Carboxylic acid reductase mutants and their applications in the synthesis of amino fatty acids

Abstract

The present invention belongs to the field of bioengineering technology, and in particular relates to a carboxylic acid reductase mutant, a nucleic acid encoding the carboxylic acid reductase mutant, a recombinant expression vector containing the nucleic acid, a recombinant expression transformant containing the recombinant expression vector, the preparation of a mutant enzyme catalyst, and the application of the mutant enzyme catalyst in the synthesis of amino fatty acids. Compared with other biocatalysts for preparing medium- and long-chain amino fatty acids, the carboxylic acid reductase mutant provided by the present invention has the advantages of a broad substrate spectrum, high catalytic activity, and strong substrate specificity, and shows broad application prospects in industrial applications.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioengineering, and in particular relates to a carboxylic acid reductase mutant, a nucleic acid encoding the carboxylic acid reductase mutant, a recombinant expression vector containing the nucleic acid, a recombinant expression transformant containing the recombinant expression vector, the preparation of a mutant enzyme catalyst, and the application of the mutant enzyme catalyst in the synthesis of amino fatty acids. Background Art

[0002] Nylon is widely used in the textile industry, automotive industry, electronic and electrical industry, mechanical equipment, construction industry, etc., and nylon 6 accounts for a large proportion of nylon. 6-Aminohexanoic acid (6-ACA) is the monomer of nylon 6 (with an annual global output of nearly 7 million tons), and the market demand is huge. Currently, it is mainly synthesized by chemical methods, but chemical synthesis methods generally have disadvantages such as harsh reaction conditions, expensive catalysts, and large environmental pollution. In order to respond to the country's call for clear waters and green mountains and green bio-manufacturing, choosing an enzyme catalyst with good catalytic performance and mild reaction conditions for the synthesis of 6-aminohexanoic acid is a very promising synthetic route.

[0003] Currently, a variety of different bio-enzymatic methods have been reported for the synthesis of 6-aminohexanoic acid. As early as 2014, Sattler et al. used cyclohexanol (50 mM) as the starting substrate and synthesized 6-aminohexanoic acid through multi-enzyme cascade catalysis under the conditions of only consuming oxygen and ammonia (Angewandte Chemie, 2014, 126(51): 14377-14381), but the final yield was only 24%. Subsequently, in 2018, Hyungdon Yun et al. from Konkuk University in South Korea used 6-hydroxy fatty acid as the substrate and synthesized 6-aminohexanoic acid through cascade catalysis of aldehyde reductase and transaminase, with the highest yield reaching 96.7%, but the substrate of this route was still relatively expensive (Green Chemistry, 2018, 20(20): 4591-4595). In 2020, Fedorchuk et al. used adipic acid as the substrate and converted adipic acid into 6-aminohexanoic acid by using carboxylic acid reductase (CARs) and transaminase (ω-TAs) (Journal of the American Chemical Society, 2019, 142(2): 1038-1048), but the substrate concentration in the cascade reaction process was only 10 mM, and at the same time, due to the poor substrate specificity of the carboxylic acid reductase, the product was further converted into 6-oxohexylamine, so the yield of 6-aminohexanoic acid was low. In summary, there is an urgent need to obtain a carboxylic acid reductase with high conversion efficiency for the substrate adipic acid and strong substrate specificity for the efficient synthesis of medium and long-chain amino fatty acids such as 6-aminohexanoic acid. Summary of the Invention

[0004] In view of the problems such as low catalytic activity and poor substrate specificity of carboxylic acid reductase in the system of synthesizing medium- and long-chain amino fatty acids (C6-C12) by biocatalytic conversion of dicarboxylic acids at present, the present invention provides a carboxylic acid reductase (NCBI Reference Sequence: WP_198151610.1) derived from Kibdelosporangium sp. MJ126-NF4 and its mutant gene. The amino acid sequence of this enzyme has only 53.8% homology with the carboxylic acid reductase MabCAR derived from Mycobacterium abscessus. The present invention also provides a recombinant expression vector and a recombinant expression transformant containing this gene, a preparation method of the carboxylic acid reductase mutant and a culture of the recombinant transformant capable of expressing the carboxylic acid reductase mutant, and an application of the carboxylic acid reductase mutant or the culture of the recombinant transformant in catalyzing the synthesis of medium- and long-chain amino fatty acids from dicarboxylic acids.

[0005] Compared with other carboxylic acid reductases, the carboxylic acid reductase mutant provided by the present invention has higher catalytic efficiency and substrate specificity for the substrate dicarboxylic acid, and thus has better synthesis effect of medium- and long-chain amino fatty acids.

[0006] The object of the present invention can be achieved by the following technical solutions:

[0007] One of the technical solutions of the present invention is:

[0008] Provide a carboxylic acid reductase mutant, that is, provide an isolated protein, which is a protein corresponding to a new amino acid sequence formed by replacing one or more amino acid residues at positions 276 Ile, 299 Gly, 335 Leu, 386 Thr, and 411 Gly in the amino acid sequence shown in SEQ ID No. 2 with other amino acid residues. The mutant protein has significantly improved activity against α,ω-dicarboxylic acids and also improved substrate specificity.

[0009] The protein having the amino acid sequence shown in SEQ ID No. 2 is named KiCAR. The nucleotide sequence of the nucleic acid encoding KiCAR is shown in SEQ ID No. 1.

[0010] Preferably, the amino acid sequence of the carboxylic acid reductase mutant is one of the following:

[0011] (1) Replace isoleucine Ile at position 276 in the amino acid sequence shown in SEQ ID No. 2 with proline Pro;

[0012] (2) Replace isoleucine Ile at position 276 in the amino acid sequence shown in SEQ ID No. 2 with arginine Arg;

[0013] (3) Replace glycine (Gly) at position 299 in the amino acid sequence shown in SEQ ID No. 2 with isoleucine (Ile).

[0014] (4) Replace glycine (Gly) at position 299 in the amino acid sequence shown in SEQ ID No. 2 with lysine (Lys).

[0015] (5) Replace glycine (Gly) at position 299 in the amino acid sequence shown in SEQ ID No. 2 with arginine (Arg).

[0016] (6) Replace leucine (Leu) at position 335 in the amino acid sequence shown in SEQ ID No. 2 with lysine (Lys).

[0017] (7) Replace leucine (Leu) at position 335 in the amino acid sequence shown in SEQ ID No. 2 with arginine (Arg).

[0018] (8) Replace threonine (Thr) at position 386 in the amino acid sequence shown in SEQ ID No. 2 with lysine (Lys).

[0019] (9) Replace threonine (Thr) at position 386 in the amino acid sequence shown in SEQ ID No. 2 with arginine (Arg).

[0020] (10) Replace glycine (Gly) at position 411 in the amino acid sequence shown in SEQ ID No. 2 with alanine (Ala).

[0021] (11) Replace glycine (Gly) at position 411 in the amino acid sequence shown in SEQ ID No. 2 with arginine (Arg).

[0022] (12) Replace glycine (Gly) at position 411 in the amino acid sequence shown in SEQ ID No. 2 with lysine (Lys).

[0023] (13) Replace glycine (Gly) at position 411 in the amino acid sequence shown in SEQ ID No. 2 with glutamic acid (Glu).

[0024] The second technical solution of the present invention is to provide a nucleic acid encoding the carboxylic acid reductase mutant.

[0025] The nucleic acid encodes any one of the carboxylic acid reductase mutants described in Technical Solution 1.

[0026] The nucleotide sequence encoding the carboxylic acid reductase mutant is the nucleic acid sequence encoding the carboxylic acid reductase mutant described in Technical Solution 1.

[0027] The third technical solution of the present invention is: to provide a recombinant expression vector.

[0028] The recombinant expression vector contains the nucleic acid as described in the second technical solution.

[0029] The recombinant expression vector can be obtained by connecting the coding nucleic acid sequence of the carboxylic acid reductase mutant of the present invention to the pET30b plasmid by conventional methods in the art.

[0030] Furthermore, in order to successfully express the carboxylic acid reductase mutant and exhibit catalytic activity, the coding nucleic acid sequence of the phosphopantetheinyl transferase Bssfp (NCBI Reference Sequence: WP_003234549.1) derived from Bacilus subtilis needs to be connected to the multiple cloning site I (MCS I) of the pCDFDuet plasmid.

[0031] The fourth technical solution of the present invention is: to provide a recombinant expression transformant.

[0032] The recombinant expression transformant contains the recombinant expression vector as described in the third technical solution. The recombinant expression transformant can be prepared by simultaneously transforming the two recombinant expression vectors of the present invention into the Escherichia coli E.coli BL21(DE3) host cell by conventional methods in the art.

[0033] The fifth technical solution of the present invention is: to provide a carboxylic acid reductase mutant catalyst, and the recombinant carboxylic acid reductase mutant catalyst is any one of the following forms:

[0034] (1) Culturing the recombinant expression transformant of the present invention and separating the transformed cells containing the carboxylic acid reductase mutant and the phosphopantetheinyl transferase sfp;

[0035] (2) Disrupting the transformed cells as described in (1) and separating the crude enzyme solution containing the carboxylic acid reductase mutant;

[0036] (3) Purifying the crude enzyme solution as described in (2) to obtain a pure enzyme;

[0037] (4) The carboxylic acid reductase mutant as described in the first technical solution.

[0038] Among them, for obtaining the recombinant carboxylic acid reductase mutant catalyst, the culturing methods and conditions of the recombinant expression transformant are conventional methods and conditions in the art.

[0039] In some embodiments, the method for culturing the recombinant expression transformant may include the following steps: culturing the recombinant expression transformant of the present invention to obtain a recombinant carboxylic acid reductase mutant. For recombinant Escherichia coli, the preferred medium is LB medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH 6.5 - 7.0. The preferred culturing method is: inoculating the recombinant Escherichia coli constructed as described above into an LB medium containing Streptomycin and Kanamycin, and culturing it overnight at 37°C with shaking at 180 rpm. Inoculate the LB medium (containing Streptomycin and Kanamycin) at an inoculation amount of 1% (v / v) and culture it on a shaker at 37°C with shaking at 180 rpm. When the OD of the culture broth reaches 0.6 - 0.8, add isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.2 mM as an inducer and induce it at 16°C for 16 - 24 h. Centrifuge the culture broth, wash the precipitate twice with physiological saline to obtain recombinant expression transformant cells. Store the harvested recombinant cells in a -80°C refrigerator, or suspend the harvested recombinant cells in 5 - 10 times the volume (v / w) of buffer, ultrasonically disrupt them, centrifuge to collect the supernatant, and thus obtain the crude enzyme solution of the recombinant carboxylic acid reductase mutant.

[0040] The sixth technical solution of the present invention is: to provide a purification method for the carboxylic acid reductase mutant. Further purify the crude enzyme solution of the recombinant carboxylic acid reductase mutant obtained as in Technical Solution Five. The following is the formulation of the protein purification buffer: Buffer A: 25 mM Hepes-Na, pH 7.5, 500 mM NaCl, 20 mM imidazole; Buffer B: 25 mM Hepes-Na, pH 7.5, 500 mM NaCl, 500 mM imidazole; Buffer C: 25 mM Hepes-Na, pH 7.5, 150 mM NaCl, 1 mM DTT, 5% glycerol. First, load the crude enzyme solution of the carboxylic acid reductase mutant obtained by disruption onto a nickel column equilibrated with Solution A, then elute the miscellaneous proteins with 10% Solution B, then elute the target protein with 50% Solution B, and use SDS-PAGE to verify whether the target protein is contained. Then collect the eluate containing the target protein, ultrafiltrate and concentrate it using an ultrafiltration tube (30 kDa), and then replace it twice with Solution C to remove the imidazole in the protein solution. Measure the activity and substrate specificity of the purified protein, then aliquot it, quickly freeze it in liquid nitrogen, and store it at -80°C for standby.

[0041] The seventh technical solution of the present invention is: to provide the application of the recombinant carboxylic acid reductase mutant catalyst as described in Technical Solution Five in the catalytic synthesis of long-chain amino fatty acids from α,ω-dicarboxylic acids.

[0042] Schematic diagram of the synthesis of medium- and long-chain amino fatty acids by multi-enzyme cascade catalysis of α,ω-dicarboxylic acids is as follows Figure 1 shown

[0043] In some embodiments of the present invention, the medium- and long-chain α,ω-dicarboxylic acid substrates are adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, undecanedioic acid, and dodecanedioic acid.

[0044] In some embodiments of the present invention, the enzymatic reaction is carried out in a Hepes-Na buffer at pH 7.5 and 30 °C. The reaction system includes α,ω-dicarboxylic acid (C6-C12) with a final concentration of 5 mM, 0.5% (v / v) DMSO, 1 mM NADPH, 10 mM ATP, 10 mM MgCl 2 , 0.05 mM PLP, 30 mM glucose, and 30 mM D / L-alanine, as well as 0.05 - 0.2 U / mL of the recombinant carboxylic acid reductase mutant described in Technical Solution Five, 0.06 - 0.25 U / mL of transaminase, and 0.2 - 1 U / mL of glucose / formate dehydrogenase.

[0045] Preferably, the reaction is carried out in a shaking reactor (1000 rpm), and the reaction time is based on the complete conversion of the substrate or the concentration of the product no longer increasing. During the reaction process, 50 - 100 μL of the reaction solution is sampled at intervals, and 5 μL of 6 M HCl is added to terminate the reaction. The product amino fatty acid is detected by high performance liquid chromatography. The sample treatment method is as follows: Take 20 μL of the sample, add 20 μL of 1 M NaOH and 20 μL of acetonitrile, mix well, place it in a -20 °C refrigerator and freeze for more than half an hour, then centrifuge at 1000 rpm for 1 min. Take 20 μL of the supernatant, add 36 μL of 1 M NaHCO 3 , 20 μL of 28 mM Marfey, and 100 μL of DMSO, mix well, stand at 40 °C for 1 h for derivatization, and finally add 40 μL of 1 M HCl to quench, mix well and filter through an organic nylon membrane.

[0046] The specific analysis conditions are as follows:

[0047] The chromatographic column is Elite C18, the mobile phase is methanol: water (0.1% trifluoroacetic acid) = 50:50 (v / v), the flow rate is 1 mL / min; the column temperature is 35 °C, and the ultraviolet detection wavelength is 340 nm.

[0048] The substrate α,ω-dicarboxylic acid was detected by gas chromatography. 30 μL of the sample was added to 300 μL of ethyl acetate (containing internal standard), shaken well for 5 - 10 min, then centrifuged at 12000 rpm for 1 min. 150 μL of the organic phase was taken, anhydrous sodium sulfate was added and shaken for drying for more than 2 h, then centrifuged at 12000 rpm for 1 min. 50 μL of the organic phase was taken and added to an inner tube, 10 μL of trimethylsilyldiazomethane and 50 μL of methanol-ether (v / v = 1:1) were added, and derivatization was carried out at room temperature for 20 min.

[0049] The specific analysis conditions are as follows:

[0050] The chromatographic column was SH-Rxi-5Sil MS column (30 m × 0.25 mm × 0.25 μm), nitrogen was used as the carrier gas, and the flow rate was 3 mL / min. The temperatures of the injector and detector were 250 °C and 280 °C respectively. The specific GC detection program was to hold at 85 °C for 3 minutes, then increase the temperature to 100 °C at a rate of 5 °C / min, then increase the temperature to 160 °C at a rate of 10 °C / min, and finally increase the temperature to 280 °C at a rate of 20 °C / min, and hold at 280 °C for 2 minutes.

[0051] In some embodiments of the present invention, since coenzyme NADPH is oxidized to NADP during the reaction + , it is necessary to use dehydrogenase to catalyze the reduction and regeneration of NADP + to NADPH.

[0052] In some embodiments of the present invention, the dehydrogenase is one of formate dehydrogenase or glucose dehydrogenase.

[0053] Furthermore, the dehydrogenase of the present invention is any one of the following dehydrogenases:

[0054] Formate dehydrogenase FDH (Appl Biochem Biotech 2020, 192, 530–543), using formate and NADP + as substrates, catalyzing the oxidation of formate, and at the same time reducing NADP + to NADPH;

[0055] Glucose dehydrogenase GDH (ChemBioChem 2020, 21:2680–2688), using glucose and NADP + as substrates, catalyzing the oxidation of glucose, and at the same time reducing NADP + to NADPH.

[0056] Compared with the prior art, the technical effects of the present invention are mainly reflected in the following aspects:

[0057] Compared with other enzymes for preparing long-chain amino fatty acids, the carboxylic acid reductase mutant provided by the present invention has the advantages of high catalytic activity, strong substrate specificity, and a wide range of catalytic substrates, showing broad application prospects in industrial applications. Brief Description of the Drawings

[0058] Figure 1 : Schematic diagram of the synthesis of long-chain amino fatty acids in multi-enzyme cascade catalysis of α,ω-dicarboxylic acids. Detailed Embodiments

[0059] The following will combine specific embodiments to clearly and completely describe the technical solutions and technical effects in the present invention, so that those skilled in the art can better understand the present invention and be able to implement it. However, the protection scope of the present invention is not limited to these embodiments.

[0060] Example 1: Preparation of the KiCAR mutant of carboxylic acid reductase

[0061] First, the present invention obtained a gene encoding a carboxylic acid reductase KiCAR (NCBI Reference Sequence: WP_198151610.1) derived from Kibdelosporangium sp. MJ126-NF4 through gene mining. The amino acid sequence encoded by this gene has only 53.8% homology with the carboxylic acid reductase MabCAR derived from Mycobacterium abscessus. The recombinant expression vector pET30b and the recombinant expression transformant E. coli BL21(DE3) containing this gene were obtained. Site-directed mutagenesis was performed on the 276th Ile, 299th Gly, 335th Leu, 386th Thr, and 411th Gly of this enzyme. The site-directed mutagenesis was carried out according to the protocol described in the IISite-Directed Mutegenesis Kit (Stratagenem Catalog#200502). First, mutagenic primers containing the mutation sites were designed. The mutagenic primers containing the mutation sites required in the present invention are common primers in the art, and only need to mutate the amino acid residues in SEQ ID No. 2 into the amino acid residues required in the present invention through general genetic engineering techniques. The specific mutation codes of the nucleic acids are not limited. The introduction of mutations at the 276th, 299th, 335th, 386th, and 411th positions in the shown sequence are all site-directed mutagenic primers.

[0062] PCR reaction system (10 μL): 20 - 50 ng of template, 0.5 μL of each of a pair of mutagenic primers (10 μM), 5 μL of PrimeStar mix, and sterilized double-distilled water was added to make up the system to 10 μL. The template therein is the carboxylic acid reductase (KiCAR, WP_198151610.1, SEQ ID No. 2).

[0063] PCR reaction procedure: (1) Denaturation at 95°C for 3 min; (2) Denaturation at 98°C for 10 s; (3) Annealing at 55°C for 5 s; (4) Extension at 72°C for 9 min; Steps (2)-(4) are extended for 25 cycles in total, and finally extended at 72°C for 10 min, and the product is stored at 4°C.

[0064] The amplified PCR product was digested with restriction endonuclease Dpn I at 37°C for 0.5 - 2 h and then transformed into competent cells of Escherichia coli BL21(DE3), and evenly spread on an LB medium (peptone: 10 g / L, yeast extract: 5 g / L, sodium chloride: 10 g / L, agar powder: 20 g / L) agar plate containing 50 μg / mL kanamycin. After culturing at 37°C for 10 - 16 h, monoclonal colonies were selected, and thus the Escherichia coli BL21(DE3) strain containing the mutant expression plasmid was obtained, and it was sent to Shanghai Qingke Biotechnology Co., Ltd. for sequencing analysis. The sequencing results were compared with the wild-type carboxylic reductase gene sequence using SnapGene software to confirm the differences in the gene sequence and the corresponding amino acid sequence before and after the mutation.

[0065] Example 2: Co-expression of carboxylic reductase KiCAR mutant gene and phosphopantetheinyl transferase sfp gene

[0066] The plasmid was extracted from the Escherichia coli BL21(DE3) strain containing the mutant plasmid using the Qiagen miniprep plasmid extraction kit, and at the same time, the plasmid pCDFDuet expressing sfp was extracted in large quantities from the transformant containing sfp. The obtained dual plasmids were simultaneously chemically transformed into competent cells of Escherichia coli BL21(DE3), and evenly spread on an LB medium agar plate (peptone: 10 g / L, yeast extract: 5 g / L, sodium chloride: 10 g / L, agar powder: 20 g / L) containing 50 μg / mL kanamycin and streptomycin double resistance, and cultured at 37°C for 12 - 18 h. Monoclonal colonies were picked, and thus the expression strain expressing different carboxylic reductase mutants was obtained, and this strain could simultaneously ensure the co-expression of sfp, and its function was to modify the end of the carboxylic reductase to ensure its catalytic efficiency.

[0067] Example 3: Preparation of pure enzymes of KiCAR and its mutants

[0068] The monoclonal bodies obtained in Example 2 were picked into 4 mL of LB medium (containing streptomycin and kanamycin) and cultured with shaking at 37 °C and 180 rpm for 12 - 16 h. Subsequently, they were inoculated into 100 mL of LB medium (containing streptomycin and kanamycin) at an inoculation amount of 1% (v / v) and cultured with shaking at 37 °C and 180 rpm. When the OD of the culture broth reached 0.6 - 0.8, isopropyl-β-D-thiogalactoside (IPTG) with a final concentration of 0.2 mM was added as an inducer and induced at 16 °C for 16 - 24 h. The culture broth was centrifuged at 10000 rpm for 3 min, and the precipitate was washed twice with physiological saline to obtain recombinant expression transformed somatic cells. Then the harvested recombinant cells were suspended in 10 mL of Buffer A buffer, sonicated, and the supernatant was collected by centrifugation to obtain the crude enzyme solution of the recombinant carboxylic acid reductase mutant.

[0069] The obtained crude enzyme solution of the recombinant carboxylic acid reductase mutant was further purified. The following is the formulation of the protein purification buffer: Buffer A: 25 mM Hepes-Na, pH 7.5, 500 mM NaCl, 20 mM imidazole; Buffer B: 25 mM Hepes-Na, pH 7.5, 500 mM NaCl, 500 mM imidazole; Buffer C: 25 mM Hepes-Na, pH 7.5, 150 mM NaCl, 1 mM DTT, 5% glycerol. The crude enzyme solution of carboxylic acid reductase obtained by fragmentation was loaded onto a nickel column equilibrated with Solution A. After loading, the impurity proteins in the column were eluted with 10% Solution B, and then the target protein was eluted with 50% Solution B. SDS-PAGE was used to verify whether the target protein was contained, and then the eluate containing the target protein was collected, ultrafiltered and concentrated using an ultrafiltration tube (30 kDa), and then replaced twice with Solution C to remove the imidazole in the protein solution. The purified protein was subjected to activity and substrate specificity determination, and then aliquoted, quickly frozen in liquid nitrogen and stored at -80 °C for standby.

[0070] Example 4: Chromatographic detection method for substrate α,ω-dicarboxylic acid and product amino fatty acid

[0071] The reaction was carried out in a shaking reactor (1000 rpm), and the reaction time was based on the complete conversion of the substrate or the concentration of the product no longer rising. During the reaction process, 50 - 100 μL of the reaction solution was sampled at intervals and the reaction was terminated by adding 5 μL of 6M HCl. The product amino fatty acid was detected by high performance liquid chromatography. The sample treatment method was as follows: 20 μL of the sample was added with 20 μL of 1M NaOH and 20 μL of acetonitrile, mixed well and frozen in a -20 °C refrigerator for more than half an hour, then centrifuged at 1000 rpm for 1 min, 20 μL of the supernatant was taken, and 36 μL of 1M NaHCO 3, 20 μL of 28 mM Marfey and 100 μL of DMSO were mixed. After that, the mixture was allowed to stand at 40 °C for derivatization for 1 h. Finally, 40 μL of 1 M HCl was added to quench the reaction. After mixing, it was filtered through an organic nylon membrane.

[0072] The specific analysis conditions are as follows:

[0073] The chromatographic column was Elite C18, the mobile phase was methanol:water (0.1% trifluoroacetic acid) = 50:50 (v / v), the flow rate was 1 mL / min; the column temperature was 35 °C, and the UV detection wavelength was 340 nm.

[0074] The substrate α,ω-dicarboxylic acid was detected by gas chromatography. 30 μL of the sample was added to 300 μL of ethyl acetate (containing internal standard), and it was shaken vigorously for 5 - 10 min. Subsequently, it was centrifuged at 12000 rpm for 1 min. 150 μL of the organic phase was taken, anhydrous sodium sulfate was added, and it was shaken and dried for more than 2 h. Then it was centrifuged at 12000 rpm for 1 min. 50 μL of the organic phase was taken and added to an internal memory tube. 10 μL of trimethylsilyldiazomethane and 50 μL of methanol-ether (v / v = 1:1) were added, and it was allowed to stand at room temperature for derivatization for 20 min.

[0075] The specific analysis conditions are as follows:

[0076] The chromatographic column was SH-Rxi-5Sil MS column (30 m × 0.25 mm × 0.25 μm). Nitrogen was used as the carrier gas with a flow rate of 3 mL / min. The temperatures of the injector and detector were 250 °C and 280 °C respectively. The specific GC detection program was to hold at 85 °C for 3 minutes, then increase the temperature to 100 °C at a rate of 5 °C / min, then increase the temperature to 160 °C at a rate of 10 °C / min, and finally increase the temperature to 280 °C at a rate of 20 °C / min, and hold at 280 °C for 2 minutes.

[0077] Example 5: Enzymatic activity assay of KiCAR and its mutants against the model substrate adipic acid

[0078] Since the single-step reaction product 6-oxohexanoic acid of KiCAR catalyzing the model substrate adipic acid is unstable and it is not convenient to analyze and quantify by chromatography, and the sample treatment method of the substrate adipic acid and the gas chromatography detection method are as described in Example 4, the enzymatic activities of KiCAR and its mutants were determined by detecting the reduction amount of adipic acid after reacting for a period of time (reacting for 5 min) by gas chromatography.

[0079] The determination system of the specific enzyme activity of KiCAR and its mutants against adipic acid is as follows: The total reaction system is 200 μL. The buffer used is 100 mM Hepes-Na buffer (pH 7.5). The reaction is carried out at 30 °C. The reaction system includes adipic acid with a final concentration of 5 mM, 0.5% (v / v) DMSO, 1 mM NADPH, 10 mM ATP, 10 mM MgCl 2 and 30 mM glucose, as well as 0.05 U / mL of the pure enzyme catalyst of the recombinant carboxylic acid reductase mutant as described in Technical Solution 5 and 0.5 U / mL of the pure enzyme of glucose dehydrogenase.

[0080] To more accurately determine the enzyme activity, the conversion rate needs to be controlled within 10%. Therefore, the reaction is only carried out for 5 min, and at the same time, the concentration of NADPH is ensured to be higher than 20%. The specific enzyme activity of the wild-type KiCAR pure enzyme is measured to be 0.359 U / mg. Among them, the specific enzyme activities of the mutants G299I, G299K, T386K, and G411K are increased more significantly. Among them, the specific enzyme activity of the mutant KiCAR_G299K can reach up to 2.55 times (Table 1). "+" indicates that the activity of the mutant protein against adipic acid is increased to 1.5 - 2.0 times; "++" indicates that the activity of the mutant protein against lansoprazole sulfide is increased to 2.0 - 5 times.

[0081] Table 1 Specific enzyme activities of KiCAR and its mutants against the substrate adipic acid

[0082]

[0083]

[0084] Example 6: Multi-enzyme cascade synthesis of 6-aminocaproic acid by KiCAR, glucose dehydrogenase and transaminase

[0085] In this example, glucose dehydrogenase is used as the coenzyme recycling system. The multi-enzyme cascade catalytic system for synthesizing 6-aminocaproic acid from adipic acid is carried out in Hepes-Na buffer at pH 7.5 and 30 °C. The reaction system includes adipic acid with a final concentration of 5 mM, 0.5% (v / v) DMSO, 1 mM NADPH, 10 mM ATP, 10 mM MgCl 2 , 0.05 mM PLP, 30 mM glucose and 30 mM D / L-alanine, as well as 0.1 U / mL of the recombinant carboxylic acid reductase catalyst, 0.125 U / mL of transaminase and 1 U / mL of glucose dehydrogenase. Samples are taken after 18 h of reaction.

[0086] As described in Example 4, the amount of the product 6-aminocaproic acid formed is detected by HPLC, and the consumption of the substrate adipic acid is detected by GC. The results show that the conversion rate of adipic acid is 78.20%, and the yield of 6-aminocaproic acid reaches 71.30%.

[0087] Example 7: Multi-enzymatic cascade synthesis of 6-aminohexanoic acid using KiCAR, formate dehydrogenase and transaminase

[0088] In this example, formate dehydrogenase was used as the coenzyme recycling system. The multi-enzymatic cascade reaction for the synthesis of 6-aminohexanoic acid from adipic acid was carried out in a Hepes-Na buffer at pH 7.5 and 30 °C. The reaction system included adipic acid with a final concentration of 5 mM, 0.5% (v / v) DMSO, 1 mM NADPH, 10 mM ATP, 10 mM MgCl 2 , 0.05 mM PLP, 30 mM ammonium formate and 30 mM D / L-alanine, as well as 0.1 U / mL of the recombinant carboxylic acid reductase catalyst, 0.06 U / mL transaminase and 0.2 U / mL formate dehydrogenase. Samples were taken after 18 h of reaction.

[0089] As described in Example 4, the amount of 6-aminohexanoic acid produced in the product was detected by HPLC, and the consumption of the substrate adipic acid was detected by GC. The final yield of 6-aminohexanoic acid reached 66.3%.

[0090] Example 8: Multi-enzymatic cascade synthesis of 6-aminohexanoic acid using KiCAR_G299K, glucose dehydrogenase and transaminase

[0091] In this example, glucose dehydrogenase was used as the coenzyme recycling system. The multi-enzymatic cascade reaction for the synthesis of 6-aminohexanoic acid from adipic acid was carried out in a Hepes-Na buffer at pH 7.5 and 30 °C. The reaction system included adipic acid with a final concentration of 5 mM, 0.5% (v / v) DMSO, 1 mM NADPH, 10 mM ATP, 10 mM MgCl 2 , 0.05 mM PLP, 30 mM glucose and 30 mM D / L-alanine, as well as 0.2 U / mL of the recombinant carboxylic acid reductase mutant KiCAR_G299K catalyst, 0.25 U / mL transaminase and 1 U / mL glucose dehydrogenase. Samples were taken after 18 h of reaction.

[0092] As described in Example 4, the amount of 6-aminohexanoic acid produced in the product was detected by HPLC, and the consumption of the substrate adipic acid was detected by GC. The yield of 6-aminohexanoic acid was further increased to 81.23%.

[0093] Example 9: Multi-enzymatic cascade synthesis of 7-aminoheptanoic acid using KiCAR_G411K, glucose dehydrogenase and transaminase

[0094] In this example, glucose dehydrogenase was used as the coenzyme recycling system. The multi-enzyme cascade catalysis system for synthesizing 7-aminoheptanoic acid from pimelic acid was carried out in a Hepes-Na buffer solution with a pH of 7.5 at 30 °C. The reaction system included pimelic acid with a final concentration of 5 mM, 0.5% (v / v) DMSO, 1 mM NADPH, 10 mM ATP, 10 mM MgCl 2 , 0.05 mM PLP, 30 mM glucose, and 30 mM D / L-alanine, as well as 0.1 U / mL of the recombinant carboxylic acid reductase mutant KiCAR_G411K catalyst, 0.125 U / mL of transaminase, and 1 U / mL of glucose dehydrogenase. Samples were taken after 18 h of reaction.

[0095] As described in Example 4, the amount of the product 7-aminoheptanoic acid formed was detected by HPLC, and the consumption of the substrate pimelic acid was detected by GC. In this example, pimelic acid was used as the substrate. When catalyzed by the mutant KiCAR_G411K, the conversion rate of pimelic acid was 51.95%, and the yield of 7-aminoheptanoic acid reached 46.72%.

[0096] Example 10: Multi-enzyme cascade synthesis of 8-aminooctanoic acid by KiCAR_G411K, glucose dehydrogenase, and transaminase

[0097] In this example, glucose dehydrogenase was used as the coenzyme recycling system. The multi-enzyme cascade catalysis system for synthesizing 8-aminooctanoic acid from suberic acid was carried out in a Hepes-Na buffer solution with a pH of 7.5 at 30 °C. The reaction system included suberic acid with a final concentration of 5 mM, 0.5% (v / v) DMSO, 1 mM NADPH, 10 mM ATP, 10 mM MgCl 2 , 0.05 mM PLP, 30 mM glucose, and 30 mM D / L-alanine, as well as 0.1 U / mL of the recombinant carboxylic acid reductase mutant KiCAR_G411K catalyst, 0.125 U / mL of transaminase, and 1 U / mL of glucose dehydrogenase. Samples were taken after 18 h of reaction.

[0098] As described in Example 4, the amount of the product 8-aminooctanoic acid formed was detected by HPLC, and the consumption of the substrate suberic acid was detected by GC. In this example, suberic acid was used as the substrate. When catalyzed by the mutant KiCAR_G411K, the conversion rate of suberic acid was 55.75%, and the yield of 7-aminoheptanoic acid reached 48.38%.

[0099] Example 11: Multi-enzyme cascade synthesis of 9-aminononanoic acid by KiCAR_G299K, glucose dehydrogenase, and transaminase

[0100] In this example, glucose dehydrogenase was used as the coenzyme recycling system. The multi-enzyme cascade catalysis system for synthesizing 9-aminononanoic acid from azelaic acid was carried out in a Hepes-Na buffer solution with a pH of 7.5 at 30 °C. The reaction system included azelaic acid with a final concentration of 5 mM, 0.5% (v / v) DMSO, 1 mM NADPH, 10 mM ATP, 10 mM MgCl 2 , 0.05 mM PLP, 30 mM glucose, and 30 mM D / L-alanine, as well as 0.1 U / mL of the recombinant carboxylic acid reductase mutant KiCAR_G299K catalyst, 0.125 U / mL of transaminase, and 1 U / mL of glucose dehydrogenase. Samples were taken after 18 h of reaction.

[0101] As described in Example 4, the amount of the product 9-aminononanoic acid formed was detected by HPLC, and the consumption of the substrate azelaic acid was detected by GC. In this example, azelaic acid was used as the substrate. When catalyzed by the mutant KiCAR_G299K, the conversion rate of azelaic acid was 69.95%, and the yield of 9-aminononanoic acid reached 66.72%.

[0102] Example 12: Multi-enzyme cascade synthesis of 10-aminodecanoic acid using KiCAR_G299R, glucose dehydrogenase, and transaminase

[0103] In this example, glucose dehydrogenase was used as the coenzyme recycling system. The multi-enzyme cascade catalysis system for synthesizing 10-aminodecanoic acid from sebacic acid was carried out in a Hepes-Na buffer solution with a pH of 7.5 at 30 °C. The reaction system included sebacic acid with a final concentration of 5 mM, 0.5% (v / v) DMSO, 1 mM NADPH, 10 mM ATP, 10 mM MgCl 2 , 0.05 mM PLP, 30 mM glucose, and 30 mM D / L-alanine, as well as 0.1 U / mL of the recombinant carboxylic acid reductase mutant KiCAR_G299R catalyst, 0.125 U / mL of transaminase, and 1 U / mL of glucose dehydrogenase. Samples were taken after 18 h of reaction.

[0104] As described in Example 4, the amount of the product 10-aminodecanoic acid formed was detected by HPLC, and the consumption of the substrate sebacic acid was detected by GC. In this example, sebacic acid was used as the substrate. When catalyzed by the mutant KiCAR_G299R, the yield of 10-aminodecanoic acid reached 57.24%.

[0105] Example 13: Multi-enzyme cascade synthesis of 11-aminoundecanoic acid using KiCAR_G299K, glucose dehydrogenase, and transaminase

[0106] In this example, a coenzyme recycling system using glucose dehydrogenase was employed. The multi-enzyme cascade catalysis system for synthesizing 11-aminoundecanoic acid from undecanedioic acid was carried out in a Hepes-Na buffer solution at pH 7.5 and 30 °C. The reaction system included undecanedioic acid with a final concentration of 5 mM, 0.5% (v / v) DMSO, 1 mM NADPH, 10 mM ATP, 10 mM MgCl 2 2, 0.05 mM PLP, 30 mM glucose, and 30 mM D / L-alanine, as well as 0.1 U / mL of the recombinant carboxylic acid reductase mutant KiCAR_G299K catalyst, 0.125 U / mL of transaminase, and 1 U / mL of glucose dehydrogenase. Samples were taken after 18 h of reaction.

[0107] As described in Example 4, the amount of the product 11-aminoundecanoic acid formed was detected by HPLC, and the consumption of the substrate undecanedioic acid was detected by GC. In this example, using undecanedioic acid as the substrate, when catalyzed by the mutant KiCAR_G299K, the yield of 11-aminoundecanoic acid reached 73.46%.

[0108] Example 14: Multi-enzyme cascade synthesis of 12-aminododecanoic acid by KiCAR_G411K, glucose dehydrogenase, and transaminase

[0109] In this example, a coenzyme recycling system using glucose dehydrogenase was employed. The multi-enzyme cascade catalysis system for synthesizing 12-aminododecanoic acid from dodecanedioic acid was carried out in a Hepes-Na buffer solution at pH 7.5 and 30 °C. The reaction system included dodecanedioic acid with a final concentration of 5 mM, 0.5% (v / v) DMSO, 1 mM NADPH, 10 mM ATP, 10 mM MgCl 2 2, 0.05 mM PLP, 30 mM glucose, and 30 mM D / L-alanine, as well as 0.1 U / mL of the recombinant carboxylic acid reductase mutant KiCAR_G411K catalyst, 0.125 U / mL of transaminase, and 1 U / mL of glucose dehydrogenase. Samples were taken after 18 h of reaction.

[0110] As described in Example 4, the amount of the product 12-aminododecanoic acid formed was detected by HPLC, and the consumption of the substrate dodecanedioic acid was detected by GC. In this example, using dodecanedioic acid as the substrate, when catalyzed by the mutant KiCAR_G411K, the yield of 12-aminododecanoic acid reached 59.34%.

[0111] SEQ ID No.1 sequence

[0112]

[0113]

[0114] SEQ ID No. 2 sequence

[0115]

[0116]

[0117]

[0118] The above description of the embodiments is provided to enable those of ordinary skill in the art to understand and use the invention. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative efforts. Therefore, the present invention is not limited to the above embodiments, and all improvements and modifications made by those skilled in the art without departing from the scope of the present invention as disclosed should be within the protection scope of the present invention.

Claims

1. A carboxylic acid reductase mutant, characterized in that it is: replacing glycine (Gly) at position 299 in the amino acid sequence shown in SEQ ID No. 2 with lysine (Lys).

2. An isolated nucleic acid, characterized in that the nucleic acid encodes the carboxylic acid reductase mutant according to claim 1.

3. A recombinant expression vector, characterized in that the recombinant expression vector contains the nucleic acid according to claim 2.

4. A recombinant expression transformant, characterized in that it contains the recombinant expression vector according to claim 3.

5. A recombinant carboxylic acid reductase mutant catalyst, characterized in that the recombinant carboxylic acid reductase mutant catalyst is any one of the following forms: (1) Culturing the recombinant expression transformant according to claim 4 and separating the transformed cells containing the carboxylic acid reductase mutant; (2) Disrupting the transformed cells as described in (1) and separating the crude enzyme solution containing the carboxylic acid reductase mutant; (3) The pure enzyme obtained by purifying the crude enzyme solution as described in (2).

6. Use of the carboxylic acid reductase mutant according to claim 1 or the recombinant carboxylic acid reductase mutant catalyst according to claim 5 in the catalytic reduction for synthesizing amino fatty acids from medium and long-chain α,ω-dicarboxylic acids; the medium and long-chain α,ω-dicarboxylic acid substrates are adipic acid, pimelic acid, suberic acid, azelaic acid, sebacic acid, undecanedioic acid, and dodecanedioic acid.

7. According to the use described in claim 6, characterized in that The reaction system includes α,ω-dicarboxylic acid, DMSO, NADPH, ATP, MgCl 2 , PLP, glucose and D / L-alanine, and 10-40 U of the recombinant carboxylic acid reductase mutant or the recombinant carboxylic acid reductase mutant catalyst / mmol of α,ω-dicarboxylic acid, 12-50 U of transaminase / mmol of α,ω-dicarboxylic acid and 40-200 U of glucose / formate dehydrogenase / mmol of α,ω-dicarboxylic acid.

8. According to the use described in claim 6, characterized in that coenzyme NADPH is required to participate in the reaction during the reaction process, and formate dehydrogenase or glucose dehydrogenase is used to achieve in-situ regeneration of coenzyme NADPH.

9. According to the use described in claim 8, characterized in that the dehydrogenase is any one of the following dehydrogenases: Formate dehydrogenase FDH, using formate and NADP + as substrates, catalyzes the oxidation of formate while NADP + is reduced to NADPH; Glucose dehydrogenase GDH, using glucose and NADP + as substrates, catalyzes the oxidation of glucose while NADP + is reduced to NADPH.

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