A thermostable transaminase or a mutant thereof and its use in the preparation of chiral amine compounds

Abstract

The present application provides a kind of thermostable transaminase or its mutant and its application in preparing chiral amine compound.The amino acid sequence of the thermostable transaminase is selected from any one of the following:(1) the amino acid sequence shown as SEQ ID NO:1;(2) the amino acid sequence shown as SEQ ID NO:1 is derived by substitution, deletion or addition of one or more amino acids and has transaminase activity;(3) the amino acid sequence shown as SEQ ID NO:1 has at least 95% identity and has transaminase activity.The present application also improves the catalytic activity and stability of the transaminase from thermophilic microorganism by modification, thereby improving the catalytic efficiency and being suitable for the preparation process of chiral amine.

Description

Technical Field

[0001] This invention belongs to the field of biocatalysis technology, specifically relating to a thermally stable transaminase or its mutant and its application in the preparation of chiral amine compounds, especially the application of the transaminase and its mutant in the preparation of (S)-1-methoxy-2-propane. Background Technology

[0002] Chiral amines are important intermediates in the synthesis of pharmaceuticals, pesticides, and fine chemicals, and are widely used in the preparation of active pharmaceutical molecules, chiral catalysts, and functional materials. Traditional chemical synthesis methods for chiral amines often suffer from cumbersome procedures, poor selectivity, and environmental pollution. In contrast, biocatalysis, due to its high efficiency, high selectivity, and environmental friendliness, has become a crucial pathway for chiral amine synthesis. Transaminases, as a key class of biocatalysts, can efficiently synthesize chiral amines through asymmetric aminotransfer reactions, and thus have significant industrial application value.

[0003] However, traditional transaminases face a key challenge in catalyzing the synthesis of chiral amines: the limitation of reaction equilibrium. Transaminase-catalyzed reactions are typically reversible processes with an equilibrium constant close to 1, resulting in limited conversion rates. To improve conversion, it is usually necessary to add an excess of amino donor or drive the reaction towards the product through other means (such as byproduct removal). Driving the volatilization of byproducts (such as acetone) under high-temperature conditions is an efficient and environmentally friendly strategy. However, traditional transaminases are easily inactivated under high-temperature conditions, making them unsuitable for this requirement.

[0004] (S)1-Methoxy-2-propylamine is an organic compound with significant applications, belonging to the amine class of compounds containing a nitrogen atom. Its chemical structure contains a methoxy (-OCH3) and an amino (-NH2) substituent group, and it is commonly used as an important intermediate in the synthesis of other organic compounds. In particular, 1-methoxy-2-propylamine has wide applications in pharmaceuticals, chemical synthesis, pesticides, and many other fields.

[0005] In the pesticide field, 1-methoxy-2-propylamine is widely used in the synthesis of herbicides. As a crucial intermediate in herbicide synthesis, 1-methoxy-2-propylamine plays a vital role in the design and production of herbicides. Specifically, 1-methoxy-2-propylamine is mainly used to synthesize herbicide molecules that can selectively target specific types of weeds. For example, some herbicides can interfere with plant growth and development by inhibiting specific enzymes within the plant, and 1-methoxy-2-propylamine, as a key intermediate in these compounds, plays a decisive role in their synthesis.

[0006] Traditional methods for synthesizing 1-methoxy-2-propylamine typically involve chemical synthesis routes, such as the reaction of the corresponding aldehydes with amino compounds. Although these chemical synthesis methods have achieved some success in laboratory settings, they often face the following challenges in industrial production: (1) Poor stereoselectivity: Many chemical synthesis methods have certain stereoselectivity issues, which may result in the presence of unnecessary enantiomers or isomers in the product, thus affecting the purity and efficacy of the final product; (2) Byproduct generation: Traditional chemical synthesis routes often require the use of toxic or environmentally harmful chemical reagents, and the generation of byproducts and environmental pollution are also quite serious; (3) Harsh reaction conditions: Most chemical synthesis methods need to be carried out under harsh conditions such as high temperature and high pressure, and the reaction steps are numerous, energy consumption is high, and production costs are high.

[0007] Therefore, developing a greener, more efficient, and stereoselective biocatalytic method for the synthesis of 1-methoxy-2-propane, especially for industrial production, has become an urgent need.

[0008] Compared with traditional chemical synthesis methods, the synthesis of 1-methoxy-2-propane using transaminase catalysis has the following advantages: (1) High stereoselectivity: Transaminase-catalyzed reactions usually have good stereoselectivity, which can effectively generate the target product (S)-1-methoxy-2-propane and avoid the unnecessary generation of enantiomers or isomers. (2) Mild reaction conditions: Unlike high-temperature and high-pressure chemical synthesis reactions, biocatalytic reactions are usually carried out under mild temperature and pH conditions, which reduces energy consumption and equipment requirements and lowers production costs. (3) Environmentally friendly: Transaminase catalysis usually does not require the use of toxic or hazardous chemical reagents, and the byproducts are few and degradable, resulting in less negative impact on the environment. (4) Simple operation: Transaminase catalysis is generally simple to operate and can achieve relatively simple scale-up production, making it suitable for large-scale applications.

[0009] However, existing transaminases exhibit poor stability under high-temperature conditions, limiting their application in large-scale industrial production. Therefore, improving the thermal stability of transaminases, especially maintaining high catalytic activity at high temperatures, is crucial for advancing the application of biocatalysis. Summary of the Invention

[0010] To address the shortcomings of existing technologies, the present invention aims to provide a thermostable transaminase or its mutant and its application in the preparation of chiral amine compounds. The transaminase exhibits high activity in catalyzing the synthesis of chiral amines. The transaminase mutant can withstand heat treatment at 60°C while maintaining its activity, and can be efficiently expressed in *E. coli*, yielding an enzyme catalyst with significantly improved purity after downstream heat treatment. Compared to the wild-type enzyme, this mutant exhibits significantly improved enzyme activity, stability, and substrate tolerance. Its unique thermostability enables the enzyme to efficiently catalyze the synthesis of chiral amines in high-temperature reaction systems, while simultaneously utilizing high temperatures to drive the volatilization of the byproduct acetone, thereby disrupting reaction equilibrium and significantly improving reaction conversion. The transaminase and its mutant maintain good catalytic activity under high-temperature enzymatic catalytic reaction conditions, particularly demonstrating significant catalytic efficiency in the preparation of (S)-chiral amine compounds.

[0011] To achieve this objective, the present invention adopts the following technical solution:

[0012] In a first aspect, the present invention provides a thermostable transaminase, wherein the amino acid sequence of the transaminase is selected from any of the following:

[0013] (1) The amino acid sequence as shown in SEQ ID NO:1;

[0014] (2) An amino acid sequence with transaminase activity derived from the amino acid sequence shown in SEQ ID NO:1 by substitution, deletion or addition of one or more amino acids.

[0015] (3) An amino acid sequence that has at least 95% identity with the amino acid sequence shown in SEQ ID NO:1 and has transaminase activity.

[0016] "Having at least 95% identity" can be, for example, 95%, 96%, 97%, 98%, or 99%, etc.

[0017] This invention screens a thermostable transaminase from thermophilic microorganisms, which exhibits a high conversion rate in the synthesis of 1-methoxy-2-propanone from 1-methoxy-2-propanone.

[0018] Secondly, the present invention provides a thermostable transaminase mutant, wherein the transaminase mutant is based on the amino acid sequence shown in SEQ ID NO:1, and the mutation site is selected from any one or a combination of at least two of S25W, S25F, Y60W or A241V.

[0019] This invention selects transaminases derived from heat-stable microorganisms (such as thermophilic bacteria or extreme thermophilic microorganisms) as the basis and optimizes them through gene mutation technology.

[0020] This invention modifies transaminases derived from thermophilic microorganisms to obtain mutants, significantly improving the thermal stability of the transaminases and enabling them to maintain good catalytic activity at high temperatures of 55°C and above. This technological innovation provides a new solution for the efficient production of chiral amine compounds such as (S)-1-methoxy-2-propane at high temperatures, and has broad prospects for industrial application.

[0021] Using the thermostable transaminase mutant of this invention in industrial production can significantly increase the rate of catalytic reactions, reduce reaction time, improve product yield, and reduce energy consumption, thereby effectively reducing production costs. Furthermore, due to the high selectivity and environmental friendliness of biocatalysis, it can play a greater role in the synthesis of pesticides such as herbicides, promoting the development of green chemistry and sustainable agriculture.

[0022] Preferably, the mutation site is a combination of S25F and Y60W, or a combination of S25F and A241V, or a combination of Y60W and A241V, or a combination of S25W and A241V.

[0023] Specifically, this invention utilizes site-directed mutagenesis, random mutagenesis, or molecular evolution to enhance the catalytic activity and temperature tolerance of the reaction substrate methoxyacetone. Mutations are primarily concentrated in hydrophobic regions of the protein, secondary structural stability, hydrogen bond networks, and substrate-binding sites.

[0024] Thirdly, the present invention provides a nucleic acid molecule that encodes the thermostable transaminase described in the first aspect, or encodes a thermostable transaminase mutant described in the second aspect.

[0025] Fourthly, the present invention provides an expression vector containing the nucleic acid molecule described in the third aspect.

[0026] Fifthly, the present invention provides an engineered bacterium, wherein the genome of the engineered bacterium integrates the nucleic acid molecules described in the third aspect, or the engineered bacterium contains the expression vector described in the fourth aspect.

[0027] In this invention, wild-type enzyme genes or mutant genes are cloned into a suitable expression vector and transformed into an E. coli system for expression. The gene can be expressed efficiently in E. coli and subsequently prepared into enzyme solution or enzyme powder. In downstream processes, heat treatment can significantly improve the purity of the enzyme catalyst.

[0028] In a sixth aspect, the present invention provides the use of the thermostable transaminase described in the first aspect and / or the thermostable transaminase mutant described in the second aspect in the preparation of chiral amine compounds.

[0029] In this invention, the transaminase and its mutants exhibit good stereoselectivity and high catalytic efficiency, and can maintain high catalytic activity even at high temperatures.

[0030] In this invention, the chiral amine compound is selected from any one of the following:

[0031] m = 1, 2, or 3;

[0032] n1 = 1, 2 or 3; R1 is selected from -H, -CH3, -CH2CH3, -CH2CH2CH3 or -CH(CH3)2;

[0033] n2 = 1, 2 or 3; R2 is selected from -H, -CH3, -CH2CH3, -CH2CH2CH3, -CH(CH3)2;

[0034] R is selected from -H, -CH3, -CH2CH3, -CH2CH2CH3 or -CH(CH3)2;

[0035] n is 1, 2, 3 or 4, and R″ is selected from H, -CH3, Boc, Bz, Cbz or Ac;

[0036] R′ is selected from -H, -CH3, Boc, Bz, Cbz, or Ac.

[0037] In a seventh aspect, the present invention provides an immobilized transaminase, the immobilized transaminase comprising a resin and the thermostable biological transaminase described in the first aspect and / or the thermostable biological transaminase mutant described in the second aspect.

[0038] Eighthly, the present invention provides a method for preparing (S)-1-methoxy-2-propane, the method comprising: using 1-methoxy-2-propanone as a substrate, and performing biocatalysis using the thermostable transaminase described in the first aspect and / or the thermostable transaminase mutant described in the second aspect to prepare (S)-1-methoxy-2-propane.

[0039] The method for preparing (S)-1-methoxy-2-propane in this invention has high catalytic efficiency and good stereoselectivity. The catalytic efficiency is significantly improved compared with ordinary methods, and the thermal stability and product stereoselectivity are superior to those of traditional catalysts.

[0040] The modified transaminase mutant of this invention significantly improves the thermal stability of the transaminase, enabling it to maintain good catalytic activity at temperatures of 55°C and above. This invention is the first to use a transaminase mutant derived from thermophilic microorganisms for the high-temperature catalytic preparation of (S)-1-methoxy-2-propane, significantly improving its thermal stability and catalytic efficiency. This invention provides a new solution for the efficient high-temperature production of (S)-1-methoxy-2-propane, with broad prospects for industrial application.

[0041] In this invention, the biocatalytic reaction formula is as follows:

[0042]

[0043] Preferably, the biocatalytic step includes: preparing enzyme solution or enzyme powder from the fermentation broth of engineered bacteria containing a transaminase or its mutant encoding gene; using the enzyme solution or enzyme powder as a catalyst, 1-methoxy-2-propanone as a substrate, and isopropylamine as an amino donor, carrying out a biocatalytic reaction in a buffer system; and after the reaction is completed, separating and purifying the reaction solution to obtain (S)-1-methoxy-2-propanamine.

[0044] Preferably, the buffer system is selected from: PBS buffer system or triethanolamine buffer system; the pH value is 7.5-8.5, for example, it can be 7.5, 7.6, 7.8, 8.0, 8.2, 8.4 or 8.5, etc.; preferably 7.8-8.2, more preferably 8.

[0045] Preferably, the temperature of the biocatalytic reaction is 55-60°C, for example, 55°C, 56°C, 57°C, 58°C, 59°C, or 60°C.

[0046] This invention enables high-temperature catalytic reactions. In the experiments of this invention, the catalytic activity and stability of mutant transaminase were tested at temperatures of 55°C and above, and its application effect in the preparation of (S)-1-methoxy-2-propane was determined.

[0047] The numerical range described in this invention includes not only the point values ​​listed above, but also any point values ​​within the numerical ranges not listed above. Due to space limitations and for the sake of brevity, this invention will not exhaustively list all the specific point values ​​included in the range.

[0048] Compared with the prior art, the present invention has the following beneficial effects:

[0049] (1) Transaminase is derived from thermostable microorganisms: This invention is the first to use a transaminase mutant derived from thermophilic microorganisms to catalyze the preparation of (S)-1-methoxy-2-propane under high temperature conditions, which significantly improves its thermal stability and catalytic efficiency.

[0050] (2) Optimization of the mutant: This invention significantly improves the thermostability of transaminase through precise molecular modification, enabling it to withstand high-temperature treatment at 60°C and maintain 100% residual activity. The purity of the enzyme is greatly improved after heat treatment. It can still maintain good catalytic activity at temperatures of 55°C and above, and side reactions are greatly reduced.

[0051] (3) Broad application prospects: Its unique thermal stability enables the enzyme to efficiently catalyze the synthesis of chiral amines in high-temperature reaction systems. Simultaneously, the high temperature conditions drive the volatilization of the byproduct acetone, thereby disrupting the reaction equilibrium and significantly improving the reaction conversion rate. This invention can not only be used for the preparation of (S)-1-methoxy-2-propane, but can also be extended to other biocatalytic reactions of chiral amine compounds requiring high-temperature catalysis, demonstrating broad industrial application prospects.

[0052] In summary, the transaminase mutant of the present invention exhibits significant thermal stability at temperatures above 60°C and demonstrates high catalytic efficiency and good stereoselectivity in the preparation of (S)-1-methoxy-2-propane. Experimental results show that, compared with transaminases in the prior art, the transaminase of the present invention can maintain higher activity at higher temperatures and achieve higher product yields in industrial production. Attached Figure Description

[0053] Figure 1 The results are SDS-PAGE of crude enzyme solutions of wild type and mutant (treated at 35°C and 60°C for one hour).

[0054] Figure 2 The results show the comparison of residual enzyme activity after heat treatment between wild-type WT and the double mutant S25F / A241V.

[0055] Figure 3 This is the result of a 5g reaction of the mutant S25F / A241V.

[0056] Figure 4 This is the result of supercritical fluid chromatography (SFC) detection of the product. Detailed Implementation

[0057] The technical solution of the present invention will be further illustrated below through specific embodiments. Those skilled in the art should understand that the embodiments described are merely illustrative of the present invention and should not be construed as limiting the invention in any way.

[0058] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0059] The analysis methods used in the following examples are:

[0060] 1. Supercritical fluid chromatography (SFC) was used to determine the optical purity of the product and calculate the conversion rate.

[0061] 1) Column type: CHIRALPAK IC-3 (3μm, 4.6mm*150m). Mobile phase: Carbon dioxide:methanol = 75:25. Detection wavelength: 338nm. Flow rate: 2.5mL / min. Column temperature: 35℃.

[0062] 2) Derivatization reagent: Weigh 0.03g of o-phthalaldehyde and 0.04g of N-acetyl-L-cysteine, respectively, and dissolve them with 400μL of ethanol. Then add 4mL of 0.2M borate-sodium hydroxide buffer (weigh 15.25g of Na2B4O7·10H2O and 0.66g of NaOH, make up to 200mL, sonicate to dissolve, pH=10.0), shake to dissolve completely, and store at 4℃ for later use (not exceeding 4 days).

[0063] 3) Derivatization reaction and determination: Take 10 μL of sample and add 100 μL of derivatization reagent, mix well, and incubate at 25℃ for 10 min. Inject 0.5 μL for analysis. The amount of product (s)-1-methoxy-2-propylamine generated is detected by high performance liquid chromatography after derivatization, and the conversion rate is calculated.

[0064] 2. The product conversion rate was determined by gas chromatography (GC). The specific method was as follows: Chromatography model: CP-Volamine 30m*0.32mm. Detector temperature: 250℃, injector temperature: 180℃, column temperature: 60℃.

[0065] 3. Product conversion was determined using liquid chromatography (LC). The specific method was as follows: Column type: WatersXBridge C18 (150*4.6mm, 3.5μm). Mobile phase: 0.05% trifluoroacetic acid dissolved in water: acetonitrile = 96:4. Detection wavelength: 265nm. Flow rate: 1.2mL / min. Column temperature: 30℃. Conversion was calculated based on the consumption of the substrate methoxyacetone.

[0066] Example 1

[0067] Construction of wild-type transaminase engineered strains

[0068] 1. Obtaining transaminase genes

[0069] By reviewing existing literature on transaminases, we searched for their amino acid sequences in the gene database (https: / / www.ncbi.nlm.nih.gov / ) and synthesized their entire genomes. The relevant information is shown in Table 1.

[0070] Table 1

[0071] Transaminase number NCBI number source ATA-01 WP_018063493 Martelella mediterranea ATA-11 WP_023048105 Pseudomonas sp. ATA-12 ABL72050.1 Paracoccus denitrificans ATA-16 WP_046128662.1 Bacillus thermotolerans

[0072] 2. Construction of transaminase strains

[0073] The transaminase gene sequence was submitted to a gene synthesis company for whole-gene synthesis and constructed into the plasmid vector pET-30a(+). The restriction sites were EcoRI and NdeI. The constructed plasmid was then introduced into the expression host E. coli BL21(DE3). Recombinant strains were obtained, and crude enzyme solutions were prepared by expressing the proteins of the recombinant bacteria.

[0074] The successfully constructed recombinant bacteria were inoculated into 5 mL of LB medium containing 50 μg / mL kanamycin and activated overnight at 37°C and 220 rpm. Subsequently, the activated bacterial culture was transferred at a 1% inoculum (V / V) to 50 mL of LB medium containing 50 μg / mL kanamycin and cultured at 37°C and 220 rpm until the logarithmic growth phase (OD50). 600 When the concentration was 0.6-0.8%, IPTG was added to a final concentration of 0.1 mmol / L, and the cells were induced and cultured at 25°C and 220 rpm for 18 h. After the induction period, the bacterial cells were collected by centrifugation at 4000 rpm, resuspended in 50 mM triethanolamine buffer (pH 8.0), the bacterial suspension was sonicated and the supernatant was collected by centrifugation at 12000 rpm, which is the crude enzyme solution.

[0075] 3. Determination of wild-type transaminase conversion rate

[0076] The enzyme activity of the recombinant transaminase-producing engineered bacteria was determined using 1-methoxy-2-propanone and isopropylamine as model substrates. The reaction system was as follows: 10 mmol / L 1-methoxy-2-propanone, 10 mmol / L isopropylamine, 0.5 mmol / L pyridoxal phosphate, 10 wt (weight equivalent, substrate mass ratio) crude enzyme solution, and 50 mmol / L pH 8.0 triethanolamine buffer, with a total reaction volume of 1 mL. The sample was incubated at 30℃ with shaking at 200 rpm for 16 h, then quenched with 100 μL of 1M NaOH. The inactivated enzyme protein was removed by centrifugation at 12000 rpm for 10 min. Finally, the sample was extracted three times with an equal volume of ethyl acetate, dried with anhydrous sodium sulfate, and detected by gas chromatography (GC). Among them, the transaminase ATA-16 from Bacillus thermotolerans showed the highest conversion rate for the synthesis of 1-methoxy-2-propanone from 1-methoxy-2-propanone. Table 2 shows the conversion rate results of the wild-type transaminase.

[0077] Table 2

[0078] Transaminase number NCBI number Conversion rate ATA-01 WP_018063493 0.00％ ATA-11 WP_023048105 13.95％ ATA-12 ABL72050.1 1.55％ ATA-16 WP_046128662.1 99.85％

[0079] 4. Recombinant expression of wild-type transaminase (also known as S-transaminase)

[0080] Single clones of the strain were picked from the resistance plate and cultured overnight at 37°C and 200 rpm in LB medium containing 50 μg / mL kanamycin resistance. The resulting seed culture was then transferred at a 1% inoculum to 20 mL of LB medium containing 50 μg / mL kanamycin resistance and cultured at 37°C and 200 rpm for 2-3 hours to allow OD to develop. 600 IPTG was added at 0.6-0.8 to a final concentration of 0.1 mM, and the culture was transferred to 22°C and incubated at 200 rpm for 16 h. After incubation, the bacterial suspension was centrifuged at 4500 rpm for 10 min, the supernatant was discarded, the bacterial cells were collected, and then resuspended in 50 mM, pH 8.0 triethanolamine buffer (containing 1 mM pyridoxal phosphate). The bacterial suspension was sonicated and centrifuged at 12000 rpm for 10 min to obtain the supernatant as crude enzyme solution.

[0081] Example 2

[0082] 1. Construction of strains expressing mutant S-transaminase

[0083] (1) Obtaining the wild-type S-transaminase template gene

[0084] Glycerol-containing Ecoli BL21(DE3) / pET-30a(+)-ATA16 was streaked onto LB agar plates containing 50 μg / mL kanamycin resistance and incubated overnight. Single colonies were picked from the plates and inoculated into LB medium containing 50 μg / mL kanamycin resistance, and incubated at 37°C and 200 rpm for 12 h. After obtaining the culture medium, plasmid extraction was performed according to the instructions of the plasmid extraction kit.

[0085] (2) Site-directed mutagenesis of the S-transaminase gene

[0086] Using the pET-30a(+)-ATA16 plasmid extracted in step 1 as a template, site-directed mutations were introduced into the ATA16 gene by whole-plasmid PCR. The PCR reaction system is shown in Table 3.

[0087] Table 3

[0088] Components volume 2×Phanta UniFi reaction mixture 25μL Upstream primer (10 μM) 1μL Downstream primer (10 μm) 1μL template XμL <![CDATA[ddH2O]]> Add to 50μL

[0089] PCR amplification conditions: 1) Pre-denaturation: 98℃ for 2 min; 2) Denaturation: 98℃ for 15 s; Annealing: 55-60℃ for 15 s; Extension: 72℃ for 1.75 min; 30-35 cycles; 3) Extension: 72℃ for 10 min; 4) Incubation at 12℃.

[0090] After PCR amplification, the products were detected by 1% agarose gel electrophoresis. The successfully detected PCR products were diluted 10-fold with ddH2O. The diluted products were then digested with Dpn I to remove the pET-30a(+)-ATA16 plasmid template from the PCR products. The digestion system is shown in Table 4.

[0091] Table 4

[0092] Components volume Diluted PCR products 8.5μL Dpn I 0.5μL 10×CutOne buffer 1μL

[0093] Digestion conditions: 1) 37℃ for 1.5h; 2) 80℃ for 20min; 3) Store at 4℃.

[0094] (3) Construction of strains expressing mutant S-transaminase

[0095] The digested product was transformed into *E. coli* BL21(DE3) competent cells. The transformed samples were sequenced and verified to obtain recombinant strains with correct sequencing. The verified recombinant strains were induced to express (using the same method as step 2 in Example 1), ultimately yielding 37 site-directed S-transaminase mutants: S25F, N26S, M46T, D48G, Y60W, V64I, V64L, V64T, M75I, A95P, E108P, A112P, F142I, F142V, H144R, and G14. 7D, G147A, Y164F, T175S, P194S, K195N, S229Q, V233F, A241V, A244T, A244I, H246P, F248Y, N267S, N267A, N267V, K375P, K375E, K395E, S408R, T411M, L413P, wild type WT.

[0096] The ATA-16 nucleotide sequence is shown in SEQ ID NO:2.

[0097] The amino acid sequence of ATA-16 is shown in SEQ ID NO:1.

[0098] The nucleotide sequence of the ATA-16(G147D) mutant is shown in SEQ ID NO:3.

[0099] The amino acid sequence of the ATA-16(G147D) mutant is shown in SEQ ID NO:4.

[0100] The nucleotide sequence of the ATA-16(A244T) mutant is shown in SEQ ID NO:5.

[0101] The amino acid sequence of the ATA-16(A244T) mutant is shown in SEQ ID NO:6.

[0102] The nucleotide sequence of the ATA-16(N267A) mutant is shown in SEQ ID NO:7.

[0103] The amino acid sequence of the ATA-16(N267A) mutant is shown in SEQ ID NO:8.

[0104] The nucleotide sequence of the ATA-16(S25W) mutant is shown in SEQ ID NO:9.

[0105] The amino acid sequence of the ATA-16(S25W) mutant is shown in SEQ ID NO:10.

[0106] The nucleotide sequence of the ATA-16(S25F) mutant is shown in SEQ ID NO:11.

[0107] The amino acid sequence of the ATA-16(S25F) mutant is shown in SEQ ID NO:12.

[0108] The nucleotide sequence of the ATA-16(Y60W) mutant is shown in SEQ ID NO:13.

[0109] The amino acid sequence of the ATA-16(Y60W) mutant is shown in SEQ ID NO:14.

[0110] The nucleotide sequence of the ATA-16(A241V) mutant is shown in SEQ ID NO:15.

[0111] The amino acid sequence of the ATA-16(A241V) mutant is shown in SEQ ID NO:16.

[0112] 2. Site-directed combinatorial mutations in the S-transaminase gene

[0113] (1) Combining three site-directed mutations: S25F, Y60W, and A241V.

[0114] Combination mutations were performed on the three site-directed mutations S25F, Y60W, and A241V. Glycerol strains carrying the recombinant strain Ecoli BL21(DE3) / pET-30a(+)-ATA87 with the three site-directed mutations S25F, Y60W, and A241V were subjected to step 1 to obtain single-point mutant plasmids containing S25F, Y60W, and A241V. Using the S25F mutant plasmid as a template and AK-ATA-8F and AK-ATA-8R as primers, full-plasmid PCR amplification was performed. The reaction system and conditions were the same as in step 2 of this embodiment. Sequencing confirmed the presence of a recombinant strain with the S25F / Y60W double mutation. Using the S25F mutant plasmid as a template, and AK-ATA-24F and AK-ATA-24R as primers, full-plasmid PCR amplification was performed. The reaction system and conditions were the same as in step 2 of this embodiment. Sequencing confirmed the presence of a recombinant strain with the S25F / A241V double mutation. Using the Y60W mutant plasmid as a template, and AK-ATA-24F and AK-ATA-24R as primers, full-plasmid PCR amplification was performed. The reaction system and conditions were the same as in step 2 of this embodiment. Sequencing confirmed the presence of a recombinant strain with the Y60W / A241V double mutation.

[0115] Using the S25F mutant plasmid as a template and AK-ATA-1F and AK-ATA-8R as primers, PCR amplification was performed to obtain fragment 1 containing two mutation sites, S25F and Y60W; using the S25F mutant plasmid as a template and AK-ATA-8F and AK-ATA-24R as primers, PCR amplification was performed to obtain fragment 2 containing two mutation sites, Y60W and A241V; using the S25F mutant plasmid as a template and AK-ATA-24F and AK-ATA-1R as primers, PCR amplification was performed to obtain fragment 3 containing the A241V mutation site; using PCR product fragments 1, 2, and 3 as templates and AK-ATA-1F and AK-ATA-1R as primers, PCR amplification was performed to obtain the ATA mutant gene fragment S25F / Y60W / A241V containing three mutation sites, S25F, Y60W, and A241V.

[0116] The linear vector pET-30a(+) was amplified by PCR using the S25F mutant plasmid as a template and PF and PR as primers.

[0117] AK-ATA-8F: SEQ ID NO:21gttggacttcttcaaccagctgtggtgcgtgaacgtc.

[0118] AK-ATA-8R: SEQ ID NO:22gacgttcacgcaccacagctggttgaagaagtccaac.

[0119] AK-ATA-24F: SEQ ID NO:23aaggtgtcggcagcgcaatgcac.

[0120] AK-ATA-24R: SEQ ID NO:24cgctgccgacaccttgtgacacttcagtaataaccg.

[0121] AK-ATA-1F: (SEQ ID NO:25)agaaggagatatacatatgacaaatcagaaacaagaaataaac.

[0122] AK-ATA-1R: (SEQ ID NO:26)tgtcgacggagctcgaattcttaggcttgaccc.

[0123] PF: SEQ ID NO:27ctaagaattcgagctccgtcg.

[0124] PR: SEQ ID NO:28ttgtcatatgtatatctccttcttaaagttaaac.

[0125] Gene fragments containing the three mutation sites S25F / Y60W / A241V were digested and ligated with the linear vector pET-30a(+) using Gibson Assembly Master Mix (purchased from NEB). The reaction system is shown in Table 5.

[0126] Table 5

[0127]

[0128] Reaction conditions: 1) Incubate at 50℃ for 15 min; 2) Store at 4℃.

[0129] The ligation product was transferred into Escherichia coli BL21(DE3) competent cells, and the correct S25F / Y60W / A241V three-point combination mutant recombinant strain was obtained by sequencing verification.

[0130] (2) Combining two site-directed mutations, S25W and A241V.

[0131] Using ATA-16(A241V) as a template, the ATA-16(S25W / A241V) mutant was obtained by saturation mutagenesis.

[0132] The nucleotide sequence of the ATA-16(S25F / A241V) mutant is shown in SEQ ID NO:17.

[0133] The amino acid sequence of the ATA-16(S25F / A241V) mutant is shown in SEQ ID NO:18.

[0134] The nucleotide sequence of the ATA-16(S25W / A241V) mutant is shown in SEQ ID NO:19.

[0135] The amino acid sequence of the ATA-16(S25W / A241V) mutant is shown in SEQ ID NO:20.

[0136] Example 3

[0137] 1. Verification of enzyme activity and stability of mutant strains

[0138] Preparation of crude enzyme solution: Seed culture of mutants with better performance selected from well plates was inoculated at 0.5% into 30 mL of TB medium, and kanamycin (final concentration 50 μg / mL) was added. The culture was incubated at 37℃ and 200 rpm for 3-4 h. When the OD reached 0.8-1.0, the culture was cooled to room temperature, and 0.1 mM IPTG was added for induction. Induction was carried out at 22℃ and 220 rpm for 16 h. The induced culture was transferred to centrifuge tubes (weighed beforehand), centrifuged at 4000 rpm for 30 min, the supernatant was discarded, and the centrifuge tubes containing wet cells were weighed again to calculate the wet cell mass. Based on the calculated wet cell mass, the cells were resuspended at 10% with 50mM triethanolamine (containing 1mM PLP). The cells were then disrupted on ice using an ultrasonic disruptor (200W, 2s sonication, 4s pause, 10min total). The bacterial culture was centrifuged at 15000rpm for 30min, and the supernatant was transferred to centrifuge tubes for storage. The default concentration was 100mg / mL.

[0139] The above-mentioned wild-type S-transaminase or mutant crude enzyme solution was used for catalytic reaction: In each 1 mL reaction system, 225 mM of 1-methoxy-2-acetone (corresponding to 20 g / L), 340 mM (32.5 g / L) isopropylamine hydrochloride, and 1 mM PLP were added, along with 0.2 wt% (4 mg / mL) of the crushed wild-type S-transaminase or mutant crude enzyme solution. The reaction system buffer was 50 mM triethanolamine, pH 8.0. The reaction conditions were controlled at 35℃ and 200 rpm using a shaker. After 3 h, the reaction was terminated with 1 M NaOH solution (final concentration approximately 0.1 M). The amount of product (S)-1-methoxy-2-propylamine generated was detected by high-performance liquid chromatography (HPLC), and the conversion rate and ee value were calculated.

[0140] In a high-concentration substrate reaction system, 1 mL of reaction solution contained 1.14 M (corresponding to 100 g / L) of 1-methoxy-2-acetone, 1.71 M (163.4 g / L isopropylamine hydrochloride), and 1 mM PLP. Wild-type S-transaminase or mutant crude enzyme solution (0.2 wt, 20 mg / mL) was added, respectively. The reaction buffer was 50 mM triethanolamine at pH 8.0. The reaction was carried out in a 10 mL sealed container, with the reaction conditions controlled by shaking at 35 °C and 200 rpm. After 3 h, the reaction was terminated with 1 M NaOH solution (final concentration approximately 0.1 M). The conversion rate was calculated by detecting the amount of (S)-1-methoxy-2-propylamine derivatives formed by derivatization using high-performance liquid chromatography.

[0141] Table 6 shows the conversion rate and ee value of the reaction at a substrate concentration of 225 mM, 35 °C, 0.2 wt, and 3 hours. As can be seen from Table 6, compared with the wild type, mutants S25F, Y60W, A241V, G147D, N267A, and A244T exhibited better activity.

[0142] Table 6

[0143] Wild type or mutant Conversion rate (%) ee H144R 2.28％ NA V64L 3.08％ NA P194S 4.39％ 99.77％ L413P 6.05％ 99.67％ K375P 6.51％ 99.66％ S408R 7.68％ 99.65％ F142I 9.07％ 99.60％ H246P 9.86％ 99.51％ T175S 10.22％ 99.38％ D48G 11.79％ 99.62％ F248Y 11.82％ 99.37％ A112P 12.73％ 99.40％ V64I 13.01％ 99.41％ F142V 13.09％ 99.19％ E108P 13.23％ 99.30％ K195N 14.05％ 99.22％ K375E 14.51％ 99.17％ K395E 15.58％ 99.37％ N26S 15.94％ 98.87％ M46T 16.76％ 99.54％ M75I 18.83％ 99.05％ S229Q 18.94％ 98.81％ Y60W 24.26％ 98.50％ A241V 25.29％ 99.25％ Y60W 27.09％ 98.63％ S25F 29.36％ 99.10％ G147D 40.55％ 99.50％ N267A 35.69％ 99.82％ A244T 30.27％ 99.84％ G147A 28.45％ 99.90％ N267V 24.35％ 99.78％ T411M 23.69％ 99.76％ A244I 23.68％ 99.68％ N267S 21.36％ 99.36％ E108P 6.68％ 99.59％ V64T 0.46％ 99.54％ Y164F 26.86％ 99.74％ 0.1wt (WT) 6.03％ 99.74％ 0.2wt (WT) 11.33％ 99.39％ 0.5wt (WT) 25.71％ 98.51％ 1wt (WT) 36.47％ 97.17％

[0144] Table 7 shows the conversion rate and ee value of the reaction at a substrate concentration of 1.14M (corresponding to 100 g / L), 35℃, 0.2 wt, and 3 hours. As can be seen from Table 7, the mutants S25F, A241V, G147D, E108P, K375E, K395E, A244T, or Y60W have higher reaction efficiency than the wild type under high substrate concentration of 100 g / L, and they still have good tolerance under high substrate concentration.

[0145] Table 7

[0146]

[0147]

[0148] The reaction system consisted of 1 mL of a 1-molecular-weight (100 g / L) substrate 1-methoxy-2-acetone, 1.71 M (163.4 g / L) isopropylamine hydrochloride, and 1 mM PLP. Crushed wild-type S-transaminase or crude enzyme solution of a multi-site mutant (0.1 wt, 10 mg / mL or 0.2 wt, 20 mg / mL) was added, respectively. The reaction buffer was 50 mM triethanolamine at pH 8.0. The reaction was carried out in a 1 mL sealed container under shaking conditions of 35 °C and 200 rpm for 3 h. The reaction was terminated with 1 M NaOH solution (final concentration approximately 0.1 M). The product (S)-1-methoxy-2-propylamine) was derivatized and the amount produced was determined by high-performance liquid chromatography (HPLC), and the conversion rate was calculated.

[0149] Table 8 shows the conversion rate and ee value of the combined mutant enzyme at 100 g / L substrate, 35℃, 0.1 wt, and 3 hours. As shown in Table 8, the double mutant S25F / A241V exhibits nearly 6-fold increased enzyme activity compared to the wild type, while maintaining high selectivity. At 0.2 wt of enzyme, the conversion rate reaches 83.82%, approaching the reaction equilibrium under closed reaction conditions.

[0150] Table 8

[0151] Wild type or mutant Enzyme amount Conversion rate (%) / 3h ee WT 0.1wt 8.28％ 99.78％ S25F / Y60W 0.1wt 32.28％ 100％ Y60W / A241V 0.1wt 4.66％ NA S25F / Y60W / A241V 0.1wt 1.25％ NA S25F / A241V 0.1wt 46.57％ 99.55％ S25F / A241V 0.2wt 83.82％ 99.53％

[0152] In Table 6-8, NA indicates that it cannot be detected.

[0153] Table 9 shows the enzyme activity verification results of S25W / A241V and S25F / A241V, with a substrate concentration of 100 g / L, an enzyme dosage of 0.1 wt, a reaction temperature of 55 °C, and a pH of 8.0.

[0154] Table 9

[0155]

[0156]

[0157] As shown in Table 9, the double mutant S25W / A241V exhibits significantly higher activity than the wild type, with a conversion rate of 73.27% after 3 hours. While its enzyme activity is slightly lower than that of the S25F / A241V double mutant, it demonstrates better chiral selection, and no R-type product was detected.

[0158] 2. Thermal stability test

[0159] Crude enzyme solution pretreatment: 1 mL of the broken wild-type S-transaminase and the selected mutant crude enzyme solutions were placed into three separate EP tubes, labeled, and heated in water baths at 35℃ and 60℃ for 1 h respectively. The tubes were then centrifuged at 12000 rpm for 10 min for subsequent thermostability testing. SDS-PAGE was performed on the enzyme solutions before and after heating to verify protein changes.

[0160] The reaction system consisted of 1 mL of 1-methoxy-2-acetone substrate, 340 mM isopropylamine hydrochloride, and 1 mM PLP. Crushed wild-type S-transaminase or mutant crude enzyme solution (0.2 wt, 4 mg / mL) was added separately. The reaction system solution contained 50 mM triethanolamine at pH 8.0. The reaction conditions were controlled at 35℃ and 200 rpm using a shaker. After 3 h, the reaction was terminated with 1 M NaOH solution (final concentration approximately 0.1 M). The amount of product (S)-1-methoxy-2-propylamine generated was detected by high-performance liquid chromatography (HPLC), and the conversion rate was calculated. The conversion results are shown in Table 10.

[0161] Table 10

[0162] Wild type or mutant Conversion rate (35℃) Conversion rate (55℃) wild type 48.82％ 0.30％ S25F 39.05％ 34.61％ A241V 61.34％ 62.81％ S25F / A241V 55.23％ 55.14％

[0163] Figure 1 SDS-PAGE results for crude enzyme solutions of wild type and mutant (treated at 35℃ and 60℃ for one hour), where S1 represents heat treatment at 35℃ for 1 hour and S2 represents heat treatment at 60℃ for 1 hour. Figure 1 It can be seen that after treatment at 60℃, the target enzyme protein bands of mutants (S25F, A241V, S25F / 241V) did not change significantly, and some impurities precipitated, while most of the wild-type target enzyme protein precipitated.

[0164] Crude enzyme solution pretreatment: 0.5 mL of wild-type S-transaminase and double mutant (S25F / A241V) crude enzyme solutions were aliquoted into EP tubes and labeled with the corresponding temperatures. Wild-type S-transaminase was heated in water baths at 35℃, 45℃, 50℃, 55℃ or 60℃ for 1 h, while double mutant crude enzyme solutions were heated in water baths at 35℃, 55℃, 60℃, 63℃, 66℃, 70℃, 75℃ or 80℃ for 1 h. The crude enzyme solutions treated at different temperatures were centrifuged at 12000 rpm for 10 min, and the supernatant was collected, labeled, and used for subsequent thermal stability testing.

[0165] The reaction system consisted of 1 mL of 1-methyl-2-acetone substrate, 340 mM isopropylamine hydrochloride, and 1 mM PLP. Crushed wild-type S-transaminase and its mutant crude enzyme solution (0.1 wt, 2 mg / mL) were added separately. The reaction buffer was 50 mM triethanolamine (pH 8.0). The reaction was carried out in a 1 mL sealed container, with the reaction conditions controlled by shaking at 35 °C and 200 rpm. After 3 h, the reaction was terminated with 1 M NaOH solution (final concentration approximately 0.1 M). The conversion rate was calculated by detecting the amount of (S)-1-methoxy-2-propylamine produced by derivatization using high-performance liquid chromatography.

[0166] Figure 2The results show a comparison of residual enzyme activity after heat treatment of WT and the double mutant S25F / A241V. Figure 2 It can be seen that S25F / 241V retained its original activity after heat treatment at 60℃ and 65℃ for one hour, until the enzyme activity began to decrease significantly and eventually became completely inactive after 70℃, with Tm values ​​between 70-75℃. In contrast, the wild-type enzyme could tolerate heat treatment at 50℃ for one hour, and almost completely lost its activity after treatment at 60℃ for one hour, with Tm values ​​between 50-55℃. The conversion results corresponding to the residual enzyme activity after heat treatment are shown in Table 11.

[0167] Table 11

[0168]

[0169]

[0170] Example 4

[0171] Gram-scale reaction of (S)-1-methoxy-2-propane

[0172] In a 100 mL four-necked reaction flask, add 5.0 g of methoxyacetone, 8.14 g of isopropylamine hydrochloride, and 35 mL of buffer solution (pH 8.0, 50 mM triethanolamine aqueous solution). Start stirring and add PLP (12.5 mg) and 10 mL of crude ATA-16 enzyme solution sequentially. Make up the volume to 50 mL, stir and heat to 55 °C. After 1 hour, maintain the system under vacuum (200 mmHg). Take samples every hour to monitor the optical purity of the product and calculate the conversion rate using supercritical fluid chromatography (SFC).

[0173] Figure 3 The results are for a 5g-scale reaction of the mutant S25F / A241V (100g / L, 55℃, 0.2wt enzyme); For example... Figure 3 As shown, the reaction tends to reach equilibrium after one to two hours, with a conversion rate of 78.6%. Under conditions of 55°C and a vacuum of 200 mmHg, the acetone volatilization-driven conversion rate increases to 92.7% after 6 hours, with an ee value of 99.6%. Figure 4 As shown, Figure 4 The peak times for isopropylamine were 1.693, for R-1-methoxy-2-propylamine 1.959, and for S-1-methoxy-2-propylamine 2.424.

[0174] In summary, the experimental results of this invention demonstrate that the thermostable transaminase mutant of this invention can withstand heat treatment at 60°C during enzyme preparation, and the purity of the enzyme catalyst is significantly improved after heat treatment. When preparing (S)-1-methoxy-2-propane at temperatures above 55°C, the catalytic efficiency is significantly improved, and both thermal stability and product stereoselectivity are superior to traditional catalysts. Especially in industrial production, the transaminase mutant of this invention can reduce reaction time, increase reaction rate, and significantly reduce energy consumption and reaction costs.

[0175] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A heat-stable transaminase, characterized in that, The amino acid sequence of the transaminase is selected from any of the following: (1) The amino acid sequence as shown in SEQ ID NO:1; (2) An amino acid sequence with transaminase activity derived from the amino acid sequence shown in SEQ ID NO:1 by substitution, deletion or addition of one or more amino acids. (3) An amino acid sequence that has at least 95% identity with the amino acid sequence shown in SEQ ID NO:1 and has transaminase activity.

2. A heat-stable transaminase mutant, characterized in that, The transaminase mutant is based on the amino acid sequence shown in SEQ ID NO:1, and the mutation site is selected from any one or a combination of at least two of S25W, S25F, Y60W or A241V.

3. The heat-stable transaminase mutant according to claim 2, characterized in that, The mutation site is a combination of S25F and Y60W, or a combination of S25F and A241V, or a combination of Y60W and A241V, or a combination of S25W and A241V.

4. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes the thermostable transaminase of claim 1, or encodes a thermostable transaminase mutant of claim 2 or 3.

5. An expression carrier, characterized in that, The expression vector contains the nucleic acid molecule as described in claim 4.

6. An engineered bacterium, characterized in that, The genome of the engineered bacteria is integrated with the nucleic acid molecule of claim 4, or the engineered bacteria contains the expression vector of claim 5.

7. The use of the thermostable transaminase of claim 1 and / or the thermostable transaminase mutant of claim 2 or 3 in the preparation of chiral amine compounds.

8. An immobilized transaminase, characterized in that, The immobilized transaminase comprises a resin and the thermostable biological transaminase of claim 1 and / or the thermostable biological transaminase mutant of claim 2 or 3.

9. A method for preparing (S)-1-methoxy-2-propylamine, characterized in that, The method includes: using 1-methoxy-2-propanone as a substrate, and performing biocatalysis with the thermostable transaminase of claim 1 and / or the thermostable transaminase mutant of claim 2 or 3 to prepare (S)-1-methoxy-2-propanamine.

10. The method for preparing (S)-1-methoxy-2-propylamine according to claim 9, characterized in that, The biocatalytic steps include: preparing enzyme solution or enzyme powder from the fermentation broth of engineered bacteria containing the gene encoding transaminase or its mutant; using the enzyme solution or enzyme powder as a catalyst, 1-methoxy-2-propanone as a substrate, and isopropylamine as an amino donor, carrying out a biocatalytic reaction in a buffer system; and after the reaction is completed, separating and purifying the reaction solution to obtain (S)-1-methoxy-2-propanamine.

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