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

Abstract

The present invention discloses an R-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid synthetic protein and its mutants and applications. The present invention relates to the field of biotechnology and provides a method for preparing R-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid, comprising: 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 a protein shown in SEQ ID No.1 or a variant thereof. The present invention is of great significance for preparing high-purity (S)-3-(aminomethyl)-5-methylhexanoic acid by a biological incubation method.

Description

Technical Field

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

[0002] Pregabalin (PGB) is a novel γ-aminobutyric acid (GABA) receptor agonist with excellent anxiolytic and neuropathic effects. It is widely used in the treatment of epilepsy and peripheral neuropathy. The S-pregabalin is 10 times more effective than the R-pregabalin, making obtaining S-pregabalin of high optical purity crucial for drug synthesis.

[0003] Currently, 3-isobutylglutarimide (hereafter referred to as cyclic imine) is used as a substrate, which is hydrolyzed in a strongly alkaline environment to produce equal proportions of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid (hereafter referred to as R-monoamide) and (S)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid (hereafter referred to as S-monoamide). The R-monoamide can be directly synthesized into S-pregabalin ((S)-3-(aminomethyl)-5-methylhexanoic acid) via Hofmann degradation. However, the byproduct S-monoamide requires racemization and recovery using the resolving agent R-α-phenylethylamine. This process requires the use of a large amount of resolving agent, is time-consuming, and produces low yields. 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 method 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;

[0007] Wherein, the specific protein may be any of the following:

[0008] (A1) a protein having an amino acid sequence of SEQ ID No. 1;

[0009] (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;

[0010] (A3) mutant protein obtained by subjecting the protein represented by SEQ ID No. 1 to the following double-point mutations: M63V / L159Y;

[0011] (A4) mutant protein obtained by subjecting the protein represented by SEQ ID No. 1 to the following three point mutations: M63V / L159Y / M125E;

[0012] (A5) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein defined in (A1)-(A4).

[0013] In the above (A1), the protein shown in SEQ ID No. 1 is derived from Bacillus ferrooxidans ( Alicyclobacillus ferrooxydans ), the proteins defined in (A2) to (A4) are variants thereof.

[0014] In (A5), the tag refers to a polypeptide or protein that is fused and expressed with the target protein using in vitro DNA recombination technology to facilitate the expression, detection, tracing, and / or purification of the target protein. The tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0015] 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 a nucleic acid molecule encoding the specific protein into a pET28a plasmid) into Escherichia coli BL21 (DE3).

[0016] 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 (e.g., recombinant E. coli) obtained by introducing the gene encoding the specific protein into a recipient cell (e.g., E. coli). Furthermore, the gene encoding the specific protein can be introduced into the recipient cell in the form of a recombinant vector. Accordingly, the reaction system of the catalytic reaction can be: the recombinant cell (e.g., recombinant E. coli) is resuspended in 50mM Trsi-HCl (pH 7.5) so that the OD 600nm= 10,3-isobutylglutarimide 10 mM. The reaction conditions of the catalytic reaction can be: 50° C. for 2 hours.

[0017] In another embodiment of the present invention, the catalyst is an enzyme (i.e., enzyme catalysis). Specifically, the specific protein serves as the catalyst in the form of an immobilized enzyme. Accordingly, the reaction system for the catalytic reaction can be: the immobilized enzyme, 3-isobutylglutarimide, and water are mixed in a ratio of 20 g:100 g:500 mL. The reaction temperature for the catalytic reaction can be 50°C, the pH controlled at 8.5 during the reaction, and the reaction time is 24 hours. The immobilized enzyme can be prepared according to a method comprising the following steps: ultrasonically disrupting a suspension of recombinant cells (e.g., 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; and mixing the supernatant enzyme solution II with an amino carrier in a ratio of 80 mL:20 g, and incubating at 18°C for 18 hours to obtain the immobilized enzyme.

[0018] In a second aspect, the present invention claims a protein.

[0019] The protein claimed in the present invention may be any of the following:

[0020] (B1) the protein obtained by replacing amino acid 63 of SEQ ID No. 1 from M to V;

[0021] (B2) a protein obtained by replacing amino acid position 63 of SEQ ID No. 1 from M to V and amino acid position 159 from L to Y;

[0022] (B3) a protein obtained by replacing amino acid position 63 of SEQ ID No. 1 from M to V, amino acid position 159 from L to Y, and amino acid position 125 from M to E;

[0023] (B4) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein defined in (B1)-(B3).

[0024] In (B1) to (B3), the protein is derived from Bacillus circulans ( Alicyclobacillus ferrooxydans ) is a variant of the dihydropyrimidinase (SEQ ID No. 1).

[0025] In (B4), the tag refers to a polypeptide or protein that is fused and expressed with the target protein using in vitro DNA recombination technology to facilitate the expression, detection, tracing, and / or purification of the target protein. The tag may be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0026] In a third aspect, the present invention claims protection for a nucleic acid molecule encoding the protein described in the second aspect above.

[0027] 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.

[0028] Furthermore, the nucleic acid molecule may be any of the following:

[0029] (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);

[0030] (C2) 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), and replacing nucleotides 475-477 from ctg (encoding amino acid L) to tac (encoding amino acid Y);

[0031] (C3) 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), nucleotides 475-477 from ctg (encoding amino acid L) to tac (encoding amino acid Y), and nucleotides 373-375 from atg (encoding amino acid M) to gaa (encoding amino acid E).

[0032] In a fourth aspect, the present invention claims protection for a biological material.

[0033] 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.

[0034] 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.

[0035] The recombinant vector may be a recombinant plasmid carrying the expression cassette.

[0036] In one embodiment of the present invention, the recombinant vector is a recombinant plasmid obtained by cloning the nucleic acid molecule into the pET28a plasmid.

[0037] The recombinant cell may be a recombinant cell carrying the recombinant plasmid.

[0038] The cell may be a prokaryotic cell or a eukaryotic cell.

[0039] 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).

[0040] In a fifth aspect, the present invention claims protection for 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:

[0041] (D1) Production of R-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid;

[0042] (D2) Preparation of products for the production of R-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid.

[0043] In a sixth aspect, the present invention claims a product for producing R-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid.

[0044] The product for producing R-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid claimed in the present invention may be Product 1 or Product 2;

[0045] The product 1 is composed of the protein described in the second aspect above and 3-isobutylglutarimide;

[0046] The product 2 is composed of the recombinant cells (such as recombinant Escherichia coli) described in the fourth aspect above and 3-isobutylglutarimide.

[0047] Wherein, 3-isobutylglutarimide is a substrate for producing R-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid, and the protein or the recombinant cell is a catalyst for producing R-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid.

[0048] In the seventh aspect, the present invention claims protection for the use of the method described in the first aspect above, or the protein described in the second aspect above, or the nucleic acid molecule described in the third aspect above, or 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.

[0049] The present invention identifies an amidohydrolase that catalyzes the conversion of 3-isobutylglutarimide to a single chiral (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid. Furthermore, through protein structure analysis and point mutagenesis, a mutant capable of catalyzing the synthesis of a high-purity R-type product was obtained. This invention is of great significance for the preparation of high-purity (S)-3-(aminomethyl)-5-methylhexanoic acid via a biological incubation method. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] 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-conserved amino acids M63, F65, G66, G67, M94, M125, and L159 were used as modification targets for subsequent experiments. DETAILED DESCRIPTION

[0051] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0052] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0053] The determination methods of the various parameters involved in the following embodiments are as follows:

[0054] 1. Determination of 3-isobutylglutarimide and (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid

[0055] An Agilent C18 reversed-phase column (250 × 4.6 mm, 5 μm) was used with a detection wavelength of 210 nm, a column temperature of 35°C, a flow rate of 1.0 ml / min, an injection volume of 20 μl, and a run time of 45 min. Mobile phase A consisted of a buffer solution (approximately 3.5 g of dipotassium hydrogen phosphate was dissolved in 1000 ml of water, mixed, and then the pH was adjusted to 3.0 with phosphoric acid and filtered). Mobile phase B consisted of acetonitrile. The linear gradient elution program is shown in Table 1. The peaks of the 3-isobutylglutarimide standard were eluted at 26.5 min, and the peak of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid was eluted at 8.0 min. The standards used were 3-isobutylglutarimide (Product No. I856241-100g) and (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid (Product No. R843024-5g), produced by Shanghai MacLean Biochemical Technology Co., Ltd.

[0056]

[0057] Note: % indicates volume percentage.

[0058] 2. Optical purity test of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid

[0059] Chromatographic column: Chiralpak AD-H column, 250×4.6 mm, 5 μm;

[0060] Detection wavelength: 210 nm; column temperature: 25°C; flow rate: 0.5 ml / min;

[0061] Mobile phase: n-hexane: ethanol: trifluoroacetic acid = 880:120:1 (volume ratio).

[0062] After the reaction, the fermentation broth was centrifuged, the supernatant was collected, lyophilized, and dissolved in 1 / 2 volume of ethanol. The product's optical purity was determined by HPLC. The standard used was a racemic mixture of 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 No. S62775). The peak elution time for the R-type product was 15.23 min, and the peak elution time for the S-type product was 16.21 min.

[0063] Product optical purity ee . p =(RS) / (R+S)*100.

[0064] Where R is the yield of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid, and S is the yield of (S)-(+)-3-(carbamoylmethyl)-5-methylhexanoic acid. A higher optical purity value calculated using this formula indicates a higher proportion of (R)-(-)-3-(carbamoylmethyl)-5-methylhexanoic acid in the product.

[0065] Example 1. Screening of key amino acid sites in AfHase

[0066] After sequence alignment, the strains from Bacillus ferrooxidans ( Alicyclobacillus ferrooxydans A dihydropyrimidinase (AfHase) from Escherichia coli (Genebank: WP_20665865.1, abbreviated as AfHase, with an amino acid sequence as shown in SEQ ID No. 1) was synthesized and optimized based on 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 expression vector pET28a between the EcoRI and HindIII restriction sites, resulting in a recombinant vector named pET28a-AfHase.

[0067] The recombinant vector pET28a-AfHase was transformed into Escherichia coli BL21 (DE3). A single positive clone was picked and inoculated into 10 mL LB medium containing 40 μg / mL kanamycin. After incubation at 37°C for 12 h, a 1% (volume percentage) inoculum was inoculated into 200 mL LB medium containing 40 μg / mL kanamycin and incubated at 37°C until the OD 600nm The 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 Tris-HCl (pH7.5) to remove the culture medium on the surface of the cells. The catalytic activity was verified according to the whole cell catalytic method. The collected cells were resuspended in 50mM Tris-HCl (pH7.5) and the OD value was adjusted. 600nm To 10, the substrate 3-isobutylglutarimide 10mM, 50℃ for 2h. A strain containing the empty pET28a vector served as a control. The results confirmed that AfHase catalyzed the conversion of 3-isobutylglutarimide to 3-(carbamoylmethyl)-5-methylhexanoic acid with an optical purity of 52.8%.

[0068] Molecular docking of the AfHase protein with the substrate 3-isobutylglutarimide was performed using the Autodock software to identify the active pocket and key amino acid positions. Seven sites, M63, F65, G66, G67, L159, M125, and M94, showed significant interactions with the substrate's isobutyl side chain, so saturation mutagenesis experiments were performed on these sites. Overlapping PCR (primer sequences are shown in Table 2) was performed using a plasmid containing wild-type AfHase (i.e., pET28a-AfHase) as a template. After PCR amplification, the wild-type template was removed by digestion with the endonuclease DpnI. The clones were transformed into Escherichia coli BL21(DE3) and sequenced to obtain positive clones. Whole-cell catalytic assays and the optical purity of the product (3-(carbamoylmethyl)-5-methylhexanoic acid) were performed according to the above experimental procedures. Changes in AfHase enzymatic activity after mutation were also measured, and the relative activities of the AfHase variants relative to the wild-type were calculated. Relative activity = product yield of mutant group / product yield of wild-type group (%), the product is 3-(carbamoylmethyl)-5-methylhexanoic acid.

[0069]

[0070] Note: N represents any base, which can be A, T, C or G.

[0071] Results showed that amino acid mutations at position 65 decreased catalytic activity and product optical purity to varying degrees, so this position was excluded from subsequent experiments. Mutations at other positions, including M63A, M63V, G66C, G66F, G67T, G67V, M125E, M125I, L159Y, L159H, M94A, M94H, M94N, M94R, and M94W, improved both catalytic activity and product optical purity. Therefore, pET28a recombinant vectors carrying the corresponding single-point mutation genes (i.e., recombinant plasmids obtained by inserting the genes encoding the aforementioned single-point mutations between the EcoR I and Hind III restriction sites of the pET-28a vector) were used as templates for the next step of screening for double-point combination mutations. Details are shown in Tables 3 and 4.

[0072]

[0073]

[0074] After the above screening, the amino acid sequences and corresponding coding gene sequences (nucleotide sequences) of the AfHase single mutants finally obtained are as follows:

[0075] 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 nucleotides 187-189 of SEQ ID No. 2 from atg (encoding amino acid M) to gca (encoding amino acid A).

[0076] 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 nucleotides at positions 187-189 of SEQ ID No. 2 from atg (encoding amino acid M) to gtt (encoding amino acid V).

[0077] 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).

[0078] 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).

[0079] 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).

[0080] 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 nucleotides at positions 199-201 of SEQ ID No. 2 from ggc (encoding amino acid G) to gtt (encoding amino acid V).

[0081] 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 nucleotides at positions 373-375 of SEQ ID No. 2 from atg (encoding amino acid M) to gaa (encoding amino acid E).

[0082] 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 nucleotides at positions 373-375 of SEQ ID No. 2 from atg (encoding amino acid M) to atc (encoding amino acid I).

[0083] 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 nucleotides at positions 475-477 of SEQ ID No. 2 from ctg (encoding amino acid L) to tac (encoding amino acid Y).

[0084] 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. The corresponding nucleotide sequence is obtained by replacing nucleotides at positions 475-477 of SEQ ID No. 2 from ctg (encoding amino acid L) to cac (encoding amino acid H).

[0085] 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 nucleotides at positions 280-282 of SEQ ID No. 2 from atg (encoding amino acid M) to gca (encoding amino acid A).

[0086] 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 nucleotides at positions 280-282 of SEQ ID No. 2 from atg (encoding amino acid M) to cac (encoding amino acid H).

[0087] 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 nucleotides at positions 280-282 of SEQ ID No. 2 from atg (encoding amino acid M) to aac (encoding amino acid N).

[0088] 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).

[0089] 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 nucleotides at positions 280-282 of SEQ ID No. 2 from atg (encoding amino acid M) to tgg (encoding amino acid W).

[0090] Example 2: Screening of AfHase double-point combination mutations

[0091] In this example, G66C, G66F, G67T, G67V, M125E, M125I, L159Y, L159H, M94A, M94H, M94N, M94R, or M94W were added to M63A and M63V in Example 1 to screen for double-point combination mutations that significantly improved both catalytic activity and the optical purity of the product (3-(carbamoylmethyl)-5-methylhexanoic acid). The experimental procedures were carried out in accordance with Example 1.

[0092] The results showed that M63V / L159Y showed the most significant improvements in catalytic activity and product optical purity. Therefore, the pET28a recombinant vector carrying the M63V / L159Y encoding gene (i.e., the recombinant plasmid obtained by inserting the M63V / L159Y encoding gene between the EcoRI and HindIII restriction sites of the pET-28a vector) was used as a template for subsequent screening of triple-point combination mutations. Detailed results are shown in Tables 5 and 6.

[0093]

[0094] Note: Relative activity = product yield of double-point mutant group / product yield of M63A group (%). The product is 3-(carbamoylmethyl)-5-methylhexanoic acid.

[0095]

[0096] Note: Relative activity = product yield of the double-point mutant group / product yield of the M63V group (%). The product is 3-(carbamoylmethyl)-5-methylhexanoic acid.

[0097] After the above screening, the amino acid sequence and corresponding coding gene sequence (nucleotide sequence) of the AfHase double-point mutant M63V / L159Y were finally obtained as follows:

[0098] The amino acid sequence of M63V / L159Y is obtained by mutating amino acid position 63 of SEQ ID No.1 from M to V, and amino acid position 159 from L to Y. The corresponding nucleotide sequence is obtained by replacing nucleotides 187-189 of SEQ ID No.2 from atg (encoding amino acid M) to gtt (encoding amino acid V), and replacing nucleotides 475-477 from ctg (encoding amino acid L) to tac (encoding amino acid Y).

[0099] Example 3: Screening of AfHase triple-point combination mutations

[0100] In this example, the AfHase double-point mutant M63V / L159Y obtained in Example 2 was screened and superimposed with G66C, G66F, G67T, G67V, M125E, M125I, M94A, M94H, M94N, M94R, and M94W, respectively, to screen for a triple-point combination mutation that significantly improved both catalytic activity and the optical purity of the product (3-(carbamoylmethyl)-5-methylhexanoic acid). The experimental procedures were similar to those in Example 1.

[0101] The results showed that M63V / L159Y / M125E showed the most significant improvements in catalytic activity and product optical purity. Therefore, the pET28a recombinant vector carrying the M63V / L159Y / M125E encoding gene (the recombinant plasmid obtained by inserting the M63V / L159Y / M125E encoding gene between 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. Detailed results are shown in Table 7.

[0102]

[0103] Note: Relative activity = product yield of triple-point mutant group / product yield of M63V / L159Y group (%). The product is 3-(carbamoylmethyl)-5-methylhexanoic acid.

[0104] After the above screening, the amino acid sequence and corresponding coding gene sequence (nucleotide sequence) of the AfHase triple-point mutant M63V / L159Y / M125E were finally obtained as follows:

[0105] The amino acid sequence of M63V / L159Y / M125E is obtained by mutating amino acid position 63 of SEQ ID No.1 from M to V, amino acid position 159 from L to Y, and amino acid position 125 from M to E; the corresponding nucleotide sequence is obtained by replacing nucleotides 187-189 of SEQ ID No.2 from atg (encoding amino acid M) to gtt (encoding amino acid V), nucleotides 475-477 from ctg (encoding amino acid L) to tac (encoding amino acid Y), and nucleotides 373-375 from atg (encoding amino acid M) to gaa (encoding amino acid E).

[0106] Example 4: Immobilization Process Development

[0107] Referring to Example 1, the recombinant vector pET-28a-M63V / L159Y / M125E (i.e., a recombinant plasmid obtained by inserting the M63V / L159Y / M125E encoding gene between the EcoRI and HindIII restriction sites of the pET-28a vector) was introduced into Escherichia coli BL21(DE3) to produce recombinant E. coli. 20 g of wet cells of this recombinant E. coli were weighed and dissolved in 100 mL of Tris-HCl buffer (50 mmol / L, pH 8.5) to obtain a bacterial suspension. The bacterial suspension was sonicated using a sonicator at low temperature at 400 W, 3 s on / off, 6 s off / on, for a total of 30 min to obtain crude enzyme solution I. The crude enzyme solution was then centrifuged in a high-speed centrifuge at 12,000 g for 30 min to remove impurities, yielding supernatant enzyme solution II. Mix 80 mL of supernatant enzyme solution II with 20 g of amino carrier (Xi'an Lanxiao Technology New Materials Co., Ltd., Cat. No. LXTE-700S) at 120 rpm and 18°C for 18 hours. Rinse the immobilized carrier particles three times with distilled water to remove any enzyme molecules that are not stably fixed to the carrier, thereby obtaining the immobilized enzyme. Add 20 g of immobilized enzyme to 500 mL of water and 100 g of the substrate, 3-isobutylglutarimide. The reaction temperature was 50°C, and the pH was controlled to 8.5 with ammonia. The reaction lasted for 24 hours. A total of 10 batches were performed. After the reaction, the concentration of the product (3-(carbamoylmethyl)-5-methylhexanoic acid) was measured in each batch.

[0108] The results are shown in Table 8, which show that the immobilized enzyme can be stably used for 9 batches, with 67.9% of the activity remaining in the 10th batch.

[0109]

[0110] 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 further improvements can be made to the present invention. In short, according to the principles of the present invention, this application is intended to include any changes, uses or improvements to the present invention, including changes that depart from the disclosed scope in this application and are made using 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 one 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 represented by SEQ ID No. 1 to the following double-point mutations: M63V / L159Y; (A4) mutant protein obtained by subjecting the protein represented by 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)-(A4).

2. The method according to claim 1, wherein: The recombinant cell is recombinant Escherichia coli.

3. Protein, any of the following: (B1) the protein obtained by replacing amino acid 63 of SEQ ID No. 1 from M to V; (B2) a protein obtained by replacing amino acid position 63 of SEQ ID No. 1 from M to V and amino acid position 159 from L to Y; (B3) a protein obtained by replacing amino acid position 63 of SEQ ID No. 1 from M to V, amino acid position 159 from L to Y, and amino acid 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 one 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 of SEQ ID No. 2 from atg to gtt, and nucleotides 475-477 from ctg to tac; (C3) A DNA molecule obtained by replacing nucleotides 187-189 of SEQ ID No. 2 from atg to gtt, nucleotides 475-477 from ctg to tac, and nucleotides 373-375 from atg to gaa.

6. Biomaterial, characterized in that: The biological material is an expression cassette, 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 products for the production of 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 is composed 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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