CN114686451B - Amine dehydrogenase mutant and application thereof in preparation of (S) -5-methyl-2-pyrrolidone - Google Patents
Amine dehydrogenase mutant and application thereof in preparation of (S) -5-methyl-2-pyrrolidone Download PDFInfo
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- CN114686451B CN114686451B CN202210185409.XA CN202210185409A CN114686451B CN 114686451 B CN114686451 B CN 114686451B CN 202210185409 A CN202210185409 A CN 202210185409A CN 114686451 B CN114686451 B CN 114686451B
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- serine
- glycine
- threonine
- substitution
- amine dehydrogenase
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- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
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- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/10—Nitrogen as only ring hetero atom
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Abstract
The invention relates to an amine dehydrogenase mutant and application thereof in preparation of (S) -5-methyl-2-pyrrolidone. Specifically disclosed are an amine dehydrogenase mutant with improved activity, a coding gene thereof, a recombinant expression vector and a recombinant expression transformant containing the gene sequence, and application of the amine dehydrogenase mutant as a catalyst for catalyzing asymmetric reductive amination of a latent chiral carbonyl compound, in particular for catalyzing asymmetric reduction of levulinic acid to prepare optically pure 5-methyl-2-pyrrolidone. Compared with the prior art, the method has the advantages of high concentration of the enzymatic reaction substrate, mild reaction condition, environmental friendliness, high yield, high optical purity of the product and the like, and has good application prospect.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to an amine dehydrogenase mutant with obviously improved catalytic performance, a coding gene thereof, a recombinant expression vector and a recombinant expression transformant containing the gene sequence, and application of the amine dehydrogenase mutant or the recombinant expression transformant in catalyzing a prochiral carbonyl compound to prepare optically pure amino acid, in particular in catalyzing asymmetric reduction of levulinic acid to prepare optically pure lactam (S) -5-methyl-2-pyrrolidone.
Background
Lactams are key compounds in organic chemistry, as they are found in many biologically active products, and can also serve as valuable intermediates for more complex structures including synthetic polymers. In addition to the β -lactams (2-azetidinones), which constitute the most important class of current antibiotics, the synthesis of γ -lactams (2-pyrrolidone) and δ -lactams (2-piperidone) has also received attention for several years. Secondly, the lactam is a natural product fragment widely existing in the nature, and researches show that the lactam is mostly existing on land and in marine organisms (bacteria, fungi, blue algae and sponges). Natural source lactam derivatives are reported to have very remarkable and wide biological activities such as insecticidal, herbicidal, antiviral, antitumor, anti-inflammatory and the like. Therefore, it would be of great economic value to find a lactam synthesis route with high yield and high stereoselectivity.
The 5-methyl-2-pyrrolidone is an important chemical and intermediate for the synthesis of medicines, pesticides and the like, and can be prepared from biomass renewable resource derivatives levulinic acid and esters thereof through catalytic hydrogenation and reductive amination. The common synthesis method is to catalyze levulinic acid (ester) under the action of a metal catalyst by taking hydrogen as a hydrogen source and ammonia or organic amine as a nitrogen source.
For example, shilling et al uses hydrogen as a hydrogen source, ammonia as a nitrogen source, diatomite-supported nickel as a catalyst, and performs reductive amination on levulinic acid to synthesize 5-methyl-2-pyrrolidone at 200 ℃ in a yield of 87% (Shilling, wilburL. Pyrrolidinones: U.S. Pat. No.3,235,562 [ P ] 1966.2.15.).
In 2017, king et al studied the reaction route and mechanism of preparing 5-methyl-2-pyrrolidone by directly reductive amination of levulinic acid with ammonium formate as a hydrogen source and N, N-dimethylformamide as a solvent, and optimized the reaction conditions, but had moderate stereoselectivity (biomass chemistry engineering, 2017,51 (02): 19-25.).
Chinese patent application publication No. CN1764376a discloses a process for producing 5-methyl-N-aryl-2-pyrrolidone, 5-methyl-N-cycloalkyl-2-pyrrolidone, and 5-methyl-N-alkyl-2-pyrrolidone by reductive amination of levulinic acid with nitro compounds using an optionally supported metal catalyst, also requiring hydrogen as a reducing agent, the product stereoselectivity is poor, even below 10%.
Chinese patent application publication No. CN110615754a discloses a process for preparing 5-methyl-2-pyrrolidone, which comprises using biomass derivative levulinic acid as starting material, ammonium formate as hydrogen source and nitrogen source, supported bimetallic catalyst as hydrogenation catalyst, and synthesizing 5-methyl-2-pyrrolidone in water by one pot method, wherein the supported metal of the supported bimetallic catalyst is bimetallic composed of two noble metals, bimetallic composed of one noble metal and one non-noble metal a or non-noble metal B, or bimetallic composed of one non-noble metal a and one non-noble metal B. The conversion rate of levulinic acid can reach 100%, and the yield of 5-methyl-2-pyrrolidone can reach more than 94%.
In 2018 Angela et al mainly described a biosynthetic route to gamma-and delta-lactams by reductive amination of ketoester substrates (isopropylamine IPA as amine donor) by catalytic action of transaminases, followed by intramolecular self-cyclization of the intermediate product to form the lactam. All substrates tested were subjected to preparation experiments with a conversion of the product of 66% -89% and an ee value of greater than 91%. This paper describes a simple one-pot two-step process for converting different gamma-and delta-keto esters to the corresponding optically active lactams. This strategy is based on a selective biotransformation reaction which allows the spontaneous intramolecular cyclization of the amino ester intermediate in its own aqueous medium without the addition of external reagents, but the first step of reductive amination requires the addition of an amine donor, so that the overall reaction is by-product and has a low yield (Advanced Synthesis & Catalysis,2018,360 (4): 686-695).
In summary, many methods for synthetically obtaining 5-methyl-2-pyrrolidone have been reported, which can be obtained by chemical or biocatalytic methods, but chemical methods have many disadvantages, such as: the diastereoselectivity is poor, the reaction environment is not mild, and byproducts are more. Transaminases catalyze the reductive amination of ketoester substrates, require the provision of amine donors, and have low enzymatic activity.
Disclosure of Invention
Aiming at the defects of the prior art amine dehydrogenase, the invention provides an amine dehydrogenase mutant and application thereof in preparing (S) -5-methyl-2-pyrrolidone.
Specifically, the invention aims at the defects of the amine dehydrogenase in the prior art, reforms the amine dehydrogenase by means of protein engineering and directed evolution, further improves the activity of the mutant on levulinic acid, provides an amine dehydrogenase mutant with obviously improved catalytic performance, a coding gene thereof, a recombinant expression vector and a recombinant expression transformant containing the gene sequence, and uses the amine dehydrogenase mutant or the recombinant expression transformant to catalyze a prochiral carbonyl compound to prepare optically pure amino acid, in particular to the application of catalyzing the asymmetric reduction of levulinic acid to prepare optically pure lactam (S) -5-methyl-2-pyrrolidone.
Based on the technical scheme of the invention, the commercial value of the amine dehydrogenase mutant can be fully realized, the application cost of the enzyme catalyst is reduced, and the synthesis process for synthesizing the lactam compound is effectively simplified.
The aim of the invention can be achieved by the following technical scheme:
according to one of the technical schemes of the invention, the amine dehydrogenase mutant with obviously improved catalytic performance is obtained.
In the present invention, the amine dehydrogenase TtherAmDH derived from Thermoanaerobacter thermohydrosulfuricus is used WT As a female parent, mutation strategies such as error-prone PCR, site-directed saturation mutation, iterative combination and the like are adopted to directionally evolve, and the amine dehydrogenase with improved catalytic performance is identified by combining high-throughput primary screening of an enzyme-labeling instrument and secondary screening of an ultraviolet spectrophotometer.
The amine dehydrogenase mutant (TtherAmDH mutant) is a derivative protein of a novel amino acid sequence, wherein the novel amino acid sequence is formed by replacing one or more amino acid residues in leucine 11, phenylalanine 24, arginine 41, valine 82, threonine 102, glycine 198, proline 226, glutamine 268 or glutamic acid 298 of the amino acid sequence shown in SEQ ID No.2 with other amino acid residues, and the derivative protein has higher catalytic performance than the protein consisting of the amino acid sequence shown in SEQ ID No. 2.
The amino acid sequence of the amine dehydrogenase mutant is selected from one of the following:
(1) Substitution of glycine for glutamic acid at position 298 of the amino acid sequence shown in SEQ ID No. 2;
(2) Substitution of glutamic acid at position 298 with glycine and valine at position 82 with threonine of the amino acid sequence shown in SEQ ID No. 2;
(3) Substitution of glycine for glutamic acid at position 298, threonine for valine at position 82, serine for phenylalanine at position 24 of the amino acid sequence shown in SEQ ID No. 2;
(4) Substitution of glycine for glutamic acid at position 298, threonine for valine at position 82, serine for phenylalanine at position 24, serine for glycine at position 198 of the amino acid sequence shown in SEQ ID No. 2;
(5) Substitution of glycine for glutamic acid at position 298, threonine for valine at position 82, serine for phenylalanine at position 24, serine for glycine at position 198, serine for proline at position 226 of the amino acid sequence shown in SEQ ID No. 2;
(6) Substitution of glycine for glutamic acid at position 298, threonine for valine at position 82, serine for phenylalanine at position 24, serine for glycine at position 198, serine for proline at position 226, and cysteine for threonine at position 102 of the amino acid sequence shown in SEQ ID No. 2;
(7) Substitution of glycine for glutamic acid at position 298, threonine for valine at position 82, serine for phenylalanine at position 24, serine for glycine at position 198, serine for proline at position 226, and alanine for leucine at position 11 of the amino acid sequence shown in SEQ ID No. 2;
(8) Substitution of glycine for glutamic acid at position 298, threonine for valine at position 82, serine for phenylalanine at position 24, serine for glycine at position 198, serine for proline at position 226, cysteine for threonine at position 102, and alanine for leucine at position 11 of the amino acid sequence shown in SEQ ID No. 2;
(9) Substitution of glycine for the 298 th glutamic acid, threonine for the 82 nd valine, serine for the 24 th phenylalanine, serine for the 198 th glycine, serine for the 226 th proline, cysteine for the 102 th threonine, alanine for the 11 th leucine, and threonine for the 268 th glutamine;
(10) Substitution of glycine at position 298, threonine at position 82, serine at position 24, serine at position 198, serine at position 226, cysteine at position 102, alanine at position 11, threonine at position 268, and serine at position 41.
In a second aspect of the present invention, there are provided a gene encoding an amine dehydrogenase (ttheramadh) mutant, and a recombinant expression vector comprising the same.
The coding gene codes and expresses the amine dehydrogenase mutant obtained by evolution modification according to the first technical scheme, and the sources of the amine dehydrogenase mutant comprise: cloning the gene sequence of the TtherAmDH mutant series in the first technical scheme by a genetic engineering technology; alternatively, the nucleic acid molecule encoding the ttheramadh mutant according to claim one can be obtained by artificial total sequence synthesis.
The recombinant expression vector can be constructed by connecting the nucleotide sequence of the amine dehydrogenase mutant gene to various commercially available empty vectors by a conventional method in the field. The commercially available empty vector may be various plasmid vectors conventional in the art, so long as the recombinant expression vector can normally replicate in a corresponding expression host and express a corresponding reductase. Preferred plasmid vectors are different for different expression hosts. It will be clear to a person of ordinary skill in the art how to select appropriate vectors, promoters, enhancers and host cells. For E.coli hosts, the preferred plasmid vector is the pET-28a (+) plasmid. The escherichia coli recombinant expression vector can be prepared by the following method: the TtherAmDH mutant gene fragment obtained by PCR amplification is digested with restriction enzymes EcoR I and Hind III, simultaneously the empty plasmid pET-28a (+) is digested with restriction enzymes EcoR I and Hind III, the DNA fragment of the digested TtherAmDH mutant and the empty plasmid are recovered, and the DNA fragment and the empty plasmid are connected by using T4 DNA ligase, so that a recombinant expression vector containing the TtherAmDH mutant coding nucleic acid molecule for expressing escherichia coli is constructed.
In a third aspect of the present invention, there is provided a recombinant expression transformant comprising the ttheramadh mutant gene of the present invention or a recombinant expression vector thereof. Recombinant expression transformants can be prepared by transforming an already constructed recombinant expression vector into a host cell. The host cell is a variety of conventional host cells in the art, and it is required that the recombinant expression vector is capable of stably self-replicating and efficiently expressing after induction by an inducer. The invention takes escherichia coli as host cells, and more preferably escherichia coli E.coli BL21 (DE 3) is used for high-efficiency expression of target proteins by recombinant expression vectors.
The fourth technical scheme of the invention provides a recombinant amine dehydrogenase mutant catalyst (TtherAmDH mutant catalyst for short), wherein the recombinant amine dehydrogenase mutant catalyst is in any one of the following forms:
(1) Culturing the recombinant expression transformant of the present invention, and isolating a transformant cell containing the amine dehydrogenase (ttheramadh) mutant;
(2) Culturing the recombinant expression transformant of the present invention, and isolating a crude enzyme solution containing the amine dehydrogenase (ttheramadh) mutant;
(3) And (3) drying the crude enzyme solution of the amine dehydrogenase (TtherAmDH) mutant to obtain crude enzyme powder.
Wherein the culture method and conditions of the recombinant expression transformant are those conventional in the art, comprising the steps of: the recombinant expression transformant of the present invention is cultured to obtain a recombinant amine dehydrogenase.
For recombinant E.coli, the preferred medium is LB medium: 10g/L peptone, 5g/L yeast extract, 10g/L NaCl and pH 6.5-7.0. The preferred cultivation method is: recombinant E.coli constructed as described above was inoculated into LB medium containing kanamycin, and cultured overnight at 37℃with shaking at 220 rpm. Inoculating 1% (v/v) of the recombinant expression transformant into a 500ml Erlenmeyer flask containing 100ml of LB medium (containing kanamycin), placing the Erlenmeyer flask in a shaking table at 37 ℃ and 220rpm for shaking culture, adding isopropyl-beta-D-thiogalactoside (IPTG) with a final concentration of 0.1-0.5mmol/L as an inducer when the OD600 of the culture solution reaches 0.6-0.8, centrifuging the culture solution after 16-24h induction at 16-25 ℃, collecting the precipitate, and washing twice with physiological saline to obtain the recombinant expression transformant cells. And freeze-drying the obtained recombinant cells to obtain the freeze-dried cells containing the TtherAmDH mutant. Suspending the obtained recombinant cells in buffer solution with the volume of 5-10 times (v/w), performing ultrasonic crushing, and centrifugally collecting supernatant to obtain crude enzyme solution of the recombinant TtherAmDH mutant. And (3) placing the collected crude enzyme solution at the temperature of minus 80 ℃ for freezing, and then drying at low temperature by using a vacuum freeze dryer to obtain the freeze-dried enzyme powder. The obtained freeze-dried enzyme powder is stored in a refrigerator at the temperature of 4 ℃ and can be conveniently used.
The method for measuring the activity of the TtherAmDH mutant comprises the following steps: 1ml of a reaction system (3 mol/L ammonium formate/ammonia water buffer, pH 6.5) containing 8mmol/L levulinic acid and 0.2mmol/L NADH was preheated to 40℃and then an appropriate amount of TtherAmDH mutant was added thereto, the reaction was incubated at 40℃and the change in absorbance of NADPH at 340nm was detected on a spectrophotometer and the change in absorbance within 1 minute was recorded.
The enzyme activity was calculated using the following formula:
enzyme activity (U) =ew×v×10 3 /(6220×l)
Wherein EW is the change in absorbance at 340nm within 1 minute; v is the volume of the reaction solution, and the unit is mL;6220 is the molar extinction coefficient of NADH, in L/(mol cm); l is the optical path distance in cm.1 enzyme activity unit (U) is defined as the amount of enzyme required to catalyze the oxidation of 1. Mu. Mol NADH per minute under the above conditions.
The fifth technical scheme of the invention provides application of the TtherAmDH mutant or the TtherAmDH mutant catalyst as a catalyst in asymmetric reduction of a latent chiral carbonyl compound.
In one embodiment of the present invention, the latent chiral carbonyl compound has the structural formula:
wherein R is 1 Alkyl of 1 to 6 carbon atoms, n is 1 or 2,
preferably, the latent chiral carbonyl compound is selected from one or more of the following compounds:
compound 1: n=1, r 1 ＝CH 3 ；
Compound 2: n=1, r 1 ＝C 2 H 5 ；
Compound 3: n=1, r 1 ＝C 3 H 7 ；
Compound 4: n=1, r 1 ＝C 4 H 9 ；
Compound 5: n=1, r 1 ＝C 6 H 13 ；
Compound 6: n=2, r 1 ＝CH 3 。
Further, the concentration of the latent chiral carbonyl compound is 20-500 mmol/L, and the dosage of the amine dehydrogenase mutant or the recombinant amine dehydrogenase mutant catalyst is selected to be 50-200U/mmol of the latent chiral carbonyl compound.
Further, the invention provides application of the TtherAmDH mutant or the TtherAmDH mutant catalyst in synthesis of (S) -5-methyl-2-pyrrolidone, namely a method for preparing (S) -5-methyl-2-pyrrolidone by asymmetrically reducing a latent chiral carbonyl compound levulinic acid by using the TtherAmDH mutant or the TtherAmDH mutant catalyst.
The specific synthetic route for (S) -5-methyl-2-pyrrolidone is shown in the following scheme, as shown in FIG. 1:
in the application, the concentration of the latent chiral carbonyl compound levulinic acid can be 20-500 mmol/L, and the TtherAmDH mutant or the TtherAmDH mutant catalyst can be used in an amount of 50-200U/mmol of the latent chiral carbonyl compound.
In one embodiment of the invention, formate dehydrogenase, ammonium formate, the coenzyme NADH or NAD is additionally added to the asymmetric reduction + 。
In one embodiment of the invention, the NADPH or NAD required in the reaction + The dosage of (C) is 0.1-0.5 mmol/L. In the reaction process, ammonium formate can be used as an auxiliary substrate, and the coenzyme circulation of NADH in the reaction system is realized through the catalysis of formate dehydrogenase, wherein the consumption of the formate dehydrogenase can be 50-200U/mmol of potential chiral carbonyl compoundThe concentration of the ammonium formate is 3mol/L.
In one embodiment of the invention, the concentration of ammonium formate buffer required in the asymmetric reduction process is preferably 3mol/L.
In one embodiment of the invention, the asymmetric reduction reaction is carried out under shaking or stirring conditions.
In one embodiment of the invention, the asymmetric reduction reaction is carried out at a temperature of 35 to 45 ℃, preferably 40 ℃. The time of the asymmetric reduction reaction is based on the time when the substrate is completely reacted or the reaction is automatically stopped, and the reaction time is preferably less than 24 hours.
The application discloses a synthetic route of (S) -5-methyl-2-pyrrolidone, which is characterized in that a substrate levulinic acid is subjected to reductive amination through amine dehydrogenase, free ammonia is used as an amine donor, NADH coenzyme factor is recycled, a byproduct of NADH coenzyme factor is only generated by water, and finally an optically pure product 5-methyl-2-pyrrolidone is synthesized through intramolecular cyclization. In the research of synthesizing the lactam of the optical pure 5-methyl-2-pyrrolidone, the known wild amine dehydrogenase TtherAmDH has the problems of low catalytic activity on the substrate levulinic acid, low substrate loading, low product yield and the like. Therefore, the enzyme catalyst with better catalytic performance is based on the invention to carry out asymmetric reduction on the substrate levulinic acid, and can meet the industrialized demands of high reaction efficiency, high substrate concentration, simple operation and high yield.
Compared with the prior art, the innovation and technical effects of the invention are as follows:
the invention provides an amine dehydrogenase mutant with better catalytic performance, which efficiently catalyzes asymmetric reductive amination of gamma-carbonyl in levulinic acid to prepare optically pure (S) -5-methyl-2-pyrrolidone. The 5-methyl-2-pyrrolidone is an important chemical and an intermediate for synthesizing medicines, pesticides and the like. The invention couples the amine dehydrogenase mutant with formate dehydrogenase to realize the in-situ regeneration of the coenzyme, thereby greatly reducing the consumption of the coenzyme. For levulinic acid as a small molecule substrate, the carbonyl reductase is capable of achieving 98% conversion at 10h at catalytic concentrations up to 500mM, with space time yields up to 75.3g -1 L - 1 day -1 . Compared with the parent amine dehydrogenase (TtherAmDH) WT ) The amine dehydrogenase mutant has the advantages of high catalytic activity, high-concentration substrate tolerance, high optical purity of reaction products and the like when aiming at levulinic acid, so that the amine dehydrogenase mutant has better industrial application prospect.
Drawings
FIG. 1 is a schematic diagram of a pathway for synthesizing (S) -5-methyl-2-pyrrolidone.
Detailed Description
The individual reaction or detection conditions described in the context of the present invention may be combined or modified in accordance with common general knowledge in the art and may be verified experimentally.
The technical solutions and technical effects of the present invention will be clearly and completely described in the following in conjunction with specific embodiments, but the scope of the present invention is not limited to these embodiments, and all changes or equivalent substitutions without departing from the concept of the present invention are included in the scope of the present invention.
The sources of materials in the following examples are:
the female parent recombinant plasmid pET28a-TtherAmDH contains a nucleic acid sequence shown as a sequence table SEQ ID No.1, is self-constructed by the inventor, and can be constructed based on the disclosure of the invention by adopting a conventional construction method in the biotechnology field.
Plasmid vector pET28a was purchased from Novagen.
E. coli BL21 (DE 3) competent cells, 2X Taq PCR MasterMix, agarose gel DNA recovery kit were purchased from Beijing Tiangen Biochemical technology Co.
The restriction enzymes EcoR I and Hind III are commercial products of the company New England Biolabs (NEB).
Unless otherwise indicated, the specific experiments in the following examples were performed according to methods and conditions conventional in the art, or following the commercial specifications of the kit.
Example 1
TtherAmDH mutant with improved random mutation screening activity
The error-prone PCR technique is used to randomly mutate TtherAmDH which contains the amino acid sequence shown in SEQ ID No. 2.
The primers used were:
the upstream primer is shown as SEQ ID No. 3:
CCGGAATTCATGGAAAATATAAAAGTCGTAGTTTGGGG
the downstream primer is shown as SEQ ID No. 4:
CCCAAGCTTTTAACGGCGACGAATCATAT
wherein the sequence shown by the upper primer underlined is EcoR I cleavage site and the sequence shown by the lower primer underlined is Hind III cleavage site.
Error-prone PCR was performed using pET28a-TtherAmDH as template and rTaq DNA polymerase to construct a random mutant library. PCR System (50. Mu.L): rTaq DNA polymerase 0.5. Mu.l, 10 XPCR buffer (Mg 2+ Plus) 5.0. Mu.l, dNTP mix (2.0 mM each) 4.0. Mu.l, mnCl at a final concentration of 250. Mu. Mol/L 2 pET28a-TtherAmDH plasmid (100 ng), 2. Mu.l each of the upstream and downstream primers (10. Mu.M), was supplemented with sterile distilled water to 50. Mu.l. PCR reaction procedure: (1) pre-denaturation at 95℃for 5min; (2) denaturation at 94℃for 30s; (3) annealing at 54 ℃ for 30s; (4) extending at 72 ℃ for 1min; steps (2) - (4) are carried out for 30 cycles altogether; finally, the product is preserved at 72 ℃ for 10min and 4 ℃. The PCR product is purified and recovered by agarose gel electrophoresis analysis and verification, and the recovered target gene and empty plasmid pET28a are respectively subjected to double enzyme digestion for 1h at 37 ℃ by using restriction enzymes EcoR I and Hind III. And (3) performing agarose gel electrophoresis analysis and verification on the double-enzyme-digested product, then performing gel-digested purification and recovery, and connecting the obtained linearized pET28a plasmid with the purified target gene fragment at 16 ℃ by using T4 DNA ligase for overnight. The ligation product was transformed into E.coli BL21 (DE 3) competent cells, and uniformly spread on LB agar plates containing 50. Mu.g/ml kanamycin, and placed in a 37℃incubator for stationary culture for about 12 hours.
Mutants on the transformation plate were picked with toothpicks into 96-well deep well plates and incubated overnight at 37℃in a 220rpm shaker. And (3) sucking 10 mu L of bacterial liquid from the holes of the primary plate, inoculating the bacterial liquid into the corresponding holes of the secondary plate, culturing for 2-3 h in a shaking table at 37 ℃ and 220rpm, and then adding IPTG with the final concentration of 0.2mM, and culturing for 24h at 16 ℃. Then, centrifugation was performed at 3500 Xg for 10min at 4℃and the upper medium was removed, 300. Mu.L of lysozyme solution (1000 mg of lysozyme and 10mg of DNase were dissolved in 1L of ammonium formate buffer) was added to each well, and the mixture was stirred and mixed well, followed by treatment on a shaking table at 37℃for 2 hours. Centrifugation was performed at 3500 Xg for 15min at 4℃and 100. Mu.L of the cell disruption supernatant was transferred to a 96-well ELISA plate to which 100. Mu.L of the reaction solution consisting of 3M ammonium formate/ammonia water (pH 6.5) and 8mM levulinic acid and 0.4mM NADH was added per well. Shaking and mixing at 40 ℃, and reading the decrease of absorbance at 340nm on an enzyme labeling instrument. The expressed protein is subjected to high-flux activity screening in a 96-well plate, mutants with higher activity are subjected to purification characterization, and the corresponding genes are sequenced.
Through the high throughput screening of the enzyme-labeled instrument described in example 1, the activity of substituting glycine (F24K) for phenylalanine at position 24 for levulinic acid is improved, and the soluble expression of the protein is obviously improved.
Example 2 semi-rational construction of TtherAmDH mutant
In example 1, mutation sites (F24K) with significantly improved catalytic activity and soluble expression were successfully screened using error-prone PCR techniques and high throughput screening methods. And carrying out homologous modeling on the TtherAmDH, then carrying out molecular docking on the modeled mutant model and levulinic acid, and selecting a proper docking posture model according to the catalysis mechanism and the binding energy condition of the amine dehydrogenase. The activity of the enzyme is further improved by site-directed saturation mutation and combined mutation of amino acids near the substrate pocket. In the TtherAmDH steric space structure of the amino acid sequence shown in SEQ ID No.2, amino acid residues around the substrate levulinic acid binding site are mainly selected from amino acid sites on peripheral loop, and the amino acid residues at the sites are subjected to saturation mutation.
PCR was performed using pET28a-TtherAmDH as template and PrimeStar HS premix. The PCR system is as follows: 2X PrimeStar HS premix. Mu.l each of the upstream and downstream primers, 40ng of pET28a-TtherAmDH plasmid, 1. Mu.l of DMSO, and the addition of sterile distilled water was made up to 20. Mu.l. PCR reaction procedure: (1) pre-denaturation at 95℃for 5min; (2) denaturation at 94℃for 30s; (3) annealing at 55 ℃ for 30s; (4) extending at 72 ℃ for 6.5min; steps (2) - (4) are carried out for 30 cycles altogether; finally, the extension is carried out for 10min at 72 ℃. Then, 1. Mu.l of Dpn I enzyme was added to 20. Mu.l of the PCR product and incubated at 37℃for 3 hours to digest the template, and the digested product was transformed into E.coli BL21 (DE 3) competent cells, which were uniformly spread on LB agar plates containing 50. Mu.g/ml kanamycin, and placed in a 37℃incubator for stationary culture for about 12 hours. The obtained monoclonal colony is picked into a 96-hole deep hole plate for culture, the wall of the cultured cells is broken, NADH is used as coenzyme, high-flux activity screening is carried out on the expressed protein in the 96-hole plate, the mutant with higher activity is purified and characterized, and the corresponding gene is sequenced.
Through the high throughput screening of the microplate reader described in example 2, it was found that the activity of mutants such as substitution of 298 th glutamic acid with glycine (E298G), substitution of 82 nd valine with threonine (V82T), substitution of 198 th glycine with serine (G198S), substitution of 226 th proline with serine (P226S), substitution of 102 th threonine with cysteine (T102C), substitution of 11 th leucine with alanine (L11A), substitution of 268 th glutamine with threonine (Q268T), and mutation of 41 st arginine with serine (R41S) on levulinic acid was improved.
The ttheramadh mutant enzyme activity assay methods described in examples 1 and 2: 1ml reaction system (3 mol/L ammonium formate/ammonia water buffer solution, pH 6.5) containing 8mmol/L levulinic acid and 0.2mmol/L NADH is preheated to 40 ℃, then a proper amount of TtherAmDH mutant enzyme solution is added, the reaction is carried out at 40 ℃ in a heat-preserving manner, the absorbance change at 340nm is detected on a spectrophotometer, the absorbance change value within 1 minute is recorded, and the enzyme activity is calculated.
High throughput viability screening assay methods for TtherAmDH mutants described in examples 1 and 2: packaging ammonium formate/ammonia water buffer solution (3 mol/L, pH 6.5) containing 8mmol/L levulinic acid and 0.1mmol/L NADH into a 96-well plate, preheating to 40 ℃, then adding a proper amount of TtherAmDH mutant enzyme solution respectively, carrying out oscillation reaction at 40 ℃, detecting the absorbance change of NADH at 340nm on an enzyme label instrument, recording the absorbance change value within 10 minutes, and calculating the corresponding enzyme activity.
Through the screens of examples 1 and 2, mutants with increased levulinic acid were obtained, the sequences of which mutants and the levulinic acid activities of these mutants are listed in table 1. A list of mutants of the specific sequences disclosed herein having relevant activity is provided in table 1, in which the specific activity of each generation of mutants on levulinic acid and fold increases are reflected.
TABLE 1 amino dehydrogenase mutant sequences and corresponding list of activity improvements
The amine dehydrogenase mutant amino acid has one of the following sequences:
(1) Substitution of glycine for glutamic acid at position 298 of the amino acid sequence shown in SEQ ID No. 2;
(2) Substitution of glutamic acid at position 298 with glycine and valine at position 82 with threonine of the amino acid sequence shown in SEQ ID No. 2;
(3) Substitution of glycine for glutamic acid at position 298, threonine for valine at position 82, serine for phenylalanine at position 24 of the amino acid sequence shown in SEQ ID No. 2;
(4) Substitution of glycine for glutamic acid at position 298, threonine for valine at position 82, serine for phenylalanine at position 24, serine for glycine at position 198 of the amino acid sequence shown in SEQ ID No. 2;
(5) Substitution of glycine for glutamic acid at position 298, threonine for valine at position 82, serine for phenylalanine at position 24, serine for glycine at position 198, serine for proline at position 226 of the amino acid sequence shown in SEQ ID No. 2;
(6) Substitution of glycine for glutamic acid at position 298, threonine for valine at position 82, serine for phenylalanine at position 24, serine for glycine at position 198, serine for proline at position 226, and cysteine for threonine at position 102 of the amino acid sequence shown in SEQ ID No. 2;
(7) Substitution of glycine for glutamic acid at position 298, threonine for valine at position 82, serine for phenylalanine at position 24, serine for glycine at position 198, serine for proline at position 226, and alanine for leucine at position 11 of the amino acid sequence shown in SEQ ID No. 2;
(8) Substitution of glycine for glutamic acid at position 298, threonine for valine at position 82, serine for phenylalanine at position 24, serine for glycine at position 198, serine for proline at position 226, cysteine for threonine at position 102, and alanine for leucine at position 11 of the amino acid sequence shown in SEQ ID No. 2;
(9) Substitution of glycine for the 298 th glutamic acid, threonine for the 82 nd valine, serine for the 24 th phenylalanine, serine for the 198 th glycine, serine for the 226 th proline, cysteine for the 102 th threonine, alanine for the 11 th leucine, and threonine for the 268 th glutamine;
(10) Substitution of glycine at position 298, threonine at position 82, serine at position 24, serine at position 198, serine at position 226, cysteine at position 102, alanine at position 11, threonine at position 268, and serine at position 41.
EXAMPLE 3 recombinant E.coli BL21 (DE 3)/pET 28a-TtherAmDH V9 Expression of (2) and enzymatic method
Recombinant E.coli BL21 (DE 3)/pET 28a-TtherAmDH of mutant V9 obtained in example 2 V9 Inoculated into LB medium containing 50. Mu.g/ml kanamycin, shake cultured at 37℃for 12 hours, then inoculated into 500ml Erlenmeyer flask containing 100ml LB medium (containing 50. Mu.g/ml kanamycin) according to an inoculum size of 1% (v/v), placed in shaking culture at 37℃and 220rpm, when OD600 of the culture solution reaches 0.6, IPTG with a final concentration of 0.2mmol/L is added as an inducer, and induced at 16℃for 24 hours. The culture broth was centrifuged at 8000 Xg for 10min, and the cells were collected and washed twice with physiological saline to obtain resting cells. Cells obtained in 100ml of the culture were suspended in 15ml of potassium phosphate buffer (100 mM, pH 7.0) and subjected to the following ultrasonication in an ice-water bath: the crude enzyme was collected by centrifugation at 12000 Xg for 40 minutes at 4℃under a power of 350W for 4s at a time of 6s intermittently for 10min. In addition, the obtained crude enzyme solution is freeze-dried, and freeze-dried enzyme powder can be obtained.
Examples 4-9 recombinant TtherAmDH V9 Catalyzing asymmetric reduction reactions of different potential chiral substrates
The reaction was performed in a 2mL centrifuge tube, and 8mM of different prochiral substrates, 0.2mM NAD, were added to 1mL ammonium formate/ammonia buffer (3M, pH 6.5) + 0.5U of recombinant TtherAmDH obtained in example 3 V9 Crude enzyme solution, 1U formate dehydrogenase. The reaction was carried out on a shaker at 1000rpm and 35 ℃. After 24 hours of reaction, EDCI aqueous solution was added to cyclize at room temperature, the mixture was extracted with an equal volume of ethyl acetate, and then dried overnight with anhydrous sodium sulfate, and the ee value of the lactam product was measured. The results are shown in Table 2.
TABLE 2 TtherAmDH V9 Results of asymmetric reduction reactions catalyzing different potential chiral substrates
TABLE 2 TtherAmDH of examples 4-9 V9 The analysis conditions of the ee values of the end products obtained by catalyzing the different latent chiral substrates are shown in Table 3.
TABLE 3 TtherAmDH V9 Analysis conditions for the ee values of the end products obtained by catalyzing different latent chiral substrates
EXAMPLE 10 recombinant TtherAmDH V9 Catalytic synthesis of (S) -5-methyl-2-pyrrolidone
In a 100mL jacketed reactor, 20g/L of the mutant recombinant expression transformant (E.coli BL21 (DE 3)/pET 28 a-TtherAmDH) as described in example 3 was added to 100mL of ammonium formate/ammonia buffer (3M, pH 6.5) containing 500mM substrate levulinic acid V9 ) Simultaneously adding 25g/L of formate dehydrogenase and finally adding 0.2mM of NAD + Is used for cyclic regeneration of coenzyme. The reaction was carried out in a 40℃water bath with stirring by a stirring paddle, and the pH was controlled at 6.5 by controlling the fed-batch of 3mol/L formic acid solution by an automatic potentiometric titrator. After 10h of reaction, 1-ethyl-3 (3-dimethylpropylamine) carbodiimide (EDCI) was added to subject the intermediate amino acid to self-cyclization to form lactam (S) -5-methyl-2-pyrrolidone, which was stirred at 30℃for 2h. Five times of extraction is carried out by using 3 times of ethyl acetate, the extracts are combined, the solvent ethyl acetate is removed by rotary evaporation, the solvent ethyl acetate is dried by using anhydrous sodium sulfate for overnight, finally, the crude product is purified by using a silica gel chromatographic column, and the target product (S) -5-methyl-2-pyrrolidone is eluted by using a mobile phase of dichloromethane and methanol (9:1), so that 3.2g of product is obtained, and the purity is 99%. Measured by gas chromatography: the substrate conversion was 98% and the ee value of the product was greater than 99% (S).
Example 4 above-example 10 was performed in recombinant TtherAmDH V9 Specific applications of the chiral catalyst in catalyzing asymmetric reduction reactions of different potential chiral substrates, catalyzing synthesis of (S) -5-methyl-2-pyrrolidone and the like are illustrated. It should be noted that: as shown in Table 1, recombinant TtherAmDH V9 However, as shown in Table 1, other amine dehydrogenase mutants have much higher specific activities than the protein composed of the amino acid sequence shown in SEQ ID No.2, so those skilled in the art know that other amine dehydrogenase mutants can catalyze asymmetric reduction reactions of different chiral substrates and catalytic synthesis of (S) -5-methyl-2-pyrrolidone, and have equivalent technical effects. In the embodiment of the invention only byBy recombinant TtherAmDH V9 For purposes of illustration.
It can be seen that the recombinant mutant enzyme preparation obtained by the method can efficiently catalyze the substrate levulinic acid, and then is cyclized by using 1-ethyl-3 (3-dimethylpropylamine) carbodiimide (EDCI) to form lactam (S) -5-methyl-2-pyrrolidone by combining a chemical method, so that the chiral compound has great application value and is an important intermediate for synthesis of chemicals, medicines, pesticides and the like.
The previous description of the embodiments is provided to facilitate a person of ordinary skill in the art in order to make and use the present invention. It will be apparent to those skilled in the art that various modifications can be readily made to these embodiments and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above-described embodiments, and those skilled in the art, based on the present disclosure, should make improvements and modifications without departing from the scope of the present invention.
Sequence listing
<110> university of Industy of Huadong
<120> amine dehydrogenase mutant and its use in preparation of (S) -5-methyl-2-pyrrolidone
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tacgtggaga aagtgttccc gctgattaag ttggcggtgg agaatgggat taacgtgatc 300
actacagcgg aagagatggc atatccgtcc gcacagcacc tggagctggc gaaagagatc 360
gaccgtttgg ctcgcgagaa cggtgtctcc gtgttgggca ccggcattaa cccgggattc 420
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gcggctcgca ttaacgatct ctctccgttt ggcaaggctg tgatggaaga gcagggtgtt 540
ggtctgaccc cagaggaatt tgaagagggt gtgaagaacg gcaccgttgc cggtcatatt 600
ggctttccgg aaagcatcag catgatctgc gatgcgctgg gttggaaact gtctggtatc 660
gagcagaccc gtgaaccgat tgtgagcaag acctatcgtg aaaccccgta tgcccgtgtt 720
gagccaggct acgttgcagg ttgccgtcag atcggttacg gcaaagttga cggcgaagtt 780
aaaatcgaat tggagcaccc gcaacagatt ctgccgcaaa aagaaggtgt cgagactggc 840
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ggtggtttgg gcacgatcgc gctgtgcgta aatatgattc cgcatgttat caacgcagaa 960
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Met Glu Asn Ile Lys Val Val Val Trp Gly Leu Gly Ala Met Gly Ser
1 5 10 15
Gly Ile Ala Lys Met Ile Leu Phe Lys Lys Gly Met Glu Ile Val Gly
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Ala Ile Asp Thr Asp Pro Asn Lys Arg Gly Lys Asp Leu Asn Glu Ile
35 40 45
Leu Gly Thr Asn Ser Lys Pro Val Tyr Ile Thr Ser Glu Pro Gln Asp
50 55 60
Ile Ile Lys Lys Gly Ser Ala Asp Ile Ala Val Ile Val Thr Ser Ser
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Tyr Val Glu Lys Val Phe Pro Leu Ile Lys Leu Ala Val Glu Asn Gly
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Ile Asn Val Ile Thr Thr Ala Glu Glu Met Ala Tyr Pro Ser Ala Gln
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His Leu Glu Leu Ala Lys Glu Ile Asp Arg Leu Ala Arg Glu Asn Gly
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Val Ser Val Leu Gly Thr Gly Ile Asn Pro Gly Phe Val Leu Asp Tyr
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Leu Ile Ile Ala Leu Thr Gly Val Cys Val Asp Val Asp Ser Ile Lys
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Ala Ala Arg Ile Asn Asp Leu Ser Pro Phe Gly Lys Ala Val Met Glu
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Glu Gln Gly Val Gly Leu Thr Pro Glu Glu Phe Glu Glu Gly Val Lys
180 185 190
Asn Gly Thr Val Ala Gly His Ile Gly Phe Pro Glu Ser Ile Ser Met
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Ile Cys Asp Ala Leu Gly Trp Lys Leu Ser Gly Ile Glu Gln Thr Arg
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Glu Pro Ile Val Ser Lys Thr Tyr Arg Glu Thr Pro Tyr Ala Arg Val
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Glu Pro Gly Tyr Val Ala Gly Cys Arg Gln Ile Gly Tyr Gly Lys Val
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Asp Gly Glu Val Lys Ile Glu Leu Glu His Pro Gln Gln Ile Leu Pro
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Gln Lys Glu Gly Val Glu Thr Gly Asp Tyr Ile Glu Ile Lys Gly Thr
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Pro Asn Ile Lys Leu Ser Ile Lys Pro Glu Ile Pro Gly Gly Leu Gly
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Thr Ile Ala Leu Cys Val Asn Met Ile Pro His Val Ile Asn Ala Glu
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<213> Artificial sequence (Artificial Sequence)
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<213> Artificial sequence (Artificial Sequence)
<400> 27
gagcagaccc gtgaannkat tgtgagcaag acc 33
<210> 28
<211> 33
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 28
ggtcttgctc acaatmnntt cacgggtctg ctc 33
<210> 29
<211> 33
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 29
aaaatcgaat tggagnnkcc gcaacagatt ctg 33
<210> 30
<211> 33
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 30
cagaatctgt tgcggmnnct ccaattcgat ttt 33
<210> 31
<211> 33
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 31
atcgaattgg agcacnnkca acagattctg ccg 33
<210> 32
<211> 33
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 32
cggcagaatc tgttgmnngt gctccaattc gat 33
<210> 33
<211> 33
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 33
gaattggagc acccgnnkca gattctgccg caa 33
<210> 34
<211> 33
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 34
ttgcggcaga atctgmnncg ggtgctccaa ttc 33
<210> 35
<211> 33
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 35
ctgtctataa agccgnnkat cccgggtggt ttg 33
<210> 36
<211> 33
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 36
caaaccaccc gggatmnncg gctttataga cag 33
<210> 37
<211> 33
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 37
aagccggaga tcccgnnkgg tttgggcacg atc 33
<210> 38
<211> 33
<212> DNA
<213> Artificial sequence (Artificial Sequence)
<400> 38
gatcgtgccc aaaccmnncg ggatctccgg ctt 33
Claims (7)
1. An amine dehydrogenase mutant, characterized in that the amino acid sequence of the amine dehydrogenase mutant is selected from one of the following:
(1) Substitution of glycine for glutamic acid at position 298 of the amino acid sequence shown in SEQ ID No. 2;
(2) Substitution of glutamic acid at position 298 with glycine and valine at position 82 with threonine of the amino acid sequence shown in SEQ ID No. 2;
(3) Substitution of glycine for glutamic acid at position 298, threonine for valine at position 82, and glycine for phenylalanine at position 24 of the amino acid sequence shown in SEQ ID No. 2;
(4) Substitution of glycine for glutamic acid at position 298, threonine for valine at position 82, serine for phenylalanine at position 24, serine for glycine at position 198 of the amino acid sequence shown in SEQ ID No. 2;
(5) Substitution of glycine for glutamic acid at position 298, threonine for valine at position 82, glycine for phenylalanine at position 24, serine for glycine at position 198, serine for proline at position 226 of the amino acid sequence shown in SEQ ID No. 2;
(6) Substitution of glycine for glutamic acid at position 298, threonine for valine at position 82, serine for phenylalanine at position 24, serine for glycine at position 198, serine for proline at position 226, and cysteine for threonine at position 102 of the amino acid sequence shown in SEQ ID No. 2;
(7) Substitution of glycine for glutamic acid at position 298, threonine for valine at position 82, glycine for phenylalanine at position 24, serine for glycine at position 198, serine for proline at position 226, and alanine for leucine at position 11 of the amino acid sequence shown in SEQ ID No. 2;
(8) Substitution of glycine for glutamic acid at position 298, threonine for valine at position 82, serine for phenylalanine at position 24, serine for glycine at position 198, serine for proline at position 226, cysteine for threonine at position 102, and alanine for leucine at position 11 of the amino acid sequence shown in SEQ ID No. 2;
(9) Substitution of glycine for the 298 th glutamic acid, threonine for the 82 nd valine, serine for the 24 th phenylalanine, serine for the 198 th glycine, serine for the 226 th proline, cysteine for the 102 th threonine, alanine for the 11 th leucine, and threonine for the 268 th glutamine;
(10) Substitution of glycine at position 298, threonine at position 82, serine at position 24, serine at position 198, serine at position 226, cysteine at position 102, alanine at position 11, threonine at position 268, and serine at position 41.
2. An isolated nucleic acid encoding the amine dehydrogenase mutant of claim 1.
3. A recombinant expression vector comprising the nucleic acid of claim 2.
4. A recombinant expression transformant comprising the recombinant expression vector of claim 3.
5. A recombinant amine dehydrogenase mutant catalyst, wherein the recombinant amine dehydrogenase mutant catalyst is in any one of the following forms:
(1) Culturing the recombinant expression transformant according to claim 4, and isolating a transformant cell containing the amine dehydrogenase mutant according to claim 1;
(2) Culturing the recombinant expression transformant according to claim 4, and isolating a crude enzyme solution containing the amine dehydrogenase mutant according to claim 1;
(3) And (3) drying the crude enzyme solution obtained in the step (2) to obtain crude enzyme powder.
6. Use of the amine dehydrogenase mutant according to claim 1 or the recombinant amine dehydrogenase mutant catalyst according to claim 5 as a catalyst for the asymmetric reduction of a prochiral carbonyl compound, wherein the prochiral carbonyl compound is present in a concentration of 20 to 500mmol/L, and the amine dehydrogenase mutant according to claim 1 or the recombinant amine dehydrogenase mutant catalyst according to claim 5 is used in an amount of 50 to 200U/mmol prochiral carbonyl compound;
the structural general formula of the latent chiral carbonyl compound is as follows:
wherein R is 1 Alkyl of 1 to 6 carbon atoms, n being 1 or 2;
adding formate dehydrogenase, ammonium formate, and coenzyme NADH or NAD into asymmetric reduction reaction + The dosage of the formate dehydrogenase is 50-200U/mmol of the potential chiral carbonyl compound, the concentration of the ammonium formate is 3M, and the coenzyme NADH or NAD + The dosage of (C) is 0.1-0.5 mmol/L.
7. The use according to claim 6, wherein the latent chiral carbonyl compound is selected from one or more of the following compounds:
compound 1: n=1, r 1 ＝CH 3 ；
Compound 2: n=1, r 1 ＝C 2 H 5 ；
Compound 3: n=1, r 1 ＝C 3 H 7 ；
Compound 4: n=1, r 1 ＝C 4 H 9 ；
Compound 5: n=1, r 1 ＝C 6 H 13 ；
Compound 6: n=2, r 1 ＝CH 3 。
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| CN110628739A (en) * | 2019-08-14 | 2019-12-31 | 华东理工大学 | Amine dehydrogenase mutant and application thereof in synthesis of chiral amine and amino alcohol |
| CN110846291A (en) * | 2020-01-14 | 2020-02-28 | 中国科学院苏州生物医学工程技术研究所 | Amine dehydrogenase mutant with improved thermal stability and construction and application of genetically engineered bacterium thereof |
| CN110951705A (en) * | 2019-12-20 | 2020-04-03 | 中国科学院苏州生物医学工程技术研究所 | Amine dehydrogenase mutant, enzyme preparation, recombinant vector, recombinant cell and preparation method and application thereof |
| CN112852894A (en) * | 2020-06-04 | 2021-05-28 | 中国科学院天津工业生物技术研究所 | Amine dehydrogenase mutant and application thereof in synthesis of chiral amine alcohol compound |
| CN113846069A (en) * | 2021-10-28 | 2021-12-28 | 华东理工大学 | Amphetamine dehydrogenase mutant and application thereof in chiral amine synthesis |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110628739A (en) * | 2019-08-14 | 2019-12-31 | 华东理工大学 | Amine dehydrogenase mutant and application thereof in synthesis of chiral amine and amino alcohol |
| CN110951705A (en) * | 2019-12-20 | 2020-04-03 | 中国科学院苏州生物医学工程技术研究所 | Amine dehydrogenase mutant, enzyme preparation, recombinant vector, recombinant cell and preparation method and application thereof |
| CN110846291A (en) * | 2020-01-14 | 2020-02-28 | 中国科学院苏州生物医学工程技术研究所 | Amine dehydrogenase mutant with improved thermal stability and construction and application of genetically engineered bacterium thereof |
| CN112852894A (en) * | 2020-06-04 | 2021-05-28 | 中国科学院天津工业生物技术研究所 | Amine dehydrogenase mutant and application thereof in synthesis of chiral amine alcohol compound |
| CN113846069A (en) * | 2021-10-28 | 2021-12-28 | 华东理工大学 | Amphetamine dehydrogenase mutant and application thereof in chiral amine synthesis |
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