A (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid synthetic protein and its mutant and application

Abstract

The present invention discloses a (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid synthetic protein and its mutants and applications. The (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid synthetic protein disclosed in the present invention is a protein of the 37-508th amino acid of SEQ ID No.1, which can catalyze 3-isobutylglutarimide to generate a single chiral (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid amidohydrolase, and can simultaneously obtain a mutant having a catalytic synthesis of a high-purity R-type product by using a point mutation technique, and realize the specific conversion of cyclic imine to R-monoamide by a biological method, thereby reducing the chemical reaction steps, and finally the R-monoamide prepared by the present invention can be used to further obtain S-pregabalin with high optical purity. The present invention is of great significance for the production of S-pregabalin with high optical purity.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid synthetic protein and a mutant thereof and application thereof. Background Art

[0002] Pregabalin (PGB) is a new type of γ-aminobutyric acid (GABA) receptor agonist that can effectively block voltage-dependent calcium channels, reduce the release of neurotransmitters, and has good anti-anxiety and neuropathic pain treatment effects. It is widely used in the treatment of epilepsy and peripheral neuropathy.

[0003] Pregabalin is a chiral drug. The pharmacological activities of different configurations vary greatly. The pharmacological activity of the S-isomer is 10 times that of the R-isomer. Therefore, optically pure S-pregabalin can effectively enhance the efficacy of the drug, reduce the occurrence of adverse reactions and avoid potential health hazards. Currently, multiple synthetic routes for pregabalin are known and reported, and each has its own advantages and disadvantages. The synthetic route for pregabalin has mild reaction conditions, short reaction time, and high purity of the target product, which is suitable for large-scale preparation of pregabalin. Obtaining high optical purity (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid (hereinafter referred to as R-monoamide) is a key step in the synthesis of S-pregabalin.

[0004] Compared with chemical synthesis, biocatalysis has many advantages, such as mild reaction conditions, high stereoselectivity, and environmental friendliness. The cyclic amide hydrolase (EC.3.5.2) family is a hydrolase that hydrolyzes amide bonds in organic rings other than peptides and proteins. It is widely present in animals, plants, and microorganisms, and most of them are metal-dependent hydrolases. Summary of the invention

[0005] The first object of the present invention is to provide a method for preparing (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid.

[0006] The present invention provides a method for preparing (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid, comprising: using 3-isobutylglutarimide as a substrate and using protein to perform a catalytic reaction to obtain (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid;

[0007] The protein is C1, C2, C3, C4 or C5:

[0008] C1, a protein derived from Bacillus stearothermophilus SD-1 (denoted as R-monoamide synthetic protein, abbreviated as RSP), whose sequence is amino acids 37-508 of SEQ ID No.1;

[0009] C2, a protein comprising amino acids 37-508 of SEQ ID No. 1;

[0010] C3, RSP mutant protein, which is a protein obtained by mutating the 63rd, 65th and / or 317th amino acid residues of RSP;

[0011] C4, RSP mutant protein, is a protein obtained by mutating the amino acid residues other than positions 63, 65 and 317 of the RSP mutant protein described in C3;

[0012] C5. A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein described in C1, C2, C3 or C4.

[0013] In the above method, the RSP mutant protein C3 is a protein obtained by modifying the RSP comprising the following A1, A2 and / or A3:

[0014] A1, mutating the methionine residue at position 63 of the RSP to an alanine residue, a cysteine ​​residue, a glutamic acid residue, an isoleucine residue, an asparagine residue, a glutamine residue, a serine residue, a threonine residue, a valine residue, an aspartic acid residue, a phenylalanine residue, a glycine residue, a histidine residue, a lysine residue, a leucine residue, a proline residue, an arginine residue, a tryptophan residue or a tyrosine residue;

[0015] A2, mutating the leucine residue at position 65 of the RSP to a histidine residue, an asparagine residue, a proline residue, an alanine residue, a cysteine ​​residue, an aspartic acid residue, a glutamic acid residue, a phenylalanine residue, a glycine residue, an isoleucine residue, a lysine residue, a methionine residue, a glutamine residue, an arginine residue, a serine residue, a threonine residue, a valine residue, a tryptophan residue or a tyrosine residue;

[0016] A3. The cysteine ​​residue at position 317 of the RSP is mutated to an alanine residue, an aspartic acid residue, a glutamic acid residue, a phenylalanine residue, a glycine residue, a histidine residue, an isoleucine residue, a lysine residue, a leucine residue, a methionine residue, an asparagine residue, a proline residue, a glutamine residue, an arginine residue, a serine residue, a threonine residue, a valine residue, a tryptophan residue or a tyrosine residue.

[0017] Specifically, the RSP mutant protein C3 may be a protein obtained by subjecting the RSP to the above-mentioned transformations of A1, A2 and / or A3.

[0018] In one embodiment of the present invention, the fusion protein is a protein obtained by connecting the N-terminus of C1 or C2 to positions 1 to 36 of SEQ ID No. 1 in the sequence list.

[0019] In the above method, the catalytic reaction system may be: buffer 50mM Trsi-HCl (pH 7.5), 10μg of protein C1, C2, C3, C4 or C5, 1mM MnCl2, 5mM 3-isobutylglutarimide.

[0020] The reaction temperature may be 50-75° C. The reaction time may be determined based on the actual situation and may generally be 0.5 hours.

[0021] The present invention also provides another method for preparing (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid, comprising: using 3-isobutylglutarimide as a substrate and using a recombinant cell expressing the protein C1, C2 or C3 to carry out a catalytic reaction to obtain (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid.

[0022] In the above method, the recombinant cell can be obtained by introducing a recombinant vector capable of expressing the protein into a biological cell.

[0023] The biological cell may be a microorganism. The microorganism may be Escherichia coli or other bacteria. In one embodiment of the present invention, the microorganism is Escherichia coli BL21 (DE3).

[0024] The recombinant vector may be a recombinant plasmid obtained by replacing the small DNA fragment between the EcoRI and HindIII recognition sequences in the pET-28a(+) vector with the RSP gene or the gene encoding the mutant protein.

[0025] The RSP gene may be the nucleic acid molecule shown in positions 109-1527 of SEQ ID No. 2 in the sequence list.

[0026] The gene encoding the mutant protein may be a DNA molecule obtained by the following transformation of E1, E2 and / or E3:

[0027] E1. Mutating positions 187-189 of the gene shown in positions 109-1527 of SEQ ID No. 2 from ATG to codons for alanine, cysteine, glutamic acid, isoleucine, asparagine, glutamine, serine, threonine, valine, aspartic acid, phenylalanine, glycine, histidine, lysine, leucine, proline, arginine, tryptophan or tyrosine;

[0028] E2. Mutating positions 193-195 of the gene shown in positions 109-1527 of SEQ ID No. 2 from CTG to codons for histidine, asparagine, proline, alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine, isoleucine, lysine, methionine, glutamine, arginine, serine, threonine, valine, tryptophan or tyrosine;

[0029] E3. Mutate positions 949-951 in the gene shown by positions 109-1527 of SEQ ID No. 2 from TGT to codons of alanine, aspartic acid, glutamic acid, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan or tyrosine.

[0030] In an embodiment of the present invention, the codons for each amino acid are preferred codons for E. coli. Specifically, the codon for alanine may be GCG; the codon for cysteine ​​may be TGT; the codon for aspartic acid may be GAT; the codon for glutamic acid may be GAA; the codon for phenylalanine may be TTT; the codon for glycine may be GGT; the codon for histidine may be CAT; the codon for isoleucine may be ATT; the codon for lysine may be AAA; the codon for leucine may be CTG; the codon for methionine may be A TG; the codon for asparagine may be AAT; the codon for glutamine may be CAG; the codon for proline may be CCG; the codon for arginine may be CGC; the codon for serine may be AGT; the codon for threonine may be ACC; the codon for valine may be GTT; the codon for tryptophan may be TGG; the codon for tyrosine may be TAT; the codon for alanine may be GCG; and the codon for cysteine ​​may be GCG.

[0031] The recombinant cell may be a recombinant bacterium obtained by introducing the recombinant vector into Escherichia coli BL21 (DE3).

[0032] In the above method, the catalytic reaction system can be: buffer 50mM Trsi-HCl (pH 7.5), 1.8±0.7×10 8 cfu / L of the recombinant cells, 1 mM MnCl2, 10 mM 3-isobutylglutarimide.

[0033] The reaction temperature may be 45-75° C., such as 50-75° C. The reaction time may be determined based on the actual situation, and may generally be 0.5-2 hours.

[0034] The proteins described in C1, C2, C3, C4 or C5 also fall within the protection scope of the present invention.

[0035] Biological materials related to the proteins described in C1, C2, C3, C4 or C5 also fall within the scope of protection of the present invention, and the biological materials are any one of the following B1) to B4):

[0036] B1) a nucleic acid molecule encoding the protein described in C1, C2, C3, C4 or C5;

[0037] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0038] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0039] B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3).

[0040] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA.

[0041] The expression cassette of the nucleic acid molecule refers to a DNA capable of expressing the protein C1, C2, C3, C4 or C5 in a host cell, and the DNA may include not only a promoter for initiating transcription of the gene encoding the protein C1, C2, C3, C4 or C5, but also a terminator for terminating transcription of the gene encoding the protein C1, C2, C3, C4 or C5. Furthermore, the expression cassette may also include an enhancer sequence.

[0042] The recombinant vector of the nucleic acid molecule can be a bacterial plasmid (such as an expression vector based on a T7 promoter expressed in bacteria, such as pET-28a, etc.), a bacteriophage, a yeast plasmid (such as a YEp series vector, etc.) or a retrovirus packaging plasmid carrying the protein encoding gene of C1, C2, C3, C4 or C5.

[0043] The recombinant vector is a vector obtained by inserting the nucleic acid molecule into an expression vector.

[0044] The present invention also provides any of the following products:

[0045] Y1. A complete product consisting of the protein described in C1, C2, C3, C4 or C5 and 3-isobutylglutarimide;

[0046] Y2. A complete set of products consisting of the biomaterial and 3-isobutylglutarimide.

[0047] The present invention also provides application of the method for preparing (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid in preparing pregabalin or 3-(carbamoylmethyl)-5-methylhexanoic acid.

[0048] The present invention also provides any of the following uses of the protein, biomaterial, or product described in C1, C2, C3, C4, or C5:

[0049] Z1, production of pregabalin;

[0050] Z2. Preparation and production of pregabalin products;

[0051] Z3, production of 3-(carbamoylmethyl)-5-methylhexanoic acid;

[0052] Z4, preparation and production of 3-(carbamoylmethyl)-5-methylhexanoic acid product;

[0053] Z5, production of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid;

[0054] Z6. Preparation and production of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid product.

[0055] The use of the protein described in C1, C2, C3, C4 or C5 as an enzyme also falls within the protection scope of the present invention.

[0056] The present invention selects the step of cyclic imine to monoamide as the starting point of transformation, screens an amide hydrolase that can catalyze 3-isobutylglutarimide to generate a single chiral (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid, and simultaneously uses point mutation technology to obtain a mutant that can catalyze the synthesis of high-purity R-type products, and achieves the specific conversion of cyclic imine to R-monoamide by biological method, thereby reducing the chemical reaction steps, and the R-monoamide prepared by the present invention can be used to further obtain S-pregabalin with high optical purity. The present invention is of great significance for the production of S-pregabalin with high optical purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] Figure 1 The results of SDS-PAGE electrophoresis of pure enzyme solution. Lanes 1-5 are pure enzyme solutions obtained from BL21 / pET-28a-RSP-O, BL21 / pET-28a-M63A, BL21 / pET-28a-M63A / L65H, BL21 / pET-28a-M63A / L65N, and BL21 / pET-28a-M63A / L65A, respectively; M indicates protein marker.

[0058] Figure 2 The optimum temperature test results of each enzyme. The ordinate is the relative enzyme activity (%), that is, the relative value of the total amount of 3-(carbamoylmethyl)-5-methylhexanoic acid generated at each temperature when the total amount of 3-(carbamoylmethyl)-5-methylhexanoic acid generated at the optimum temperature is defined as 100. M63AL65H, M63AL65N, M63AL65A, M63A, RSP represent M63AL65H fusion protein, M63AL65N fusion protein, M63AL65A fusion protein, M63A fusion protein, RSP fusion protein, respectively. DETAILED DESCRIPTION

[0059] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0060] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are carried out according to the techniques or conditions described in the literature in the art or according to the product instructions. The materials, reagents, instruments, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources. The quantitative tests in the following examples were all repeated three times, and the results were averaged. In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence table is the 5' terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA / RNA.

[0061] 3-Isobutylglutarimide: CAS No.: 916982-10-0; the 3-isobutylglutarimide used in the examples is a Macklin product.

[0062] (R)-(-)-3-(Carbamoylmethyl)-5-methylhexanoic acid: CAS No.: 181289-33-8; the (R)-(-)-3-(Carbamoylmethyl)-5-methylhexanoic acid used in the examples is a product of Shanghai Yuanye Biotechnology Co., Ltd.

[0063] In the following examples, 3-isobutylglutarimide and (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid were detected by high performance liquid chromatography, and the detection conditions were as follows: Agilent C18 reverse phase column (250×4.6 mm, 5 μm), detection wavelength: 210 nm; column temperature: 35° C.; flow rate: 1.0 ml / min; injection volume: 20 μl; running time: 45 min; mobile phase A: buffer salt (weigh about 3.5 g of dipotassium hydrogen phosphate, add 1000 ml of water to dissolve, mix well, adjust the pH to 3.0 with phosphoric acid, and filter), mobile phase B: acetonitrile; program settings are shown in Table 1. The elution time of the 3-isobutylglutarimide standard was 26.5 min, and the elution time of the (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid was 8.0 min. The standards used were Macklin's 3-isobutylglutarimide and (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid.

[0064] Table 1. HPLC detection conditions

[0065] Time (min) Flow rate (ml / min) Mobile phase A% Mobile phase B% 0 1.0 80 20 20 1.0 80 20 30 1.0 60 40 35 1.0 60 40 40 1.0 80 20 45 1.0 80 20

[0066] In the following examples, the chiral purity of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid (i.e., the proportion of R-type product in the total product) was first detected by high performance liquid chromatography to determine the yields of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid and (S)-(+)-3-carbamoylmethyl-5-methylhexanoic acid. The detection conditions were as follows: chromatographic column: Chiralpak AD-H column, 250×4.6 mm, 5 μm; detection wavelength: 210 nm; column temperature: 25° C.; flow rate: 0.5 ml / min; mobile phase: n-hexane: ethanol: trifluoroacetic acid = 880:120:1.5 (volume ratio). The standard substance used was mixed racemic 3-(carbamoylmethyl)-5-methylhexanoic acid (i.e., a mixture of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid and (S)-(+)-3-carbamoylmethyl-5-methylhexanoic acid) (Shanghai Yuanye Biotechnology Co., Ltd., catalog number: S62775).

[0067] Then calculate the chiral purity of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid, (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid chiral purity = (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid yield / ((R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid yield + (S)-(+)-3-carbamoylmethyl-5-methylhexanoic acid yield).

[0068] Example 1: Enzyme activity detection

[0069] In this example, a protein derived from Bacillus stearothermophilus SD-1 (denoted as R-monoamide synthetic protein, abbreviated as RSP) was found to be able to hydrolyze 3-isobutylglutarimide. The amino acid sequence of RSP is positions 37-508 of sequence 1 in the sequence list. In this example, the RSP gene was optimized according to the codon preference of Escherichia coli to obtain the RSP-O gene. The sequence of the RSP-O gene is positions 109-1527 of SEQ ID No. 2 in the sequence list.

[0070] 1. Construction of recombinant vector

[0071] The DNA fragment between the EcoRI and HindIII recognition sequences of the expression vector pET-28a(+) (Novagen) was replaced with the RSP-O gene shown at positions 109-1527 of SEQ ID No.2 to obtain a recombinant vector, which was recorded as pET-28a-RSP-O. pET-28a-RSP-O contained the DNA fragment shown in SEQ ID No.2 in the sequence table (recorded as RSP-O fusion gene), and was capable of expressing a fusion protein formed by RSP and a His tag (whose sequence is sequence 1 in the sequence table, recorded as RSP fusion protein).

[0072] The DNA fragment shown in SEQ ID No. 2 encodes the fusion protein shown in sequence 1. Positions 37-508 of sequence 1 are RSP, and positions 5-10 are His tags. Positions 109-1527 of SEQ ID No. 2 are RSP-O genes, and positions 13-30 are the encoding DNA sequence of the His tag.

[0073] 2. Preparation of recombinant bacteria

[0074] The pET-28a-RSP-O obtained in step 1 was introduced into Escherichia coli BL21 (DE3) to obtain the recombinant bacteria BL21 / pET-28a-RSP-O; the expression vector pET-28a (+) was introduced into Escherichia coli BL21 (DE3) to obtain the control recombinant bacteria.

[0075] 3. Detection of enzyme activity

[0076] The recombinant bacteria BL21(DE3) / pET-28a-RSP-O prepared in step 2 and the control recombinant bacteria were used as test strains and the experiment was carried out according to the following steps:

[0077] 1) The strain to be tested was inoculated into 10 mL LB liquid culture medium containing 40 μg / mL kanamycin, cultured at 37°C until OD600 = 0.6-0.8, and the inducer IPTG was added to a concentration of 0.5 mM in the culture system, and then cultured overnight at 24°C and 200 rpm, and the cells were collected and washed once with 50 mM Trsi-HCl (pH 7.5) to remove the culture medium on the cell surface to obtain cells.

[0078] 2) Resuspend the washed cells in 50 mM Trsi-HCl (pH 7.5) and adjust the OD600 to 30 (viable cell content 1.8 ± 0.7 × 10 8 cfu / L) to obtain a bacterial suspension, and then add the substrate 3-isobutylglutarimide (its concentration in the reaction system is 10 mM) to the bacterial suspension, react at 45°C and 120 rpm for 2 h to obtain a reaction product.

[0079] 3) After completing step 2), the reaction product was centrifuged at 12000 rpm for 2 min, and the supernatant was subjected to HPLC detection. The mixed racemic 3-(carbamoylmethyl)-5-methylhexanoic acid was used as a standard, and the catalytic activity of the recombinant strain was determined according to the total amount of 3-(carbamoylmethyl)-5-methylhexanoic acid produced.

[0080] The results showed that BL21 / pET-28a-RSP-O had catalytic activity for the substrate 3-isobutylglutarimide, and could catalyze the production of about 8 mM 3-(carbamoylmethyl)-5-methylhexanoic acid at the end of the reaction. The control recombinant bacteria had no catalytic activity for the substrate 3-isobutylglutarimide.

[0081] Example 2, point mutation

[0082] In this example, the optimized RSP-O gene was mutated to obtain RSP amino acids to obtain mutant proteins, and the names of the mutant proteins were M63A, C317A, C317G, L65A, L65D and L65Y.

[0083] Among them, M63A is a mutant protein obtained by mutating the methionine residue at position 63 of RSP to an alanine residue; C317A is a mutant protein obtained by mutating the cysteine ​​residue at position 317 of RSP to an alanine residue; C317G is a mutant protein obtained by mutating the cysteine ​​residue at position 317 of RSP to a glycine residue; L65A is a mutant protein obtained by mutating the leucine residue at position 65 of RSP to an alanine residue; L65D is a mutant protein obtained by mutating the leucine residue at position 65 of RSP to an aspartic acid residue; L65Y is a mutant protein obtained by mutating the leucine residue at position 65 of RSP to a tyrosine residue.

[0084] Here are the steps:

[0085] 1. PCR amplification was performed using the conventional overlapping PCR method with BL21 / pET-28a-RSP-O in Example 1 as a template, and the resulting PCR product was digested with endonuclease DpnI to remove the template, and the digestion product was transformed into Escherichia coli BL21 (DE3), and then the strain was cultured, and the plasmid was extracted for sequencing to obtain a recombinant bacterium capable of expressing a fusion protein (denoted as the corresponding fusion protein) formed by the target mutant protein and positions 1-36 of sequence 1. The primer sequences used to construct each mutant protein are shown in Table 2.

[0086] Table 2. Primer sequences

[0087]

[0088]

[0089] The recombinant bacteria expressing the M63A fusion protein were recorded as BL21 / pET-28a-M63A. BL21 / pET-28a-M63A contained the recombinant vector pET-28a-M63A. In the obtained BL21 / pET-28a-M63A and pET-28a-M63A, positions 187-189 of the RSP-O gene were mutated from ATG to GCG, and the mutated gene was recorded as the M63A gene.

[0090] The recombinant bacteria expressing C317A fusion protein were recorded as BL21 / pET-28a-C317A. BL21 / pET-28a-C317A contained the recombinant vector pET-28a-C317A. In the obtained BL21 / pET-28a-C317A and pET-28a-C317A, positions 949-951 of the RSP-O gene were mutated from TGT to GCG, and the mutated gene was recorded as the C317A gene.

[0091] The recombinant bacteria expressing C317G fusion protein were recorded as BL21 / pET-28a-C317G. BL21 / pET-28a-C317G contained the recombinant vector pET-28a-C317G. In the obtained BL21 / pET-28a-C317G and pET-28a-C317G, the 949-951 positions of the RSP-O gene were mutated from TGT to GGT, and the mutated gene was recorded as the C317G gene.

[0092] The recombinant bacteria expressing L65A fusion protein were recorded as BL21 / pET-28a-L65A. BL21 / pET-28a-L65A contained the recombinant vector pET-28a-L65A. In the obtained BL21 / pET-28a-L65A and pET-28a-L65A, positions 193-195 of the RSP-O gene were mutated from CTG to GCG, and the mutated gene was recorded as the L65A gene.

[0093] The recombinant bacteria expressing L65D fusion protein were recorded as BL21 / pET-28a-L65D. BL21 / pET-28a-L65D contained the recombinant vector pET-28a-L65D. In the obtained BL21 / pET-28a-L65D and pET-28a-L65D, the 193-195 positions of the RSP-O gene were mutated from CTG to GAT, and the mutated gene was recorded as the L65D gene.

[0094] The recombinant bacteria expressing the L65Y fusion protein were recorded as BL21 / pET-28a-L65Y. BL21 / pET-28a-L65Y contained the recombinant vector pET-28a-L65Y. In the obtained BL21 / pET-28a-L65Y and pET-28a-L65Y, the 193-195 positions of the RSP-O gene were mutated from CTG to TAT, and the mutated gene was recorded as the L65Y gene.

[0095] 2. The recombinant bacteria prepared in step 1 were inoculated into 10 mL LB liquid medium containing 40 μg / mL kanamycin, cultured at 37°C to OD600 = 0.6-0.8, and the inducer IPTG was added to a final concentration of 0.5 mM. After overnight culture at 24°C and 200 rpm, the cells were collected and washed once with 50 mM Trsi-HCl (pH 7.5) to remove the culture medium on the cell surface.

[0096] 3. Resuspend the washed bacteria in 50 mM Trsi-HCl (pH 7.5) and adjust the OD600 to 30 (viable bacteria content is 1.8 ± 0.7 × 10 8 cfu / L) to obtain a bacterial suspension, and then add the substrate 3-isobutylglutarimide (its concentration in the reaction system is 10 mM) to the bacterial suspension, react at 45°C and 120 rpm for 2 h to obtain a reaction product.

[0097] 4. After completing step 3, the reaction system was centrifuged at 12000 rpm for 2 min, the supernatant was taken, and the sample was freeze-dried using a vacuum freeze dryer. After drying, 1 mL of ethanol was added to dissolve the sample, and the chiral purity of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid in the product was determined by HPLC (i.e., the proportion of R-type product in the total product).

[0098] BL21 (DE3) / pET-28a-RSP-O of Example 1 was used as a control, and the results are shown in Table 3. The results in Table 3 show that the chiral purity of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid in the product of the recombinant bacteria expressing the M63A fusion protein is the highest, indicating that the mutation of methionine at position 63 of RSP to alanine can improve the chiral purity of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid in the product, and C317A, C317G, G66A, K334A, I335A, and L65D can also improve the chiral purity of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid in the product to varying degrees.

[0099] Table 3. Chiral purity of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid

[0100] Protein name Mean ± standard deviation (%) P-value RSP fusion protein 70.91±4.70 M63A fusion protein 94.73±0.32 0.0186 C317A fusion protein 78.77±3.96 0.2121 C317G fusion protein 73.02±2.09 0.619 L65A fusion protein 64.52±15.72 0.6372 L65D fusion protein 83.35±4.70 0.1179 L65Y fusion protein 44.50±2.73 0.0205

[0101] Note: In Table 3, the P value is the significance analysis result of the RSP fusion protein; in each mutation site listed in Table 3, the number indicates the position of the mutation site, the letter before the number is the amino acid residue of the wild type, and the letter after the number is the amino acid residue after the mutation, for example: M63A is obtained by mutating the 63rd position M of the wild-type protein to A.

[0102] Example 3: Saturation mutation of the M63 site of RSP

[0103] In this embodiment, saturation mutation was performed on the M63 site, and PCR amplification was performed using BL21 / pET-28a-RSP-O in Example 1 as a template. The resulting PCR product was digested with endonuclease DpnI to remove the template, and the digestion product was transformed into Escherichia coli BL21 (DE3), and then the strain was cultured, and the plasmid was extracted for sequencing verification. Recombinant bacteria that can express M63H, M63N, M63P, M63A, M63C, M63D, M63E, M63F, M63G, M63I, M63K, M63M, M63Q, M63R, M63S, M63T, M63V, M63W, and M63Y mutant proteins (the number before the unmutated amino acid residue and the number after the mutation) and the fusion protein formed by positions 1-36 of sequence 1 (denoted as the corresponding fusion protein) were obtained.

[0104] The primers used are as follows:

[0105] F: CGCATACCCATCTGGATNNNCCTCTGGGTGGTACCGTGAC (5'-->3' direction);

[0106] R: GTCACGGTACCACCCAGAGGNNNATCCAGATGGGTATGCG (5'-->3' direction), N represents A, T, C or G.

[0107] According to the method of steps 2-4 in Experimental Example 2, the recombinant bacteria were replaced with the recombinant bacteria obtained in this example, and the other steps remained unchanged. The chiral purity of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid in the products obtained by each recombinant bacteria was detected, and BL21 / pET-28a-RSP-O in Example 1 was used as a control.

[0108] The results showed that the chirality of the reactions catalyzed by all mutants except M63G was improved to varying degrees (Table 4).

[0109] Table 4. Chiral purity of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid

[0110]

[0111] In Table 4, the P value is the significance analysis result of the fusion protein with RSP.

[0112] The recombinant bacteria expressing the M63C fusion protein were recorded as BL21 / pET-28a-M63C. BL21 / pET-28a-M63C contained the recombinant vector pET-28a-M63C. In the obtained BL21 / pET-28a-M63C and pET-28a-M63C, positions 187-189 of the RSP-O gene were mutated from ATG to TGT, and the mutated gene was recorded as the M63C gene.

[0113] The recombinant bacteria expressing the M63E fusion protein were recorded as BL21 / pET-28a-M63E. BL21 / pET-28a-M63E contained the recombinant vector pET-28a-M63E. In the obtained BL21 / pET-28a-M63E and pET-28a-M63E, positions 187-189 of the RSP-O gene were mutated from ATG to GAA, and the mutated gene was recorded as the M63E gene.

[0114] The recombinant bacteria expressing the M63I fusion protein were recorded as BL21 / pET-28a-M63I. BL21 / pET-28a-M63I contained the recombinant vector pET-28a-M63I. In the obtained BL21 / pET-28a-M63I and pET-28a-M63I, positions 187-189 of the RSP-O gene were mutated from ATG to ATT, and the mutated gene was recorded as the M63I gene.

[0115] The recombinant bacteria expressing the M63N fusion protein were recorded as BL21 / pET-28a-M63N. BL21 / pET-28a-M63N contained the recombinant vector pET-28a-M63N. In the obtained BL21 / pET-28a-M63N and pET-28a-M63N, positions 187-189 of the RSP-O gene were mutated from ATG to AAT, and the mutated gene was recorded as the M63N gene.

[0116] The recombinant bacteria expressing the M63Q fusion protein were recorded as BL21 / pET-28a-M63Q. BL21 / pET-28a-M63Q contained the recombinant vector pET-28a-M63Q. In the obtained BL21 / pET-28a-M63Q and pET-28a-M63Q, positions 187-189 of the RSP-O gene were mutated from ATG to CAG, and the mutated gene was recorded as the M63Q gene.

[0117] The recombinant bacteria expressing the M63S fusion protein were recorded as BL21 / pET-28a-M63S. BL21 / pET-28a-M63S contained the recombinant vector pET-28a-M63S. In the obtained BL21 / pET-28a-M63S and pET-28a-M63S, positions 187-189 of the RSP-O gene were mutated from ATG to AGT, and the mutated gene was recorded as the M63S gene.

[0118] The recombinant bacteria expressing the M63T fusion protein were recorded as BL21 / pET-28a-M63T. BL21 / pET-28a-M63T contained the recombinant vector pET-28a-M63T. In the obtained BL21 / pET-28a-M63T and pET-28a-M63T, positions 187-189 of the RSP-O gene were mutated from ATG to ACC, and the mutated gene was recorded as the M63T gene.

[0119] The recombinant bacteria expressing the M63V fusion protein were recorded as BL21 / pET-28a-M63V. BL21 / pET-28a-M63V contained the recombinant vector pET-28a-M63V. In the obtained BL21 / pET-28a-M63V and pET-28a-M63V, positions 187-189 of the RSP-O gene were mutated from ATG to GTT, and the mutated gene was recorded as the M63V gene.

[0120] The recombinant bacteria expressing the M63D fusion protein were recorded as BL21 / pET-28a-M63D. BL21 / pET-28a-M63D contained the recombinant vector pET-28a-M63D. In the obtained BL21 / pET-28a-M63D and pET-28a-M63D, positions 187-189 of the RSP-O gene were mutated from ATG to GAT, and the mutated gene was recorded as the M63D gene.

[0121] The recombinant bacteria expressing the M63F fusion protein were recorded as BL21 / pET-28a-M63F. BL21 / pET-28a-M63F contained the recombinant vector pET-28a-M63F. In the obtained BL21 / pET-28a-M63F and pET-28a-M63F, positions 187-189 of the RSP-O gene were mutated from ATG to TTT, and the mutated gene was recorded as the M63F gene.

[0122] The recombinant bacteria expressing the M63H fusion protein were recorded as BL21 / pET-28a-M63H. BL21 / pET-28a-M63H contained the recombinant vector pET-28a-M63H. In the obtained BL21 / pET-28a-M63H and pET-28a-M63H, positions 187-189 of the RSP-O gene were mutated from ATG to CAT, and the mutated gene was recorded as the M63H gene.

[0123] The recombinant bacteria expressing the M63K fusion protein were recorded as BL21 / pET-28a-M63K. BL21 / pET-28a-M63K contained the recombinant vector pET-28a-M63K. In the obtained BL21 / pET-28a-M63K and pET-28a-M63K, positions 187-189 of the RSP-O gene were mutated from ATG to AAA, and the mutated gene was recorded as the M63K gene.

[0124] The recombinant bacteria expressing the M63L fusion protein were recorded as BL21 / pET-28a-M63L. BL21 / pET-28a-M63L contained the recombinant vector pET-28a-M63L. In the obtained BL21 / pET-28a-M63L and pET-28a-M63L, positions 187-189 of the RSP-O gene were mutated from ATG to CTG, and the mutated gene was recorded as the M63L gene.

[0125] The recombinant bacteria expressing the M63P fusion protein were recorded as BL21 / pET-28a-M63P. BL21 / pET-28a-M63P contained the recombinant vector pET-28a-M63P. In the obtained BL21 / pET-28a-M63P and pET-28a-M63P, positions 187-189 of the RSP-O gene were mutated from ATG to CCG, and the mutated gene was recorded as the M63P gene.

[0126] The recombinant bacteria expressing the M63R fusion protein were recorded as BL21 / pET-28a-M63R. BL21 / pET-28a-M63R contained the recombinant vector pET-28a-M63R. In the obtained BL21 / pET-28a-M63R and pET-28a-M63R, positions 187-189 of the RSP-O gene were mutated from ATG to CGC, and the mutated gene was recorded as the M63R gene.

[0127] The recombinant bacteria expressing the M63W fusion protein were recorded as BL21 / pET-28a-M63W. BL21 / pET-28a-M63W contained the recombinant vector pET-28a-M63W. In the obtained BL21 / pET-28a-M63W and pET-28a-M63W, positions 187-189 of the RSP-O gene were mutated from ATG to TGG, and the mutated gene was recorded as the M63W gene.

[0128] The recombinant bacteria expressing the M63Y fusion protein were recorded as BL21 / pET-28a-M63Y. BL21 / pET-28a-M63Y contained the recombinant vector pET-28a-M63Y. In the obtained BL21 / pET-28a-M63Y and pET-28a-M63Y, positions 187-189 of the RSP-O gene were mutated from ATG to TAT, and the mutated gene was recorded as the M63Y gene.

[0129] Example 4, double mutation of RSP

[0130] In this example, the leucine at position 65 was mutated on the basis of M63A in Example 2, and PCR amplification was performed using the recombinant vector pET-28a-M63A in Example 2 as a template. The resulting PCR product was digested with endonuclease DpnI to remove the template, and the digested product was transformed into Escherichia coli BL21 (DE3), and then the strain was cultured, and the plasmid was extracted for sequencing to obtain plasmids that can express M63AL65H, M63AL65N, M63AL65P, M63AL65A, M63AL65C, M63AL65D, M63AL65E, M63AL65F, M63AL65G, M63AL65I, M63AL65K, M63AL65M, M63AL65Q, M63AL65R, M63AL65S, M63AL65T, M63AL65V, The recombinant bacteria of the fusion protein (denoted as the corresponding fusion protein) formed by the double mutant protein M63AL65W and M63AL65Y (the amino acid residue before the number is not mutated, and the amino acid residue after the number is mutated) and the 1st to 36th positions of sequence 1. The primers used are as follows:

[0131] F: GTCACGGTACCACCCAGAGGNNNATCCAGATGGGTATGCG (5'-->3' direction);

[0132] R: CGCATACCCATCTGGATNNNCCTCTGGGTGGTACCGTGAC (5'-->3' direction), N represents A, T, C or G.

[0133] According to the method of steps 2-4 in Experimental Example 2, the recombinant bacteria were replaced with the recombinant bacteria obtained in this example, and the other steps remained unchanged. The chiral purity of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid in the products obtained by each recombinant bacteria was detected, and BL21 / pET-28a-M63A in Example 2 was used as a control.

[0134] The results are shown in Table 5. The results showed that the chiral purity of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid in the products obtained from BL21 / pET-28a-M63AL65H, BL21 / pET-28a-M63AL65N and BL21 / pET-28a-M63AL65A was above 97%.

[0135] Table 5. Chiral purity of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid

[0136]

[0137] In Table 5, the P value is the significance analysis result of the M63A fusion protein.

[0138] M63AL65H is a mutation of the leucine residue at position 65 in M63A to a histidine residue. The recombinant bacteria expressing the M63AL65H fusion protein are recorded as BL21 / pET-28a-M63AL65H. BL21 / pET-28a-M63AL65H contains the recombinant vector pET-28a-M63AL65H. In the resulting BL21 / pET-28a-M63AL65H and pET-28a-M63AL65H, positions 187-189 of the RSP-O gene are mutated from ATG to GCG, and positions 193-195 are mutated from CTG to CAT. The mutated genes are recorded as M63AL65H genes.

[0139] M63AL65N is a mutation of the leucine residue at position 65 in M63A to an asparagine residue. The recombinant bacteria expressing the M63AL65N fusion protein are recorded as BL21 / pET-28a-M63AL65N. BL21 / pET-28a-M63AL65N contains the recombinant vector pET-28a-M63AL65N. In the obtained BL21 / pET-28a-M63AL65N and pET-28a-M63AL65N, positions 187-189 of the RSP-O gene are mutated from ATG to GCG, and positions 193-195 are mutated from CTG to AAT. The mutated genes are recorded as M63AL65N genes.

[0140] M63AL65P is a mutation of the leucine residue at position 65 in M63A to a proline residue. The recombinant bacteria expressing the M63AL65P fusion protein are recorded as BL21 / pET-28a-M63AL65P. BL21 / pET-28a-M63AL65P contains the recombinant vector pET-28a-M63AL65P. In the obtained BL21 / pET-28a-M63AL65P and pET-28a-M63AL65P, positions 187-189 of the RSP-O gene are mutated from ATG to GCG, and positions 193-195 are mutated from CTG to CCG. The mutated genes are recorded as M63AL65P genes.

[0141] M63AL65A is a mutation of the leucine residue at position 65 in M63A to an alanine residue. The recombinant bacteria expressing the M63AL65A fusion protein are recorded as BL21 / pET-28a-M63AL65A. BL21 / pET-28a-M63AL65A contains the recombinant vector pET-28a-M63AL65A. In the obtained BL21 / pET-28a-M63AL65A and pET-28a-M63AL65A, positions 187-189 of the RSP-O gene are mutated from ATG to GCG, and positions 193-195 are mutated from CTG to GCG. The mutated genes are recorded as M63AL65A genes.

[0142] M63AL65C is a mutation of the leucine residue at position 65 in M63A to a cysteine ​​residue. The recombinant bacteria expressing the M63AL65C fusion protein are recorded as BL21 / pET-28a-M63AL65C. BL21 / pET-28a-M63AL65C contains the recombinant vector pET-28a-M63AL65C. In the obtained BL21 / pET-28a-M63AL65C and pET-28a-M63AL65C, positions 187-189 of the RSP-O gene are mutated from ATG to GCG, and positions 193-195 are mutated from CTG to TGT. The mutated gene is recorded as the M63AL65C gene.

[0143] M63AL65D is a mutation of the leucine residue at position 65 in M63A to an aspartic acid residue. The recombinant bacteria expressing the M63AL65D fusion protein are recorded as BL21 / pET-28a-M63AL65D. BL21 / pET-28a-M63AL65D contains the recombinant vector pET-28a-M63AL65D. In the obtained BL21 / pET-28a-M63AL65D and pET-28a-M63AL65D, positions 187-189 of the RSP-O gene are mutated from ATG to GCG, and positions 193-195 are mutated from CTG to GAT. The mutated genes are recorded as M63AL65D genes.

[0144] M63AL65E is a mutation of the leucine residue at position 65 in M63A to a glutamic acid residue. The recombinant bacteria expressing the M63AL65E fusion protein are recorded as BL21 / pET-28a-M63AL65E. BL21 / pET-28a-M63AL65E contains the recombinant vector pET-28a-M63AL65E. In the obtained BL21 / pET-28a-M63AL65E and pET-28a-M63AL65E, positions 187-189 of the RSP-O gene are mutated from ATG to GCG, and positions 193-195 are mutated from CTG to GAA. The mutated genes are recorded as M63AL65E genes.

[0145] M63AL65F is a mutation of the leucine residue at position 65 in M63A to a phenylalanine residue. The recombinant bacteria expressing the M63AL65F fusion protein are recorded as BL21 / pET-28a-M63AL65F. BL21 / pET-28a-M63AL65F contains the recombinant vector pET-28a-M63AL65F. In the resulting BL21 / pET-28a-M63AL65F and pET-28a-M63AL65F, positions 187-189 of the RSP-O gene are mutated from ATG to GCG, and positions 193-195 are mutated from CTG to TTT. The mutated gene is recorded as the M63AL65F gene.

[0146] M63AL65G is a mutation of the leucine residue at position 65 in M63A to a glycine residue. The recombinant bacteria expressing the M63AL65G fusion protein are recorded as BL21 / pET-28a-M63AL65G. BL21 / pET-28a-M63AL65G contains the recombinant vector pET-28a-M63AL65G. In the obtained BL21 / pET-28a-M63AL65G and pET-28a-M63AL65G, positions 187-189 of the RSP-O gene are mutated from ATG to GCG, and positions 193-195 are mutated from CTG to GGT. The mutated genes are recorded as M63AL65G genes.

[0147] M63AL65I is a mutation of the leucine residue at position 65 in M63A to an isoleucine residue. The recombinant bacteria expressing the M63AL65I fusion protein are recorded as BL21 / pET-28a-M63AL65I. BL21 / pET-28a-M63AL65I contains the recombinant vector pET-28a-M63AL65I. In the resulting BL21 / pET-28a-M63AL65I and pET-28a-M63AL65I, positions 187-189 of the RSP-O gene are mutated from ATG to GCG, and positions 193-195 are mutated from CTG to ATT. The mutated genes are recorded as M63AL65I genes.

[0148] M63AL65K is a mutation of the leucine residue at position 65 in M63A to a lysine residue. The recombinant bacteria expressing the M63AL65K fusion protein are recorded as BL21 / pET-28a-M63AL65K. BL21 / pET-28a-M63AL65K contains the recombinant vector pET-28a-M63AL65K. In the resulting BL21 / pET-28a-M63AL65K and pET-28a-M63AL65K, positions 187-189 of the RSP-O gene are mutated from ATG to GCG, and positions 193-195 are mutated from CTG to AAA. The mutated genes are recorded as M63AL65K genes.

[0149] M63AL65M is a mutation of the leucine residue at position 65 in M63A to a methionine residue. The recombinant bacteria expressing the M63AL65M fusion protein are recorded as BL21 / pET-28a-M63AL65M. BL21 / pET-28a-M63AL65M contains the recombinant vector pET-28a-M63AL65M. In the resulting BL21 / pET-28a-M63AL65M and pET-28a-M63AL65M, positions 187-189 of the RSP-O gene are mutated from ATG to GCG, and positions 193-195 are mutated from CTG to ATG. The mutated genes are recorded as M63AL65M genes.

[0150] M63AL65Q is a mutation of the leucine residue at position 65 in M63A to a glutamine residue. The recombinant bacteria expressing the M63AL65Q fusion protein are recorded as BL21 / pET-28a-M63AL65Q. BL21 / pET-28a-M63AL65Q contains the recombinant vector pET-28a-M63AL65Q. In the resulting BL21 / pET-28a-M63AL65Q and pET-28a-M63AL65Q, positions 187-189 of the RSP-O gene are mutated from ATG to GCG, and positions 193-195 are mutated from CTG to CAG. The mutated genes are recorded as M63AL65Q genes.

[0151] M63AL65R means that the leucine residue at position 65 in M63A is mutated to an arginine residue. The recombinant bacteria expressing the M63AL65R fusion protein are recorded as BL21 / pET-28a-M63AL65R. BL21 / pET-28a-M63AL65R contains the recombinant vector pET-28a-M63AL65R. In the obtained BL21 / pET-28a-M63AL65R and pET-28a-M63AL65R, positions 187-189 of the RSP-O gene are mutated from ATG to GCG, and positions 193-195 are mutated from CTG to CGC. The mutated gene is recorded as the M63AL65R gene.

[0152] M63AL65S is a mutation of the leucine residue at position 65 in M63A to a serine residue. The recombinant bacteria expressing the M63AL65S fusion protein are recorded as BL21 / pET-28a-M63AL65S. BL21 / pET-28a-M63AL65S contains the recombinant vector pET-28a-M63AL65S. In the resulting BL21 / pET-28a-M63AL65S and pET-28a-M63AL65S, positions 187-189 of the RSP-O gene are mutated from ATG to GCG, and positions 193-195 are mutated from CTG to AGT. The mutated genes are recorded as M63AL65S genes.

[0153] M63AL65T is a mutation of the leucine residue at position 65 in M63A to a threonine residue. The recombinant bacteria expressing the M63AL6T fusion protein are recorded as BL21 / pET-28a-M63AL65T. BL21 / pET-28a-M63AL65T contains the recombinant vector pET-28a-M63AL65T. In the obtained BL21 / pET-28a-M63AL65T and pET-28a-M63AL65T, positions 187-189 of the RSP-O gene are mutated from ATG to GCG, and positions 193-195 are mutated from CTG to ACC. The mutated genes are recorded as M63AL65T genes.

[0154] M63AL65V is a mutation of the leucine residue at position 65 in M63A to a valine residue. The recombinant bacteria expressing the M63AL65V fusion protein are recorded as BL21 / pET-28a-M63AL65V. BL21 / pET-28a-M63AL65V contains the recombinant vector pET-28a-M63AL65V. In the resulting BL21 / pET-28a-M63AL65V and pET-28a-M63AL65V, positions 187-189 of the RSP-O gene are mutated from ATG to GCG, and positions 193-195 are mutated from CTG to GTT. The mutated genes are recorded as M63AL65V genes.

[0155] M63AL65W is a mutation of the leucine residue at position 65 in M63A to a tryptophan residue. The recombinant bacteria expressing the M63AL65W fusion protein are recorded as BL21 / pET-28a-M63AL65W. BL21 / pET-28a-M63AL65W contains the recombinant vector pET-28a-M63AL65W. In the obtained BL21 / pET-28a-M63AL65W and pET-28a-M63AL65W, positions 187-189 of the RSP-O gene are mutated from ATG to GCG, and positions 193-195 are mutated from CTG to TGG. The mutated genes are recorded as M63AL65W genes.

[0156] M63AL65Y means that the leucine residue at position 65 in M63A is mutated to a tyrosine residue. The recombinant bacteria expressing the M63AL65Y fusion protein are recorded as BL21 / pET-28a-M63AL65Y. BL21 / pET-28a-M63AL65Y contains the recombinant vector pET-28a-M63AL65Y. In the obtained BL21 / pET-28a-M63AL65Y and pET-28a-M63AL65Y, positions 187-189 of the RSP-O gene are mutated from ATG to GCG, and positions 193-195 are mutated from CTG to TAT. The mutated genes are recorded as M63AL65Y genes.

[0157] Example 5. Determination of Enzymatic Parameters of M63AL65H, M63AL65N, and M63AL65A

[0158] This example detects the enzymatic parameters of the fusion proteins expressed by some recombinant bacteria in Example 4. The bacteria to be tested include BL21 / pET-28a-M63AL65H, BL21 / pET-28a-M63AL65N, BL21 / pET-28a-M63AL65A, BL21 / pET-28a-M63A in Example 2, and BL21 / pET-28a-RSP-O in Example 1.

[0159] Definition of enzyme activity: The amount of enzyme required to generate 1 μmol of 3-(carbamoylmethyl)-5-methylhexanoic acid per unit time under specific conditions is defined as 1U.

[0160] 1. Recombinant protein expression and purification

[0161] 1. The test bacteria were inoculated into 10 mL LB liquid medium containing 40 μg / mL kanamycin, cultured at 37°C to OD600 = 0.6-0.8, and the inducer IPTG was added to a final concentration of 0.5 mM. After overnight culture at 24°C and 200 rpm, the bacteria were collected by refrigerated centrifugation for more than 12 hours, and the bacteria were resuspended in a binding solution (sodium chloride: 500 mM, imidazole: 10 mM, Tris-HCl: 20 mM, pH 7.5, and the balance was water), treated with lysozyme on ice for half an hour, and ultrasonically disrupted (10% power for 10 min, working for 3 s, and resting for 5 s). After disruption, the supernatant solution was centrifuged at 12000 rpm for 10 min and passed through a 0.22 μm membrane to obtain a crude enzyme solution.

[0162] 2. The crude enzyme solution in step 1 was purified using a conventional Ni-Agarose His tag protein purification kit (soluble protein) (Beijing Kangwei Century Biotechnology Co., Ltd., CW0894A) as follows:

[0163] (1) Mix the filler (His nickel column) and add it to the chromatography column (nickel column), and rinse the filled column with 5 times the bed volume of deionized water;

[0164] (2) Equilibrate the column with 2.5 bed volumes of binding buffer (sodium chloride: 500 mM, imidazole: 10 mM, Tris-HCl: 20 mM, pH 7.5, the balance is water);

[0165] (3) Add the crude enzyme solution to the chromatography column and bind on ice for 1.5 h;

[0166] (4) performing step elution using elution buffers containing different concentrations of imidazole (sodium chloride: 500 mM, imidazole: 80 mM-300 mM, Tris-HCl: 20 mM, pH 7.5, the balance being water), and collecting the elution peak of the buffer with an imidazole concentration of 300 mM;

[0167] (5) The elution peak collected in step (4) was concentrated using a Millipore ultrafiltration centrifuge tube (30KD), 4 mL of 50 mM TrisSi-HCl (pH 7.5) was added, and the supernatant was collected after centrifugation again, which was the pure enzyme solution. The concentration of the enzyme (i.e., fusion protein) was detected and further experiments were performed;

[0168] (6) Wash 5 column volumes with binding buffer and pure water respectively, and then wash 3 column volumes with 20% (volume percentage) ethanol. Store the column in an environment of 4-8°C.

[0169] The collected pure enzyme solution was subjected to SDS-PAGE electrophoresis. Figure 1 As shown, the target proteins were obtained.

[0170] 2. Optimum temperature

[0171] The optimum temperature of the enzyme prepared in step 1 was detected respectively.

[0172] The following reaction system (1 ml) was prepared: 50 mM Trsi-HCl (pH 7.5) buffer, 10 μg enzyme, 1 mM MnCl2, 5 mM 3-isobutylglutarimide.

[0173] The reaction system was reacted at different temperatures (45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C) for 30 min, and the total amount of 3-(carbamoylmethyl)-5-methylhexanoic acid produced (i.e., the total amount of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid and (S)-(+)-3-carbamoylmethyl-5-methylhexanoic acid) was detected.

[0174] The results are as follows Figure 2The optimum temperature of RSP fusion protein and other proteins is 75℃.

[0175] 3. Enzyme activity determination

[0176] The specific enzyme activity of each enzyme prepared in step 1 was detected.

[0177] The following reaction system (1 ml) was prepared: 50 mM Trsi-HCl (pH 7.5) buffer, 10 μg enzyme, 1 mM MnCl2, 5 mM 3-isobutylglutarimide.

[0178] The RSP fusion protein and other proteins were reacted at 75°C, and the reaction time was set to 10 min, 15 min, 20 min, and 30 min. The total amount of 3-(carbamoylmethyl)-5-methylhexanoic acid generated was detected, and the specific enzyme activity was calculated.

[0179] The results are shown in Table 6, which show that M63A has high catalytic activity, and the M63AL65N fusion protein has the highest specific enzyme activity among the double-point mutations.

[0180] Table 6. Detection results of specific enzyme activity

[0181]

[0182] Example 6: Mutation of C317 based on M63AL65A and M63AL65H

[0183] In this example, the cysteine ​​residue at position 317 of M63AL65A and M63AL65H in Example 4 was mutated respectively:

[0184] 1. Mutation of cysteine ​​at position 317 based on M63AL65A

[0185] The recombinant vector pET-28a-M63AL65A of Example 4 was used as a template for PCR amplification. The resulting PCR product was digested with endonuclease DpnI to remove the template. The digestion product was transformed into Escherichia coli BL21 (DE3), and then the strain was cultured. The plasmid was extracted for sequencing to obtain plasmids that can express the three mutant proteins of RSP (M63AL65AC317A, M63AL65AC317D, M63AL65AC317E, M63AL65AC317F, M63AL65AC317G, M63AL65AC317H, M63AL65AC317I, M63AL65AC317K, M63AL65AC317L, M63AL65AC317M, M63AL65AC317N, M63AL65AC317P, M63AL65AC317Q, M63AL65AC317R, M63AL65AC317S, M63AL65AC317T, M63AL65AC317V, M63AL65AC317W, M63AL65AC317Y) and the 1st to 36th positions of sequence 1 to form a fusion protein (denoted as the corresponding fusion protein). The primers used are as follows:

[0186] F: CGCATACCCATCTGGATNNNCCTCTGGGTGGTACCGTGAC (5'-->3' direction);

[0187] R: GTCACGGTACCACCCAGAGGNNNATCCAGATGGGTATGCG (5'-->3' direction), N represents A, T, C or G.

[0188] Among them, M63AL65AC317T, M63AL65AC317A, M63AL65AC317D, M63AL65AC317E, M63AL65AC317F, M63AL65AC317G, M63AL65AC317H, M63AL65AC317I, M63AL65AC317K, M63AL65AC317L, M63AL65AC317M, M63AL65AC317N, M63AL65AC317P, M63AL65AC317Q, M63AL65AC317R, M63AL65AC317S, M63AL65AC317V, M63AL65AC317W, and M63AL65AC317Y are proteins obtained by mutating the cysteine ​​residue at position 317 in M63AL65A to an alanine residue, an aspartic acid residue, a glutamic acid residue, a phenylalanine residue, a glycine residue, a histidine residue, an isoleucine residue, a lysine residue, a leucine residue, a methionine residue, an asparagine residue, a proline residue, a glutamine residue, an arginine residue, a serine residue, a threonine residue, a valine residue, a tryptophan residue, and a tyrosine residue, respectively. The genes encoding these proteins are denoted as M63AL65AC317A gene, M63AL65AC317D gene, M63AL65AC317E gene, M63AL65AC317F gene, M63AL65AC317G gene, and M63AL65AC317Y gene, respectively. M63AL65AC317H gene, M63AL65AC317I gene, M63AL65AC317K gene, M63AL65AC317L gene, M63AL65AC317M gene, M63AL65AC317N gene, M63AL65AC317P gene, M63AL65AC317Q gene, M63AL65AC317R gene, M63AL65AC317S gene, M63AL65AC317T gene, M63AL65AC317V gene, M63AL65AC317W gene, and M63AL65AC317Y gene, respectively, mutate the 949-951 positions of the M63AL65A gene from TGT to ACC, GCG, GAT, GAA, TTT, GGT, CAT, ATT, AAA, Genes obtained from CTG, ATG, AAT, CCG, CAG, CGC, AGT, GTT, TGG, and TAT.

[0189] "BL21 / pET-28a-" and "pET-28a-" are added before the name of the RSP mutant protein, and "-O" is added after it to indicate the recombinant bacteria and recombinant vector expressing the RSP mutant protein fusion protein.

[0190] According to the method of steps 2-4 in Experimental Example 2, the recombinant bacteria were replaced with the above-mentioned recombinant bacteria, the reaction temperature was 75° C., and the other steps remained unchanged. The chiral purity of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid in the products obtained by each recombinant bacteria was detected, and BL21 / pET-28a-M63AL65A in Example 4 was used as a control.

[0191] The results are shown in Table 7. The chirality of the M63AL65AC317T fusion protein is improved to a certain extent compared with the M63AL65A double point mutation.

[0192] Table 7. Chiral purity of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid

[0193]

[0194] 2. Mutation of cysteine ​​at position 317 based on M63AL65H

[0195] The recombinant vector pET-28a-M63AL65H of Example 4 was used as a template for PCR amplification. The resulting PCR product was digested with endonuclease DpnI to remove the template. The digestion product was transformed into Escherichia coli BL21 (DE3), and then the strain was cultured. The plasmid was extracted for sequencing to obtain the recombinant vectors that can express RSP mutant proteins (M63AL65HC317A, M63AL65HC317D, M63AL65HC317E, M63AL65HC317F, M63AL65HC317G, M63AL65HC317H, M63AL65HC317I, M63AL65HC317K, M63AL65HC317L, M63AL65HC317M, M63AL65HC317N, M63AL65HC317P, M63AL65HC317Q, M63AL65HC317R, M63AL65HC317S, M63AL65HC317T, M63AL65HC317V, M63AL65HC317W, M63AL65HC317Y) and the 1st to 36th positions of sequence 1 to form a fusion protein (denoted as the corresponding fusion protein). The primers used are as follows:

[0196] F: CGCATACCCATCTGGATNNNCCTCTGGGTGGTACCGTGAC (5'-->3' direction);

[0197] R: GTCACGGTACCACCCAGAGGNNNATCCAGATGGGTATGCG (5'-->3' direction), N represents A, T, C or G.

[0198] Among them, M63AL65HC317T, M63AL65HC317A, M63AL65HC317D, M63AL65HC317E, M63AL65HC317F, M63AL65HC317G, M63AL65HC317H, M63AL65HC317I, M63AL65HC317K, M63AL65HC317L, M63AL65HC317M, M63AL65HC317N, M63AL65HC317P, M63AL65HC317Q, M63AL65HC317R, M63AL65HC317S, M63AL65HC317V, M63AL65HC317W, and M63AL65HC317Y are proteins obtained by mutating the cysteine ​​residue at position 317 in M63AL65H to an alanine residue, an aspartic acid residue, a glutamic acid residue, a phenylalanine residue, a glycine residue, a histidine residue, an isoleucine residue, a lysine residue, a leucine residue, a methionine residue, an asparagine residue, a proline residue, a glutamine residue, an arginine residue, a serine residue, a threonine residue, a valine residue, a tryptophan residue, and a tyrosine residue, respectively. The genes encoding the proteins are denoted as M63AL65HC317A gene, M63AL65HC317D gene, M63AL65HC317E gene, M63AL65HC317F gene, M63AL65HC317G gene, and M63AL65HC317Y gene, respectively. M63AL65HC317H gene, M63AL65HC317I gene, M63AL65HC317K gene, M63AL65HC317L gene, M63AL65HC317M gene, M63AL65HC317N gene, M63AL65HC317P gene, M63AL65HC317Q gene, M63AL65HC317R gene, M63AL65HC317S gene, M63AL65HC317T gene, M63AL65HC317V gene, M63AL65HC317W gene, and M63AL65HC317Y gene, respectively, mutate the 949-951st position of the M63AL65H gene from TGT to ACC, GCG, GAT, GAA, TTT, GGT, CAT, ATT, AAA, Genes obtained from CTG, ATG, AAT, CCG, CAG, CGC, AGT, GTT, TGG, and TAT.

[0199] "BL21 / pET-28a-" and "pET-28a-" are added before the name of the RSP mutant protein, and "-O" is added after it to indicate the recombinant bacteria and recombinant vector expressing the RSP mutant protein fusion protein.

[0200] According to the method of steps 2-4 in Experimental Example 2, the recombinant bacteria were replaced with the above-mentioned recombinant bacteria, the reaction temperature was 75° C., and the other steps remained unchanged. The chiral purity of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid in the products obtained by each recombinant bacteria was detected, and BL21 / pET-28a-M63AL65H in Example 4 was used as a control.

[0201] The results are shown in Table 8. The chirality values ​​of M63AL65HC317I fusion protein, M63AL65HC317N fusion protein, M63AL65HC317T fusion protein, and 63AL65HC317V fusion protein are basically consistent with or slightly improved from the double-point mutation of M63AL65H fusion protein.

[0202] Table 8. Chiral purity of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid

[0203]

[0204] Example 7: Determination of Enzymatic Parameters of M63AL65HC317T and M63AL65AC317T

[0205] The recombinant protein was expressed and purified according to the method of step 1 in Example 5, and the enzymatic parameters of the obtained M63AL65HC317T fusion protein and the M63AL65AC317T fusion protein with three-point mutations were determined according to the method of step 3 in Example 5. The results are shown in Table 9.

[0206] Table 9. Detection results of specific enzyme activity

[0207] Protein name Specific enzyme activity (μmol / min / mg) <![CDATA[Kcat(S -1 )]]> <![CDATA[Kcat / Km(M -1 .S -1 )]]> Chirality (%) M63AL65AC317T fusion protein 0.64±0.02 0.94±0.06 542.19 98.26 M63AL65HC317T fusion protein 1.08±0.06 1.26±0.05 857.03 98.50

[0208] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims. Sequence Listing <110> Institute of Microbiology, Chinese Academy of Sciences <120> A (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid synthetic protein and its mutant and application <160> 2 <170> PatentIn version 3.5 <210> 1 <211> 508 <212> PRT <213> Artificial sequence <400> 1 Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro 1 5 10 15 Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg 20 25 30 Gly Ser Glu Phe Met Thr Lys Ile Ile Lys Asn Gly Thr Ile Val Thr 35 40 45 Ala Thr Asp Thr Tyr Glu Ala His Leu Leu Ile Lys Asp Gly Lys Ile 50 55 60 Ala Met Ile Gly Gln Asn Leu Glu Glu Lys Gly Ala Glu Val Ile Asp 65 70 75 80 Ala Lys Gly Cys Tyr Val Phe Pro Gly Gly Ile Asp Pro His Thr His 85 90 95 Leu Asp Met Pro Leu Gly Gly Thr Val Thr Lys Asp Asp Phe Glu Ser 100 105 110 Gly Thr Ile Ala Ala Ala Phe Gly Gly Thr Thr Thr Ile Ile Asp Phe 115 120 125 Cys Leu Thr Asn Lys Gly Glu Pro Leu Lys Lys Ala Ile Glu Thr Trp 130 135 140 His Asn Lys Ala Asn Gly Lys Ala Val Ile Asp Tyr Gly Phe His Leu 145 150 155 160 Met Ile Ser Glu Ile Thr Asp Asp Val Leu Glu Glu Leu Pro Lys Val 165 170 175 Leu Glu Glu Glu Gly Ile Thr Ser Leu Lys Val Phe Met Ala Tyr Lys 180 185 190 Asn Val Phe Gln Ala Asp Asp Gly Thr Leu Tyr Cys Thr Leu Leu Ala 195 200 205 Ala Lys Glu Leu Gly Ala Leu Val Met Val His Ala Glu Asn Gly Asp 210 215 220 Val Ile Asp Tyr Leu Thr Lys Lys Ala Leu Ala Asp Gly Asn Thr Asp 225 230 235 240 Pro Ile Tyr His Ala Leu Thr Arg Pro Pro Glu Leu Glu Gly Glu Ala 245 250 255 Thr Gly Arg Ala Cys Gln Leu Thr Glu Leu Ala Gly Ser Gln Leu Tyr 260 265 270 Val Val His Val Thr Cys Ala Gln Ala Val Glu Lys Ile Ala Glu Ala 275 280 285 Arg Asn Lys Gly Leu Asp Val Trp Gly Glu Thr Cys Pro Gln Tyr Leu 290 295 300 Val Leu Asp Gln Ser Tyr Leu Glu Lys Pro Asn Phe Glu Gly Ala Lys 305 310 315 320 Tyr Val Trp Ser Pro Pro Leu Arg Glu Lys Trp His Gln Glu Val Leu 325 330 335 Trp Asn Ala Leu Lys Asn Gly Gln Leu Gln Thr Leu Gly Ser Asp Gln 340 345 350 Cys Ser Phe Asp Phe Lys Gly Gln Lys Glu Leu Gly Arg Gly Asp Phe 355 360 365 Thr Lys Ile Pro Asn Gly Gly Pro Ile Ile Glu Asp Arg Val Ser Ile 370 375 380 Leu Phe Ser Glu Gly Val Lys Lys Gly Arg Ile Thr Leu Asn Gln Phe 385 390 395 400 Val Asp Ile Val Ser Thr Arg Ile Ala Lys Leu Phe Gly Leu Phe Pro 405 410 415 Lys Lys Gly Thr Ile Val Val Gly Ser Asp Ala Asp Leu Val Ile Phe 420 425 430 Asp Pro Asn Ile Glu Arg Val Ile Ser Ala Glu Thr His His Met Ala 435 440 445 Val Asp Tyr Asn Ala Phe Glu Gly Met Lys Val Thr Gly Glu Pro Val 450 455 460 Ser Val Leu Cys Arg Gly Glu Phe Val Val Arg Asp Lys Gln Phe Val 465,470,475,480 Gly Lys Pro Gly Tyr Gly Gln Tyr Leu Lys Arg Ala Lys Tyr Gly Thr 485 490 495 Ser Thr Ile Ser Lys Gln Ser Glu Glu Leu Thr Ile 500 505 <210> 2 <211> 1527 <212> DNA <213> Artificial sequence <400> 2 atgggcagca gccatcatca tcatcatcac agcagcggcc tggtgccgcg cggcagccat 60 atggctagca tgactggtgg acagcaaatg ggtcgcggat ccgaattcat gaccaaaatt 120 attaaaaatg gtaccatcgt taccgccacc gatacctatg aagcccatct gctgattaaa 180 gatggtaaaa ttgcaatgat tggccagaat ctggaagaaa aaggtgccga agttattgat 240 gcgaaaggct gttatgtgtt tccgggcggt attgatccgc atacccatct ggatatgcct 300 ctgggtggta ccgtgaccaa agatgatttt gaatcaggta ccattgccgc cgcgtttggc 360 ggtaccacca ccattattga tttttgcctg accaataaag gtgaaccgct gaaaaaagca 420 attgaaacct ggcataataa agccaatggc aaagcggtga tcgattatgg ttttcatctg 480 atgattagtg aaattactga tgatgtttta gaagaactgc cgaaagttct tgaagaagaa 540 ggtattacct cactgaaagt gtttatggcg tataaaaatg tttttcaggc tgatgatggt 600 accctgtatt gcaccctgct ggccgcaaaa gaactgggtg ccctggtgat ggtgcatgcc 660 gaaaacggtg atgttattga ttatctgacg aaaaaagcac tggcagatgg taataccgat 720 ccgatctatc atgcgctgac ccgtccgccg gaactggaag gtgaagctac aggtcgcgca 780 tgccagctga cagaactggc aggctcacag ctgtatgtgg tgcatgtgac atgtgcccag 840 gccgtggaaa aaattgcgga agcgcgcaat aaaggtctgg atgtttgggg cgaaacctgc 900 ccgcagtatt tagttctgga tcagagctat ctggaaaaac caaattttga aggtgcaaaa 960 tatgtgtgga gtccgccgct gcgcgaaaaa tggcatcagg aagttctgtg gaatgcgctg 1020 aaaaatggtc agctgcagac cctgggtagt gatcagtgta gttttgattt taaaggtcag 1080 aaagaactgg gacgtggtga ttttaccaaa attccgaatg gtggtccgat tattgaagat 1140 cgtgtgagta ttctgtttag cgaaggtgtt aaaaaaggtc gcattaccct gaaccagttt 1200 gttgatattg tgagcacccg cattgcaaaa ctgtttggcc tgtttccgaa aaaaggcacc 1260 attgttgtgg gtagcgatgc ggatctggtg atttttgatc cgaatattga acgtgtgatt 1320 agtgccgaaa cacatcatat ggccgttgat tataatgcgt ttgaaggcat gaaagtgacc 1380 ggtgaaccgg tgagcgttct gtgtcgtggt gaatttgtgg tgcgtgataa acagtttgtt 1440 ggcaaaccgg gttatggtca gtatctgaaa cgcgcgaaat atggcaccag caccattagt 1500 aaacagagcg aagaactgac catttaa 1527

Claims

1. A method for preparing (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid, comprising: Using 3-isobutylglutarimide as a substrate and using protein to catalyze the reaction, (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid was obtained; The protein is C1 or C2: C1, RSP mutant protein, is a protein obtained by subjecting RSP to the following transformation of A1, A2, or A3, wherein the sequence of RSP is positions 37-508 of SEQ ID No. 1; A1, mutating the methionine residue at position 63 of the RSP to an alanine residue, a cysteine ​​residue, a glutamic acid residue, an isoleucine residue, an asparagine residue, a glutamine residue, a serine residue, a threonine residue, a valine residue, an aspartic acid residue, a phenylalanine residue, a histidine residue, a lysine residue, a leucine residue, a proline residue, an arginine residue, a tryptophan residue or a tyrosine residue; A2, mutating the methionine residue at position 63 of the RSP to an alanine residue, mutating the leucine residue at position 65 of the RSP to a histidine residue, an asparagine residue, a proline residue, an alanine residue, a cysteine ​​residue, an aspartic acid residue, a glutamic acid residue, a phenylalanine residue, a glycine residue, an isoleucine residue, a lysine residue, a methionine residue, a glutamine residue, an arginine residue, a serine residue, a threonine residue, a valine residue, a tryptophan residue or a tyrosine residue; A3, mutating the methionine residue at position 63 of the RSP to an alanine residue, mutating the leucine residue at position 65 of the RSP to an alanine residue or a histidine residue, mutating the cysteine ​​residue at position 317 of the RSP to an alanine residue, an aspartic acid residue, a glutamic acid residue, a phenylalanine residue, a glycine residue, a histidine residue, an isoleucine residue, a lysine residue, a leucine residue, a methionine residue, an asparagine residue, a glutamine residue, an arginine residue, a serine residue, a threonine residue, a valine residue, a tryptophan residue or a tyrosine residue; The RSP mutant protein excludes the modification of RSP with M63AL65HC317K, M63AL65HC317Q and M63AL65HC317W; C2: A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein described in C1.

2. A method for preparing (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid, comprising: Using 3-isobutylglutarimide as a substrate, a recombinant cell expressing the protein of claim 1 is used to carry out a catalytic reaction to obtain (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid.

3. The method according to claim 2, characterized in that: The recombinant cell is obtained by introducing a recombinant vector capable of expressing the protein of claim 1 into a biological cell.

4. Protein, either C1 or C2: C1, RSP mutant protein, is a protein obtained by modifying RSP as follows, the sequence of RSP is positions 37-508 of SEQ ID No.1: The methionine residue at position 63 of RSP is mutated to an alanine residue, and the leucine residue at position 65 of RSP is mutated to a histidine residue, and the cysteine ​​residue at position 317 of RSP is mutated to an alanine residue, aspartic acid residue, glutamic acid residue, phenylalanine residue, glycine residue, histidine residue, isoleucine residue, leucine residue, methionine residue, asparagine residue, arginine residue, serine residue, threonine residue, valine residue or tyrosine residue; or, the methionine residue at position 63 of RSP is mutated to an alanine residue, and the leucine residue at position 65 of RSP is mutated to an alanine residue, and the cysteine ​​residue at position 317 of RSP is mutated to an alanine residue, aspartic acid residue, glutamic acid residue, phenylalanine residue, glycine residue, histidine residue, isoleucine residue, lysine residue, leucine residue, methionine residue, asparagine residue, glutamine residue, arginine residue, serine residue, threonine residue, valine residue, tryptophan residue or tyrosine residue; C2: A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein described in C1.

5. The biological material related to the protein according to claim 4, which is any one of the following B1) to B4): B1) a nucleic acid molecule encoding the protein according to claim 4; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1) or a recombinant vector containing the expression cassette described in B2); B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3).

6. A product for preparing (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid, which comprises the protein according to claim 4 and 3-isobutylglutarimide.

7. Use of any one of the methods described in claims 1 to 3 in the preparation of 3-(carbamoylmethyl)-5-methylhexanoic acid.

8. Any of the following uses of the protein of claim 1, or a biological material related to the protein of claim 1, or the product of claim 6: X1. Production of 3-(carbamoylmethyl)-5-methylhexanoic acid; X2. Preparation and production of 3-(carbamoylmethyl)-5-methylhexanoic acid product; The biological material is any one of the following B1) to B4): B1) a nucleic acid molecule encoding the protein according to claim 1; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1) or a recombinant vector containing the expression cassette described in B2); B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3).

9. Any of the following uses of the protein of claim 1, or a biological material related to the protein of claim 1, or the product of claim 6: Z1, production of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid; Z2, preparation and production of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid product; The biological material is any one of the following B1) to B4): B1) a nucleic acid molecule encoding the protein according to claim 1; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1) or a recombinant vector containing the expression cassette described in B2); B4) A recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3).

10. Use of the protein according to claim 4 as an enzyme.

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