CN114134126B

CN114134126B - Use of aminotransferase and mutants thereof in the preparation of (S) -1-methoxy-2-propylamine

Info

Publication number: CN114134126B
Application number: CN202111259352.5A
Authority: CN
Inventors: 杨立荣; 张涛; 周海胜; 张红玉; 吴坚平
Original assignee: ZJU Hangzhou Global Scientific and Technological Innovation Center
Current assignee: ZJU Hangzhou Global Scientific and Technological Innovation Center
Priority date: 2021-10-28
Filing date: 2021-10-28
Publication date: 2023-12-05
Anticipated expiration: 2041-10-28
Also published as: CN114134126A

Abstract

The application discloses an application of aminotransferase and a mutant thereof in preparation of (S) -1-methoxy-2-propylamine. According to the application, the transaminase with extremely high activity for preparing (S) -1-methoxy-2-propylamine by catalyzing substrate 1-methoxy-2-acetone is obtained through screening, and the transaminase is applied to (S) -1-methoxy-2-propylamine, so that the ee value of a product obtained through reaction reaches more than 99%. The application also carries out mutation on the wild aminotransferase by a site-directed mutagenesis technology, so that the enzyme activity of the mutant obtained by screening is further improved, and the conversion rate is obviously improved. The method takes 1-methoxy-2-acetone as a substrate, and directly prepares chiral pure (S) -1-methoxy-2-propylamine through the amine group transfer reaction of the substrate catalyzed by omega-aminotransferase in the presence of isopropylamine or isopropylamine salt and cofactor.

Description

Use of aminotransferase and mutants thereof in the preparation of (S) -1-methoxy-2-propylamine

Technical Field

The application relates to the technical field of biochemical engineering, in particular to application of wild type aminotransferase in preparation of (S) -1-methoxy-2-propylamine, and an aminotransferase mutant and application thereof in preparation of (S) -1-methoxy-2-propylamine.

Background

(S) -1-methoxy-2-propylamine is a key chiral intermediate for the synthesis of the chloroacetamide herbicides Metolachlor (Metolachhlor) and Dimethenamid (Dimethenamid) (FIG. 1). Metolachlor 1970 was found by the Ciba-Jia group company (now Zhengda), and was marketed in 1975. The metolachlor is an active S-body separated on the basis of the metolachlor, has higher activity, lower toxicity and better safety, and is marketed in the United states in 1997. The metolachlor is the largest variety in amide herbicides, global sales in 2018 is 6.45 hundred million dollars, the global herbicide market is sixth, and the composite annual growth rate in 2013-2018 is 0.9%. The metolachlor is widely applied to crops such as corn, sunflower, soybean, cotton, beet, sugarcane and the like. In recent years, the efficient isomer and the compound product of the compound are continuously developed and marketed, and the market sales of the compound product is further promoted. In 2018, the Zhengda company registers mesotrione and metolachlor compound agent to the grape teeth and spanish application, and is used for preventing and killing annual gramineous weeds in corn fields; the dicamba and metolachlor compound Tavium Plus Vapor Grip Technology of the same year company is registered in the United states and is used before planting of glyphosate-resistant soybeans and in the early emergence stage. In 2019, the compound preparation of norflurazon, mesotrione and prim (Acuron Flexi) is marketed in Canada for preventing and killing important weeds in corn fields, such as quinoa (Chenopodium album), amaranth (Amaranthus retroflexus) and the like, and annual grassy weeds. The dimethenamid is developed and marketed by basf company in 1993, and has global sales of dollars of 2.19 million in 2018 and a compound annual growth rate of 6.0% in 2013-2018. The dimethenamid can be used for preventing and killing annual gramineous weeds and partial broadleaf weeds of corns, rapes, soybeans, grains, sunflowers and the like, and is an important herbicide for corns. The market benefits from numerous complex products and efficient isomers. In 2019, dimethenamid-spermate acquired the eu renewal registration of 15 years, with a validity period of 9 months from 2034.

The synthesis of (S) -1-methoxy-2-propylamine with high optical purity is important for the production and preparation of the two herbicides. The chinese patent application publication No. CN110066223a discloses a chemical process for preparing 1-methoxy-2-propylamine: under the condition of high temperature and high pressure, a metal catalyst is adopted to reduce and aminate the 1-methoxy-2-acetone into the 1-methoxy-2-propylamine. The method has the advantages of expensive catalyst, harsh reaction conditions, low product yield and lack of optical purity data, and does not accord with the trend of green development of pesticide chemical industry, so that the method is not suitable for large-scale industrialization.

The Chinese patent application publication No. CN1292828 discloses a method for preparing (S) -1-methoxy-2-propylamine by catalyzing 1-methoxy-2-acetone with transaminase, which has the advantages of mild reaction conditions and high optical purity of the product. However, the lack of a key catalyst, the gene source of the transaminase, and the process for preparing the transaminase, limits the further large-scale application of this process.

Disclosure of Invention

Aiming at the defects of the existing (S) -1-methoxy-2-propylamine synthesis process, the application provides the application of a wild type transaminase in the preparation of (S) -1-methoxy-2-propylamine, and the application of a transaminase mutant and the transaminase mutant in the preparation of (S) -1-methoxy-2-propylamine.

The specific technical scheme is as follows:

the application provides application of aminotransferase in preparation of (S) -1-methoxy-2-propylamine, wherein the amino acid sequence of the aminotransferase is shown as SEQ ID NO. 2.

Further, the nucleotide sequence of the gene encoding the aminotransferase is shown in SEQ ID NO. 1.

In order to obtain the transaminase, the application constructs a omega-transaminase enzyme library, which comprises the following steps: (1) Collecting the source of omega-aminotransferase and related information of gene sequences through literature investigation and multi-sequence alignment; (2) Inquiring omega-aminotransferase gene sequences or amino acid sequences of different sources through a gene database (https:// www.ncbi.nlm.nih.gov/genome /) to perform total gene synthesis; (3) integrating the synthesized gene fragment into an expression plasmid; (4) Transforming an expression plasmid comprising a further ω -transaminase gene into an expression host cell; (5) sequencing to verify whether the recombinant bacteria are successfully constructed; (6) Successfully constructing recombinant bacteria, numbering, and preserving in a refrigerator at-80 ℃ for later use.

The above aminotransferase is a wild-type aminotransferase from Bacillus sp.soil768D1, having NCBI accession number KRF52528.1.

The application provides application of genetically engineered bacteria in preparation of (S) -1-methoxy-2-propylamine, wherein the genetically engineered bacteria comprise aminotransferase genes with encoding nucleotide sequences shown as SEQ ID No. 1.

The application also provides a transaminase mutant, which is one of the following:

(1) Mutating serine at position 303 of the amino acid shown in SEQ ID NO.2 into glycine;

(2) Mutating phenylalanine at 381 st position of amino acid shown in SEQ ID NO.2 into valine;

(3) Mutating aspartic acid at 382 th position of the amino acid shown in SEQ ID NO.2 into arginine;

(4) Mutation of aspartic acid at 391 st position of amino acid shown in SEQ ID NO.2 to glutamic acid;

(5) The 392 rd isoleucine of the amino acid shown in SEQ ID NO.2 is mutated into leucine.

The application also provides a coding gene of the aminotransferase mutant.

The application also provides a recombinant vector containing the coding gene; recombinant vectors include pET series vectors, shuttle vectors, phage or viral vectors; more preferably, the expression vector is pET-28a (+).

The application also provides a genetic engineering bacterium containing the coding gene; the host cell can be a prokaryotic cell, a yeast cell or a eukaryotic cell; more preferably, the host cell is E.coli BL21 (DE 3).

The application also provides application of the transaminase mutant or the genetically engineered bacterium in preparation of (S) -1-methoxy-2-propylamine.

The application also provides a method for preparing (S) -1-methoxy-2-propylamine, which comprises the following steps: the (S) -1-methoxy-2-propylamine is obtained by using 1-methoxy-2-propanone as a substrate and performing non-para-aminogenesis transfer reaction by using a catalyst in the presence of isopropylamine or isopropylamine salt and cofactor, wherein the reaction formula is shown in figure 2.

In the reaction system, the use form of omega-aminotransferase as a catalyst is crude enzyme liquid after cell disruption, engineering bacteria resting cells expressing recombinant omega-aminotransferase, purified pure enzyme or immobilized enzyme. Therefore, the catalyst is wild type aminotransferase or immobilized enzyme thereof, or aminotransferase mutant or immobilized enzyme thereof, or genetically engineered bacterium.

Further, the cofactor is pyridoxal phosphate, or pyridoxamine phosphate, or pyridoxine phosphate; preferably, the cofactor is added in an amount of 0.1 to 100mM.

Further, in the reaction system, the addition amount of the catalyst is 0.5 to 15% of the weight of the reaction solution based on the wet weight of the cells after centrifugation at 4000rpm for 30 min. The concentration of the substrate 1-methoxy-2-acetone is 0.1-2.0M. The addition amount of the amino donor isopropylamine or isopropylamine salt is 0.15-10.0M.

Further, the temperature of the non-para-aminogenesis transfer reaction is 10-70 ℃, the time is 6-72 h, and the pH value of the reaction solution is 6-10. More preferably, the temperature is 20 to 60 ℃ and the time is 12 to 36 hours.

Compared with the prior art, the application has the following beneficial effects:

(1) According to the application, the transaminase with extremely high activity for preparing (S) -1-methoxy-2-propylamine by catalyzing substrate 1-methoxy-2-acetone is obtained through screening, and the transaminase is applied to (S) -1-methoxy-2-propylamine, so that the ee value of a product obtained through reaction reaches more than 99%.

(2) The application also carries out mutation on the wild aminotransferase by a site-directed mutagenesis technology, so that the enzyme activity of the mutant obtained by screening is further improved, and the conversion rate is obviously improved.

(3) The method takes 1-methoxy-2-acetone as a substrate, and directly prepares chiral pure (S) -1-methoxy-2-propylamine through the amine group transfer reaction of the substrate catalyzed by omega-aminotransferase in the presence of isopropylamine or isopropylamine salt and cofactor.

Drawings

FIG. 1 shows the molecular structures of (S) -1-methoxy-2-propylamine, metolachlor and dithiacet.

FIG. 2 is a reaction scheme for the preparation of (S) -1-methoxy-2-propylamine using omega-aminotransferase to catalyze 1-methoxy-2-propanone.

FIG. 3 shows the HPLC detection spectrum of 1-methoxy-2-propylamine: wherein the retention time 25.432min is (S) -1-methoxy-2-propylamine and 27.212min is (R) -1-methoxy-2-propylamine.

FIG. 4 is a QC detection spectrum of a reaction process for preparing (S) -1-methoxy-2-propylamine by catalyzing 1-methoxy-2-propanone with omega-aminotransferase, wherein: the retention time was 2.232min for isopropylamine, 2.340min for acetone, 2.740min for ethyl acetate as solvent, 3.982min for 1-methoxy-2-propylamine, 4.648min for internal standard n-nonane, 5.482min for 1-methoxy-2-propanone.

Detailed Description

The experimental methods in the application are all conventional methods unless otherwise specified, and the gene cloning operation can be specifically found in the "molecular cloning Experimental guidelines" by J.Sam Broker et al. The application relates to recombinant escherichia coli Escherichia coli BL (DE 3) with omega-aminotransferase gene, and the vector used is pET-28a (+) purchased from TAKARA company. Reagents for downstream catalytic processes: 1-methoxy-2-propanone, (S) -1-methoxy-2-propylamine, (R) -1-methoxy-2-propylamine. Purchased from ala Ding Huaxue reagents ltd; other commonly used reagents are purchased from national pharmaceutical group chemical reagent limited. The three-letter or one-letter expression of amino acids used in the context of the present application employs the amino acid codes specified by IUPAC (eur.j. Biochem.,138:9-37,1984).

The optical purity of the product is detected by adopting High Performance Liquid Chromatography (HPLC), and the specific method comprises the following steps: 1) Chromatographic conditions: chromatographic column model:QS-C18,5 μm,4.6 mm. Times.250 mm. Mobile phase: 50mM sodium acetate solution, acetonitrile=8:2. Detection wavelength: 338nm. Flow rate: 1.0mL/min. Column temperature: 30 ℃. 2) Derivatizing agent: 0.03g of phthalic dicarboxaldehyde and 0.1N-acetyl-L-cysteine were weighed out separately, dissolved in 400. Mu.L of ethanol, and 4mL of 0.1M boric acid-sodium hydroxide buffer (Na was weighed out ₂ B ₄ O ₇ ·10H ₂ 15.25g of O and 0.66g of NaOH, the volume is fixed to 200mL, the ultrasonic volume is dissolved, the pH=10.0), the solution is fully dissolved by shaking, and the solution is stored in a refrigerator at 4 ℃ for standby (not more than 4 days). 3) Derivatization reaction and assay: 100. Mu.L of sample is taken, 100. Mu.L of derivatization reagent is added, and the mixture is uniformly mixedThen, the sample was incubated at 25℃for 5min, and 20. Mu.L of the sample was introduced for analysis.

The conversion of each substance in the reaction process is detected by adopting gas chromatography (QC), and the specific method comprises the following steps: chromatographic column model: GT-A capillary column, detector temperature: 240 ℃, injector temperature: 240 ℃, column furnace temperature: 70 ℃.

EXAMPLE 1 construction of genetically engineered bacterium expressing wild type ω -transaminase and Activity determination

1. Acquisition of the ω -transaminase Gene

Collecting the source of omega-aminotransferase and related information of gene sequences through literature investigation and multi-sequence alignment; inquiring omega-aminotransferase gene sequences or amino acid sequences of different sources through a gene database (https:// www.ncbi.nlm.nih.gov/genome /) to perform total gene synthesis; specific information of the nitrile hydratase obtained is shown in Table 1.

TABLE 1 omega-aminotransferase library information

2. Construction of strains expressing wild-type omega-aminotransferase

Submitting the omega-aminotransferase gene sequence to a gene synthesis company for total gene synthesis, and constructing the gene sequence on a plasmid vector pET-28a (+) with enzyme cutting sites of EcoRI/BamHI and HindIII; and then the constructed plasmid is led into an expression host E.coli BL21 (DE 3) strain, namely the genetically engineered bacterium E.coli BL21 (DE 3)/pET-28 a (+) -TAx.

3. Recombinant expression of wild-type omega-aminotransferase

And inoculating the successfully constructed engineering bacteria into an LB liquid culture medium, shake culturing for 2-3 hours at 200rpm in a shaking table at 37 ℃, cooling to 18 ℃ when the density OD600 value of the bacteria reaches 0.8, and adding IPTG to the final concentration of 0.5mM. The flasks were then transferred to an 18℃shaker at 200rpm for 16h. After the completion of the culture, the culture broth was centrifuged at 4000rpm for 30min, the supernatant was discarded, the cells were collected, and then resuspended in 100mM phosphate buffer, pH8.0, the bacterial suspension was sonicated, and the precipitate was removed by centrifugation to obtain a supernatant as a crude enzyme solution.

4. Enzyme activity assay for recombinant ω -transaminase

The enzyme activity of the recombinant omega-aminotransferase is detected by using a 1-methoxy-2-acetone substrate, and the detection system is as follows: the total reaction system was 1mL, and it comprises 200. Mu.L of 500mM isopropylamine hydrochloride solution, 200. Mu.L of 100mM substrate solution, 100. Mu.L of 10mM pyridoxal phosphate (PLP) solution, and 500. Mu.L of crude enzyme solution for cell disruption of engineering bacteria, and the above materials were prepared by 100mM phosphate buffer solution at pH 8.0. The reaction was quenched by shaking at 40℃for 15min and 100. Mu.L of 1M NaOH was added. The reaction mixture was centrifuged at 12000rpm for 5min to remove cells and enzyme proteins. High performance liquid chromatography was used to determine the (S) -1-methoxy-2-propylamine produced in the reaction system, enzyme activity definition: the amount of enzyme capable of converting the substrate to 1. Mu. Mol of product at 40℃for 1min was 1U. The activity of all recombinant ω -transaminases was determined and the results are shown in table 4. As can be seen from Table 4, the ω -transaminase activity from Bacillus sp.Soil768d1 is highest, nearly two orders of magnitude higher than other enzymes.

TABLE 2 omega-transaminase Activity and optical Selectivity assay

EXAMPLE 2 construction of genetically engineered bacterium expressing mutant ω -transaminase and Activity measurement

1. Acquisition of omega-aminotransferase template Gene

Glycerol tubes storing recombinant E.coli BL21 (DE 3)/pET-28 a (+) -TA15 were streaked onto plates containing LB solid medium (50. Mu.g/mL kanamycin) and incubated at 37℃for 12h. Single colonies were picked from the plates and inoculated into 5mL of LB medium containing 50. Mu.g/mL kanamycin, and cultured at 37℃for 12 hours at 200 rpm. After the culture broth was obtained, plasmid extraction was performed according to the instructions of the plasmid extraction kit.

2. Site-directed mutagenesis of omega-aminotransferase genes

And (3) introducing site-directed mutation into the TA15 gene by using the pET-28a (+) -TA15 plasmid extracted in the step one as a template through full plasmid PCR, wherein the used primers and a PCR reaction system are shown in Table 1 and Table 2 respectively.

TABLE 1 primers for PCR

TABLE 2 PCR amplification System

Component (A)	Volume (mu L)
		PrimeSTAR	20
Upstream primer	0.8
		Downstream primer	0.8
Plasmid template	0.4
		ddH ₂ O	18

Point mutation PCR was performed using the pET-28a (+) -TA15 plasmid as a template and the primers shown in Table 1, and the PCR amplification system is shown in Table 2, and the PCR amplification conditions are as follows:

1) Pre-denaturation: 98 ℃ for 5min;

2) Denaturation: 98 ℃ for 10s; annealing: 15s at 60 ℃; extension: 1min at 72℃for 30s; cycling for 35 times;

3) Extension: 72 ℃ for 10min;

4) Preserving at 4 ℃ for 2.0h.

After the PCR amplification, the amplified product was detected by 1.0% agarose gel electrophoresis, and the target band was purified and recovered by using a DNA recovery and purification kit. The recovered PCR product was digested with DPN I and the template was removed. The digestion system is shown in Table 3.

TABLE 3 digestive system

Reagent(s)	Volume (mu L)
		PCR amplified products/plasmids	8.5
DPNI	0.5
		10×Buffer	1

Digestion conditions:

1)37℃：1h；

2)75℃：15min；

3) Preserving at 4 ℃ for 2.0h.

3. Construction of strains expressing mutant omega-aminotransferase

And (3) after digestion, converting the obtained product into competent cells of escherichia coli BL21 (DE 3), sequencing the competent cells by Beijing qingke new industry biotechnology Co., ltd, and expressing plasmids with correct sequencing results to obtain the recombinant strain.

Through induced expression (the operation is the same as that of the third step in the example 1), nine single-point mutation aminotransferase mutants TA15-S244G, TA15-T259C, TA-S303G, TA15-A307L, TA-V341A, TA15-F381V, TA-D382R, TA15-D391E, TA15-I392L are finally obtained; the wild type was designated TA15-WT.

4. Determination of enzymatic Activity and thermal stability of mutant omega-aminotransferase

The method and procedure for measuring the enzyme activity refer to the fourth step in example 1.

Characterization of the thermostability of the enzyme: the enzyme solutions are respectively placed in hot water bath at 40 ℃ for treatment for 60min, and then the enzyme activity detection system is utilized to determine the residual enzyme activity, wherein the initial enzyme activity which is not treated is defined as 100% in the residual relative enzyme activity. The enzyme activity measurement data are shown in Table 3 below.

TABLE 4 results of enzyme activity assays for TA15 aminotransferase wild-type and mutant

Example 3

The E.coli genetically engineered strain expressing the ω -transaminase of TA15 obtained in example 1 was cultured in the same manner as in example 1 to obtain a crude enzyme solution.

The reaction system was 10mL, containing 100mM substrate 1-methoxy-2-propanone, 200mM isopropylamine, 1mM PLP, and TA15 crude enzyme solution (0.05 g in wet bacteria) was added, the reaction temperature was controlled to 35℃by a metal bath, and the pH was controlled to 7.5 by 100mM phosphate buffer during the reaction. Detecting the generation amount of the (S) -1-methoxy-2-propylamine by gas chromatography for 12 hours; the concentration of the raw material 1-methoxy-2-acetone is 10.1mM, and the conversion rate is 89.9%; the high performance liquid chromatography detection product mainly comprises (S) -1-methoxy-2-propylamine, and the ee value is 99.5%.

Example 4

The E.coli genetically engineered bacterium expressing ω -transaminase of the number TA15-D382R obtained in example 1 was cultured in the same manner as in example 1 to obtain a crude enzyme solution.

The reaction system was 10mL, containing 100mM substrate 1-methoxy-2-propanone, 200mM isopropylamine, 1mM PLP, and TA15 crude enzyme solution (0.05 g in wet bacteria) was added, the reaction temperature was controlled to 35℃by a metal bath, and the pH was controlled to 7.5 by 100mM phosphate buffer during the reaction. Detecting the generation amount of the (S) -1-methoxy-2-propylamine by gas chromatography for 12h to 90.3mM; the concentration of the raw material 1-methoxy-2-acetone is 8.7mM, and the conversion rate is 91.3%; the high performance liquid chromatography detection product mainly comprises (S) -1-methoxy-2-propylamine, and the ee value is 99.7%.

Example 5

The E.coli genetically engineered bacterium expressing ω -transaminase of No. TA15-S303G obtained in example 1 was cultured in the same manner as in example 1 to obtain resting cells.

The reaction system was 100mL, containing 500mM substrate 1-methoxy-2-propanone, 500mM isopropylamine, 0.1mM PLP, and TA15-S303G resting cells (wet weight of cells after centrifugation at 4000rpm for 30min 1.0G) were added, the reaction temperature was controlled to 40℃by water bath, and the pH was controlled to 8.5 by 20% aqueous isopropylamine solution during the reaction. Detecting the yield 373.3mM of the (S) -1-methoxy-2-propylamine by gas chromatography after 24 hours of reaction; the concentration of the 1-methoxy-2-acetone of the raw material is 124.1mM, and the conversion rate is 75.2%; the high performance liquid chromatography detection product mainly comprises (S) -1-methoxy-2-propylamine, and the ee value is 99.6%.

Example 6

The E.coli genetically engineered strain expressing ω -aminotransferase of TA15-F381V obtained in example 1 was cultured in the same manner as in example 1 to obtain about 2.0g of wet cells, and the wet cells were subjected to cell disruption to obtain a crude enzyme solution, which was then subjected to nickel column affinity chromatography (report of bioengineering, 2016,32 (7): 912-926) to obtain pure enzyme of TA 15-F381V.

The reaction system was 100mL, containing 500mM substrate 1-methoxy-2-propanone, 1000mM isopropylamine, 0.2mM PLP, adding TA15-F381V pure enzyme, controlling the reaction temperature to 30℃by water bath, and controlling the pH to 8.0 by 20% isopropylamine aqueous solution during the reaction. Detecting the generation amount 449.1mM of (S) -1-methoxy-2-propylamine by gas chromatography after 48h of reaction; the concentration of the raw material 1-methoxy-2-acetone is 44.3mM, and the conversion rate is 91.1%; the high performance liquid chromatography detection product mainly comprises (S) -1-methoxy-2-propylamine, and the ee value is 99.9%.

Example 7

The E.coli genetically engineered strain expressing TA15-I392L obtained in example 1 was cultured in the same manner as in example 1 to obtain about 2.0g of wet cells, and the wet cells were subjected to cell disruption to obtain a crude enzyme solution.

The reaction system was 100mL, containing 700mM substrate 1-methoxy-2-propanone, 750mM isopropylamine, 0.1mM PLP, adding TA15-I392L crude enzyme solution, controlling the reaction temperature to 30℃by water bath, and controlling the pH to 8.5 by 20% isopropylamine aqueous solution during the reaction. Detecting 589.3mM of the (S) -1-methoxy-2-propylamine by gas chromatography after 96 hours of reaction; raw material 1-methoxy-2-acetone concentration 104.0mM, conversion rate 85.1%; the high performance liquid chromatography detection product mainly comprises (S) -1-methoxy-2-propylamine, and the ee value is 99.8%.

Example 8

The E.coli genetically engineered strain expressing ω -aminotransferase of TA15-D391E obtained in example 1 was cultured in the same manner as in example 1 to obtain about 15.0g of wet cell, the wet cell was disrupted to obtain a crude enzyme solution, and then subjected to nickel column affinity chromatography (journal of bioengineering, 2016,32 (7): 912-926) to obtain a purified TA15-D391E enzyme, and the purified TA15-D391E enzyme was immobilized (immobilization method reference: chemCatchem,2012,4,1071-1074) to obtain an immobilized TA15-D391E.

1000mL of the reaction system, 1.0mM PLP, TA15-D391E immobilized enzyme, 1500mM substrate 1-methoxy-2-acetone and 3000mM isopropylamine are added continuously, the reaction temperature is controlled to be 30 ℃ through a water bath, and the pH is controlled to be 8.0 through 20% isopropylamine water solution in the reaction process. Detecting the generation amount of the (S) -1-methoxy-2-propylamine by using a gas chromatography for 36h to obtain 1029.3mM; the concentration of the raw material 1-methoxy-2-acetone is 404.0mM, and the conversion rate is 73.0%; the high performance liquid chromatography detection product mainly comprises (S) -1-methoxy-2-propylamine, and the ee value is 99.9%.

Sequence listing

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Gly Gln Lys Asn Pro Lys Val Asn Ala Ala Ile Lys Glu Ala Leu Asp

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Gly Ala Gly Ser Ala Met Pro Pro Tyr Glu Tyr Ile Pro Gln Ile Arg

245 250 255

Lys Met Thr Lys Glu Leu Gly Val Leu Trp Ile Thr Asp Glu Val Leu

260 265 270

Thr Gly Phe Gly Arg Thr Gly Lys Trp Phe Gly Tyr Gln His Tyr Gly

275 280 285

Val Gln Pro Asp Ile Ile Thr Met Gly Lys Gly Leu Ser Ser Ser Ser

290 295 300

Leu Pro Ala Gly Ala Val Leu Val Ser Lys Glu Ile Ala Glu Phe Met

305 310 315 320

Asp Arg His Arg Trp Glu Ser Val Ser Thr Tyr Ala Gly His Pro Val

325 330 335

Ala Met Ala Ala Val Cys Ala Asn Leu Glu Val Met Met Glu Glu Asn

340 345 350

Phe Val Glu Gln Ala Lys Asn Ser Gly Glu Tyr Ile Arg Ser Lys Leu

355 360 365

Glu Leu Leu Gln Glu Lys His Lys Ser Ile Gly Asn Phe Asp Gly Tyr

370 375 380

Gly Leu Leu Trp Ile Val Asp Ile Val Asn Ala Lys Thr Lys Thr Pro

385 390 395 400

Tyr Val Lys Leu Asp Arg Asn Phe Thr His Gly Met Asn Pro Asn Gln

405 410 415

Ile Pro Thr Gln Ile Ile Met Lys Lys Ala Leu Glu Lys Gly Val Leu

420 425 430

Ile Gly Gly Val Met Pro Asn Thr Met Arg Ile Gly Ala Ser Leu Asn

435 440 445

Val Ser Arg Glu Asp Ile Asp Lys Ala Met Asp Ala Leu Asp Tyr Ala

450 455 460

Leu Asp Tyr Leu Glu Ser Gly Glu Trp Gln Gln Ser

465 470 475

<210> 3

<211> 32

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 3

agcaggcggt gcaatgccgc cgtacgaata ta 32

<210> 4

<211> 32

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 4

gcattgcacc gcctgctccc tgacttacct cc 32

<210> 5

<211> 32

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

aatgtgtaag gagctgggtg tgctgtggat ta 32

<210> 6

<211> 41

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 6

ccagctcctt acacatttta cgaatctgag gaatatattc g 41

<210> 7

<211> 32

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 7

tgagcagtgg tagcctgccg gccggtgcag tt 32

<210> 8

<211> 32

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 8

caggctacca ctgctcagtc ccttacccat tg 32

<210> 9

<211> 32

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 9

ctgggtgcag ttctggtttc taaagaaatt gc 32

<210> 10

<211> 38

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 10

accagaactg cacccagcgg caggctacta ctgctcag 38

<210> 11

<211> 34

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 11

ccgcagcatg tgcaaattta gaagttatga tgga 34

<210> 12

<211> 32

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 12

atttgcacat gctgcggcca ttgcaaccgg at 32

<210> 13

<211> 32

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 13

aggtaacgtt gacggttatg gactgctgtg ga 32

<210> 14

<211> 37

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 14

aaccgtcaac gttacctatg cttttatgtt tttcctg 37

<210> 15

<211> 32

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 15

ctttcgtggt tatggactgc tgtggattgt gg 32

<210> 16

<211> 41

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 16

gtccataacc acgaaagtta cctatgcttt tatgtttttc c 41

<210> 17

<211> 42

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 17

gctgtggatt gtggaaattg ttaatgcaaa gacaaaaaca cc 42

<210> 18

<211> 32

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 18

tttccacaat ccacagcagt ccataaccgt ca 32

<210> 19

<211> 34

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 19

tgtggacctg gttaatgcaa agacaaaaac accg 34

<210> 20

<211> 32

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 20

cattaaccag gtccacaatc cacagcagtc ca 32

Claims

1. A transaminase mutant, characterized in that the transaminase mutant is one of the following:

(4) The 392 rd isoleucine of the amino acid shown in SEQ ID NO.2 is mutated into leucine.

2. A gene encoding the transaminase mutant according to claim 1.

3. A recombinant vector comprising the coding gene of claim 2.

4. A genetically engineered bacterium comprising the coding gene of claim 2.

5. The transaminase mutant of claim 1 or the genetically engineered bacterium of claim 4S) -1-methoxy-2-propylamine.

6. Preparation [ ]S) A process for the preparation of-1-methoxy-2-propylamine comprising: using 1-methoxy-2-acetone as substrate, under the condition of isopropyl amine or isopropyl amine salt and cofactor existence utilizing catalyst to make asymmetric amino transfer reaction so as to obtain the invented productS) -1-methoxy-2-propaneAn amine;

the catalyst is the transaminase mutant or immobilized enzyme thereof of claim 1 or the genetically engineered bacterium of claim 4.

7. The preparation of claim 6S) -a process for the preparation of 1-methoxy-2-propylamine, characterized in that the cofactor is pyridoxal phosphate, pyridoxamine phosphate, or pyridoxine phosphate.