CpLTA)). Six amino acid sequences are converted into nucleotide sequences (the nucleotide sequences are shown as SEQ ID No. 1-6) according to the codon preference of escherichia coli. Six nucleotide sequences were chemically synthesized (Anhui Utility. Anhui) and integrated between the multiple cloning sites BamH I and Hind III of the expression vector pET-28 a. Finally, the constructed plasmid is introduced into Escherichia coli BL21(DE3) to construct a wild L-threonine aldolase engineering bacterium.
Example 2: construction of mutant enzymes
Activation of engineering bacteria and plasmid extraction
All engineering bacteria (constructed in example 1) are activated and cultured by using an LB culture medium, and the formula is as follows: 10g/L of peptone, 5g/L of yeast powder and 10g/L of NaCl, dissolving with deionized water, fixing the volume, and sterilizing at 115 ℃ for 30min for later use. The solid culture medium is LB culture medium added with 2% agar.
The glycerol tube of the preserved engineering bacteria is put into a test tube containing 10mL of LB culture medium and cultured for 12h at 37 ℃ and 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 and can also be stored at-20 ℃ for a long time.
Second, site-directed mutagenesis of gene
The gene mutation adopts a whole plasmid PCR method to obtain a mutant gene. FIG. 4 is a diagram showing the alignment of threonine aldolase sequences from different sources and the corresponding mutation sites.
PCR amplification System:
PCR amplification conditions:
1) pre-denaturation: 5min at 98 ℃;
2) denaturation: 30s at 98 ℃; annealing: 30s at 60 ℃; extension: 90s at 72 ℃; circulating for 30 times;
3) and (3) post-extension: 10min at 72 ℃;
4) storing at 4 ℃.
After the PCR amplification is finished, the amplification product is detected by 0.9% agarose gel electrophoresis, and the result shows that the amplification product is a single band with the size of about 7000 bp. And (3) purifying and recovering the amplified product by using a DNA recovery and purification kit, wherein the specific steps refer to the specification of the purification kit.
The primer for mutating lysine 5 of L-threonine aldolase DdLTA into phenylalanine is as follows:
an upstream primer: ATGATCAGTTTCTTTAACGATTACAGCGA
A downstream primer: TCGCTGTAATCGTTAAAGAAACTGATCAT
Other mutation site primers were designed according to this principle.
Third, construction of mutant engineering bacteria
The purified gene fragment is digested with DpnI to remove the template and then recombined with a recombinase. And (3) transforming the recombinant product into an E.coli BL21(DE3) competent cell, coating a flat plate, selecting a single colony to LB liquid for culture, identifying a successfully constructed positive transformant by a PCR method, and verifying the correctness of the mutation site by sequencing. After verification, sterile glycerol with the final concentration of 25% is added, and the mixture is numbered and stored at minus 80 ℃ for later use.
Example 3: culture of cells and preparation of crude enzyme solution
First, culture of the cells
Composition of LB liquid medium: 10g/L of peptone, 5g/L of yeast powder and 10g/L of NaCl, dissolving with deionized water, fixing the volume, and sterilizing at 115 ℃ for 30min for later use.
After the engineering bacteria containing the L-threonine aldolase gene are activated by plate streaking, a single colony is selected and inoculated into 5mL LB liquid culture medium containing 50 ug/mL kanamycin, and shake culture is carried out at 37 ℃ for 12 h. Transferring the strain into 50mL of fresh LB liquid culture medium containing 50 mu g/mL Kan according to the inoculation amount of 2%, shaking and culturing at 37 ℃ until OD600 reaches about 0.6, adding IPTG until the final concentration is 0.5mM, and carrying out induction culture at 28 ℃ for 10 h. 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 ultra-low temperature refrigerator at minus 80 ℃ for standby.
Second, preparation of crude enzyme solution
The collected cells after the completion of the culture were washed twice with 50mM phosphate buffer solution having pH 8.0. Then, the cells were resuspended in phosphate buffer at pH 8.0 and disrupted by sonication at 400W for 30 cycles, each for 3 seconds, with a pause of 7 seconds. The cell disruption solution was centrifuged at 12000rpm at 4 ℃ for 3min to remove precipitates, and the resulting supernatant was a crude enzyme solution containing recombinant L-threonine aldolase.
Example 4: l-threonine aldolase and mutant thereof for preparing L-syn-p-methylsulfonyl phenyl serine in aqueous solution
The engineered bacteria capable of expressing L-threonine aldolase and mutants thereof were cultured in the same manner as in example 3 to obtain a crude enzyme solution. 0.1M of p-methylsulfonylbenzaldehyde and 1M of glycine were quantitatively weighed into a 1L reactor, and the volume was adjusted to 1L with 100mM of a pH 8.0NaOH-Gly buffer solution, the final concentration of pyridoxal phosphate was 1. mu.M, and the wet cell concentration was 10 g/L. Controlling the reaction temperature to be 30 ℃ through water bath, magnetically stirring, and detecting the concentration of the substrate and the product and the value of L-syn-p-methylsulfonylphenylserine de after reacting for 10 min.
The end of reaction data are shown in Table 1. FIG. 9 shows a high performance liquid chromatography detection profile (chiral analysis) of a reaction solution (after completion of the reaction) of wild-type L-threonine aldolase DdLTA: wherein, the retention time: the content of L-anti-p-methylsulfonylphenylserine is 6.286min, and the content of L-syn-p-methylsulfonylphenylserine is 7.436 min.
TABLE 1 conversion and de (%) values of L-threonine aldolase and its mutants in different systems
Example 5: l-threonine aldolase and mutant thereof in water-DMF solution to prepare L-syn-p-methylsulfonylphenylserine
The engineered bacteria capable of expressing L-threonine aldolase and mutants thereof were cultured in the same manner as in example 3 to obtain a crude enzyme solution. 0.1M of p-methylsulfonylbenzaldehyde and 1M of glycine are quantitatively weighed into a 1L reactor, the volume fraction of DMF in the reaction system is 20%, 100mM of pH 8.0NaOH-Gly buffer solution is used for constant volume of 1L, the final concentration of pyridoxal phosphate is 1 mu M, and the wet thallus concentration is 10 g/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 value of L-syn-p-methylsulfonylphenylserine de by using a chiral column.
The data of the end of the reaction are shown in Table 1.
Example 6: l-threonine aldolase and mutant thereof in water-DMSO solution to prepare L-syn-p-methylsulfonylphenylserine
The engineered bacteria capable of expressing L-threonine aldolase and mutants thereof were cultured in the same manner as in example 3 to obtain a crude enzyme solution. 0.1M of p-methylsulfonylbenzaldehyde and 1M of glycine are quantitatively weighed into a 1L reactor, the volume fraction of DMSO in a reaction system is 20%, 100mM of pH 8.0NaOH-Gly buffer solution is used for constant volume to 1L, the final concentration of pyridoxal phosphate is 1mM, and the wet thallus concentration is 10 g/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 value of L-syn-p-methylsulfonylphenylserine de by using a chiral column.
The data of the end of the reaction are shown in Table 1.
Sequence listing
<110> Zhejiang university
<120> L-threonine aldolase mutant, gene and method for preparing L-syn-p-methylsulfonylphenylserine
<160>8
<170>SIPOSequenceListing 1.0
<210>1
<211>1023
<212>DNA
<213> Desulfodichlorobacillus (Desulfobacter dichloreeliiminans)
<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> Chryseobacterium chapronatum (Chryseobacterium chapponense)
<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> Bacillus nielsen (Bacillus nealsoni)
<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)
<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 butanoicum (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 bacterium (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 being obtained from a wild-type L-threonine aldolase mutation having an accession number of WP _015261381.1, SIS82838.1, WP _016204489.1, WP _023973138.1, WP _077361571.1 or WP _069998496.1, in particular one of the following mutations:
(1) resulting from a mutation in the wild-type L-threonine aldolase with accession number WP _015261381.1 at 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/8H/31H/227R/305R, and/227R, 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) resulting from a mutation in the wild-type L-threonine aldolase with accession number SIS82838.1 at one of the following mutation sites: 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/138H/R/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/319/323H, 21F/44M/138V/157R/266F/319R/323K;
(3) resulting from a mutation in the wild-type L-threonine aldolase with accession number WP _016204489.1 at 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/8H/31H/227R/305R, and/227R, 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) resulting from a mutation in the wild-type L-threonine aldolase with accession number WP _023973138.1 at 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/8H/31H/227R/305R, and/227R, 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) resulting from a mutation in the wild-type L-threonine aldolase with accession number WP _077361571.1 at 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/8H/31H/227R/305R, and/227R, 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) resulting from a mutation in the wild-type L-threonine aldolase with accession number WP _069998496.1 at 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/8H/31H/227R/305R, and/227R, 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. A gene encoding the L-threonine aldolase mutant as described in claim 1.
3. An expression vector comprising the gene of claim 2.
4. The expression vector of claim 3, which is pET28a plasmid into which the gene of claim 2 is inserted.
5. A genetically engineered bacterium comprising the gene of claim 2.
6. Use of one of the L-threonine aldolase mutant as defined in claim 1, the gene as defined in claim 2, or the genetically engineered bacterium as defined in claim 5 for the preparation of L-syn-p-methylsulfonylphenylserine.
7. A method for preparing L-syn-p-methylsulfonylphenylserine, characterized in that glycine and p-methylsulfonylbenzaldehyde are used as substrates, pyridoxal phosphate is used as a coenzyme, and an L-threonine aldolase mutant according to claim 1 or a cell expressing the L-threonine aldolase mutant according to claim 1 is used as a catalyst to perform a condensation reaction to produce L-syn-p-methylsulfonylphenylserine.
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 amount of organic solvent added is 20% or less of the total volume.
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| CN110272856A (en) * | 2019-05-08 | 2019-09-24 | 江南大学 | A kind of recombinant bacterium that expressing D-Thr aldolase and its construction method and application |
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| CN115611785A (en) * | 2022-12-19 | 2023-01-17 | 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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