R-(-)-3-(carbamylmethyl)-5-methylhexanoic acid synthetic protein and mutant and application thereof

Abstract

The invention discloses an R-(-)-3-(carbamylmethyl)-5-methylhexanoic acid synthetic protein as well as a mutant and application thereof. The invention relates to the technical field of biology, and provides a method for preparing R-(-)-3-(carbamylmethyl)-5-methylhexanoic acid, which comprises the following steps: carrying out catalytic reaction by taking 3-isobutyl glutarimide as a substrate and taking specific protein or a recombinant cell capable of expressing the specific protein as a catalyst to obtain the R-(-)-3-(carbamylmethyl)-5-methylhexanoic acid. The R-(-)-3-(carbamyl methyl)-5-methylhexanoic acid is obtained through a reaction; the specific protein is a protein as shown in SEQ ID No.1 or a variant thereof. The method disclosed by the invention is of great significance to preparation of high-purity (S)-3-(aminomethyl)-5-methylhexanoic acid through a biological incubation method.

Description

Technical Field

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

[0002] Pregabalin (PGB) is a new type of γ-aminobutyric acid (GABA) receptor agonist with good anti-anxiety and neuropathic effects, and is widely used in the treatment of epilepsy and peripheral neuropathy. Among them, the therapeutic effect of S-configuration pregabalin is 10 times that of R-configuration, so obtaining S-pregabalin with high optical purity is the key to drug synthesis.

[0003] In current production, 3-isobutylglutarimide (hereinafter referred to as cyclic imine) is used as a substrate, and hydrolyzed in a strong alkaline environment to obtain equal proportions of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid (hereinafter referred to as R-monoamide) and (S)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid (hereinafter referred to as S-monoamide). R-monoamide can be directly synthesized into S-pregabalin ((S)-3-(aminomethyl)-5-methylhexanoic acid) through Hofmann degradation reaction. However, the byproduct S-monoamide needs to be racemized and recycled using the resolving agent R-ɑ-phenylethylamine. This process requires the use of a large amount of resolving agent, has a long cycle and low yield. Summary of the invention

[0004] The purpose of the present invention is to provide R-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid synthetic protein and its mutant and application.

[0005] In a first aspect, the present invention claims a process for preparing R-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid.

[0006] The method for preparing R-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid claimed in the present invention may include the following steps: using 3-isobutylglutarimide as a substrate and a specific protein or a recombinant cell capable of expressing the specific protein as a catalyst to carry out a catalytic reaction to obtain R-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid; Wherein, the specific protein may be any of the following: (A1) a protein having an amino acid sequence of SEQ ID No. 1; (A2) mutant proteins obtained by subjecting the protein represented by SEQ ID No. 1 to the following single point mutations: M63A, M63V, G66C, G66F, G67T, G67V, M125E, M125I, L159Y, L159H, M94A, M94H, M94N, M94R, M94W; (A3) mutant protein obtained by subjecting the protein shown in SEQ ID No. 1 to the following double point mutations: M63V / L159Y; (A4) mutant protein obtained by subjecting the protein shown in SEQ ID No. 1 to the following three point mutations: M63V / L159Y / M125E; (A5) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein defined in (A1) to (A4).

[0007] In the above (A1), the protein shown in SEQ ID No. 1 is derived from Bacillus ferrooxidans ( Alicyclobacillus ferrooxydans ) is a dihydropyrimidinase, and the proteins defined in (A2) to (A4) are variants thereof.

[0008] In (A5), the tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag and / or a SUMO tag, etc.

[0009] Furthermore, the recombinant cell may be a recombinant Escherichia coli. In one embodiment of the present invention, the recombinant cell is specifically a recombinant Escherichia coli obtained by introducing a recombinant vector (i.e., a recombinant plasmid obtained by cloning the nucleic acid molecule encoding the specific protein into the pET28a plasmid) into Escherichia coli BL21 (DE3).

[0010] In one embodiment of the present invention, the catalyst is a recombinant cell capable of expressing the specific protein (i.e., whole-cell catalysis). Specifically, the recombinant cell capable of expressing the specific protein is a recombinant cell (such as recombinant E. coli) obtained by introducing the coding gene of the specific protein into a receptor cell (such as E. coli). Furthermore, the coding gene of the specific protein can be introduced into the receptor cell in the form of a recombinant vector. Accordingly, the reaction system of the catalytic reaction can be: resuspend the recombinant cell (such as recombinant E. coli) in 50mM Trsi-HCl (pH7.5) so that the OD 600nm =10, 3-isobutylglutarimide 10 mM. The reaction conditions of the catalytic reaction may be: 50° C. for 2 hours.

[0011] In another embodiment of the present invention, the catalyst is an enzyme (i.e., enzyme catalysis), and specifically, the specific protein is used as the catalyst in the form of an immobilized enzyme. Accordingly, the reaction system of the catalytic reaction may be: the immobilized enzyme, 3-isobutylglutarimide and water are mixed in a ratio of 20g: 100g: 500mL. The reaction temperature of the catalytic reaction may be 50°C, the pH is controlled to be 8.5 during the reaction, and the reaction time is 24h. Among them, the immobilized enzyme can be prepared according to a method comprising the following steps: ultrasonically crushing the suspension of the recombinant cells (such as recombinant Escherichia coli) to obtain a crude enzyme solution I; centrifuging the crude enzyme solution I to remove impurities by high-speed centrifugation to obtain a supernatant enzyme solution II; mixing the supernatant enzyme solution II and the amino carrier in a ratio of 80mL: 20g, and incubating at 18°C ​​for 18h to obtain the immobilized enzyme.

[0012] In a second aspect, the present invention claims a protein.

[0013] The protein claimed in the present invention may be any of the following: (B1) a protein obtained by replacing amino acid 63 of SEQ ID No. 1 from M to V; (B2) a protein obtained by replacing the amino acid at position 63 of SEQ ID No. 1 from M to V and the amino acid at position 159 from L to Y; (B3) a protein obtained by replacing the amino acid at position 63 of SEQ ID No. 1 from M to V, the amino acid at position 159 from L to Y, and the amino acid at position 125 from M to E; (B4) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein defined in (B1)-(B3).

[0014] In (B1) to (B3), the protein is derived from Bacillus ferrooxidans ( Alicyclobacillus ferrooxydans ) is a variant of the dihydropyrimidinase (SEQ ID No. 1).

[0015] In (B4), the tag refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology to facilitate the expression, detection, tracing and / or purification of the target protein. The tag can be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag and / or a SUMO tag, etc.

[0016] In a third aspect, the present invention claims a nucleic acid molecule encoding the protein described in the second aspect above.

[0017] 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.

[0018] Furthermore, the nucleic acid molecule may be any of the following: (C1) a DNA molecule obtained by replacing nucleotides 187-189 of SEQ ID No. 2 from atg (encoding amino acid M) to gtt (encoding amino acid V); (C2) a DNA molecule obtained by replacing nucleotides 187-189 from atg (encoding amino acid M) to gtt (encoding amino acid V), and nucleotides 475-477 from ctg (encoding amino acid L) to tac (encoding amino acid Y) of SEQ ID No. 2; (C3) A DNA molecule obtained by replacing nucleotides at positions 187-189 from atg (encoding amino acid M) with gtt (encoding amino acid V), nucleotides at positions 475-477 from ctg (encoding amino acid L) with tac (encoding amino acid Y), and nucleotides at positions 373-375 from atg (encoding amino acid M) with gaa (encoding amino acid E) of SEQ ID No.2.

[0019] In a fourth aspect, the present invention claims a biological material.

[0020] The biological material claimed in the present invention may be an expression cassette, a recombinant vector or a recombinant cell containing the nucleic acid molecule described in the third aspect above.

[0021] The expression cassette refers to a DNA sequence composed of promoter, target gene, terminator and other elements in genetic engineering, which can initiate the expression of the target gene in the host cell.

[0022] The recombinant vector may be a recombinant plasmid carrying the expression cassette.

[0023] In one embodiment of the present invention, the recombinant vector is a recombinant plasmid obtained by cloning the nucleic acid molecule into a pET28a plasmid.

[0024] The recombinant cell may be a recombinant cell carrying the recombinant plasmid.

[0025] The cell may be a prokaryotic cell or a eukaryotic cell.

[0026] In one embodiment of the present invention, the recombinant cell is a recombinant Escherichia coli, specifically a recombinant Escherichia coli obtained by introducing the recombinant vector (i.e., the recombinant plasmid obtained by cloning the nucleic acid molecule into the pET28a plasmid) into Escherichia coli BL21 (DE3).

[0027] In a fifth aspect, the present invention claims the use of the protein described in the second aspect above, the nucleic acid molecule described in the third aspect above, or the biomaterial described in the fourth aspect above in any of the following: (D1) Production of R-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid; (D2) Preparation of a product for producing R-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid.

[0028] In a sixth aspect, the present invention claims a product for producing R-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid.

[0029] The product for producing R-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid claimed in the present invention may be product 1 or product 2; The product 1 is composed of the protein described in the second aspect above and 3-isobutylglutarimide; The product 2 is composed of the recombinant cells (such as recombinant Escherichia coli) described in the fourth aspect above and 3-isobutylglutarimide.

[0030] Among them, 3-isobutylglutarimide is a substrate used for producing R-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid, and the protein or the recombinant cell is a catalyst used for producing R-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid.

[0031] In the seventh aspect, the present invention claims protection for the use of the method described in the first aspect above, the protein described in the second aspect above, the nucleic acid molecule described in the third aspect above, the biomaterial described in the fourth aspect above, or the product described in the sixth aspect above in the production of (S)-3-(aminomethyl)-5-methylhexanoic acid.

[0032] The present invention selects an amidohydrolase that can catalyze 3-isobutylglutarimide to generate a single chiral (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid, and simultaneously obtains a mutant that can catalyze the synthesis of a high-purity R-type product by using a point mutation technique through protein structure analysis. The present invention is of great significance for preparing high-purity (S)-3-(aminomethyl)-5-methylhexanoic acid by a biological incubation method. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the result of molecular docking, that is, the substrate small molecule 3-isobutylglutarimide (IBI) was docked to the active pocket of the enzyme, among which the non-conservative amino acids M63, F65, G66, G67, M94, M125, and L159 were used as modification targets for subsequent experiments. DETAILED DESCRIPTION

[0034] 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.

[0035] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0036] The determination methods of various parameters involved in the following embodiments are as follows: 1. Determination method of 3-isobutylglutarimide and (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid Agilent C18 reverse phase column (250×4.6mm, 5μm), detection wavelength: 210nm; column temperature: 35℃; flow rate: 1.0ml / min; injection volume: 20μl; running time: 45min. Mobile phase A: buffer salt (weigh about 3.5g of potassium dihydrogen phosphate, add 1000ml of water to dissolve, mix well, adjust pH to 3.0 with phosphoric acid, and filter). Mobile phase B: acetonitrile. The gradient linear elution program is shown in Table 1. The elution time of 3-isobutylglutarimide standard is 26.5min, and the elution time of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid is 8.0min. The standards used were produced by Shanghai MacLean Biochemical Technology Co., Ltd., 3-isobutylglutarimide (Product No. I856241-100g) and (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid (Product No. R843024-5g).

[0037]

[0038] Note: % indicates volume percentage.

[0039] 2. Optical purity test of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid Chromatographic column: Chiralpak AD-H column, 250×4.6 mm, 5 μm; Detection wavelength: 210nm; column temperature: 25℃; flow rate: 0.5ml / min; Mobile phase: n-hexane: ethanol: trifluoroacetic acid = 880:120:1 (volume ratio).

[0040] After the fermentation liquid was centrifuged, the supernatant was taken, freeze-dried and dissolved in 1 / 2 volume of ethanol, and the optical purity of the product was detected by HPLC. The standard 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., item number: S62775). The peak time of the R-type product was 15.23 min, and the peak time of the S-type product was 16.21 min.

[0041] Optical purity of product ee . p =(RS) / (R+S)*100.

[0042] Wherein, R is the yield of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid, and S is the yield of (S)-(+)-3-(carbamoylmethyl)-5-methylhexanoic acid. The higher the optical purity value calculated by this formula, the higher the proportion of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid in the product.

[0043] Example 1: Screening of key amino acid sites of AfHase After sequence alignment, the ferrooxidans from Bacillus circulans ( Alicyclobacillus ferrooxydans The dihydropyrimidinase (Genebank: WP_20665865.1, abbreviated as AfHase, the amino acid sequence is shown in SEQ ID No.1) of Escherichia coli was synthesized by sequence optimization according to the codon preference of Escherichia coli to obtain the DNA sequence shown in SEQ ID No.2 (SEQ ID No.2 encodes the amino acid sequence shown in SEQ ID No.1). The DNA fragment shown in SEQ ID No.2 was cloned into the middle of the EcoR I and Hind III restriction sites of the expression vector pET28a to obtain a recombinant vector named pET28a-AfHase.

[0044] The recombinant vector pET28a-AfHase was transformed into Escherichia coli BL21 (DE3). A single colony of positive clones was selected and inoculated in 10 mL LB medium containing 40 μg / mL kanamycin. After culturing at 37°C for 12 h, 1% (volume percentage) of the inoculum was inoculated in 200 mL LB medium containing 40 μg / mL kanamycin and incubated at 37°C until OD 600nmThe OD value was 0.6-0.8, and the inducer IPTG was added to a final concentration of 0.5mM. After overnight culture at 24℃ and 200rpm, the cells were collected and washed once with 50mM Trsi-HCl (pH7.5) to remove the culture medium on the surface of the cells. The catalytic activity was verified according to the whole cell catalysis method. The collected cells were resuspended in 50mMT Trsi-HCl (pH7.5) and the OD value was adjusted. 600nm to 10, substrate 3-isobutylglutarimide 10mM, 50℃ for 2h. In this experiment, the strain containing the pET28a empty vector was used as the control group. The experimental results confirmed that AfHase can catalyze 3-isobutylglutarimide to produce 3-(carbamoylmethyl)-5-methylhexanoic acid with an optical purity of 52.8%.

[0045] The AfHase protein was molecularly docked with the substrate 3-isobutylglutarimide using the software Autodock to find the active pocket and key amino acid sites. The seven sites M63, F65, G66, G67, L159, M125, and M94 have obvious interaction forces with the isobutyl side chain of the substrate, so these sites were verified by saturation mutation experiments. The overlapping PCR method (primer sequences are shown in Table 2) was used as a template for the plasmid containing wild-type AfHase (i.e., pET28a-AfHase). After PCR amplification, the wild-type template was digested with the endonuclease DpnI, transformed into Escherichia coli BL21 (DE3), and sequenced to verify the positive clones. According to the above experimental steps, the whole cell catalytic experiment and the optical purity of the product (3-(carbamoylmethyl)-5-methylhexanoic acid) were carried out. The changes in the enzymatic activity of AfHase after mutation were detected, and the relative activity of the AfHase variant relative to the wild type was calculated. Relative activity = product yield of mutant group / product yield of wild-type group (%), the product is 3-(carbamoylmethyl)-5-methylhexanoic acid.

[0046]

[0047] Note: N represents any base, which can be A, T, C or G.

[0048] The results showed that the catalytic activity and product optical purity decreased to varying degrees after the amino acid mutation at position 65, so this position was excluded in subsequent experiments. The catalytic activity and product optical purity were improved after mutations at other positions such as M63A, M63V, G66C, G66F, G67T, G67V, M125E, M125I, L159Y, L159H, M94A, M94H, M94N, M94R, and M94W, so the pET28a recombinant vector carrying the corresponding single-point mutation gene (i.e., the recombinant plasmid obtained by inserting the above-mentioned single-point mutation encoding gene into the middle of the EcoR I and Hind III restriction sites of the pET-28a vector) was used as a template for the next step of screening double-point combination mutations. The details are shown in Tables 3 and 4.

[0049]

[0050]

[0051] After the above screening, the amino acid sequences and corresponding coding gene sequences (nucleotide sequences) of each AfHase single mutant finally obtained are as follows: The amino acid sequence of M63A is obtained by mutating the amino acid at position 63 of SEQ ID No.1 from M to A, and the corresponding nucleotide sequence is obtained by replacing the nucleotides at positions 187-189 of SEQ ID No.2 from atg (encoding amino acid M) to gca (encoding amino acid A).

[0052] The amino acid sequence of M63V is obtained by mutating the amino acid at position 63 of SEQ ID No.1 from M to V, and the corresponding nucleotide sequence is obtained by replacing the nucleotides at positions 187-189 of SEQ ID No.2 from atg (encoding amino acid M) to gtt (encoding amino acid V).

[0053] The amino acid sequence of G66C is obtained by mutating the amino acid at position 66 of SEQ ID No. 1 from G to C, and the corresponding nucleotide sequence is obtained by replacing the nucleotides at positions 186-198 of SEQ ID No. 2 from ggt (encoding amino acid G) to tgc (encoding amino acid C).

[0054] The amino acid sequence of G66F is obtained by mutating the amino acid at position 66 of SEQ ID No. 1 from G to F, and the corresponding nucleotide sequence is obtained by replacing the nucleotides at positions 186-198 of SEQ ID No. 2 from ggt (encoding amino acid G) to ttt (encoding amino acid F).

[0055] The amino acid sequence of G67T is obtained by mutating the amino acid at position 67 of SEQ ID No.1 from G to T, and the corresponding nucleotide sequence is obtained by replacing the nucleotides at positions 199-201 of SEQ ID No.2 from ggc (encoding amino acid G) to acg (encoding amino acid T).

[0056] The amino acid sequence of G67V is obtained by mutating the amino acid at position 67 of SEQ ID No.1 from G to V, and the corresponding nucleotide sequence is obtained by replacing the nucleotides at positions 199-201 of SEQ ID No.2 from ggc (encoding amino acid G) to gtt (encoding amino acid V).

[0057] The amino acid sequence of M125E is obtained by mutating the amino acid at position 125 of SEQ ID No. 1 from M to E, and the corresponding nucleotide sequence is obtained by replacing the nucleotides at positions 373-375 of SEQ ID No. 2 from atg (encoding amino acid M) to gaa (encoding amino acid E).

[0058] The amino acid sequence of M125I is obtained by mutating the amino acid at position 125 of SEQ ID No.1 from M to I, and the corresponding nucleotide sequence is obtained by replacing the nucleotides at positions 373-375 of SEQ ID No.2 from atg (encoding amino acid M) to atc (encoding amino acid I).

[0059] The amino acid sequence of L159Y is obtained by mutating the amino acid at position 159 of SEQ ID No. 1 from L to Y, and the corresponding nucleotide sequence is obtained by replacing the nucleotides at positions 475-477 of SEQ ID No. 2 from ctg (encoding amino acid L) to tac (encoding amino acid Y).

[0060] The amino acid sequence of L159H is obtained by mutating the amino acid at position 159 of SEQ ID No. 1 from L to H, and the corresponding nucleotide sequence is obtained by replacing the nucleotides at positions 475-477 of SEQ ID No. 2 from ctg (encoding amino acid L) to cac (encoding amino acid H).

[0061] The amino acid sequence of M94A is obtained by mutating the amino acid at position 94 of SEQ ID No.1 from M to A, and the corresponding nucleotide sequence is obtained by replacing the nucleotides at positions 280-282 of SEQ ID No.2 from atg (encoding amino acid M) to gca (encoding amino acid A).

[0062] The amino acid sequence of M94H is obtained by mutating the amino acid at position 94 of SEQ ID No.1 from M to H, and the corresponding nucleotide sequence is obtained by replacing the nucleotides at positions 280-282 of SEQ ID No.2 from atg (encoding amino acid M) to cac (encoding amino acid H).

[0063] The amino acid sequence of M94N is obtained by mutating the amino acid at position 94 of SEQ ID No.1 from M to N, and the corresponding nucleotide sequence is obtained by replacing the nucleotides at positions 280-282 of SEQ ID No.2 from atg (encoding amino acid M) to aac (encoding amino acid N).

[0064] The amino acid sequence of M94R is obtained by mutating the amino acid at position 94 of SEQ ID No.1 from M to R, and the corresponding nucleotide sequence is obtained by replacing the nucleotides at positions 280-282 of SEQ ID No.2 from atg (encoding amino acid M) to cgt (encoding amino acid R).

[0065] The amino acid sequence of M94W is obtained by mutating the amino acid at position 94 of SEQ ID No.1 from M to W, and the corresponding nucleotide sequence is obtained by replacing the nucleotides at positions 280-282 of SEQ ID No.2 from atg (encoding amino acid M) to tgg (encoding amino acid W).

[0066] Example 2: Screening of AfHase double-point combination mutations In this example, G66C, G66F, G67T, G67V, M125E, M125I, L159Y, L159H, M94A, M94H, M94N, M94R or M94W are superimposed on M63A and M63V in Example 1, so as to screen for double-point combination mutations that significantly improve the catalytic activity and the optical purity of the product (3-(carbamoylmethyl)-5-methylhexanoic acid). The experimental operation is carried out with reference to Example 1.

[0067] The results showed that M63V / L159Y had the most significant improvement in catalytic activity and product optical purity. Therefore, the pET28a recombinant vector carrying the M63V / L159Y coding gene (i.e., the recombinant plasmid obtained by inserting the M63V / L159Y coding gene into the middle of the EcoR I and Hind III restriction sites of the pET-28a vector) was used as a template for subsequent screening of three-point combination mutations. See Tables 5 and 6 for specific results.

[0068]

[0069] Note: Relative activity = product yield of double-point mutant group / product yield of M63A group (%), the product is 3-(carbamoylmethyl)-5-methylhexanoic acid.

[0070]

[0071] Note: Relative activity = product yield of double-point mutant group / product yield of M63V group (%), the product is 3-(carbamoylmethyl)-5-methylhexanoic acid.

[0072] After the above screening, the amino acid sequence and corresponding coding gene sequence (nucleotide sequence) of the AfHase double-point mutant M63V / L159Y obtained are as follows: The amino acid sequence of M63V / L159Y is obtained by mutating the amino acid at position 63 of SEQ ID No.1 from M to V, and the amino acid at position 159 from L to Y; the corresponding nucleotide sequence is obtained by replacing the nucleotides at positions 187-189 of SEQ ID No.2 from atg (encoding amino acid M) to gtt (encoding amino acid V), and the nucleotides at positions 475-477 from ctg (encoding amino acid L) to tac (encoding amino acid Y).

[0073] Example 3: Screening of AfHase triple-point combination mutations In this example, G66C, G66F, G67T, G67V, M125E, M125I, M94A, M94H, M94N, M94R, and M94W were superimposed on the AfHase double-point mutant M63V / L159Y obtained by screening in Example 2, thereby screening for a three-point combination mutation that significantly improves the catalytic activity and the optical purity of the product (3-(carbamoylmethyl)-5-methylhexanoic acid). The experimental operation is referred to Example 1.

[0074] The results showed that M63V / L159Y / M125E had the most significant improvement in catalytic activity and product optical purity. Therefore, the pET28a recombinant vector carrying the M63V / L159Y / M125E coding gene (i.e., the recombinant plasmid obtained by inserting the M63V / L159Y / M125E coding gene into the middle of the EcoR I and Hind III restriction sites of the pET-28a vector) was used as a template for subsequent screening of four-point combination mutations. See Table 7 for specific results.

[0075]

[0076] Note: Relative activity = product yield of triple-point mutant group / product yield of M63V / L159Y group (%), the product is 3-(carbamoylmethyl)-5-methylhexanoic acid.

[0077] After the above screening, the amino acid sequence and corresponding coding gene sequence (nucleotide sequence) of the AfHase triple-point mutant M63V / L159Y / M125E obtained are as follows: The amino acid sequence of M63V / L159Y / M125E is obtained by mutating the amino acid at position 63 of SEQ ID No.1 from M to V, the amino acid at position 159 from L to Y, and the amino acid at position 125 from M to E; the corresponding nucleotide sequence is obtained by replacing the nucleotides at positions 187-189 of SEQ ID No.2 from atg (encoding amino acid M) to gtt (encoding amino acid V), the nucleotides at positions 475-477 from ctg (encoding amino acid L) to tac (encoding amino acid Y), and the nucleotides at positions 373-375 from atg (encoding amino acid M) to gaa (encoding amino acid E).

[0078] Example 4: Immobilization process development Referring to Example 1, the recombinant vector pET-28a-M63V / L159Y / M125E (i.e., the recombinant plasmid obtained by inserting the M63V / L159Y / M125E encoding gene into the middle of the EcoR I and Hind III restriction sites of the pET-28a vector) was introduced into Escherichia coli BL21 (DE3) to obtain the recombinant Escherichia coli. Weigh 20g of the wet bacteria of the recombinant Escherichia coli and dissolve it in 100mL Tris-HCl buffer (50mmol / L, pH8.5) to obtain a bacterial suspension. The bacterial suspension was subjected to low-temperature ultrasonic disruption using an ultrasonic disruptor, with a working power of 400w, a working time of 3s, an intermittent time of 6s, and a total time of 30min to obtain a crude enzyme solution I. The crude enzyme solution was centrifuged and impurities were removed using a high-speed centrifuge, 12000g, 30min, to obtain a supernatant enzyme solution II. Take 80mL of supernatant enzyme solution II and mix it with 20g of amino carrier (Xi'an Lanxiao Technology New Materials Co., Ltd., product number: LXTE-700S), 120rpm, 18℃, 18h. Use distilled water to wash the immobilized carrier particles 3 times to remove the enzyme molecules that are not stably fixed on the carrier to obtain the immobilized enzyme. Take 20g of immobilized enzyme and add it to 500mL of water, add 100g of substrate 3-isobutylglutarimide, the reaction temperature is 50℃, and ammonia water is used to control the pH to 8.5 during the reaction. The reaction time is 24h. A total of 10 batches of experiments were carried out. After the reaction, the concentration of each batch of products (3-(carbamoylmethyl)-5-methylhexanoic acid) was detected.

[0079] The results are shown in Table 8, which show that the immobilized enzyme can be stably used for 9 batches, and the activity of the 10th batch remains 67.9%.

[0080]

[0081] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A method for preparing R-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid, comprising the following steps: using 3-isobutylglutarimide as a substrate and a specific protein or a recombinant cell capable of expressing the specific protein as a catalyst to carry out a catalytic reaction to obtain R-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid; The specific protein is any of the following: (A1) a protein having an amino acid sequence of SEQ ID No. 1; (A2) mutant proteins obtained by subjecting the protein represented by SEQ ID No. 1 to the following single point mutations: M63A, M63V, G66C, G66F, G67T, G67V, M125E, M125I, L159Y, L159H, M94A, M94H, M94N, M94R, M94W; (A3) mutant protein obtained by subjecting the protein shown in SEQ ID No. 1 to the following double point mutations: M63V / L159Y; (A4) mutant protein obtained by subjecting the protein shown in SEQ ID No. 1 to the following three point mutations: M63V / L159Y / M125E; (A5) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein defined in (A1) to (A4).

2. The method according to claim 1, characterized in that: The recombinant cell is recombinant Escherichia coli.

3. Protein, any of the following: (B1) a protein obtained by replacing amino acid 63 of SEQ ID No. 1 from M to V; (B2) a protein obtained by replacing the amino acid at position 63 of SEQ ID No. 1 from M to V and the amino acid at position 159 from L to Y; (B3) a protein obtained by replacing the amino acid at position 63 of SEQ ID No. 1 from M to V, the amino acid at position 159 from L to Y, and the amino acid at position 125 from M to E; (B4) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein defined in (B1)-(B3).

4. A nucleic acid molecule encoding the protein of claim 3.

5. The nucleic acid molecule according to claim 4, characterized in that: The nucleic acid molecule is any of the following: (C1) a DNA molecule obtained by replacing nucleotides 187-189 of SEQ ID No. 2 from atg to gtt; (C2) a DNA molecule obtained by replacing nucleotides 187-189 from atg to gtt, and nucleotides 475-477 from ctg to tac of SEQ ID No. 2; (C3) A DNA molecule obtained by replacing nucleotides 187-189 from atg to gtt, nucleotides 475-477 from ctg to tac, and nucleotides 373-375 from atg to gaga of SEQ ID No.

2.

6. Biomaterial, characterized in that: The biological material is an expression cassette or a recombinant vector or a recombinant cell containing the nucleic acid molecule according to claim 4 or 5.

7. The biomaterial according to claim 6, characterized in that: The recombinant cell is recombinant Escherichia coli.

8. Use of the protein according to claim 3, the nucleic acid molecule according to claim 4 or 5, or the biomaterial according to claim 6 or 7 in any of the following: (D1) Production of R-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid; (D2) Preparation of a product for producing R-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid.

9. A product for producing R-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid, characterized in that: The product is product 1 or product 2; The product 1 consists of the protein according to claim 3 and 3-isobutylglutarimide; The product 2 consists of the recombinant cell according to claim 6 or 7 and 3-isobutylglutarimide.

10. Use of the method according to claim 1 or 2, the protein according to claim 3, the nucleic acid molecule according to claim 4 or 5, the biomaterial according to claim 6 or 7, or the product according to claim 9 in the production of (S)-3-(aminomethyl)-5-methylhexanoic acid.

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