Recombinant omega-transaminase catalyst, coding gene thereof and application of recombinant omega-transaminase catalyst in catalytic synthesis of cyclohexylamine

Abstract

The invention belongs to the technical field of enzyme engineering and gene engineering, and provides a recombinant omega-transaminase catalyst, a coding gene thereof and application of the recombinant omega-transaminase catalyst in catalytic synthesis of cyclohexylamine. The amino acid sequence of the gene is as shown in SEQ ID NO: 1. The gene for coding the recombinant omega-transaminase catalyst has a nucleotide sequence as shown in SEQ ID NO: 2. According to the invention, codon optimization is carried out on an omega-transaminase gene to obtain a nucleotide sequence for coding omega-transaminase. The recombinant omega-transaminase obtained by the invention has high selectivity and specific activity on cyclohexanone, 5-methyl-2-hexanone and pyruvic acid. According to the present invention, the novel 2-(4-morpholino-anilino)-N6-cyclohexyladenine can be used as the key raw material for preparing the anti-cancer drug Reversine [1, 2-(4-morpholino-anilino)-N6-cyclohexyladenine] for leukemia and other tumor cells, the catalytic activity is significantly higher than the catalytic activity of the currently reported enzyme, and the application of the production practice is easily achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of enzyme engineering and gene engineering, and specifically relates to a recombinant ω-aminotransferase catalyst, a coding gene thereof and application thereof in catalytic synthesis of cyclohexylamine. Background Art

[0002] Primary amines are a common and widely used platform molecule with great application value in chemical production and life sciences. As one of the representative substances of primary amines, cyclohexylamine is in great demand in chemical production, medicine, food processing and other fields. Cyclohexylamine is a key intermediate in the synthesis of many drugs, such as an anticancer drug "Reversine" [1,2-(4-morpholineanilino)-N6-cyclohexyladenine]; cyclohexylamine sulfonate is a common and inexpensive sweetener widely used as a food additive. With the continuous development of downstream products of cyclohexylamine, the demand for cyclohexylamine in the market is also increasing. At present, the production of cyclohexylamine mainly uses nitrobenzene as raw material for reduction and hydrogenation, which has harsh conditions, difficult to handle by-products, and serious environmental pollution, which does not conform to the concept of green environmental protection.

[0003] Since the enzyme catalytic reaction conditions are mild, highly selective and environmentally friendly, the green enzymatic synthesis of cyclohexylamine is a major trend in the future industry. The ω-aminotransferase that relies on the coenzyme pyridoxal phosphate has excellent properties such as high selectivity and a wide substrate spectrum, making it the most attractive enzyme catalyst. However, most of the current wild-type ω-aminotransferases have many deficiencies in substrate specificity, stability, catalytic efficiency, etc., and cannot meet the needs of industrial applications. Therefore, the discovery and transformation of efficient, stable and low-cost transaminases for catalytic production of cyclohexylamine is of great significance for promoting the development of cyclohexylamine in the domestic market. Summary of the invention

[0004] The purpose of the present invention is to provide a recombinant ω-aminotransferase catalyst, a coding gene thereof and application thereof in catalytic synthesis of cyclohexylamine. The catalyst catalytically synthesizes a high value-added cyclohexylamine.

[0005] The present invention is achieved by the following technical scheme: a recombinant ω-aminotransferase catalyst, whose amino acid sequence is shown in SEQ ID NO: 1.

[0006] The present invention also provides a recombinant ω-aminotransferase gene, which encodes the recombinant ω-aminotransferase catalyst.

[0007] Furthermore, its nucleotide sequence is shown in SEQ ID NO:2.

[0008] The present invention also provides a recombinant vector comprising the recombinant ω-aminotransferase gene.

[0009] Furthermore, the vector is an Escherichia coli expression vector.

[0010] Furthermore, the expression vector is any one of pET-28a, pET-29a, pET-30a, pET-32a, pCold I or pCold II.

[0011] The present invention also provides a recombinant host cell comprising the recombinant vector, wherein the host cell is any one of BL21 (DE3), BL21 Star (DE3), BL21 (DE3) pLysS, C41 (DE3), C43 (DE3), BL21 (DE3) pLysE, Origami series, AD494 (DE3), BL21 Trxb (DE3), and SHuffle T7.

[0012] The present invention further provides an enzyme preparation comprising the recombinant ω-aminotransferase, wherein the enzyme preparation is a crude enzyme liquid, crude enzyme powder or pure enzyme; and is prepared by the following method: expressing the ω-aminotransferase in a host cell to obtain a recombinant cell; and lysing the recombinant cell to obtain a crude enzyme liquid, crude enzyme powder or pure enzyme.

[0013] The invention discloses an application of the recombinant ω-aminotransferase catalyst in catalyzing cyclohexanone to generate cyclohexylamine.

[0014] The specific method is as follows: the amino donor is phenylethylamine, the amino acceptor is cyclohexanone, cyclohexylamine and byproduct acetophenone are generated under the catalysis of the enzyme preparation and the coenzyme pyridoxal phosphate, the reaction temperature is 35°C, and the pH is 8.5 in a 100 mmol / L triethanolamine buffer. The present invention selects bacteria from the genus Pseudomonas putida ( Pseudomonas putida ) was used to obtain a wild protein sequence of ω-transaminase by artificial design and codon optimization using gene synthesis technology. The nucleotide sequence is shown in SEQ ID NO: 2.

[0015] The present invention also provides a recombinant expression vector comprising the nucleotide sequence, such as a conventional plasmid (pET series) in the art, preferably pET-28a. The nucleotide sequence is inserted into pET-28a by molecular biological operations such as enzyme digestion and ligation on the vector to construct a recombinant expression plasmid named pET28a-ω-TA.

[0016] The present invention also provides a recombinant host cell comprising the recombinant expression vector. E .coli BL21(DE3). The recombinant expression plasmid pET28a-TA described in the second aspect of the present invention was transformed into E. coliBL21(DE3) was used to obtain the corresponding genetically engineered bacteria E. coli BL21(DE3) pET28a-TA.

[0017] The present invention also provides a method for preparing the ω-aminotransferase catalyst. The free protein catalyst is a crude enzyme solution obtained by ultrasonically crushing whole cells and centrifuging them, and also includes pure enzymes obtained by protein purification, and also includes crude enzyme powder obtained by drying the crude enzyme solution.

[0018] The present invention also provides the use of the transaminase in the preparation of cyclohexylamine. The structural formula of the compound is as follows Figure 4 As shown, the applications include synthesis of key pharmaceutical intermediates for cancer treatment, food additives and various chemical synthesis raw materials.

[0019] The recombinant aminotransferase catalyst obtained by the invention can catalyze cyclohexanone to synthesize cyclohexylamine, has high affinity with cyclohexanone and high catalytic efficiency, and the specific activity (790 U / mg) of the catalyst is higher than that previously reported. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0021] Figure 1 This is the electrophoresis diagram of protein purification of recombinant ω-aminotransferase TA, in which: M: protein standard marker, 1: whole bacteria sample after disruption; 2: supernatant sample after centrifugation; 3: precipitate sample after centrifugation; 4: penetration sample; 5: washed sample during affinity purification; 6: eluted sample after affinity purification; Figure 2 The chemical reaction equation for the production of cyclohexylamine by recombinant ω-TA; Figure 3 The results of HPLC test for the preparation of cyclohexylamine by recombinant ω-TA. Figure A: HPLC retention time of standard sample phenylethylamine; Figure B: HPLC retention time of standard sample acetophenone; Figure C: HPLC retention time of the product; Figure 4 is the relative activity of recombinant ω-TA in catalyzing the production of cyclohexanone under different temperature conditions; Figure 5 The relative activity of recombinant ω-TA in catalyzing the production of cyclohexanone in different pH and different types of buffers; Figure 6 The thermal stability of ω-TA catalyzed production of cyclohexanone. DETAILED DESCRIPTION

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0024] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0025] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0026] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0027] Example 1: Cloning of ω-aminotransferase gene and construction of recombinant vector: Select Pseudomonas putida ( Pseudomonas putida ) wild protein sequence, using pET-28a as an expression vector, and designing at both ends of the gene sequence of the ω-aminotransferase described in SEQ ID NO: 2 Hind III and Bm I restriction site, and cloned the sequence encoding ω-transaminase by PCR technology.

[0028] Use endonuclease Hind III and BmⅠ The plasmid and the gene cloned in this example were double-digested, and the gene fragments after digestion were recovered by nucleic acid electrophoresis (0.1% agarose) and a kit, and then the target gene fragments after digestion were ligated to the plasmid after digestion.

[0029] A 10 μL ligation system, including 1 μL 10×T4 DNA ligation buffer (Takara), 6 μL target gene fragment, 2 μL plasmid fragment, and 1 μL T4 DNA ligase, was ligated overnight at 16°C and transformed into DH5α competent cells. Single clones were picked for sequencing verification, and the correctly sequenced recombinant plasmid was extracted to obtain a recombinant expression vector containing the transaminase encoding gene, which was named pET28a-TA.

[0030] Example 2: Construction of ω-transaminase genetic engineering bacteria and preparation of cell catalyst: The recombinant expression vector pET28a-TA prepared in Example 1 was transformed into competent cells using the heat shock method. E. coli BL21 (DE3), coated with LB plates containing kanamycin (50 μg / mL), cultured at 37°C overnight, picked a single colony and transferred it to LB liquid culture containing 50 μg / mL kanamycin, cultured at 37°C for 12 hours, and sent the sample for gene sequencing. The genetically engineered bacteria containing the recombinant expression vector pET28a-TA were named E. coli BL21(DE3) pET28a-TA.

[0031] The genetically engineered bacteria containing the coding sequence of ω-aminotransferase were inoculated into LB liquid medium containing kanamycin resistance (10 g / L peptone, 5 g / L yeast extract, 10 g / L sodium chloride, the solvent is deionized water, pH = 7.0), and placed in a test tube containing LB medium (5 mL, containing kanamycin at a final concentration of 50 μg / mL) in a 37°C shaker, and cultured at 200 rpm for 10 to 12 hours to obtain a seed solution.

[0032] In the clean bench, transfer the seed solution in the test tube to a shake flask containing LB medium (100 mL, containing kanamycin at a final concentration of 50 μg / mL), place it in a 37°C shaker, and culture it at 200 rpm for 2 to 3 hours. 600 When the value reaches 0.6 to 0.8, add IPTG with a final concentration of 0.1 to 0.8 mM to induce expression, and the induction temperature is 16 to 37° C. The preferred IPTG concentration is 0.2 mM, and the preferred induction temperature is 16° C. After induction for 24 hours under the preferred conditions, the cells are collected by centrifugation to obtain recombinant cells containing ω-aminotransferase.

[0033] Example 3: Purification of ω-transaminase protein: Weigh 10 g of the frozen transaminase wet cells in Example 2, add them to 100 mL of buffer A (25 mM Tris-HCl, pH 8.0; 300 mM NaCl), place them at room temperature to thaw, and then use an ultrasonic disruptor to disrupt the cells. The horn model is Φ10, the ultrasonic power is 100 W, the ultrasound is turned on for 4 seconds, and the ultrasound is turned off for 6 seconds. During the disruption process, a low-temperature circulation device is used to cool down, and the ultrasonic disruption is performed for 15 minutes. The collected disrupted liquid is centrifuged and the supernatant is collected.

[0034] Assemble the protein purification device, self-assembled nickel column (10mL Sepharose 6Fast Flow, GE) and collection device. Pass the collected supernatant through a 0.22 μm filter membrane and then add it to the equilibrated self-assembled nickel column. Bind for 0.5h at 4°C, then collect the permeate in the nickel column, and then wash the impurities in the nickel column with washing buffer (25mM Tris-HCl, pH 8.0; 300mM NaCl; 20mM imidazole). Finally, add 15ml of elution buffer (25mM Tris-HCl, pH 8.0; 300mM NaCl; 500mM imidazole), let it stand for 10min, and then collect the eluate. The collected eluate was filtered through a 0.22um filter membrane and then loaded onto a Superdex200 gel column equilibrated with 25mM Tris-HCl, pH 8.0; 300mM NaCl buffer to collect the protein sample, which was then concentrated by centrifugation at 4°C, 4000rpm using a 10-30kDa ultrafiltration tube. After concentration, the concentration of the pure protein was determined by the CBB method, and the protein was quickly frozen in liquid nitrogen and placed in a ‑80°C refrigerator. The purity of the target protein was analyzed by SDS-PAGE, as shown in Figure 2. Figure 1 shown.

[0035] Example 4: Determination of ω-aminotransferase, i.e., ω-TA enzyme activity and specific activity: Preparation of the product acetophenone standard curve, prepare 8.3uM, 16.6uM, 24.9uM, 33.2uM, 41.6uM, 83.2uM and 124.8uM standard acetophenone aqueous solutions respectively, mix them well and add them to a 1ml quartz cuvette, and measure the absorbance value at a wavelength of 245nm using an ultraviolet spectrophotometer. Draw a standard curve of acetophenone concentration and its absorbance (y=0.0003471x+0.04378, R 2 =0.9908).

[0036] The pure enzyme stored in the ‑80°C refrigerator described in Example 3 was taken and placed on ice to dissolve. The final concentration of the pure enzyme was 8 mg / mL. 400 μL of the dilution was added to 543 μL of Tris-HCl buffer (pH 8.0, 0.1 M), and then 50 μL of pyridoxal phosphate PLP (10 mM) and 5 μL of phenylethylamine (40 mM, pH 8.0) were added in sequence. After preheating on an oscillator (30°C, 1000 rpm) for 2 min, 2 μL of substrate phenylethylamine (20 mM) was added and reacted for 2 h, 100 μL of HCl (6 M) was added for quenching, and then an equal volume of n-butanol or ethyl acetate was added for extraction, and the organic phase was collected by centrifugation and dried. Add 1 ml of Tris-HCl buffer (pH 8.0, 0.1 M), dilute 100 times, and measure the absorbance of the product acetophenone at a wavelength of 245 nm using an ultraviolet spectrophotometer. Calculate the specific activity of the transaminase based on the amount of acetophenone added, the reaction time, and the amount of enzyme added.

[0037] ω-transaminase enzyme activity and specific activity calculation: enzyme activity (U) = (A 245nm x dilution factor - 0.04378) / 0.0003471*time (h); specific activity A (U / g) = U / m (g), where m is the mass of pure enzyme involved in the transamination reaction.

[0038] The ω-TA derived from Pseudomonas YZ-26 can be efficiently expressed in Escherichia coli, and about 400 mg of total protein can be harvested per liter of ordinary fermentation. The recombinant transaminase catalyst obtained by the present invention realizes the bioenzymatic catalytic synthesis of cyclohexylamine for the first time, and the specific activity reaches 790 U / g.

[0039] Example 5: HPLC determination of cyclohexylamine catalyzed by recombinant ω-TA: With phenylethylamine as the amino donor and cyclohexanone as the amino acceptor, cyclohexylamine and byproduct acetophenone were generated under the catalysis of ω-TA and coenzyme pyridoxal phosphate (PLP). Figure 2 shown.

[0040] Take 10 μL of standard acetophenone and 10 μL of standard cyclohexylamine and dissolve them in 1 mL of acetonitrile to prepare 1% standard acetophenone sample and 1% standard cyclohexylamine sample, take 1 mL of the sample after ω-TA catalytic reaction, extract it three times with the same volume of isoamyl acetate and dry it, add 1 mL of acetonitrile to dissolve it, filter the standard and reaction sample with a 0.22 μm water filter membrane, and use HPLC to detect: the chromatographic column used is Hypersil GOLD (5μm, 250x4.6), the mobile phase is: acetonitrile: water = 80:20, the flow rate is 1 ml / min, and UV detection is carried out at 245 nm. The experimental results are as follows Figure 3 As shown in A, the results show that the retention time of cyclohexylamine standard is 2.193 min ( Figure 3 A), the retention time of the standard acetophenone is 3.862 min ( Figure 3 B), after adding ω-TA, peaks appeared at similar retention times (2.202 min and 3.863 min) ( Figure 3 C), indicating that the ω-TA catalyzed product formed cyclohexylamine and the by-product acetophenone, and ω-TA has the ability to generate cyclohexylamine.

[0041] Example 6: Determination of the optimal temperature for the recombinant ω-TA to catalyze the production of cyclohexylamine: Using phenylethylamine as the amino donor and cyclohexanone as the amino acceptor as the bisubstrate, the activity of the recombinant transaminase was determined at different reaction temperatures (specifically 25°C, 30°C, 35°C, 40°C, and 45°C) according to the method described in Example 4, while other conditions remained unchanged. The results are shown in Figure 4 As shown, the relative enzyme activity of ω-TA was the highest when the reaction temperature was 30°C, and under this condition, the activity of ω-TA in catalyzing the production of cyclohexylamine was the highest.

[0042] Example 7: Determination of the optimal pH for the catalytic production of cyclohexylamine by recombinant ω-TA: Using phenylethylamine as the amino donor and cyclohexanone as the double substrate of the amino acceptor, the activity was determined in accordance with the method described in Example 4 using different types of buffers at different pH values ​​(specifically, sodium dihydrogen phosphate-citric acid buffers at pH = 5.0, pH = 5.5, pH = 6.0, pH = 6.5, and pH = 7.0; triethanolamine buffers at pH = 7.0, pH = 7.5, pH = 8.0, pH = 8.5; and Gly-NaOH buffers at pH = 8.5, pH = 9, pH = 9.5, and pH = 10), while other conditions remained unchanged. The results are shown in Table 4. Figure 5 As shown, the relative enzyme activity of ω-TA is the highest in triethanolamine buffer at pH 8.0, and the activity of catalyzing the production of cyclohexylamine is the highest under this condition.

[0043] Example 8: Determination of thermal stability of cyclohexylamine produced by recombinant ω-TA: ω-TA was incubated in a water bath at 30°C, 40°C, and 50°C, and the ω-TA enzyme solution was added to the activity test system every 12 hours. The reaction was carried out at the optimal temperature for 1 hour. Three parallel controls were set up for each group, and the enzyme activity of ω-TA in each group was measured to analyze the stability of the transaminase at different temperatures. The results are as follows: Figure 6 As shown, when incubated at 50°C, the insulation time when the enzyme activity loses half is about 12 h, which has good thermal stability and the half-life is much longer than the reported 2 h. (Yang Xiaoxiao, Li Jing, Zhang Chaofeng, et al. Synthesis of chiral β-amino alcohols catalyzed by Bacillus megaterium ω-aminotransferase [J]. Journal of Chemical Engineering of Colleges and Universities, 2021, 35(6):6.).

[0044] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A recombinant ω-aminotransferase catalyst, characterized in that: Its amino acid sequence is shown in SEQ ID NO:

1.

2. A recombinant ω-aminotransferase gene, characterized in that: Encoding the recombinant ω-aminotransferase catalyst according to claim 1.

3. The recombinant ω-aminotransferase gene according to claim 2, characterized in that: Its nucleotide sequence is shown in SEQ ID NO:

2.

4. A recombinant vector comprising the recombinant ω-aminotransferase gene according to claim 2 or 3, characterized in that: The vector is an Escherichia coli expression vector.

5. The recombinant vector according to claim 4, characterized in that: The expression vector is any one of pET-28a, pET-29a, pET-30a, pET-32a, pCold I or pCold II.

6. The recombinant vector according to claim 5, characterized in that: The expression vector is pET-28a.

7. A recombinant host cell comprising the recombinant vector according to claim 4, characterized in that: The host cell is any one of BL21(DE3), BL21 Star(DE3), BL21(DE3) pLysS, C41(DE3), C43(DE3), BL21(DE3) pLysE, Origami series, AD494(DE3), BL21 Trxb(DE3), and SHuffle T7.

8. The recombinant host cell comprising a recombinant vector according to claim 7, characterized in that: The host cell is E. coli BL21(DE3).

9. An enzyme preparation comprising the recombinant ω-aminotransferase according to claim 1, characterized in that: The enzyme preparation is a crude enzyme liquid, crude enzyme powder or pure enzyme; it is prepared by the following method: expressing the ω-aminotransferase in a host cell to obtain a recombinant cell; and lysing the recombinant cell to obtain a crude enzyme liquid, crude enzyme powder or pure enzyme.

10. Use of the recombinant ω-aminotransferase catalyst according to claim 1 in catalyzing cyclohexanone to produce cyclohexylamine, characterized in that: The specific method is as follows: the amino donor is phenylethylamine, the amino acceptor is cyclohexanone, cyclohexylamine and by-product acetophenone are generated under the catalysis of recombinant ω-aminotransferase and coenzyme pyridoxal phosphate, the reaction temperature is 35°C, and the pH is 8.5 in 100 mmol / L triethanolamine buffer.

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