High-activity heat-resistant aminotransferase mutant and its encoding gene, construction method and application

Abstract

The present invention discloses a highly active thermostable transaminase mutant and its encoding gene, construction method and application. The highly active thermostable transaminase mutant is obtained by mutating the 61st tryptophan of the wild-type transaminase MhTA amino acid sequence shown in SEQ ID NO.1 to alanine, cysteine, glycine or valine. The 5 mutants (W61A, W61C, W61G and W61V) obtained by the present invention have significantly higher activities in catalyzing prochiral ketones than the wild-type before mutation (abbreviated as WT), specifically: the activities of W61A, W61C, W61G and W61V mutants in catalyzing 5-methoxy-2-tetralinone are 43.03, 192.1, 25.87 and 77.48 times that of the wild-type, respectively. It can be seen from the catalytic activity improvement effect that the transaminase mutant disclosed in the present invention has the application potential of catalyzing the synthesis of rotigotine chiral intermediates.

Description

Technical Field

[0001] The present invention relates to the technical field of enzyme engineering, and in particular to a highly active heat-resistant aminotransferase mutant and its encoding gene, construction method and application. Background Art

[0002] Rotigotine is a second-generation non-ergot dopamine receptor agonist used to treat early Parkinson's disease and lower limb hyperactivity syndrome, and has extremely high medical value. (S) -2-Amino-5-methoxy-tetralin is a key chiral intermediate for the synthesis of rotigotine. U.S. Patent US201213570210 discloses a chemical synthesis route of the chiral intermediate, which uses 5-methoxy-2-tetralinone as a raw material and involves multiple reaction steps involving chiral auxiliary agents, sodium borohydride, palladium carbon catalyst, etc., with a low yield. The biocatalytic synthesis route based on transaminase can directly convert 5-methoxy-2-tetralinone into optically pure ( S )-2-amino-5-methoxy-tetralin has the advantages of simple steps, low cost and high synthesis efficiency.

[0003] Transaminase is a pyridoxal-5'-phosphate (PLP)-dependent enzyme that can reversibly catalyze the transfer of amino groups from donors to carbonyl acceptors. It can generate valuable chiral amines through a one-step catalytic reaction under mild reaction conditions with high theoretical yield and strict stereoselectivity. Transaminase can be divided into S Selectivity and R Selectivity, belonging to the folding class I and folding class IV of PLP-dependent enzymes, respectively, the two have obvious differences in structure, and their functional difference is S Selective transaminase recognition S Amine donor and transfer amino group to generate S Type chiral amine, R Selective transaminase recognition R Amine donor and transfer amino group to generate R Type chiral amine.

[0004] The natural substrates of transaminases are mainly small molecule amino acids, keto acids, etc. However, they have high application value such as ( S )-2-amino-5-methoxy-tetralin, a chiral amine used as a drug intermediate (herein referred to as high-value chiral amine), usually contains large steric hindrance groups on one or both sides of the chiral group. For this type of compound, natural transaminases are usually unable to synthesize it or the efficiency is extremely low, which has become the main bottleneck in the technical development and industrial application of transaminase-catalyzed synthesis of high-value chiral amines.

[0005] The key factor affecting the catalysis of transaminases on sterically hindered substrates is the spatial structure of the substrate binding region. The rational modification of the substrate binding region by protein engineering techniques can effectively improve the catalytic performance of transaminases on sterically hindered substrates, thereby broadening the scope of application of transaminases in the green synthesis of chiral amines. In addition, thermostable enzymes are widely used as the first choice for biocatalysts, and their sources are mainly thermophilic microorganisms or molecular modifications to improve the thermal stability of enzymes. The advantage of using thermostable transaminases in the synthesis of chiral amines is that they can better adapt to industrial requirements, achieve higher reaction flux, improve production efficiency and reduce process costs. Therefore, it is of great significance to develop new highly active thermostable transaminases mutants to catalyze the synthesis of high-value chiral amines. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a highly active thermostable transaminase mutant and its encoding gene, construction method and application in view of the above-mentioned deficiencies in the prior art. The present invention provides a highly active thermostable transaminase mutant with significantly improved catalytic activity on large sterically hindered substrates through enzyme engineering modification. S A type of transaminase mutant is provided to solve the problem of low activity in the prior art when synthesizing large sterically hindered chiral amines by transaminase catalysis.

[0007] To achieve the above object, the technical solution adopted by the present invention is: In the first aspect of the present invention, a high-activity thermostable transaminase mutant is provided, wherein the high-activity thermostable transaminase mutant is obtained by synthesizing a thermophilic bacterium as shown in SEQ ID NO. Meiothermus hypogaeus The amino acid sequence of the wild-type aminotransferase MhTA is obtained by mutating the tryptophan (W) at position 61 to alanine (A), cysteine ​​(C), glycine (G) or valine (V). The amino acid sequence of the wild-type aminotransferase is obtained by querying and screening from the NCBI database (https: / / www.ncbi.nlm.nih.gov / protein / WP_119342135.1 / ).

[0008] Preferably, the amino acid sequences of the mutants obtained by mutating the tryptophan at position 61 of the wild-type transaminase MhTA amino acid sequence to alanine, cysteine, glycine or valine are SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5, respectively.

[0009] The second aspect of the present invention provides a gene encoding the highly active thermostable aminotransferase mutant as described above.

[0010] Preferably, the nucleotide sequence encoding the wild-type transaminase MhTA as shown in SEQ ID NO.1 is SEQ ID NO.6. The genes encoding the highly active thermostable transaminase mutants are all obtained by site-directed mutagenesis based on the nucleotide sequence shown in SEQ ID NO.6.

[0011] The third aspect of the present invention provides a recombinant plasmid comprising the gene as described above.

[0012] Preferably, the plasmid vector of the recombinant plasmid is pET-28a(+) or pET-22b(+).

[0013] The fourth aspect of the present invention provides a host cell comprising the gene or recombinant plasmid as described above.

[0014] Preferably, the host cell is a prokaryotic cell.

[0015] More preferably, the host cell is a transaminase engineered bacterium.

[0016] More preferably, the host cell is Escherichia coli carrying the recombinant plasmid described above. coli BL21(DE3).

[0017] The fifth aspect of the present invention provides a method for constructing a highly active thermostable transaminase mutant as described above, the method comprising the following steps: culturing the host cells as described above under suitable conditions, inducing expression of the highly active thermostable transaminase mutant; collecting the host cells, crushing and centrifuging, and isolating and purifying the highly active thermostable transaminase mutant from the supernatant.

[0018] The present invention also provides a soluble protein or an engineered bacterium comprising the above transaminase mutant.

[0019] In a sixth aspect of the present invention, a highly active thermostable aminotransferase mutant or host cell as described above is provided as a biocatalyst for catalyzing the synthesis of prochiral ketone compounds. S Application of chiral amines.

[0020] The present invention also provides a method for producing ( S A green synthesis method for )-2-amino-5-methoxy-tetralin, the method comprising adding the above-mentioned transaminase mutant to a reaction system for reaction to obtain a reaction liquid; and extracting the reaction liquid to obtain a target chiral amine.

[0021] Preferably, the concentration of the transaminase mutant added to the reaction system is 0.1-1 mg / mL.

[0022] Preferably, the reaction system contains a coenzyme, an amino donor, a prochiral ketone and a buffer.

[0023] Preferably, the coenzyme is PLP, and the added concentration is 0.02-2 mmol / L.

[0024] Preferably, the amino donor is isopropylamine, L-alanine or 1-phenylethylamine, and the added concentration is 2-20 mmol / L.

[0025] Preferably, the buffer is a phosphate buffer or a Tris-HCl buffer with a concentration of 0.01-0.1 mol / L.

[0026] Preferably, the reaction temperature is 37°C to 60°C, the pH is 7 to 10, and the reaction time is 2 to 24 h.

[0027] Preferably, the prochiral ketone is 5-methoxy-2-tetralinone, and the added concentration is 0.2 to 2 mmol / L.

[0028] Preferably, the chiral amine is ( S )-2-amino-5-methoxy-tetralin.

[0029] The present invention also provides the above transaminase mutant, the above encoding gene, the above recombinant plasmid, the above host cell, the above construction method or the above green synthesis method in catalytic synthesis (S) -Application of 2-amino-5-methoxy-tetralin.

[0030] The beneficial effects of the present invention are:

[0031] The invention uses site-directed mutagenesis technology to Meiothermus hypogaeus The tryptophan (W) at position 61 of the wild-type transaminase MhTA amino acid sequence shown in SEQ ID NO.1 is mutated to alanine (A), cysteine ​​(C), glycine (G) or valine (V), and the obtained five mutants (W61A, W61C, W61G and W61V) have significantly higher catalytic activity of prochiral ketone than the wild-type before mutation (abbreviated as WT), specifically: the activity of W61A, W61C, W61G and W61V mutants in catalyzing 5-methoxy-2-tetralinone is 43.03, 192.1, 25.87 and 77.48 times that of the wild-type, respectively. From the effect of improving the catalytic activity, it can be seen that the transaminase mutant disclosed in the present invention has the application potential of catalyzing the synthesis of rotigotine chiral intermediates. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 Schematic diagram of the route for the transaminase-catalyzed synthesis of chiral intermediates of rotigotine.

[0033] Figure 2 Derived from thermophilic bacteria Meiothermus hypogaeus SDS-PAGE electrophoresis of the wild type (WT) and mutants of transaminase MhTA.

[0034] Figure 3 These are the results of thermal stability testing of transaminase MhTA and its mutants. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.

[0036] It should be understood that the terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0037] The test methods used in the following examples are conventional methods unless otherwise specified. The materials and reagents used in the following examples are all commercially available unless otherwise specified. In the following examples, if no specific conditions are specified, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, if the manufacturer is not specified, are all conventional products that can be purchased commercially.

[0038] The Escherichia coli BL21 (DE3) competent cells used in the present invention were purchased from Beijing Quanshijin Biotechnology Co., Ltd.; the transaminase wild-type plasmid based on the pET-28a (+) plasmid vector and the primers for constructing the site-directed mutation plasmid were synthesized by Beijing Qingke Biotechnology Co., Ltd.; the DNA polymerase, restriction endonuclease, Dpn I, recombinase and other molecular biological reagents used were purchased from Takara; PLP was purchased from Sigma-Aldrich; 5-methoxy-2-tetralone was purchased from Leyan Chemical Reagent Mall; and other biochemical reagents used were purchased from Sangon Biotechnology (Shanghai) Co., Ltd.

[0039] The PCR instrument used was purchased from Thermo Fisher Scientific, and the ELISA reader used was Bio Tek Synergy H1.

[0040] The culture medium involved in the following examples is as follows:

[0041] LB solid medium: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl, 15 g / L agar, and 50 mg / L kanamycin.

[0042] LB liquid medium: 5 g / L yeast extract, 10 g / L tryptone, 10 g / L NaCl, and 50 mg / L kanamycin.

[0043] Among them, the method for detecting the activity of transaminase catalyzing 5-methoxy-2-tetralinone is as follows (see Chen et al., 2020, Applied Microbiology and Biotechnology 104:2999–3009):

[0044] Using 1-(6-aminonaphthalene-2-yl)ethylamine as the amino donor substrate and 5-methoxy-2-tetralone as the amino acceptor substrate, the fluorescence intensity of the amino donor product 1-(6-aminonaphthalene-2-yl)ethanone in the catalytic reaction was measured by an enzyme marker and then converted into the molar concentration of 1-(6-aminonaphthalene-2-yl)ethanone using a standard curve.

[0045] Enzyme activity detection system (100 μL): 0.2 mM 1-(6-aminonaphthalen-2-yl)ethylamine, 0.5 mM 5-methoxy-2-tetralone, 0.1 mM PLP, 10 mM phosphate buffer (pH 8.5), 10 mg / L wild-type or mutant transaminase.

[0046] The reaction was carried out at 37°C and fluorescence was detected in real time, with an excitation wavelength of 357 nm, an emission wavelength of 525 nm, a gain of 50, and a continuous vibration of 5 s. The fluorescence was detected every 2 min for 2 hours. The amount of 1-(6-aminonaphthalene-2-yl)ethanone generated per minute in the transamination reaction (μmol) can be calculated using the concentration-fluorescence standard curve of 1-(6-aminonaphthalene-2-yl)ethanone.

[0047] Example 1: Construction and screening of transaminase MhTA mutants

[0048] According to the amino acid sequence SEQ ID NO.1 of the wild-type transaminase MhTA, protein homology modeling analysis was performed on the SWISS-MODEL website (https: / / swissmodel.expasy.org / ), and then the simulated structure was molecularly docked with the amino donor substrate and the amino acceptor substrate (AutoDock 4.2.6 software) to analyze the substrate binding pocket. It was found that W61 has a large residue, which affects the entry of large substrates into the active pocket. Therefore, it is speculated that this site may be the key site affecting the catalysis of large substrates by MhTA.

[0049] According to the primers shown in Table 1, whole-plasmid single-point mutation PCR was performed with the wild-type plasmid pET-28a(+)-TbTA as a template to construct single-point mutation recombinant plasmids containing the coding genes in which the TbTA-W61 site was mutated to have smaller side chain amino acids (A / C / G / I / L / M / S / T / V).

[0050] Table 1 Primers for constructing MhTA-W61 site mutations

[0051]

[0052] The whole plasmid single point mutation PCR system is shown in Table 2.

[0053] Table 2 PCR reaction system

[0054]

[0055] The whole plasmid single point mutation PCR program settings are:

[0056] (1) Denaturation at 98°C for 10 seconds; (2) Annealing at 55°C for 5 seconds; (3) Extension at 72°C for 1 minute; (4) Repeat steps (1) to (3) for 29 cycles; (5) Full extension at 72°C for 5 minutes; (6) Store the amplified product at 4°C.

[0057] Example 2: Preparation, expression and purification of wild-type and mutant transaminase MhTA

[0058] The mutant plasmids obtained in Example 1 were transformed into competent cells of Escherichia coli BL21 (DE3) and spread on plates for overnight growth. Single clones were then picked and placed in 15 mL of 0.1 mg / mL Kan + LB liquid medium, culture overnight; then transfer to 1 L containing 0.1 mg / mL Kan + LB liquid medium and cultured to OD 600 When the pH was about 0.8, 0.5 mM IPTG was added and the mixture was cultured at 16°C and 220 rpm for 16 hours to express the target protein.

[0059] The bacterial liquid was centrifuged, the bacterial sludge was collected, and it was resuspended with 40 mL buffer (10 mM PBS, 150 mM NaCl), and then pressure-crushed. The treatment condition was 1100 Pa for 2 min. Then, it was centrifuged at 12000 rpm for 30 min, and the supernatant was filtered again with a filter membrane and placed on ice for later use. Before purification, the nickel column was washed with 10 column volumes of pure water, and then with 10 column volumes of buffer. The filtered supernatant was slowly poured in and out, and the nickel column was washed with 10 column volumes of imidazole buffer (10 mM PBS, 150 mM NaCl and 10 μM PLP) containing 20 mM, 40mM, 160 mM and 500 mM, respectively, and the flow-through was collected. The target protein was mainly in the flow-through with an imidazole concentration of 160 mM. The flow-through containing high-purity target protein was concentrated and desalted with a desalting gravity column, and then replaced with 10 mM PBS buffer; finally, the enzyme solution concentration was adjusted to 5 mg / mL (containing 30% glycerol) and stored in a -20℃ refrigerator for later use. The protein electrophoresis results of wild-type aminotransferase MhTA and mutants are shown in Figure 2 shown. Figure 2 In the figure, the lanes with odd numbers are the supernatant of cell lysate before purification, and the lanes with even numbers are the enzyme solution after purification.

[0060] Example 3: Determination of enzyme activity of wild-type and mutant transaminase MhTA

[0061] Appropriate amounts of pure enzyme solutions of wild-type and mutant transamination MhTA prepared according to the method of Example 2 were taken to detect their catalytic activities of 5-methoxy-2-tetralinone. The activity of wild-type MhTA was taken as 100%, and the specific activities of the remaining mutants were compared with it to calculate the relative enzyme activities. The results are shown in Table 3.

[0062] Table 3 Relative enzyme activities of wild-type and W61 mutant of transaminase MhTA

[0063]

[0064] "NA" means no enzyme activity was detected.

[0065] According to the test results in Table 3, the aminotransferase mutants obtained by mutating the tryptophan (W) at position 61 to alanine (A), cysteine ​​(C), glycine (G) or valine (V) were screened out: W61A, W61C, W61G, and W61V.

[0066] Example 4: Thermal stability test of transaminase MhTA and its mutants

[0067] Take appropriate amounts of pure enzyme solutions of wild-type transaminase MhTA (denoted as WT) and mutants W61A, W61C, W61G, and W61V with enhanced activity prepared according to the method of Example 2, dilute to 1 mg / mL with 10 mM PBS (pH 8.0), and then place them in a water bath at the optimal temperature of 55°C for incubation. Starting from 0 h of incubation, the residual activity of the enzyme at different time periods (detected every 2 h) at this temperature is monitored by fluorescence activity measurement ( Figure 3 ), the incubation time when the residual activity reaches half of the initial activity is the activity half-life of the enzyme at 55°C ( t 1 / 2 ). The experimental results show that, based on the excellent heat resistance of the wild type MhTA, the above four mutants with improved activity have further improved thermal stability, as shown in Table 4:

[0068] Table 4 Thermal stability test results of transaminase MhTA and its mutants

[0069]

[0070] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes. They can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to specific details.

Claims

1. A highly active thermostable aminotransferase mutant, characterized in that: The highly active thermostable transaminase mutant is obtained by mutating the 61st tryptophan in the amino acid sequence of the wild-type transaminase MhTA as shown in SEQ ID NO.1 to alanine, cysteine, glycine or valine.

2. The highly active thermostable aminotransferase mutant according to claim 1, characterized in that: The amino acid sequences of the mutants obtained by mutating the tryptophan at position 61 of the amino acid sequence of the wild-type transaminase MhTA to alanine, cysteine, glycine or valine are SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 respectively.

3. A gene encoding the highly active thermostable aminotransferase mutant according to claim 1 or 2.

4. The gene according to claim 3, characterized in that The nucleotide sequence encoding the wild-type aminotransferase MhTA shown in SEQ ID NO.1 is SEQ ID NO.

6.

5. A recombinant plasmid comprising the gene according to claim 3 or 4.

6. The recombinant plasmid according to claim 5, characterized in that The plasmid vector of the recombinant plasmid is pET-28a(+) or pET-22b(+).

7. A host cell comprising the gene according to claim 3 or 4 or the recombinant plasmid according to claim 5 or 6.

8. The host cell according to claim 7, characterized in that The host cell is a prokaryotic cell.

9. The host cell according to claim 8, characterized in that The host cell is Escherichia coli carrying the recombinant plasmid according to claim 5 or 6. coli BL21.

10. A method for constructing a highly active thermostable aminotransferase mutant according to any one of claims 1 to 2, characterized in that: The method comprises the following steps: culturing the host cell described in any one of claims 7 to 9, and inducing expression of the highly active thermostable transaminase mutant; collecting the host cells, crushing and centrifuging, and separating and purifying the highly active thermostable transaminase mutant from the supernatant.

11. A highly active thermostable aminotransferase mutant according to any one of claims 1 to 2 or a host cell according to any one of claims 7 to 9 as a biocatalyst in catalyzing the synthesis of prochiral ketone compounds S Application of chiral amines; The prochiral ketone compound is 5-methoxy-2-tetralinone (I), and the S-type chiral amine is the rotigotine drug intermediate (S)-2-amino-5-methoxy-tetralinone (II).

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