CN108823179B - Transaminase derived from actinomycetes, mutant, recombinant bacterium and application - Google Patents
Transaminase derived from actinomycetes, mutant, recombinant bacterium and application Download PDFInfo
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Abstract
The invention discloses a transaminase, mutant and recombinant bacteria derived from actinomycetes and a biological preparation method of R-3-aminobutanol provided by the invention. The needed recombinant transaminase can be prepared in large quantities by constructing escherichia coli genetic engineering bacteria and then fermenting, and is relatively easy to obtain and cheap. The reaction is in a one-pot type, the substrate and the enzyme are added and then start to react, and the final product R-3-aminobutanol is directly obtained, so that the industrial cost is low. Compared with the wild type, the actinomycete transaminase mutant provided by the invention has better catalytic activity. Under an optimal system, the conversion rate of 100mM substrate reaches 90%, and the ee value reaches 99.9%; the conversion rate of 500mM substrate reaches 78%, the ee value reaches 99.9%, and compared with the wild type, the conversion rate of the substrate is respectively improved by 12% -25%.
Description
(I) technical field
The invention relates to a transaminase gene, a mutant and an engineering bacterium from actinomycetes (Actinobacillus) and application thereof in catalytic synthesis of R-3-aminobutanol.
(II) background of the invention
The structural formula of the R-3-aminobutanol is shown as follows, and the R-3-aminobutanol has wide application in organic synthesis and drug production.
R-3-aminobutanol is an important raw material for synthesizing dolutegravir, dolutegravir is an anti-AIDS integrase inhibitor approved by the American FDA to be on the market in 2013, the safety of the drug is improved compared with the existing HIV integrase inhibitors Letergevir and Eltegravir, and compared with the anti-HIV/AIDS drug Lateravir of Variosato, dolutegravir not only achieves the curative effect equivalent to that of the drug in a three-stage clinical test, but also does not need to be combined with a drug promoter for use, and has very strong drug resistance. The quality and price of the intermediate R-3-aminobutanol have important influence on the quality and production cost of dolutegravir. R-3-aminobutanol can be derived as a beta-lactam and can be used to synthesize penem antibiotics. Therefore, the research on the synthesis of the R-3-aminobutanol is of great significance.
At present, the literature reports that the synthesis methods of R-3-aminobutanol mainly comprise the following methods: 1. direct ester reduction by chiral R-3-aminobutyrate; although the method has few steps, chiral R-3-aminobutyrate is difficult to obtain, the price is high, the enantiomeric excess value is not high, and the reduction yield is low. 2. Chiral (R) -alanine is used as a starting material, amino protection is carried out, diazomethane is used for increasing a carbon chain to be changed into beta-amino acid ester, and deprotection reduction is carried out to obtain a target product. The disadvantage of this process is that (R) -alanine of high chiral purity is difficult to obtain and diazomethane is dangerous to use. 3. Crotonate reacts with (R) - (+) -alpha-phenylethylamine to generate a group of epimers with two chiral centers, single isomers are obtained after silica gel column chromatography separation, and then R-3-aminobutanol is obtained through ester reduction and debenzylation; the route has fewer steps and easily-obtained raw materials, but has the following problems: because the reaction selectivity of the first step is poor, two epimers with almost the same quantity are obtained, the separation and purification are difficult, the chromatographic column method is usually adopted for separation, the dosage of eluent is large, the loss is large, and the efficiency is low; meanwhile, the cost of raw materials is obviously increased because the LiAlH4 with high price is used as a reducing agent, and the column chromatography method is not suitable for large-scale industrial production because of efficiency and cost.
The biocatalytic synthesis of R-3-aminobutanol has the characteristics of mild reaction conditions, high efficiency, strong stereoselectivity and regioselectivity, environmental friendliness and the like, and is receiving more and more attention. In addition, the molecular modification of the biocatalyst by utilizing rational design and directed evolution improves the catalytic properties of the catalyst, such as activity, stereoselectivity and the like, and has important significance in promoting the industrial production process of the catalyst by further optimizing a reaction system. At present, no report of biocatalytic synthesis exists, and the actinomycete (actinobacillus) -derived transaminase disclosed by the invention is firstly applied to the synthesis application of R-3-aminobutanol after being expressed and modified in escherichia coli.
Disclosure of the invention
The invention aims to provide a transaminase and a mutant derived from actinomycetes and application of the transaminase and the mutant in catalytic synthesis of R-3-aminobutanol, wherein the transaminase and the mutant are used as substrates of butanol, and pyridoxal phosphate is used as a coenzyme to catalytically synthesize the R-3-aminobutanol (figure 1); the transaminase derived from actinomycetes is transformed by a directed evolution strategy to obtain mutant protein, the enzyme activity of the mutant protein is improved, and the synthesis capability of the mutant protein on R-3-aminobutanol is improved.
The technical scheme adopted by the invention is as follows:
in a first aspect, the invention provides an actinomycete (actinobacilla) -derived transaminase capable of catalyzing the synthesis of R-3-aminobutanol from butanols, wherein the gene sequence is shown as SEQ ID NO.1, and the amino acid sequence is shown as SEQ ID NO. 2. The invention also relates to a recombinant vector and a gene engineering bacterium constructed by the transaminase gene.
The method for synthesizing R-3-aminobutanol by transaminase catalysis comprises the following steps: taking wet thalli obtained by fermentation culture of recombinant genetic engineering bacteria containing transaminase coding genes as a catalyst, taking butyl ketol as a substrate, taking coenzyme pyridoxal phosphate as a cofactor, taking isopropylamine or D-alanine as an amino donor, taking acetonitrile or dimethyl sulfoxide as a cosolvent, taking a buffer solution (preferably Tris-HCl buffer solution with pH of 8.0-8.5) with pH of 5.0-9.0 as a reaction medium to form a reaction system, carrying out conversion reaction at the temperature of 20-45 ℃ (preferably 30 ℃), and after the reaction is finished, separating and purifying the reaction solution to obtain R-3-aminobutanol; in the reaction system, the final concentration of the substrate is 20-500mM (preferably 50-100mM), the final concentration of the cofactor is 0.1-2mM (preferably 1mM), the final concentration of the amino donor is 0.04-3M (preferably 0.1-2M, more preferably 0.2-0.4M), the volume concentration of the cosolvent is 0.1% -15% (preferably 5-10%), and the amount of the catalyst is 10-100g/L (preferably 50-100g/L, most preferably 50g/L) based on the weight of wet bacteria.
In a second aspect, the present invention provides an actinomycete (actinobacillus) -derived transaminase mutant obtained by single-or double-mutating the amino acid sequence shown in SEQ ID NO.2 at positions 80 and 294.
Furthermore, the mutant is obtained by mutating valine at position 80 of an amino acid sequence shown in SEQ ID NO.2 into glycine, tryptophan at position 203 into serine, and threonine at position 294 into serine, wherein the amino acid sequence of the mutant is shown in SEQ ID NO. 4.
The invention relates to a transaminase mutant coding gene, wherein the nucleotide sequence of the coding gene is shown in SEQ ID NO. 3.
The invention is designed and synthesized artificially aiming at the transaminase sequence from actinomycetes (Actinobacillus), and the gene is expressed after being successfully cloned in escherichia coli. Further extracting plasmid containing transaminase gene from Escherichia coli, changing the nucleotide sequence of transaminase by error-prone PCR method, connecting to expression vector, expressing in Escherichia coli, and screening to obtain mutant with improved activity for improving the catalytic activity of butanol.
The present invention can be constructed by ligating the nucleotide sequences of the transaminase and its mutant of the present invention to various vectors by a method conventional in the art. The recombinant vector of the present invention is not limited as long as it can maintain its replication or autonomous replication in various host cells of prokaryotic and/or eukaryotic cells, and it may be various vectors conventional in the art, such as various plasmids, phage or viral vectors, etc., preferably pET-28 b. Preferably, the recombinant expression vector of the present invention can be obtained by: the obtained wild transaminase and mutant gene products are connected with a vector pET-28b to construct transaminase mutant gene recombinant expression plasmids pET28b-TA0 and pET28b-TA 1.
The host cell into which the DNA encoding the transaminase of the present invention and its mutant is introduced is not limited as long as a recombinant expression system has been established therefor so long as the recombinant expression vector can stably self-replicate and the carried mutant gene of the transaminase of the present invention can be efficiently expressed. Such as Escherichia coli, Bacillus subtilis, yeast, actinomycetes, Aspergillus, and animal cells and higher plant cells. Coli BL21(DE3) is preferred in the present invention. The recombinant plasmids pET28b-TA0 and pET28b-TA1 are transformed into E.coli BL21(DE3) to obtain engineering bacteria E.coli BL21(DE3)/pET28b-TA0 and E.coli BL21(DE3)/pET28b-TA 1.
The preparation of the recombinant transaminase and the mutant thereof comprises the steps of culturing the recombinant expression transformant and inducing to obtain the recombinant transaminase mutant protein. Among them, the medium used for culturing the recombinant expression transformant may be a medium which allows the transformant to grow and produce the transaminase of the present invention in the art, and preferably LB medium: 10g/L of peptone, 5g/L of yeast extract, 10g/L of sodium chloride and deionized water as a solvent, wherein the pH value is 7.2. The culture method and culture conditions are not particularly limited as long as the transformant can grow and produce the cells. The following methods are preferred: recombinant E.coli BL21(DE3)/pET28b-TA0 and E.coli BL21(DE3)/pET28b-TA1 related to the present invention were inoculated into LB medium containing 50. mu.g/ml kanamycin and cultured at 37 ℃ to optical density OD600When the concentration reaches 0.4-1 (preferably 0.4), the transaminase and the mutant protein thereof can be efficiently expressed under the induction of isopropyl- β -D-thiogalactopyranoside (IPTG) with the final concentration of 0.1-1.0 mM (preferably 1.0 mM).
In a third aspect, the method for synthesizing R-3-aminobutanol by using the transaminase mutant comprises the following steps: the method comprises the steps of taking wet thalli obtained by fermentation culture of recombinant genetic engineering bacteria containing transaminase mutant coding genes as a catalyst, taking butyl ketol as a substrate, taking coenzyme pyridoxal phosphate (PLP) as a cofactor, taking isopropylamine or D-alanine as an amino donor, taking acetonitrile or dimethyl sulfoxide as a cosolvent, taking a buffer solution with the pH of 5.0-9.0 (preferably 8.0-8.5) as a reaction medium to form a reaction system, carrying out conversion reaction at the temperature of 20-45 ℃ (preferably 30 ℃), and after the reaction is finished, separating and purifying the reaction solution to obtain the R-3-aminobutanol.
Further, in the reaction system, the final concentration of the substrate is 20 to 500mM (preferably 50 to 100mM), the final concentration of the cofactor is 0.1 to 2mM (preferably 0.5 to 1.2mM, more preferably 1mM), the final concentration of the amino donor is 0.04 to 3M (preferably 0.15 to 2M, more preferably 0.2 to 0.4M), the volume concentration of the cosolvent is 0.1 to 15% (preferably 5 to 10%), and the amount of the catalyst is 10 to 100g/L (preferably 50g/L) based on the weight of wet cells.
Further, the buffer was 50mM Tris-HCl buffer (pH 8.0).
Further, the catalyst is prepared by the following method:
1) slant culture: inoculating the recombinant bacterium genetic engineering bacterium containing the transaminase mutant coding gene to an LB culture medium containing 50 mu g/ml kanamycin, and culturing at 37 ℃ for 16h to obtain slant thalli; the LB culture medium comprises the following components in percentage by mass: 10g/L of peptone, 5g/L of yeast extract, 10g/L of sodium chloride, 1.5% agar and deionized water as a solvent, wherein the pH value is 7.0;
2) seed culture: inoculating the slant thallus to an LB liquid culture medium containing 50 mu g/ml kanamycin, and culturing at 37 ℃ for 8-10 h to obtain a seed solution; the LB liquid culture medium has the following final concentration composition: 10g/L of peptone, 5g/L of yeast extract, 10g/L of sodium chloride, deionized water as a solvent and pH 7.0;
3) fermentation culture: inoculating the seed solution into LB liquid culture medium containing 50 ug/ml kanamycin resistance at an inoculation amount of 1% by volume, culturing at 37 ℃ until OD600 value is 0.4, adding isopropyl-beta-D-galactoside (0.1 mM final concentration) or lactose (15 g/L final concentration), culturing at 28 ℃ for 12h, centrifuging, and collecting wet cells.
Compared with the prior art, the biological preparation method of the R-3-aminobutanol provided by the invention has the advantages that the butanone alcohol is used as the initial raw material, and the price is low. The needed recombinant transaminase can be prepared in large quantities by constructing escherichia coli genetic engineering bacteria and then fermenting, and is relatively easy to obtain and cheap. The reaction is in a one-pot type, the substrate and the enzyme are added and then start to react, and the final product R-3-aminobutanol is directly obtained, so that the industrial cost is low. Compared with the wild type, the actinomycete transaminase mutant provided by the invention has better catalytic activity. Under an optimal system, the conversion rate of 100mM substrate reaches 90%, and the ee value reaches 99.9%; the conversion rate of 500mM substrate reaches 78%, the ee value reaches 99.9%, and compared with the wild type, the conversion rate of the substrate is respectively improved by 12% -25%.
(IV) description of the drawings
FIG. 1 is a reaction scheme of the biological preparation method of R-3-aminobutanol according to the present invention.
(V) detailed description of the preferred embodiments
The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:
EXAMPLE 1 construction of transaminase-containing genetically engineered bacteria
A transaminase gene (nucleotide sequence is shown as SEQ ID NO.1, and an amino acid sequence is shown as SEQ ID NO. 2) derived from an actinomycete (Actinobacillus) is selected for whole gene synthesis, is connected with a plasmid pET-28b, is transferred into Escherichia coli DH5 alpha competent cells, is coated with an LB plate containing kanamycin (50 mu g/ml), is cultured overnight at 37 ℃, and then positive transformants are picked and identified for sequencing. The positive monoclonal which is verified is inoculated into 5mL LB liquid culture medium containing 50 mug/mL kanamycin, cultured overnight at 37 ℃, plasmid is extracted, after the verification is carried out again, the recombinant expression vector is transferred into Escherichia coli BL21(DE3) strain, recombinant strain E.coli BL21(DE3)/pET28b-TA0 (namely parent strain) is obtained, and cultured overnight on LB plate containing 50 mug/mL kanamycin at 37 ℃. A single clone was selected and inoculated into 5mL of LB liquid medium containing 50. mu.g/mL kanamycin, cultured overnight at 37 ℃ and then centrifuged at 8000rpm for 10min at 4 ℃ to collect wet cells, which were stored in glycerol at-80 ℃ for further use.
Example 2 obtaining of transaminase mutants and construction of genetically engineered bacteria containing mutants
1. Construction of a mutant library
The mutant sequence was obtained by error-prone PCR amplification using the recombinant expression vector pET28b-TA0 obtained in example 1 as a template. The amplification primer is (5' GCTGA)GGATCCATGACCATCTCTAAAGACAT3 ') and (5' GCATC)AAGCTTTCAGTATTCGATAGCTTC3’)。
The amplification system is as follows: 50 μ l reaction: 10xTaq polymerase buffer: 5 mu l of the solution; mg (magnesium)2+(25mM)：2-8μl；Mm2+2-8 μ l (25 mM); 10mM dNTP mix (2.5 mM each of dATP, dCTP, dGTP and dTTP) 4. mu.L; 1. mu.L each of the forward primer and the reverse primer at a concentration of 50. mu.M, DNA template: 1 mu L of the solution; taq DNA polymerase: 10U; the system is complemented with double distilled water.
The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 1min, then temperature cycling at 95 ℃ for 10s, 56 ℃ for 90s, and 72 ℃ for 1min for 30 cycles, and finally extension at 72 ℃ for 10min, and termination at 4 ℃. The PCR product was analyzed by 1% agarose gel electrophoresis and recovered by cutting gel, digested by BamHI/HindIII, ligated with pET28b digested with the same enzyme, E.coli BL21(DE3) competent cells were transformed by electric shock with a ligation solution, coated with LB plate containing kanamycin (50. mu.g/ml), and cultured overnight at 37 ℃ to obtain a mutation library of transaminase.
2. Screening of Positive mutants
A single clone was picked from the mutant library, inoculated into 1mL LB liquid medium containing 50. mu.g/mL kanamycin in a 96-well plate, cultured at 37 ℃ for 3 hours, added with isopropyl-. beta. -D-galactoside at a final concentration of 0.1mM, placed at 28 ℃ for further culture for 12 hours, centrifuged, collected as wet cells, and studied for the catalytic synthesis of R-3-aminobutanol. The reaction system is 1mL, and contains 20mM substrate, cosolvent DMSO with the volume concentration of 2%, cofactor PLP with the final concentration of 1mM, and amino donor with the final concentration of 80 mM. After reacting for 2h, sampling and carrying out gas chromatography to determine the substrate conversion rate, screening to obtain a positive clone (recombinant bacterium E. coliBL21(DE3)/pET28b-TA1, namely a mutant strain) with improved activity, and carrying out sequencing analysis to obtain a mutant, namely a three-mutant V80G/W203S/T294S (namely, valine at the 80 th position of an amino acid sequence of SEQ ID NO.2 is mutated into glycine, tryptophan at the 203 th position is mutated into serine, and threonine at the 294 th position is mutated into serine), wherein the amino acid sequence is shown as SEQ ID NO.4, and the nucleotide sequence is shown as SEQ ID NO. 3. The enzyme activity of the mutant recombinant cell for catalyzing and synthesizing the R-3-aminobutanol reaches 592U/g, and is improved by 35 percent compared with the wild type.
The definition of the thallus enzyme activity is as follows: under the above reaction conditions, the amount of enzyme required to catalyze the conversion of 1. mu. mol of substrate butanonol to R-3-aminobutanol per hour is one unit of enzyme activity, denoted by U.
EXAMPLE 3 preparation of transaminase wild-type and mutant catalysts
1) Slant culture: respectively inoculating recombinant bacteria E.coliBL21(DE3)/pET28b-TA0 and recombinant bacteria E.coliBL21(DE3)/pET28b-TA1 containing transaminase wild-type genes and transaminase mutant genes into LB culture medium containing 50 mu g/ml kanamycin, and culturing at 37 ℃ for 16 hours to obtain slant thalli; the LB culture medium comprises the following components in percentage by mass: 10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride, 1.5% agar, deionized water as solvent, pH 7.0, and 50. mu.g/ml kanamycin before use.
2) Seed culture: inoculating the slant thallus to an LB liquid culture medium containing 50 mu g/ml kanamycin, and culturing at 37 ℃ for 8-10 h to obtain a seed solution; the LB liquid culture medium has the following final concentration composition: 10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride, deionized water as solvent, pH 7.0, and 50. mu.g/ml kanamycin before use.
3) Fermentation culture: inoculating the seed solution into LB liquid culture medium containing 50 ug/ml kanamycin resistance at an inoculation amount of 1% by volume concentration, culturing at 37 ℃ until OD600 value is 0.4, adding isopropyl-beta-D-galactoside with final concentration of 0.1mM or lactose with final concentration of 15g/L, culturing at 28 ℃ for 12h, centrifuging, collecting wet thallus, and storing at-20 ℃ for later use. The LB liquid culture medium has the following final concentration composition: 10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride, deionized water as solvent, pH 7.0, and 50. mu.g/ml kanamycin before use.
Example 4 transaminase and mutant catalyzed Synthesis of R-3-aminobutanol
1) Adding a substrate with a final concentration of 50mM, a cosolvent with a volume concentration of 5%, a cofactor with a final concentration of 0.5mM, an amino donor with a final concentration of 150mM and a catalyst with a concentration of 30g/L into a reaction medium in sequence to form a reaction system; reacting the reaction system at 30 ℃ for 24 h; in the reaction process, sampling and detecting the generation amount of the R-3-aminobutanol in a sample by adopting a high performance gas chromatography, and as a result, catalyzing and synthesizing the R-3-aminobutanol by using a parent strain cell (recombinant bacterium E.coliBL21(DE3)/pET28b-TA0) to obtain the product with the optical purity of 99 percent and the conversion rate of about 81 percent; the mutant strain cell (recombinant strain E. coliBL21(DE3)/pET28b-TA1) catalyzes and synthesizes R-3-aminobutanol with the optical purity of 99 percent and the conversion rate of 91 percent.
The substrate is butyl ketol; the cosolvent is acetonitrile; the cofactor is pyridoxal phosphate; the amino donor is D-alanine, and the catalyst is the recombinant bacterium E prepared in example 3.
coliBL21(DE3)/pET28b-TA0 wet cells and recombinant E.coliBL21(DE3)/pET28b-TA1 wet cells, the reaction medium being 100mM bicarbonate buffer at pH 8.0.
2) Adding a substrate with a final concentration of 50mM, a cosolvent with a volume concentration of 8%, a cofactor with a final concentration of 1mM, an amino donor with a final concentration of 200mM and a catalyst with a concentration of 50g/L into a reaction medium in sequence to form a reaction system; reacting the reaction system at the temperature of 28 ℃ for 24 hours; in the reaction process, sampling and detecting the generation amount of R-3-aminobutanol in a sample by adopting a high performance gas chromatography, and measuring that the optical purity of R-3-aminobutanol synthesized by the cells of the parent strain through catalysis is 99% and the conversion rate is about 85%; the mutant strain cell catalyzes and synthesizes R-3-aminobutanol with the optical purity of 99 percent and the conversion rate of about 96 percent.
The substrate is butyl ketol; the reaction medium is 50mM Tris buffer solution with the pH value of 8.5; the cosolvent is dimethyl sulfoxide; the cofactor is pyridoxal phosphate; the amino donor is D-alanine, and the catalyst is the recombinant bacterium E.coliBL21(DE3)/pET28b-TA0 wet bacterium prepared in example 3 and the recombinant bacterium E.coliBL21(DE3)/pET28b-TA1 wet bacterium.
3) Adding a reaction system consisting of a substrate with a final concentration of 100mM, a cosolvent with a volume concentration of 10%, a cofactor with a final concentration of 1mM, an amino donor with a final concentration of 400mM and a catalyst with a concentration of 50g/L into a reaction medium in sequence; reacting the reaction system at 25 ℃ for 24 h; in the reaction process, sampling and detecting the generation amount of R-3-aminobutanol in a sample by adopting a high-efficiency gas phase, and carrying out catalytic synthesis on the R-3-aminobutanol by using parental strain cells, wherein the optical purity is 99% and the conversion rate is about 80%; the mutant strain cell catalyzes and synthesizes the R-3-aminobutanol with the optical purity of 99 percent and the conversion rate of about 90 percent.
The substrate is butyl ketol; the reaction solution is a bicarbonate buffer solution with the pH of 7.8 and the concentration of 80 mM; the cosolvent is acetonitrile; the cofactor is pyridoxal phosphate; the amino donor is isopropylamine, and the catalyst is the recombinant bacterium E.coliBL21(DE3)/pET28b-TA0 wet bacterium prepared in example 3 and the recombinant bacterium E.coliBL21(DE3)/pET28b-TA1 wet bacterium.
4) Adding a substrate with the final concentration of 200mM, a cosolvent with the volume concentration of 10%, a cofactor with the final concentration of 1mM, an amino donor with the final concentration of 1M and 80g/L of a catalyst into a reaction medium in sequence to form a reaction system; reacting the reaction system at 35 ℃ for 24 h; in the reaction process, sampling and adopting a high performance gas chromatography to detect the generation amount of the R-3-aminobutanol in the sample, wherein the optical purity of R-3-aminobutanol synthesized by the parental strain cell in a catalytic mode is 99 percent, and the conversion rate is about 77 percent; the mutant strain cell catalyzes and synthesizes the R-3-aminobutanol with the optical purity of 99 percent and the conversion rate of about 86 percent.
The substrate is butyl ketol; the reaction solution is 50mM Tris buffer solution with the pH value of 8.5; the cosolvent is acetonitrile; the cofactor is pyridoxal phosphate; the amino donor is D-alanine, and the catalyst is the recombinant bacterium E.coliBL21(DE3)/pET28b-TA0 wet bacterium prepared in example 3 and the recombinant bacterium E.coliBL21(DE3)/pET28b-TA1 wet bacterium.
5) Adding a substrate with a final concentration of 400mM, a cosolvent with a volume concentration of 10%, a cofactor with a final concentration of 1.2mM, an amino donor with a final concentration of 1.6M and a catalyst with a concentration of 80g/L into a reaction medium in sequence to form a reaction system; reacting the reaction system at 30 ℃ for 24 h; in the reaction process, sampling and adopting a high performance gas chromatography to detect the generation amount of the R-3-aminobutanol in the sample, wherein the optical purity of the R-3-aminobutanol synthesized by the parental strain cell in a catalytic mode is 99 percent, and the conversion rate is about 61 percent; the mutant strain cell catalyzes and synthesizes the R-3-aminobutanol with the optical purity of 99 percent and the conversion rate of about 78 percent.
The substrate is butyl ketol; the reaction solution is 90mM Tris buffer solution with the pH value of 7.5; the cosolvent is dimethyl sulfoxide; the cofactor is pyridoxal phosphate; the amino donor is isopropylamine, and the catalyst is the recombinant bacterium E.coliBL21(DE3)/pET28b-TA0 wet bacterium prepared in example 3 and the recombinant bacterium E.coliBL21(DE3)/pET28b-TA1 wet bacterium.
6) Adding a substrate with a final concentration of 500mM, a cosolvent with a volume concentration of 15%, a cofactor with a final concentration of 1mM, an amino donor with a final concentration of 2M and a catalyst with a concentration of 100g/L in turn into a reaction medium to form a reaction system; reacting the reaction system at the temperature of 28 ℃ for 24 hours; in the reaction process, sampling and adopting a high performance gas chromatography to detect the generation amount of the R-3-aminobutanol in the sample, wherein the optical purity of R-3-aminobutanol synthesized by the parental strain cell in a catalytic mode is 99 percent, and the conversion rate is about 58 percent; the mutant strain cell catalyzes and synthesizes the R-3-aminobutanol with the optical purity of 99 percent and the conversion rate of about 72 percent.
The substrate is butyl ketol; the reaction solution is a bicarbonate buffer solution with the pH of 8.0 and the concentration of 100 mM; the cosolvent is dimethyl sulfoxide; the cofactor is pyridoxal phosphate; the amino donor is D-alanine, and the catalyst is the recombinant bacterium E.coliBL21(DE3)/pET28b-TA0 wet bacterium prepared in example 3 and the recombinant bacterium E.coliBL21(DE3)/pET28b-TA1 wet bacterium.
The invention is not limited by the specific text described above. The invention can be varied within the scope outlined by the claims and these variations are within the scope of the invention.
Sequence listing
<110> Zhejiang industrial university
<120> transaminase, mutant and recombinant bacteria derived from actinomycetes and application thereof
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<170>SIPOSequenceListing 1.0
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gaagctcgta tctctctgtt cgacaccggt ttcggtcact ctgacctgac ctacaccgtt 240
gctcacgttt ggcacggtaa catcttccgt ctgaaagacc acatcgaccg tgttttcgac 300
ggtgctcaga aactgcgtct gcagtctccg ctgaccaaag ctgaagttga agacatcacc 360
aaacgttgcg tttctctgtc tcagctgcgt gaatctttcg ttaacatcac catcacccgt 420
ggttacggtg ctcgtaaagg tgaaaaagac ctgtctaaac tgacctctca gatctacatc 480
tacgctatcc cgtacctgtg ggctttcccg ccggaagaac agatcttcgg tacctctgct 540
atcgttccgc gtcacgttcg tcgtgctggt cgtaacaccg ttgacccgac cgttaaaaac 600
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gctatcctgc tggacgctga caactgcgtt gctgaaggtc cgggtttcaa cgttgttatg 720
gttaaagacg gtaaactgtc ttctccgtct cgtaacgctc tgccgggtat cacccgtctg 780
accgttatgg aaatggctga cgaaatgggt atcgaattca ccctgcgtga catcacctct 840
cgtgaactgt acgaagctga cgaactgatc gctgttacca ccgctggtgg tatcaccccg 900
atcacctctc tggacggtga accgctgggt gacggtaccc cgggtccggt taccgttgct 960
atccgtgaca ggttctgggc tatgatggac gaaccgtctt ctctggttga agctatcgaa 1020
tactga 1026
<210>2
<211>341
<212>PRT
<213> Unknown (Unknown)
<400>2
Met Thr Ile Ser Lys Asp Ile Asp Tyr Ser Thr Ser Asn Leu Val Ser
1 5 10 15
Val Ala Pro Gly Ala Ile Arg Glu Pro Thr Pro Ala Gly Ser Val Ile
20 25 30
Gln Tyr Ser Asp Tyr Glu Leu Asp Glu Ser Ser Pro Phe Ala Gly Gly
35 40 45
Ala Ala Trp Ile Glu Gly Glu Tyr Val Pro Ala Ala Glu Ala Arg Ile
50 55 60
Ser Leu Phe Asp Thr Gly Phe Gly His Ser Asp Leu Thr Tyr Thr Val
65 70 75 80
Ala His Val Trp His Gly Asn Ile Phe Arg Leu Lys Asp His Ile Asp
85 90 95
Arg Val Phe Asp Gly Ala Gln Lys Leu Arg Leu Gln Ser Pro Leu Thr
100 105 110
Lys Ala Glu Val Glu Asp Ile Thr Lys Arg Cys Val Ser Leu Ser Gln
115 120 125
Leu Arg Glu Ser Phe Val Asn Ile Thr Ile Thr Arg Gly Tyr Gly Ala
130 135 140
Arg Lys Gly Glu Lys Asp Leu Ser Lys Leu Thr Ser Gln Ile Tyr Ile
145 150 155 160
Tyr Ala Ile Pro Tyr Leu Trp Ala Phe Pro Pro Glu Glu Gln Ile Phe
165 170 175
Gly Thr Ser Ala Ile Val Pro Arg His Val Arg Arg Ala Gly Arg Asn
180 185 190
Thr Val Asp Pro Thr Val Lys Asn Tyr Gln Trp Gly Asp Leu Thr Ala
195 200 205
Ala Ser Phe Glu Ala Lys Asp Arg Gly Ala Arg Thr Ala Ile Leu Leu
210 215 220
Asp Ala Asp Asn Cys Val Ala Glu Gly Pro Gly Phe Asn Val Val Met
225 230 235 240
Val Lys Asp Gly Lys Leu Ser Ser Pro Ser Arg Asn Ala Leu Pro Gly
245 250 255
Ile Thr Arg Leu Thr Val Met Glu Met Ala Asp Glu Met Gly Ile Glu
260 265 270
Phe Thr Leu Arg Asp Ile Thr Ser Arg Glu Leu Tyr Glu Ala Asp Glu
275 280 285
Leu Ile Ala Val Thr Thr Ala Gly Gly Ile Thr Pro Ile Thr Ser Leu
290 295 300
Asp Gly Glu Pro Leu Gly Asp Gly Thr Pro Gly Pro Val Thr Val Ala
305 310 315 320
Ile Arg Asp Arg Phe Trp Ala Met Met Asp Glu Pro Ser Ser Leu Val
325 330 335
Glu Ala Ile Glu Tyr
340
<210>3
<211>1026
<212>DNA
<213> Unknown (Unknown)
<400>3
atgaccatct ctaaagacat cgactactct acctctaacc tggtttctgt tgctccgggt 60
gctatccgtg aaccgacccc ggctggttct gttatccagt actctgacta cgaactggac 120
gaatcttctc cgttcgctgg tggtgctgct tggatcgaag gtgaatacgt tccggctgct 180
gaagctcgta tctctctgtt cgacaccggt ttcggtcact ctgacctgac ctacaccggt 240
gctcacgttt ggcacggtaa catcttccgt ctgaaagacc acatcgaccg tgttttcgac 300
ggtgctcaga aactgcgtct gcagtctccg ctgaccaaag ctgaagttga agacatcacc 360
aaacgttgcg tttctctgtc tcagctgcgt gaatctttcg ttaacatcac catcacccgt 420
ggttacggtg ctcgtaaagg tgaaaaagac ctgtctaaac tgacctctca gatctacatc 480
tacgctatcc cgtacctgtg ggctttcccg ccggaagaac agatcttcgg tacctctgct 540
atcgttccgc gtcacgttcg tcgtgctggt cgtaacaccg ttgacccgac cgttaaaaac 600
taccagtcgg gtgacctgac cgctgcttct ttcgaagcta aagaccgtgg tgctcgtacc 660
gctatcctgc tggacgctga caactgcgtt gctgaaggtc cgggtttcaa cgttgttatg 720
gttaaagacg gtaaactgtc ttctccgtct cgtaacgctc tgccgggtat cacccgtctg 780
accgttatgg aaatggctga cgaaatgggt atcgaattca ccctgcgtga catcacctct 840
cgtgaactgt acgaagctga cgaactgatc gctgttacca gcgctggtgg tatcaccccg 900
atcacctctc tggacggtga accgctgggt gacggtaccc cgggtccggt taccgttgct 960
atccgtgaca ggttctgggc tatgatggac gaaccgtctt ctctggttga agctatcgaa 1020
tactga 1026
<210>4
<211>341
<212>PRT
<213> Unknown (Unknown)
<400>4
Met Thr Ile Ser Lys Asp Ile Asp Tyr Ser Thr Ser Asn Leu Val Ser
1 5 10 15
Val Ala Pro Gly Ala Ile Arg Glu Pro Thr Pro Ala Gly Ser Val Ile
20 25 30
Gln Tyr Ser Asp Tyr Glu Leu Asp Glu Ser Ser Pro Phe Ala Gly Gly
35 40 45
Ala Ala Trp Ile Glu Gly Glu Tyr Val Pro Ala Ala Glu Ala Arg Ile
50 55 60
Ser Leu Phe Asp Thr Gly Phe Gly His Ser Asp Leu Thr Tyr Thr Gly
65 70 75 80
Ala His Val Trp His Gly Asn Ile Phe Arg Leu Lys Asp His Ile Asp
85 90 95
Arg Val Phe Asp Gly Ala Gln Lys Leu Arg Leu Gln Ser Pro Leu Thr
100 105 110
Lys Ala Glu Val Glu Asp Ile Thr Lys Arg Cys Val Ser Leu Ser Gln
115 120 125
Leu Arg Glu Ser Phe Val Asn Ile Thr Ile Thr Arg Gly Tyr Gly Ala
130 135 140
Arg Lys Gly Glu Lys Asp Leu Ser Lys Leu Thr Ser Gln Ile Tyr Ile
145 150 155 160
Tyr Ala Ile Pro Tyr Leu Trp Ala Phe Pro Pro Glu Glu Gln Ile Phe
165 170 175
Gly Thr Ser Ala Ile Val Pro Arg His Val Arg Arg Ala Gly Arg Asn
180 185 190
Thr Val Asp Pro Thr Val Lys Asn Tyr Gln Ser Gly Asp Leu Thr Ala
195 200 205
Ala Ser Phe Glu Ala Lys Asp Arg Gly Ala Arg Thr Ala Ile Leu Leu
210 215 220
Asp Ala Asp Asn Cys Val Ala Glu Gly Pro Gly Phe Asn Val Val Met
225 230 235 240
Val Lys Asp Gly Lys Leu Ser Ser Pro Ser Arg Asn Ala Leu Pro Gly
245 250 255
Ile Thr Arg Leu Thr Val Met Glu Met Ala Asp Glu Met Gly Ile Glu
260 265 270
Phe Thr Leu Arg Asp Ile Thr Ser Arg Glu Leu Tyr Glu Ala Asp Glu
275 280 285
Leu Ile Ala Val Thr Ser Ala Gly Gly Ile Thr Pro Ile Thr Ser Leu
290 295 300
Asp Gly Glu Pro Leu Gly Asp Gly Thr Pro Gly Pro Val Thr Val Ala
305 310 315 320
Ile Arg Asp Arg Phe Trp Ala Met Met Asp Glu Pro Ser Ser Leu Val
325 330 335
Glu Ala Ile Glu Tyr
340
Claims (8)
1. The application of the transaminase derived from actinomycetes in synthesizing R-3-aminobutanol is characterized in that the amino acid sequence of the transaminase is shown in SEQ ID NO. 2.
2. The use according to claim 1, wherein the R-3-aminobutanol is synthesized by: taking wet thalli obtained by fermentation culture of recombinant genetic engineering bacteria containing transaminase coding genes as a catalyst, taking butyl ketol as a substrate, taking coenzyme pyridoxal phosphate as a cofactor, taking isopropylamine or D-alanine as an amino donor, taking acetonitrile or dimethyl sulfoxide as a cosolvent, and taking a buffer solution with the pH value of 5.0-9.0 as a reaction medium to form a reaction system, carrying out conversion reaction at the temperature of 20-45 ℃, and after the reaction is finished, separating and purifying the reaction solution to obtain R-3-aminobutanol; in the reaction system, the final concentration of the substrate is 20-500mM, the final concentration of the cofactor is 0.1-2mM, the final concentration of the amino donor is 0.04-3M, the volume concentration of the cosolvent is 0.1% -15%, and the dosage of the catalyst is 10-100g/L based on the weight of wet bacteria.
3. An actinomycete-derived transaminase mutant as set forth in claim 1, which is obtained by mutating valine at position 80 to glycine, tryptophan at position 203 to serine, and threonine at position 294 to serine of the amino acid sequence shown in SEQ ID No. 2.
4. A gene encoding the transaminase mutant of claim 3, characterized in that the nucleotide sequence of the encoding gene is shown in SEQ ID No. 3.
5. A recombinant genetically engineered bacterium constructed from the gene encoding the transaminase mutant of claim 4.
6. Use of the transaminase mutant of claim 3 for the synthesis of R-3-aminobutanol.
7. The use of claim 6, wherein the R-3-aminobutanol is synthesized by: taking wet thalli obtained by fermentation culture of recombinant genetic engineering bacteria containing transaminase mutant coding genes as a catalyst, taking butyl ketol as a substrate, taking coenzyme pyridoxal phosphate as a cofactor, taking isopropylamine or D-alanine as an amino donor, taking acetonitrile or dimethyl sulfoxide as a cosolvent, and taking a buffer solution with the pH value of 5.0-9.0 as a reaction medium to form a reaction system, carrying out conversion reaction at the temperature of 20-45 ℃, and after the reaction is finished, separating and purifying the reaction solution to obtain R-3-aminobutanol; in the reaction system, the final concentration of the substrate is 20-500mM, the final concentration of the cofactor is 0.1-2mM, the final concentration of the amino donor is 0.04-3M, the volume concentration of the cosolvent is 0.1% -15%, and the dosage of the catalyst is 10-100g/L based on the weight of wet bacteria.
8. The use according to claim 7, wherein the catalyst is prepared by the following process:
1) slant culture: inoculating the recombinant bacterium genetic engineering bacterium containing the transaminase mutant coding gene to an LB culture medium containing 50 mu g/ml kanamycin, and culturing at 37 ℃ for 16h to obtain slant thalli; the LB culture medium comprises the following components in percentage by mass: 10g/L of peptone, 5g/L of yeast extract, 10g/L of sodium chloride, 1.5% agar and deionized water as a solvent, wherein the pH value is 7.0;
2) seed culture: inoculating the slant thallus to an LB liquid culture medium containing 50 mu g/ml kanamycin, and culturing at 37 ℃ for 8-10 h to obtain a seed solution; the LB liquid culture medium has the following final concentration composition: 10g/L of peptone, 5g/L of yeast extract, 10g/L of sodium chloride, deionized water as a solvent and pH 7.0;
3) fermentation culture: inoculating the seed solution into LB liquid culture medium containing 50 ug/ml kanamycin resistance at an inoculation amount of 1% by volume, culturing at 37 ℃ until OD600 value is 0.4, adding isopropyl-beta-D-galactoside (0.1 mM final concentration) or lactose (15 g/L final concentration), culturing at 28 ℃ for 12h, centrifuging, and collecting wet cells.
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| CN109609476B (en) * | 2019-01-14 | 2020-06-19 | 浙江工业大学 | α -transaminase and mutant and application thereof in asymmetric synthesis of L-glufosinate-ammonium |
| CN109897843B (en) * | 2019-03-01 | 2021-02-02 | 浙江工业大学 | Recombinant echinocandin B deacylase mutant and application thereof |
| WO2021077425A1 (en) * | 2019-10-25 | 2021-04-29 | 凯莱英医药化学（阜新）技术有限公司 | Transaminase mutant and use thereof |
| CN110724675B (en) * | 2019-10-31 | 2021-02-02 | 宁波酶赛生物工程有限公司 | Transaminase catalyst and method for synthesizing (R) -1-tert-butoxycarbonyl-3-aminopiperidine by enzyme method |
| CN111235127B (en) * | 2020-04-29 | 2020-08-14 | 凯莱英医药集团（天津）股份有限公司 | Transaminase mutants and uses thereof |
| CN112280761B (en) * | 2020-11-16 | 2022-04-12 | 清华大学 | Recombinant transaminase, mutant of recombinant transaminase and application of mutant |
| CN112941045B (en) * | 2021-02-05 | 2023-07-21 | 南京红杉生物科技有限公司 | Recombinant aminotransferase and method for synthesizing L-phenylglycinol |
| CN113088503B (en) * | 2021-04-27 | 2023-01-10 | 浙江工业大学 | O-succinyl mercaptotransferase mutant and application thereof in L-methionine synthesis |
| CN113215124A (en) * | 2021-04-27 | 2021-08-06 | 浙江工业大学 | Application of O-succinyl mercaptotransferase in L-methionine synthesis |
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