Reducing aminoenzyme mutant and application thereof

Abstract

The invention discloses a reductive aminoenzyme mutant and application thereof. The invention provides a reductive amino enzyme mutant, and compared with an amino acid sequence as shown in SEQ ID NO: 2, the reductive amino enzyme mutant comprises one or more amino acid residue differences in the following sites: the 49th site, the 144th site, the 120th site, the 130th site, the 207th site and the 237th site. The mutant provided by the invention is used for synthesizing the chiral amine compound, the reaction condition is mild, harmful byproducts are not generated, the mutant is environment-friendly, and high-level conversion rate and selectivity can be achieved on the preparation scale.

Description

Technical Field

[0001] This invention relates to a reducing aminoase mutant and its application, belonging to the fields of biotechnology and pharmaceutical intermediate synthesis. Background Technology

[0002] Chiral amines are common building blocks in the pharmaceutical industry, used to obtain a wide range of bioactive compounds. They can be used in various applications, from the development of small molecule drugs to more complex therapeutic approaches, such as chimeras targeting proteolysis or for the bioconjugation of peptides or proteins. The pharmaceutical industry's continuous search for innovative methods to produce new bioactive compounds increases the demand for novel building blocks in drug design. The structural diversity of such compounds is beneficial for studying drug properties such as permeability, potency, or secondary pharmacology. Current synthetic methods can broadly cover the available chemical space, but opportunities still exist for developing new building blocks.

[0003] Biocatalysis is a powerful synthetic approach that yields novel chemicals by utilizing mild reaction conditions and transformative catalytic properties, exhibiting high stereoselectivity driven by precise enzyme / substrate interactions. Importantly, biocatalytic synthesis can play a crucial role in drug discovery because enzymes are tunable catalysts, providing excellent control over chemoselectivity, regioselectivity, and emerging platforms for biocatalysis. Here, the present invention discovers and evolves a reducing aminoase mutant for the synthesis of 4-substituted cyclohexylamine. Summary of the Invention

[0004] To address the lack of a technical method for preparing chiral amine compounds using bio-enzymatic catalysis, this invention provides a reducing aminoase mutant and its applications. This invention is the first to utilize a reducing aminoase mutant to catalyze the synthesis of chiral amine compounds. The reaction conditions are mild, no harmful byproducts are generated, it is environmentally friendly, and it achieves high conversion rates and selectivity on a large scale.

[0005] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0006] A first aspect of the present invention provides a reducing aminoase mutant that differs from the amino acid sequence shown in SEQ ID NO: 2 by one or more amino acid residues at positions 49, 114, 120, 130, 207, and 237.

[0007] In some embodiments of the present invention, the reducing aminoase mutant contains one or more amino acid residues selected from the following, compared to the amino acid sequence shown in SEQ ID NO: 2: H49K, H114D, T120S, L130C, A207L, A237I.

[0008] In some embodiments of the present invention, the reducing aminoase mutant contains the following amino acid residue difference compared to the amino acid sequence shown in SEQ ID NO: 2: A207L;

[0009] Preferably, the reducing aminoase mutant, compared with the amino acid sequence shown in SEQ ID NO: 2, further comprises one or more of the following amino acid residue differences: H49K, H114D, T120S, L130C and A237I, and more preferably comprises the following amino acid residue difference combination: H114D / T120S / A237I;

[0010] More preferably, the reducing aminoase mutant further comprises the following amino acid residue differences compared to the amino acid sequence shown in SEQ ID NO: 2: L130C and / or H49K.

[0011] In some embodiments of the present invention, the amino acid sequence of the reducing aminoase mutant is shown in SEQ ID NO: 3, 4, 5 or 6.

[0012] A second aspect of the present invention provides an isolated nucleic acid that encodes a reductive aminoase mutant as described in the first aspect of the present invention.

[0013] A third aspect of the present invention provides a recombinant expression vector comprising isolated nucleic acids as described in the second aspect of the present invention.

[0014] In some embodiments of the present invention, the backbone of the recombinant expression vector is pET-30a.

[0015] A fourth aspect of the present invention provides a transformant comprising isolated nucleic acid as described in the second aspect of the present invention or a recombinant expression vector as described in the third aspect of the present invention.

[0016] In some embodiments of the present invention, the host cells used in the construction of the transformant are selected from Escherichia coli cells, insect cells, yeast cells and mammalian cells; the host cells are preferably Escherichia coli, such as BL21(DE3).

[0017] A fifth aspect of the present invention provides a method for preparing a reducing aminoase mutant, the method comprising: culturing a transformant as described in the fourth aspect of the present invention to obtain the reducing aminoase mutant.

[0018] A sixth aspect of the present invention provides an enzyme preparation containing a reducing aminoase mutant as described in the first aspect of the present invention.

[0019] A seventh aspect of the present invention provides an enzyme composition comprising a reducing aminoase or a mutant reducing aminoase as described in the first aspect of the present invention, and a coenzyme and glucose dehydrogenase (GDH).

[0020] In some embodiments of the present invention, the coenzyme is nicotinamide adenine dinucleotide phosphate (NADP) or nicotinamide adenine dinucleotide (NAD).

[0021] In some embodiments of the present invention, the reducing aminoase is derived from Aspergillus udagawae.

[0022] In some embodiments of the present invention, the amino acid sequence of the reducing aminoase is selected from the group consisting of:

[0023] (1) As shown in SEQ ID NO: 2;

[0024] (2) It has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 2.

[0025] In some embodiments of the present invention, the amino acid sequence of the glucose dehydrogenase is as shown in SEQ ID NO: 7 or 8.

[0026] In some embodiments of the present invention, the mass ratio of the reducing aminoase or reducing aminoase mutant, coenzyme and glucose dehydrogenase is (10-30):(0.2-1.2):(1-6); for example, 10:0.4:2, 30:1.2:6, 12:1.2:6, 10:0.2:1 or 15:0.3:3.

[0027] The eighth aspect of the present invention provides the use of a reducing amino enzyme, a reducing amino enzyme mutant as described in the first aspect of the present invention, an isolated nucleic acid as described in the second aspect of the present invention, a recombinant expression vector as described in the third aspect of the present invention, a transformant as described in the fourth aspect of the present invention, an enzyme preparation as described in the sixth aspect of the present invention, or an enzyme composition as described in the seventh aspect of the present invention in reducing compound I with structure I to obtain compound II with structure II.

[0028] ;

[0029] R1 is selected from hydrogen and hydroxyl; R2 is selected from methyl, ethyl, propyl, tert-butyl, phenyl, benzyl and thiophene.

[0030] In some embodiments of the present invention, the reducing aminoase is derived from Aspergillus udagawae.

[0031] In some embodiments of the present invention, the amino acid sequence of the reducing aminoase is selected from the group consisting of:

[0032] (1) As shown in SEQ ID NO: 2;

[0033] (2) It has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 2.

[0034] In some embodiments of the present invention, compound I is any of the following compounds:

[0035] , , , , , , , , and .

[0036] In some embodiments of the present invention, compound II is any of the following compounds:

[0037] , , , , , , , , and .

[0038] The ninth aspect of the present invention provides a method for preparing a compound of formula II, the method comprising the following steps: using a reducing amino enzyme, a reducing amino enzyme mutant as described in the first aspect of the present invention, an enzyme preparation as described in the sixth aspect of the present invention, or an enzyme composition as described in the seventh aspect of the present invention to catalyze an amino donor to contact and react with a compound of formula I to obtain a compound of formula II.

[0039] ;

[0040] R1 is selected from hydrogen and hydroxyl; R2 is selected from methyl, ethyl, propyl, tert-butyl, phenyl, benzyl and thiophene.

[0041] In some embodiments of the present invention, the amino donor is an ammonium salt solution, such as an ammonium chloride buffer solution; the molar ratio of the ammonium chloride to the compound shown in Formula I is preferably (10-15):1, more preferably 12:1; the ammonium chloride buffer solution is preferably an ammonia solution of ammonium chloride or an ammonium chloride phosphate solution.

[0042] The phosphate buffer solution is preferably a mixed aqueous solution of potassium dihydrogen phosphate and dipotassium hydrogen phosphate; the concentration of the phosphate buffer solution is preferably 100 mM.

[0043] And / or, the mass ratio of the reducing aminoase or the reducing aminoase mutant to the compound shown in Formula I is (0.01-10):1, preferably (0.5-2):1, for example 0.5:1, 0.2:1, 1:1 or 0.16:1.

[0044] In some embodiments of the present invention, compound I and compound II are as described in the eighth aspect of the present invention.

[0045] In some embodiments of the present invention, the amino acid sequence of the reducing aminoase is selected from the group consisting of:

[0046] (1) As shown in SEQ ID NO: 2;

[0047] (2) It has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 2.

[0048] In some embodiments of the present invention, the preparation method includes the following steps: in a solvent, in the presence of a coenzyme, glucose dehydrogenase (GDH) and D-glucose, the reducing amino enzyme or the reducing amino enzyme mutant catalyzes the contact and reaction of the amino donor with the compound shown in Formula I to obtain the compound shown in Formula II.

[0049] In some embodiments of the present invention, the preparation method is selected from one or more of the following conditions:

[0050] (1) The pH value of the reaction is 7-11, preferably 8.5;

[0051] (2) The coenzyme is nicotinamide adenine dinucleotide phosphate (NADP) or nicotinamide adenine dinucleotide (NAD); preferably, the coenzyme is nicotinamide adenine dinucleotide phosphate.

[0052] (3) The solvent is an organic solvent, such as dimethyl sulfoxide;

[0053] (4) The mass-to-volume ratio of the compound shown in Formula I to the solvent is 0.1~1 mg / μL, preferably 0.2 mg / μL;

[0054] (5) The mass ratio of the coenzyme to the compound shown in Formula I is (0.01-0.5):1, preferably (0.05-0.1):1, for example 0.02:1 or 0.1:1;

[0055] (6) The mass ratio of the glucose dehydrogenase to the compound shown in Formula I is (0.01-0.5):1, preferably (0.05-0.1):1, for example 0.1:1;

[0056] (7) The mass ratio of the D-glucose to the compound shown in Formula I is (0.01-10):1, preferably (1-6):1, for example 3:1;

[0057] (8) The reaction temperature is 20~35℃, preferably 30℃;

[0058] (9) The amino acid sequence of the glucose dehydrogenase is as shown in SEQ ID NO: 7 or 8; the mass ratio of the reducing aminoase or reducing aminoase mutant, coenzyme and glucose dehydrogenase is preferably (10-30):(0.2-1.2):(1-6); for example 10:0.4:2, 30:1.2:6, 12:1.2:6, 10:0.2:1 or 15:0.3:3.

[0059] In some embodiments of the present invention, the preparation method further includes the following steps: after the reaction solution is reacted overnight on a shaker, methanol is added to terminate the reaction, the pH of the reaction solution is adjusted to 8.0-10.0 (preferably 9.0), centrifuged, filtered through a membrane, and the resulting reaction solution is detected by a high-performance gas chromatograph; preferably, KOH is used to adjust the pH, and the mass ratio of KOH to the compound shown in Formula I is (0.01-10):1, preferably (1-5):1.

[0060] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0061] The reagents and raw materials used in this invention are all commercially available.

[0062] The positive and progressive effects of this invention are as follows:

[0063] (1) This invention is the first to use a reducing aminoase mutant to catalyze the synthesis of chiral amine compounds. The reaction conditions are mild, no harmful byproducts are produced, and it is environmentally friendly.

[0064] (2) The reducing aminoase mutant provided by the present invention has further improved the stereoselectivity and stereospecificity of the enzyme by saturating key active site residues, and can achieve a high level of conversion and selectivity in the preparation of 4-substituted cyclohexylamine. Detailed Implementation

[0065] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0066] Example 1: Establishment of genetically engineered bacteria

[0067] Based on the reductase mutant listed in NCBI (NCBI reference sequence number XP_043147525.1, its amino acid sequence is shown in SEQ ID NO: 2), a gene fragment of the reductase (SEQ ID NO: 1) was artificially synthesized. Using this gene fragment as a template, the fragment was amplified by PCR. The gene was inserted into the pET-30a plasmid, and the ligated vector was transformed into Escherichia coli BL21(DE3) to establish a reductase-producing genetically engineered bacterium.

[0068] Example 2: Obtaining a reducing aminoase mutant

[0069] Protein engineering was performed on reductases using site-directed saturation mutagenesis PCR and combinatorial PCR, respectively, to obtain reductase mutants with amino acid sequences as shown in SEQ ID NO: 3, 4, 5 or 6.

[0070] Example 3: Preparation of crude enzyme powder from recombinant reducing aminoase mutant

[0071] 1. Shake-flask fermentation

[0072] Glycerin-containing bacteria were inoculated at 1% (v / v) into LB medium (containing 1% peptone, 0.5% yeast extract, 1% NaCl, 50 mL). Kanamycin was added at 0.1% (v / v) of LB medium, and the culture was incubated overnight at 37°C. 3 mL of the overnight activated bacterial culture was inoculated into a 1 L shake flask containing 250 mL of TB medium (containing 1.2% peptone, 2.4% yeast extract, 0.4% glycerol, and phosphate buffer: 17 mM KH₂PO₄ and 72 mM K₂HPO₄). Kanamycin was added at 0.1% (v / v) of TB medium, and the culture was incubated at 37°C and 300 rpm until the OD reached 0.6–1. IPTG was then added to a concentration of 0.1 mM, and the culture was incubated overnight at 25°C and 250 rpm.

[0073] 2. Enzyme powder preparation

[0074] The obtained bacterial cells were centrifuged (4000 rpm, 4℃, 30 min), washed with 0.9% NaCl solution, and resuspended in 20 mL of 50 mM phosphate solution (pH 7.0). The cells were then sonicated in ice water until clear. The sonication conditions were (500 W, 2 s operation, 5 s interval). The lysate was placed in a low-temperature high-speed centrifuge (12000 rpm, 4℃, 30 min), and the supernatant was collected. The supernatant was then freeze-dried to obtain crude enzyme powder.

[0075] Example 4: Preparation of a reducing aminoase mutant and its use in the synthesis of 4-substituted cyclohexylamine compounds

[0076] This example primarily aims to test whether the conversion rate of the mutant enzyme powder is higher than that of the wild type, using 4-(methyl)cyclohexanone as an example:

[0077] Preparation Example 1:

[0078] In a system containing 3 mL of 727 mM NH4Cl buffer, 0.4 mg NADP, 2 mg GDH, and 60 mg D-glucose, an appropriate amount of ammonia was added to adjust the pH to 8.5. Then, a mixture of 20 mg 4-(methyl)cyclohexanone dissolved in 100 μL of dimethyl sulfoxide was added, followed by 10 mg of enzyme powder obtained from site-directed saturation mutagenesis (amino acid sequence as shown in SEQ ID NO: 3). The reaction was carried out overnight at 30 °C with shaking for 18 h. The reaction was terminated by adding 9 mL of methanol, and 30 μL of KOH was added to adjust the pH of the final reaction solution to approximately 9.0. The sample was centrifuged at 12000 rpm for 3 min and filtered through a membrane. 300 μL of the reaction solution was collected, and the components of the reaction mixture were analyzed using high-performance gas chromatography (GC). The mutant transformation rate was found to be 62.55%, the wild-type transformation rate was 33.6%, and the chirality was greater than 95% for both types. The results are shown in Table 1.

[0079] Table 1

[0080]

[0081] Note: In Tables 1-7 of this invention, ISO represents the product isomer; PRO represents the target configuration of the product; OH-1 and OH-2 represent impurity alcohols; and SM represents the raw material.

[0082] Preparation Example 2:

[0083] In a system containing 3 mL of 727 mM NH4Cl buffer, 0.4 mg NADP, 2 mg GDH, and 60 mg D-glucose, an appropriate amount of ammonia was added to adjust the pH to 8.5. A mixture of 20 mg 4-(methyl)cyclohexanone dissolved in 100 μL of dimethyl sulfoxide was added, followed by 10 mg of the combined mutagen-derived enzyme powder (amino acid sequence as shown in SEQ ID NO: 4). The mixture was reacted overnight at 30 °C with shaking for 18 h. The reaction was terminated by adding 9 mL of methanol, and 300 μL of KOH was added to adjust the pH of the final reaction solution to approximately 9.0. The sample was centrifuged at 12000 rpm for 3 min and filtered through a membrane. 300 μL of the reaction solution was collected, and the components of the reaction mixture were analyzed using high-performance gas chromatography (GC). The mutant conversion rate was found to be 50.51%, as shown in Table 2.

[0084] Table 2

[0085]

[0086] Preparation Example 3:

[0087] In a system containing 3 mL of 727 mM NH4Cl buffer, 1.2 mg NADP, 6 mg GDH, and 180 mg D-glucose, an appropriate amount of ammonia was added to adjust the pH to 8.5. A mixture of 60 mg 4-(methyl)cyclohexanone dissolved in 300 μL of dimethyl sulfoxide was added, followed by 30 mg of enzyme powder (amino acid sequence shown in SEQ ID NO: 5) obtained from site-directed saturation mutagenesis. The reaction was carried out overnight at 30 °C with shaking for 18 h. The reaction was terminated by adding 9 mL of methanol, and 300 μL of KOH was added to adjust the pH of the final reaction solution to approximately 9.0. The sample was centrifuged at 12000 rpm for 3 min and filtered through a membrane. 300 μL of the reaction solution was collected, and the components of the reaction mixture were analyzed using high-performance gas chromatography (GC). The mutant conversion rate was found to be 47.35%, with chirality greater than 95%. The results are shown in Table 3 below.

[0088] Table 3

[0089]

[0090] Preparation Example 4:

[0091] In a system containing 3 mL of 727 mM NH4Cl buffer, 1.2 mg NADP, 6 mg GDH, and 180 mg D-glucose, an appropriate amount of ammonia was added to adjust the pH to 8.5. A mixture of 60 mg 4-(methyl)cyclohexanone dissolved in 300 μL of dimethyl sulfoxide was added, followed by 12 mg of enzyme powder (amino acid sequence as shown in SEQ ID NO: 6) obtained from site-directed saturation mutagenesis. The reaction was carried out overnight at 30 °C with shaking for 18 h. The reaction was terminated by adding 9 mL of methanol, and 300 μL of KOH was added to adjust the pH of the final reaction solution to approximately 9.0. The sample was centrifuged at 12000 rpm for 3 min and filtered through a membrane. 300 μL of the reaction solution was collected, and the components of the reaction mixture were analyzed using high-performance gas chromatography (GC). The mutant conversion rate was found to be 21.54%, with chirality greater than 95%. The results are shown in Table 4.

[0092] Table 4

[0093]

[0094] Example 5: Preparation of reducing aminoase mutants and process optimization for the synthesis of 4-substituted cyclohexylamine compounds

[0095] This embodiment primarily aims to further determine the effect of coenzyme-dependent reducing aminoase mutants on the synthesis of 4-substituted cyclohexylamine compounds, using 4-(methyl)cyclohexanone as an example:

[0096] Preparation Example 5:

[0097] In a system containing 3 mL of 727 mM NH4Cl buffer, 1 mg GDH, and 30 mg D-glucose, 10 mg of 4-(methyl)cyclohexanone dissolved in 50 μL of dimethyl sulfoxide, 10 mg of enzyme powder (amino acid sequence as shown in SEQ ID NO: 4), and an appropriate amount of ammonia water were added to adjust the pH to 8.5. 0.2 mg NADP and 0.2 mg NAD were added, and the mixture was stirred overnight at 30 °C for 18 h. The reaction was terminated by adding 9 mL of methanol, and 300 μL of KOH was added to adjust the pH of the final reaction solution to approximately 9.0. The sample was centrifuged at 12000 rpm for 3 min and filtered through a membrane. 300 μL of the reaction solution was collected, and the components of the reaction mixture were analyzed using high-performance gas chromatography (GC).

[0098] The tests revealed that the conversion rate of NADP was 33.19%, while that of NAD was 15.38%, with both chiralities greater than 95%. NADP had a higher conversion rate, as shown in Table 5.

[0099] Table 5

[0100]

[0101] Preparation Example 6:

[0102] In a system containing 3 mL of 727 mM NH4Cl buffer, 0.2 mg NADP, and 30 mg D-glucose, 60 mg of 4-(methyl)cyclohexanone dissolved in 300 μL of dimethyl sulfoxide, 10 mg of enzyme powder (amino acid sequence as shown in SEQ ID NO: 4), and an appropriate amount of ammonia were added to adjust the pH to 8.5. Then, 1 mg of commercially available GDH and 1 mg of synthetically produced mutant GDH (amino acid sequences as shown in SEQ ID NO: 7 and SEQ ID NO: 8) were added. The mixture was reacted overnight at 30 °C with stirring for 18 h. The reaction was terminated by adding 9 mL of methanol, and 300 μL of KOH was added to adjust the pH of the final reaction solution to approximately 9.0. The sample was centrifuged at 12000 rpm for 3 min and filtered through a membrane. 300 μL of the reaction solution was collected, and the components of the reaction mixture were analyzed using high-performance gas chromatography (GC). The tests revealed that the conversion rates of the two GDHs were similar, around 10%, and the chirality was greater than 95%. However, the product synthesized from commercial GDH had fewer impurities and was therefore the preferred choice. The results are shown in Table 6.

[0103] Table 6

[0104]

[0105] Example 6: Preparation of reducing aminoase mutants and process optimization for the synthesis of 4-substituted cyclohexylamine compounds

[0106] This embodiment aims to further determine the effect of buffer type on the use of reducing aminoase mutants for the synthesis of 4-substituted cyclohexylamine compounds, taking 4-(methyl)cyclohexanone as an example:

[0107] Preparation Example 7:

[0108] In a system containing 3 mL of 727 mM NH4Cl buffer, 1.2 mg NADP, 6 mg GDH, and 180 mg D-glucose, 100 mM potassium dihydrogen phosphate and dipotassium hydrogen phosphate mixed aqueous solution were added, and an appropriate amount of KOH was added to adjust the pH to 8.5. 60 mg of 4-(methyl)cyclohexanone dissolved in 300 μL of dimethyl sulfoxide was added, along with 12 mg of enzyme powder (P18C9 amino acid sequence as shown in SEQ ID NO: 6). The mixture was reacted overnight at 30 °C with shaking for 18 h. The reaction was terminated by adding 9 mL of methanol, and 300 μL of KOH was added to adjust the pH of the final reaction solution to approximately 9.0. The sample was centrifuged at 12000 rpm for 3 min and filtered through a membrane. 300 μL of the reaction solution was collected, and the components of the reaction mixture were detected using high-performance gas chromatography (GC).

[0109] Preparation Example 8:

[0110] In a system containing 3 mL of 727 mM NH4Cl buffer, 1.2 mg NADP, 6 mg GDH, and 180 mg D-glucose, an appropriate amount of ammonia was added to adjust the pH to 8.5. Then, a mixture of 60 mg 4-(methyl)cyclohexanone dissolved in 300 μL of dimethyl sulfoxide and 12 mg enzyme powder (amino acid sequence as shown in SEQ ID NO: 6) were added. The mixture was stirred overnight at 30 °C for 18 h on a shaker. The reaction was terminated by adding 9 mL of methanol, and 300 μL of KOH was added to adjust the pH of the final reaction solution to approximately 9.0. The sample was centrifuged at 12000 rpm for 3 min and filtered through a membrane. 300 μL of the reaction solution was collected, and the components of the reaction mixture were analyzed using high-performance gas chromatography (GC).

[0111] The results showed that the conversion rate of the buffer system with added phosphate (75.39%) was twice that of the buffer system with added ammonia (39.62%). Changing the buffer had a significant impact on the system. The results are shown in Table 7.

[0112] Table 7

[0113]

[0114] Example 7: Preparation of reducing aminoase mutants and process optimization for the synthesis of 4-substituted cyclohexylamine compounds

[0115] This embodiment aims to further determine the effect of buffer pH on the use of reducing aminoase mutants for the synthesis of 4-substituted cyclohexylamine compounds, using 4-(methyl)cyclohexanone as an example:

[0116] Preparation Example 9:

[0117] In a system containing 3 mL of 727 mM NH4Cl buffer, 0.3 mg NADP, 3 mg GDH, and 90 mg D-glucose, 30 mg of 4-(methyl)cyclohexanone dissolved in 150 μL of dimethyl sulfoxide, 15 mg of enzyme powder (amino acid sequence as shown in SEQ ID NO: 4), and different volumes of ammonia were added to adjust the pH to 7.5, 8.5, 9.5, and 10.5. After reacting overnight at 30 °C with stirring for 18 h, the reaction was terminated by adding 9 mL of methanol, and 300 μL of KOH was added to adjust the pH of the final reaction solution to approximately 9.0. The sample was centrifuged at 12000 rpm for 3 min and filtered through a membrane. 300 μL of the reaction solution was taken, and the components of the reaction mixture were detected by high-performance gas chromatography (GC).

[0118] The tests revealed that the conversion rate was 41.1% at pH 7.5, 49.9% at pH 8.5, 26.8% at pH 9.5, and 9.4% at pH 10.5. The pH 8.5 concentration had the highest conversion rate and was therefore the preferred concentration.

[0119] The sequences used in this invention are as follows:

[0120] SEQ ID NO: 1, DNA sequence encoding wild-type reductase.

[0121] ATGAGCAGCGTGAGCATTTTTGGCCTGGGTGCGATGGGCAAAGCGCTGGCGAGCCGCTTTCTGGCGGAAAAATATAAAGTGGCGGTGTGGAACCGCAGCCCGGAAAAAGCGAGCCCGCTGCTGGAAAAAGGCGCGACCCTGAGCCATACCGCGGTGGATGGCATTAACGCGAGCGATCTGATTATCATTTGCCTGCTGGATAACGCGGCGGTGCAAGCGACCCTGGATAGCGCGCTGGATCGCCTGCATGGCAAAACCATTGTGAACCTGACCAACGGCACCCCGGATCAAGCGCGCAAACTGAGCGACCTGATTGTGAGTCATGGCGCGCAGTATGTGCATGGCGGCATTATGGCGACCCCGAGCATGATTGGCAGCCCGCATGCGCTGGTGCTGTATAGCGGCAGCCCGGATGCGTTTAACGCGGCGGAAGCGGATCTGAGCGTGCTGGCGAACTGCGTGTTTCTGGGCGAAGATGCGGGCAGCGCGAGCCTGCATGATCTGGCGCTGCTGAGCGGCATGTATGGCCTGTTTAGCGGCTTTCTGCATGCGACCGCGCTGGTGAAAAGCAGCACCCCGGCGGTGAAATTTCTGGATCTGCTGGTGCCGTGGCTGGGCGCGATGACCGAGTATACCAAAGGCATGGCGAAACAGATTGATGAAGGTCAGTATGCGAGCGAAGGCAGCAACCTGGCGATGCAGCTGGTGGCGGTGGAAAACATTATTGATGCGAGCGCGGCGCAGCAAGTGAGCGCGGATTTTATTCGCCCGATGAAAGAATTTATGGAAAAAGCGGTGGCGGCGGGCCATGGCGGCGATGATATTAGCAGCCTGATTGATTTTGTGAAAAGCACCTAA

[0122] SEQ ID NO: 2, wild-type reducing amino enzyme amino acid sequence

[0123] MSSVSIFGLGAMGKALASRFLAEKYKVAVWNRSPEKASPLLEKGATLSHTAVDGINASDLIIICLLDNAAVQATLDSALDRLHGKTIVNLTNGTPDQARKLSDLIVSHGAQYVHGGIMATPSMIGSPHALVLYSGSPDAFNAAEADLSVLANCVFLGEDAGSASLHDLALLSGMYGLFSGFLHATALVKSSTPAVKFLDLLVPWLGAMTEYTKGMAKQIDEGQYASEGSNLAMQLVAVENIIDASAAQQVSADFIRPMKEFMEKAVAAGHGGDDISSLIDFVKST

[0124] SEQ ID NO: 3, Amino acid sequence of the reductase aminase mutant

[0125] MSSVSIFGLGAMGKALASRFLAEKYKVAVWNRSPEKASPLLEKGATLSHTAVDGINASDLIIICLLDNAAVQATLDSALDRLHGKTIVNLTNGTPDQARKLSDLIVSHGAQYVHGGIMATPSMIGSPHALVLYSGSPDAFNAAEADLSVLANCVFLGEDAGSASLHDLALLSGMYGLFSGFLHATALVKSSTPAVKFLDLLVPWLGLMTEYTKGMAKQIDEGQYASEGSNLAMQLVAVENIIDASAAQQVSADFIRPMKEFMEKAVAAGHGGDDISSLIDFVKST

[0126] SEQ ID NO: 4, Amino acid sequence of the reductase aminase mutant

[0127] MSSVSIFGLGAMGKALASRFLAEKYKVAVWNRSPEKASPLLEKGATLSHTAVDGINASDLIIICLLDNAAVQATLDSALDRLHGKTIVNLTNGTPDQARKLSDLIVSHGAQYVDGGIMASPSMIGSPHALVLYSGSPDAFNAAEADLSVLANCVFLGEDAGSASLHDLALLSGMYGLFSGFLHATALVKSSTPAVKFLDLLVPWLGLMTEYTKGMAKQIDEGQYASEGSNLAMQLVIVENIIDASAAQQVSADFIRPMKEFMEKAVAAGHGGDDISSLIDFVKST

[0128] SEQ ID NO: 5, Amino acid sequence of the reductase amino enzyme mutant

[0129] MSSVSIFGLGAMGKALASRFLAEKYKVAVWNRSPEKASPLLEKGATLSHTAVDGINASDLIIICLLDNAAVQATLDSALDRLHGKTIVNLTNGTPDQARKLSDLIVSHGAQYVDGGIMASPSMIGSPHACVLYSGSPDAFNAAEADLSVLANCVFLGEDAGSASLHDLALLSGMYGLFSGFLHATALVKSSTPAVKFLDLLVPWLGLMTEYTKGMAKQIDEGQYASEGSNLAMQLVIVENIIDASAAQQVSADFIRPMKEFMEKAVAAGHGGDDISSLIDFVKST

[0130] SEQ ID NO: 6, Amino acid sequence of the reductase amino enzyme mutant

[0131] MSSVSIFGLGAMGKALASRFLAEKYKVAVWNRSPEKASPLLEKGATLSKTAVDGINASDLIIICLLDNAAVQATLDSALDRLHGKTIVNLTNGTPDQARKLSDLIVSHGAQYVDGGIMASPSMIGSPHACVLYSGSPDAFNAAEADLSVLANCVFLGEDAGSASLHDLALLSGMYGLFSGFLHATALVKSSTPAVKFLDLLVPWLGLMTEYTKGMAKQIDEGQYASEGSNLAMQLVIVENIIDASAAQQVSADFIRPMKEFMEKAVAAGHGGDDISSLIDFVKST

[0132] SEQ ID NO: 7, Commercial GDH Amino Acid Sequence

[0133] MAVTQTAQACDLVIFGAKGDLARRKLLPSLYQLEKAGQLNPDTRIIGVGRADWDKAAYTKVVREALETFMKETIDEGLWDTLSARLDFCNLDVNDTAAFSRLGAMLDQKNRITINYFAMPPSTFGAICKGLGEAKLNAKPARVVMEKPLGTSLATSQEINDQVGEYFEECQVYRIDHYLGKETVLNLLALRFANSLFVNNWDNRTIDHVEITVAEEVGIEGRWGYFDKAGQMRDMIQNHLLQILCMIAMSPPSDLSADSIRDEKVKVLKSLRRIDRSNVREKTVRGQYTAGFAQGKKVPGYLEEEGANKSSNTETFVAIRVDIDNWRWAGVPFYLRTGKRLPTKCSEVVVYFKTPELNLFKESWQDLPQNKLTIRLQPDEGVDIQVLNKVPGLDHKHNLQITKLDLSYSETFNQTHLADAYERLLLETMRGIQALFVRRDEVEEAWKWVDSITEAWAMDNDAPKPYQAGTWGPVASVAMITRDGRSWNEFE

[0134] SEQ ID NO: 8, Synthetic GDH Amino Acid Sequence

[0135] MYSDLAGKVVVITGSATGLGRAMGVRFAKEKAKVVINYRSRESEANDVLEEIKKVGGEAIAVKGDVTVESDVANLIQSAVKEFGTLDVMINNAGIENAVPSHEMPLEDWNRVIKTNLTGAFLGSREAIKYFVEHDIKGSV INMSSVHEKIPWPLFVHYAASKGGIKLMTETLALEYAPKGIRVNNIGPGAINTPINAEKFADPKKRADVESMIPMGYIGKPEEIAAVATWLASSEASYVTGITLFADGGMTLYPSFQAVEGKNSSSVDKLAAALEHHHHHH

[0136] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A reducing aminoase mutant, characterized in that, The reducing aminoase mutant differs from the amino acid sequence shown in SEQ ID NO:2 by one or more amino acid residues at the following sites: position 49, position 114, position 120, position 130, position 207, and position 237.

2. The reducing aminoase mutant as described in claim 1, characterized in that, The reducing aminoase mutant contains one or more amino acid residues selected from the following, compared to the amino acid sequence shown in SEQ ID NO: 2: H49K, H114D, T120S, L130C, A207L, A237. Preferably, the reducing aminoase mutant comprises the following amino acid residue difference combination compared with the amino acid sequence shown in SEQ ID NO: 2: A207L; More preferably, the reducing aminoase mutant, compared with the amino acid sequence shown in SEQ ID NO: 2, further includes one or more of the following amino acid residue differences: H49K, H114D, T120S, L130C and A237I, and preferably also includes the following combination of amino acid residue differences: H114D / T120S / A237I; More preferably, the reducing aminoase mutant further comprises the following amino acid residue differences compared to the amino acid sequence shown in SEQ ID NO: 2: L130C and / or H49K; More preferably, the amino acid sequence of the reducing aminoase mutant is shown in SEQ ID NO: 3, 4, 5 or 6.

3. An isolated nucleic acid, characterized in that, The isolated nucleic acid encodes the reductive aminoase mutant as described in claim 1 or 2.

4. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the isolated nucleic acid as described in claim 3; preferably, the backbone of the recombinant expression vector is pET-30a.

5. A transformant, characterized in that, The transformant comprises the isolated nucleic acid as described in claim 3 or the recombinant expression vector as described in claim 4; preferably, the host cell used in constructing the transformant is selected from Escherichia coli cells, insect cells, yeast cells and mammalian cells; the host cell is preferably Escherichia coli, such as BL21(DE3).

6. A method for preparing a reducing aminoase mutant, characterized in that, The method includes: culturing the transformant as described in claim 5 to obtain the reducing aminoase mutant.

7. An enzyme preparation, characterized in that, The enzyme preparation contains the reducing aminoase mutant as described in claim 1 or 2.

8. An enzyme composition, characterized in that, The enzyme composition comprises a reducing aminoase or a mutant of the reducing aminoase as described in claim 1 or 2, as well as a coenzyme and glucose dehydrogenase. The coenzyme is preferably nicotinamide adenine dinucleotide phosphate or nicotinamide adenine dinucleotide; The reducing aminoase is preferably derived from Aspergillus udagawae. For example, the amino acid sequence of the reducing aminoase is selected from the group consisting of: (1) As shown in SEQ ID NO: 2; (2) Has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO: 2; Preferably, the amino acid sequence of the glucose dehydrogenase is SEQ ID NO: 7 or 8; More preferably, the mass ratio of the reducing aminoase or reducing aminoase mutant, coenzyme and glucose dehydrogenase is (10-30):(0.2-1.2):(1-6); for example, 10:0.4:2, 30:1.2:6, 12:1.2:6, 10:0.2:1 or 15:0.3:

3.

9. The use of a reducing aminoase, a reducing aminoase mutant as claimed in claim 1 or 2, an isolated nucleic acid as claimed in claim 3, a recombinant expression vector as claimed in claim 4, a transformant as claimed in claim 5, an enzyme preparation as claimed in claim 7, or an enzyme composition as claimed in claim 8 in reducing compound I with structure I to obtain compound II with structure II; ； in, R1 is selected from hydrogen and hydroxyl; R2 is selected from methyl, ethyl, propyl, tert-butyl, phenyl, benzyl and thiophene; Preferably, the application satisfies one or more of the following conditions: (1) The reducing aminoase is derived from Aspergillus udagawae; for example, the amino acid sequence of the reducing aminoase is selected from the group consisting of: (a) As shown in SEQ ID NO: 2; (b) Has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity with SEQ ID NO: 2; (2) Compound I is any of the following compounds: , , , , , , , , and ; (3) Compound II is any of the following compounds: , , , , , , , , and .

10. A method for preparing a compound represented by formula II, characterized in that, The preparation method includes the following steps: using a reducing amino enzyme, the reducing amino enzyme mutant as described in claim 1 or 2, the enzyme preparation as described in claim 7, or the enzyme composition as described in claim 8 to catalyze the amino donor to contact and react with the compound shown in Formula I to obtain the compound shown in Formula II; ； Wherein, R1 is selected from hydrogen and hydroxyl; R2 is selected from methyl, ethyl, propyl, tert-butyl, phenyl, benzyl and thiophene; Preferably, the amino donor is an ammonium salt solution, such as an ammonium chloride buffer solution; the molar ratio of the ammonium chloride to the compound shown in Formula I is preferably (10-15):1, more preferably 12:1; And / or, the mass ratio of the reducing aminoase or the reducing aminoase mutant to the compound shown in Formula I is preferably (0.01-10):1, more preferably (0.5-2):1, for example 0.5:1, 0.2:1, 1:1 or 0.16:1; And / or, compound I and compound II as described in claim 9; And / or, the reducing aminoase is derived from Aspergillus udagawae, and preferably, the amino acid sequence of the reducing aminoase is selected from the group consisting of: (1) As shown in SEQ ID NO: 2; (2) It has at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity with SEQ ID NO:

2.

11. The preparation method according to claim 10, characterized in that, The preparation method includes the following steps: in a solvent, in the presence of a coenzyme, glucose dehydrogenase and D-glucose, the reducing amino enzyme or the reducing amino enzyme mutant is used to catalyze the amino donor to contact and react with the compound shown in Formula I to obtain the compound shown in Formula II. Preferably, the preparation method is selected from one or more of the following conditions: (1) The pH value of the reaction is 7-11, preferably 8.5; (2) The coenzyme is nicotinamide adenine dinucleotide phosphate or nicotinamide adenine dinucleotide; preferably, the coenzyme is nicotinamide adenine dinucleotide phosphate. (3) The solvent is an organic solvent, such as dimethyl sulfoxide; (4) The mass-to-volume ratio of the compound shown in Formula I to the solvent is 0.1~1 mg / μL, preferably 0.2 mg / μL; (5) The mass ratio of the coenzyme to the compound shown in Formula I is (0.01-0.5):1, preferably (0.05-0.1):1, for example 0.02:1 or 0.1:1; (6) The mass ratio of the glucose dehydrogenase to the compound shown in Formula I is (0.01-0.5):1, preferably (0.05-0.1):1, for example 0.1:1; (7) The mass ratio of the D-glucose to the compound shown in Formula I is (0.01-10):1, preferably (1-6):1, for example 3:1; (8) The reaction temperature is 20~35℃, preferably 30℃; (9) The amino acid sequence of the glucose dehydrogenase is as shown in SEQ ID NO: 7 or 8; the preferred mass ratio of the reducing aminoase or reducing aminoase mutant, coenzyme and glucose dehydrogenase is (10-30):(0.2-1.2):(1-6); for example, 10:0.4:2, 30:1.2:6, 12:1.2:6, 10:0.2:1 or 15:0.3:3; (10) The preparation method further includes the following steps: after the reaction solution is reacted overnight on a shaker, methanol is added to terminate the reaction, the pH of the reaction solution is adjusted to 8.0-10.0 (preferably 9.0), centrifuged, filtered through a membrane, and the reaction solution is detected by a high performance gas chromatograph.

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