Omega-transaminase progenitor enzyme mutant for efficient catalytic synthesis of (R)-chiral amine compound and application of omega-transaminase progenitor enzyme mutant

Abstract

The invention discloses an omega-transaminase progenitor enzyme mutant for efficient catalytic synthesis of (R)-chiral amine compounds and application, and relates to the technical field of molecular biology. The omega-transaminase mutant is obtained by mutating an ancestor enzyme Ancaa-101 of omega-transaminase from Aspergillus terreus, and the amino acid sequence of the omega-transaminase mutant is as shown in SEQ ID NO. 6. The invention also discloses a preparation method of the omega-transaminase mutant. Compared with an ancestor enzyme, the activity of the omega-transaminase ancestor enzyme mutant on a non-natural substrate, namely 1-acetyl naphthalene, N-tert-butyloxycarboryl-3-piperidone or 3-oxopyrrolidine-1-carboxylic acid tert-butyl ester, is remarkably improved.

Description

Technical Field

[0001] The present invention relates to the field of molecular biology technology, and in particular to a mutant of an ω-aminase ancestral enzyme obtained based on pocket engineering, and in particular to an ω-aminase ancestral enzyme mutant for efficiently catalyzing the synthesis of (R)-chiral amine compounds and its application. Background Art

[0002] Chiral amines refer to a class of compounds containing amino groups at the chiral center of small molecule compounds, and are an important class of drug synthesis intermediates. In recent years, with the continuous expansion of the chiral drug market, chiral amines and their derivatives have accounted for more than 70% of the chiral drugs, such as neurological drugs, cardiovascular drugs, antihypertensive drugs, anti-infective drugs and vaccines; the main ingredient of anti-diabetic drugs, sitagliptin, is an R-type amine. The market demand for chiral amine drugs is huge, making the efficient preparation of chiral amine drugs extremely important.

[0003] Transaminase is a key enzyme for the biological preparation of chiral amines. It can reversibly catalyze the amino transfer reaction between keto and amino groups to synthesize amine compounds. The ω-transaminase from Aspergillus terreus uses pyridoxal phosphate (PLP) as a coenzyme to catalyze the transfer of the amino group on the amino donor to the chiral acceptor ketone to generate chiral amines and byproduct ketones. The catalytic process is shown below:

[0004]

[0005] Although transaminases have good application prospects in the synthesis of chiral amines, wild-type enzymes have poor thermal stability, low activity on non-natural substrates, and narrow substrate spectrum, which are not conducive to their application in industrial production. In the prior art, the patent applications with application numbers CN105441404A and CN105950581A use site-directed mutagenesis technology to transform the wild-type ω-transaminases, and obtain ω-transaminases mutants with further improved thermal stability. And the invention patent with authorization number CN114134128B uses ancestral sequence reconstruction technology to transform the wild-type ω-transaminases from Aspergillus terreus, and obtains the ω-transaminases mutant Ancata-101 with significantly improved thermal stability. However, the activity of the mutant on non-natural substrates is not significantly changed compared to the wild-type enzyme.

[0006] The steric restriction of the substrate binding pocket of ω-aminotransferase is the main reason for its narrow substrate spectrum and low activity on non-natural substrates. Therefore, modifying the substrate binding pocket is one of the methods to improve the activity of ω-aminotransferase and expand its substrate spectrum.

[0007] So far, there are no reports on pocket modification of the ancestral enzyme of Aspergillus terreus ω-aminotransferase to enhance its activity and expand its substrate range. Summary of the invention

[0008] The present invention is based on a structure-guided pocket engineering strategy, through which a mutant of an ancestral enzyme of Aspergillus terreus ω-aminotransferase with significantly improved enzyme activity to a non-natural substrate is obtained.

[0009] The present invention analyzes the structure of Ancata-101, the ancestral enzyme of Aspergillus terreus ω-aminotransferase, and selects 17 amino acid sites near the substrate binding pocket for saturation iterative mutation. After screening, a mutant with significantly improved activity is obtained:

[0010] The mutant was named Ancata-1016: H55T-E117S-R128M-V150A-L183F-L188F.

[0011] The present invention provides a mutant based on an ω-aminase ancestral enzyme, which is obtained by mutating the ω-aminase ancestral enzyme Ancata-101 from Aspergillus terreus, wherein the amino acid sequence of Ancata-101 is shown in SEQ ID NO.4, and the amino acid sequence of the ω-aminase ancestral enzyme mutant is shown in SEQ ID NO.6.

[0012] The present invention further provides the use of the ω-aminotransferase ancestral enzyme mutant in catalyzing 1-acetylnaphthalene to generate (R)-(+)-1-(1-naphthyl)ethylamine.

[0013] The present invention further provides the use of the ω-aminotransferase ancestral enzyme mutant in catalyzing N-tert-butyloxycarbonyl-3-piperidone to generate ((R)-1-tert-butyloxycarbonyl-3-aminopiperidine.

[0014] The present invention further provides the use of the ω-aminotransferase ancestral enzyme mutant in catalyzing tert-butyl 3-oxopyrrolidine-1-carboxylate to generate (R)-1-tert-butyloxycarbonyl-3-aminopyrrolidine.

[0015] Compared with the ancestral enzyme Ancata-101, the mutant enzyme has better activity towards non-natural substrates and is more suitable for industrial applications.

[0016] The present invention also provides a gene encoding the ω-aminotransferase ancestral enzyme mutant.

[0017] The gene sequence of the mutant is shown in SEQ ID NO.5.

[0018] The invention further provides application of the gene in catalyzing 1-acetylnaphthalene to generate (R)-(+)-1-(1-naphthyl)ethylamine.

[0019] The present invention further provides the use of the gene in catalyzing N-tert-butyloxycarbonyl-3-piperidone to generate ((R)-1-tert-butyloxycarbonyl-3-aminopiperidine.

[0020] The present invention further provides the use of the gene in catalyzing 3-oxopyrrolidine-1-carboxylic acid tert-butyl ester to generate (R)-1-tert-butyloxycarbonyl-3-aminopyrrolidine.

[0021] The invention also provides a recombinant expression plasmid comprising the gene.

[0022] The present invention also provides a genetically engineered bacterium comprising the above-mentioned recombinant expression plasmid.

[0023] The present invention further provides the use of the genetically engineered bacteria in catalyzing 1-acetylnaphthalene to generate (R)-(+)-1-(1-naphthyl)ethylamine.

[0024] The present invention further provides the use of the genetically engineered bacteria in catalyzing N-tert-butyloxycarbonyl-3-piperidone to generate ((R)-1-tert-butyloxycarbonyl-3-aminopiperidine.

[0025] The present invention further provides the use of the genetically engineered bacteria in catalyzing tert-butyl 3-oxopyrrolidine-1-carboxylate to generate (R)-1-tert-butyloxycarbonyl-3-aminopyrrolidine.

[0026] In summary, the present invention provides the use of the ω-aminotransferase ancestral enzyme mutant obtained by the present invention, the gene encoding the mutant, or the above-mentioned genetically engineered bacteria in any of the following catalytic reactions:

[0027] (1) Catalyzes 1-acetylnaphthalene to produce (R)-(+)-1-(1-naphthyl)ethylamine;

[0028] (2) catalyzes N-tert-butyloxycarbonyl-3-piperidone to produce (R)-1-tert-butyloxycarbonyl-3-aminopiperidine;

[0029] (3) Catalyze tert-butyl 3-oxopyrrolidine-1-carboxylate to generate (R)-1-tert-butyloxycarbonyl-3-aminopyrrolidine.

[0030] The present invention also provides a method for catalyzing 1-acetylnaphthalene to generate (R)-(+)-1-(1-naphthyl)ethylamine, wherein 1-acetylnaphthalene and (R)-(+)-α-methylbenzylamine are used as substrates, respectively, and the above-mentioned ω-transaminase ancestral enzyme mutant or the above-mentioned genetically engineered bacteria are used to catalyze a transamination reaction to generate (R)-(+)-1-(1-naphthyl)ethylamine.

[0031] The present invention also provides a method for catalyzing N-tert-butyloxycarbonyl-3-piperidone to generate (R)-1-tert-butyloxycarbonyl-3-aminopiperidine, wherein N-tert-butyloxycarbonyl-3-piperidone and (R)-(+)-α-methylbenzylamine are used as substrates, and the above-mentioned ω-transaminase ancestral enzyme mutant or the above-mentioned genetically engineered bacteria are used to catalyze a transamination reaction to generate (R)-1-tert-butyloxycarbonyl-3-aminopiperidine.

[0032] The present invention also provides a method for catalyzing tert-butyl 3-oxopyrrolidine-1-carboxylate to generate (R)-1-tert-butyloxycarbonyl-3-aminopyrrolidine, wherein tert-butyl 3-oxopyrrolidine-1-carboxylate and (R)-(+)-α-methylbenzylamine are used as substrates, and the above-mentioned ω-transaminase ancestral enzyme mutant or the above-mentioned genetically engineered bacteria are used to catalyze a transamination reaction to generate (R)-1-tert-butyloxycarbonyl-3-aminopyrrolidine.

[0033] Compared with the prior art, the present invention has the following beneficial effects:

[0034] (1) Compared with the ancestral enzyme Ancata-101, the activity of mutant Ancata-1016 towards 1-acetylnaphthalene, N-tert-butyloxycarbonyl-3-piperidone, and tert-butyl 3-oxopyrrolidine-1-carboxylate was 0.106 U / mg, 0.495 U / mg, and 0.199 U / mg, respectively, while that of the ancestral enzyme Ancata-101 was only 0.016 U / mg, 0.012 U / mg, and ND (not detected). The activity of Ancata-1016 was significantly improved.

[0035] (2) Using structure-guided pocket engineering strategies, we screened for ω-aminotransferase mutants with significantly enhanced activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Schematic diagram of the catalytic process of ω-aminotransferase relying on the coenzyme PLP to reversibly catalyze the transfer of amino groups to prochiral ketones to prepare chiral amine compounds.

[0037] Figure 2 The figure shows the enzyme activity assay results of wild-type ω-aminotransferase, ancestral enzyme Ancata-101 and mutant Ancata-1016.

[0038] Figure 3 The figure shows the conversion rate measurement results using wild-type ω-aminotransferase, ancestral enzyme Ancata-101 and mutant Ancata-1016 as catalyzers; wherein a is the conversion rate result of 1-acetylanthracene; b is the conversion rate result of N-tert-butyloxycarbonyl-3-piperidone; and c is the conversion rate result of tert-butyl 3-oxopyrrolidine-1-carboxylate. DETAILED DESCRIPTION

[0039] The schematic diagram of the catalytic process of the ω-aminotransferase of the present invention reversibly catalyzing the transfer of amino groups to prochiral ketones to prepare chiral amine compounds is shown in the figure. Figure 1 The specific embodiments are shown below.

[0040] Example 1

[0041] The amino acid sequence of the wild-type ω-aminotransferase (R)-ω-TA from Aspergillus terreus is shown in SEQ ID NO.2, and the gene sequence is shown in SEQ ID NO.1; the amino acid sequence of its ancestral enzyme Ancata-101 is shown in SEQ ID NO.4, and the gene sequence is shown in SEQ ID NO.3.

[0042] The structure of Ancata-101 was analyzed, and the amino acid sites near the substrate binding pocket were selected for saturation iterative mutation. Finally, a mutant with significantly improved activity was obtained, named Ancata-1016. The mutation site and sequence are as follows:

[0043] Ancata-1016: H55T-E117S-R128M-V150A-L183F-L188F, the gene sequence is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.6.

[0044] Example 2

[0045] (1) Materials and reagents

[0046] The genes of (R)-ω-TA and Ancata-101 were synthesized by Anhui General Biotechnology Co., Ltd., the vector was pET-28a(+), and the expression host strain was E. coli BL21(DE3); the gene of Ancata-1016 was mutated from Ancata-101. Isopropyl-β-D-thiogalactoside (IPTG), kanamycin sulfate, pyrodoxal-5'-phosphate (PLP), and modified Bradford protein concentration assay kit were purchased from Shanghai Biotechnology Co., Ltd.; Ni-NTA chromatography medium was purchased from Beijing Quanshijin Biotechnology Co., Ltd.; dimethyl sulfoxide (DMSO), 1-acetylnaphthalene, N-tert-butyloxycarbonyl-3-piperidone, tert-butyl 3-oxopyrrolidine-1-carboxylate, and (R)-α-methylbenzylamine were purchased from Aladdin Biochemical Technology Co., Ltd.

[0047] (2) Combined mutation

[0048] The selected 17 sites (M54, H55, Y60, D61, V62, R79, F115, E117, R128, V148, W149, V150, L183, L188, E237, V239, and T240) were subjected to saturation mutagenesis. The primers are shown in Table 1 , where N represents A / T / C / G, K represents G / T, and M represents A / C.

[0049] Table 1 Primers and their sequences

[0050]

[0051] Note: The underline indicates the mutation site, the primer name with -F indicates the upstream primer, and the primer name with -R indicates the downstream primer.

[0052] PCR amplification was performed using pET-28a-ω-Ancata-101 plasmid as a template. The PCR amplification system was 25 μL, including: 12.5 μL Prime STAR Max DNA Polymerase, 1 μL upstream primer, 1 μL downstream primer, 1 μL plasmid template (100 ng / μL), and high-temperature sterilized ultrapure water to a total volume of 25 μL.

[0053] The PCR amplification procedure was as follows: denaturation at 98°C for 5 min, followed by amplification cycles, i.e., denaturation at 98°C for 15 s, annealing at 55°C for 15 s, and extension at 72°C for 100 s, for a total of 35 cycles, and finally extension at 72°C for 8 min. The PCR product was detected by electrophoresis, and its bands were single and clear.

[0054] The obtained site-specific PCR reaction product was enzymatically digested with DpnI at 37°C for 2 h to eliminate the paternal template. The enzymatic product was transformed into chemically competent cells E. coli BL21 (DE3) using the heat shock method. The transformation solution was coated on LB solid plates containing kanamycin (50 μg / mL) and cultured at 37°C for 12 h to obtain a mutant library.

[0055] The forward mutations obtained by screening are iterated to gradually improve the activity of the transaminase.

[0056] (3) Enzyme expression and purification

[0057] Take 10 μL of wild-type recombinant plasmid bacterial solution, Ancata-101 and mutant bacterial solution and inoculate them into 5 mL of Luria-Bertani liquid medium (LB medium) containing a final concentration of 50 μg / mL Kanamycin, and culture them in a shaking incubator at 37°C and 200 rpm for 12 h. Transfer the bacterial solution to 200 mL of LB liquid medium containing a final concentration of 50 μg / mL Kanamycin at an inoculum volume of 2% (V / V), and continue to culture at 37°C and 200 rpm for 2-3 h. When OD 600 When the p-value reached 0.8, IPTG was added to a final concentration of 0.5 mM, and protein expression was induced at 25°C and 150 rpm. After 20 h of induction, the cells were collected by centrifugation at 6000 rpm and 4°C.

[0058] The bacterial cells were washed once with 50mM PBS buffer (50mM sodium dihydrogen phosphate, 50mM sodium hydrogen phosphate, 300mM sodium chloride, pH 8.0) to remove the residual culture medium and then resuspended in the above PBS buffer. The bacterial cells were homogenized under ice bath conditions. The cell lysis solution was centrifuged at 8000rpm and 4℃ for 1h, and the supernatant collected was the crude enzyme solution containing ω-transaminase. Subsequently, the crude enzyme solution was filtered with a 0.45μm filter membrane and the target protein was separated and purified using a Ni-NTA affinity chromatography column.

[0059] The purification buffer is as follows:

[0060] 20 mM imidazole wash buffer: 50 mM sodium dihydrogen phosphate, 300 mM sodium chloride, 20 mM imidazole, pH 8.0;

[0061] 50 mM imidazole wash buffer: 50 mM sodium dihydrogen phosphate, 300 mM sodium chloride, 50 mM imidazole, pH 8.0;

[0062] 250 mM Imidazole Elution Buffer: 50 mM Sodium Hydrogen Phosphate (NaH2PO4), 300 mM Sodium Chloride, 250 mM Imidazole, pH 8.0.

[0063] Specific purification steps:

[0064] 1) Equilibrate the Ni-NTA affinity chromatography column: wash with 20% (V / V) ethanol aqueous solution, deionized water and 20 mM imidazole washing buffer, respectively, for 3 column volumes.

[0065] 2) Loading: The crude enzyme solution is taken by syringe and filtered through a 0.45 μm filter membrane. The target protein with 6-histidine tags can bind to the filler.

[0066] 3) Washing: Wash with 3 column volumes of 20 mM imidazole washing buffer and 50 mM imidazole washing buffer respectively, and use Bradford solution to detect whether the impurities are washed away.

[0067] 4) Elution: Wash with 250 mM imidazole elution buffer and collect 5 mL of flow-through.

[0068] 5) Column preservation: wash with 20 mM imidazole washing buffer, deionized water and 20% (V / V) ethanol aqueous solution for 3 column volumes each in sequence, and finally preserve in 20% (V / V) ethanol aqueous solution.

[0069] (4) Determination of enzyme activity

[0070] 100 μL of pure enzyme was mixed with 400 μL of substrate solution (0.1 mM PLP, 5 mM 1-acetylnaphthalene, N-tert-butyloxycarbonyl-3-piperidone or tert-butyl 3-oxopyrrolidine-1-carboxylate, 5 mM (R)-α-MBA ((R)-(+)-α-methylbenzylamine, 0.20% DMSO, 50 mM PBS, pH 5.0). 8.0) was reacted at 20°C for 5 min, then the reaction was terminated with 50% (V / V) acetonitrile and the amount of product generated was determined by high performance liquid chromatography. Enzyme activity (U) is defined as the amount of enzyme required per minute for transaminase to catalyze the transamination reaction of substrate (1-acetylnaphthalene, N-tert-butyloxycarbonyl-3-piperidone or tert-butyl 3-oxopyrrolidine-1-carboxylate) and (R)-α-MBA to produce 1 μmol product ((R)-(+)-1-(1-naphthyl)ethylamine, (R)-1-tert-butyloxycarbonyl-3-aminopiperidine or (R)-1-tert-butyloxycarbonyl-3-aminopyrrolidine) under certain conditions.

[0071] The enzyme activity assay results of wild type (WT), Ancata-101 and mutant (Ancata-1016) are shown in Figure 2 As shown, the enzyme activity of Ancata-1016 was significantly increased compared with that of WT and Ancata-101.

[0072] (5) Determination of conversion rate

[0073] The engineered bacteria containing wild-type enzyme, Ancata-101 and mutant recombinant expression plasmid genes were used to catalyze the transformation of 1-acetylnaphthalene, N-tert-butyloxycarbonyl-3-piperidone or tert-butyl 3-oxopyrrolidine-1-carboxylate to generate the corresponding products (R)-(+)-1-(1-naphthyl)ethylamine, (R)-1-tert-butyloxycarbonyl-3-aminopiperidine or (R)-1-tert-butyloxycarbonyl-3-aminopyrrolidine, and the conversion rate at different reaction times was determined by high performance liquid chromatography. The reaction system contained 50 mM PBS, 10 mM substrate, 0.1 mM PLP, 20% DMSO and 2 g wet bacteria, and the total volume was 50 mL.

[0074] The results of the conversion rate of the wild type (WT), Ancata-101 and mutant (Ancata-1016) engineering bacteria on the three substrates are shown in Figure 3 As shown in the figure, after 6 hours of reaction, the conversion rates of Ancata-1016 for 1-acetylnaphthalene, N-tert-butyloxycarbonyl-3-piperidone, and tert-butyl 3-oxopyrrolidine-1-carboxylate were 54.7%, 85.2%, and 66.8%, respectively, among which the catalytic efficiency for N-tert-butyloxycarbonyl-3-piperidone reached 81.3% within 40 minutes. In contrast, the catalytic effects of WT and Ancata-101 were poor. After 24 hours of reaction, the conversion rates of WT for the three substrates were 10.15%, 14.4%, and 3.7%; the conversion rates of Ancata-101 for N-tert-butyloxycarbonyl-3-piperidone and tert-butyl 3-oxopyrrolidine-1-carboxylate were similar to those of WT, but the conversion rate of Ancata-101 for 1-acetylnaphthalene reached 45.5%. In summary, the mutant Ancata-1016 has excellent catalytic performance.

Claims

1. A ω-aminotransferase ancestral enzyme mutant, characterized in that: It is obtained by mutation of the ancestral enzyme of ω-aminase from Aspergillus terreus. The amino acid sequence of the ancestral enzyme mutant of ω-aminase is shown in SEQ ID NO.

6.

2. A gene encoding the ω-aminotransferase ancestral enzyme mutant according to claim 1.

3. The gene according to claim 2, characterized in that The gene sequence encoding the ω-aminotransferase ancestral enzyme mutant is shown in SEQ ID NO.

5.

4. A recombinant expression plasmid comprising the gene according to claim 2 or 3.

5. A genetically engineered bacterium comprising the recombinant expression plasmid according to claim 4.

6. Use of the ω-aminotransferase ancestral enzyme mutant according to claim 1, the gene according to claim 2 or 3, or the genetically engineered bacteria according to claim 5 in any of the following catalytic reactions: (1) Catalyzes 1-acetylnaphthalene to produce (R)-(+)-1-(1-naphthyl)ethylamine; (2) catalyzes N-tert-butyloxycarbonyl-3-piperidone to produce (R)-1-tert-butyloxycarbonyl-3-aminopiperidine; (3) Catalyze tert-butyl 3-oxopyrrolidine-1-carboxylate to generate (R)-1-tert-butyloxycarbonyl-3-aminopyrrolidine.

7. A method for producing (R)-(+)-1-(1-naphthyl)ethylamine from 1-acetylnaphthalene, characterized in that: 1-acetylnaphthalene and (R)-(+)-α-methylbenzylamine are used as substrates, and the ω-aminotransferase ancestral enzyme mutant described in claim 1 or the genetically engineered bacteria described in claim 5 are used to catalyze a transamination reaction to generate (R)-(+)-1-(1-naphthyl)ethylamine and acetophenone.

8. A method for generating (R)-1-tert-butyloxycarbonyl-3-aminopiperidine from N-tert-butyloxycarbonyl-3-piperidone, characterized in that: N-tert-butyloxycarbonyl-3-piperidone and (R)-(+)-α-methylbenzylamine are used as substrates, and the ω-aminotransferase ancestral enzyme mutant described in claim 1 or the genetically engineered bacteria described in claim 5 are used to catalyze a transamination reaction to generate (R)-1-tert-butyloxycarbonyl-3-aminopiperidine and acetophenone.

9. A method for producing (R)-1-tert-butyloxycarbonyl-3-aminopiperidine from tert-butyl 3-oxopyrrolidine-1-carboxylate, characterized in that: Using tert-butyl 3-oxopyrrolidine-1-carboxylate and (R)-(+)-α-methylbenzylamine as substrates, the ω-aminotransferase ancestral enzyme mutant described in claim 1 or the genetically engineered bacteria described in claim 5 are used to catalyze a transamination reaction to generate (R)-1-tert-butyloxycarbonyl-3-aminopyrrolidine and acetophenone.

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