A glucosyl hydrolase mutant and its preparation method and application

Abstract

The present invention relates to a glucosyl hydrolase mutant and a preparation method and application thereof, wherein the glucosyl hydrolase mutant is a mutant in which at least one of asparagine at position 338, valine at position 340, or leucine at position 508 of the BglSK enzyme with an amino acid sequence such as that shown in SEQ ID NO: 1 is mutated to alanine having a strong hydrophobic side chain and weak steric hindrance. The mutant of the present invention can promote the hydrolysis of ginsenoside Rg1 and effectively improve the efficiency of converting ginsenoside Rg1 into ginsenoside F1.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a glucosyl hydrolase mutant and a preparation method and application thereof. Background Art

[0002] Plants of the Araliaceae family, especially ginseng, American ginseng and Panax notoginseng, have a long history of medicinal use due to their significant tonic effects. They have now become an important source of various health products, functional foods and cosmetics. Ginsenoside F1, with a molecular formula of C 36 H 62 O9, a rare saponin component of Araliaceae, is a metabolite of ginsenoside Rg1. In recent years, research on ginsenoside F1 has been further deepened. The results showed that ginsenoside F1 showed significant inhibitory effects on protecting UV-induced cell apoptosis and proliferation of skin melanoma cells, playing an anti-photoaging role. Compared with other rare saponins, ginsenoside F1 is also a pro-angiogenic agent, which can promote angiogenesis in vitro and in vivo, inhibit vascular leakage, and resist atherosclerosis, preventing vascular aging. In addition, ginsenoside F1 has a neuroprotective effect on aging human astrocytes and the adverse effects of high-fat diet on the brain, showing more significant biological activity than other rare saponins. At the same time, the beneficial effects of ginsenoside F1 can extend to protecting the brain from amyloid β-induced toxicity and preventing Alzheimer's disease. Recent studies have also shown that ginsenoside F1 can significantly improve mitochondrial function, protect telomeres and ultimately delay cell aging. Therefore, ginsenoside F1 has become a potential active substance for promoting healthy aging.

[0003] Ginsenoside F1 is usually formed by removing a molecule of glucose from ginsenoside Re or ginsenoside Rg1 by β-glucosidase. β-glucosidase can break the glycosidic bond formed by the reaction of alcohol hydroxyl and hemiacetal in an exo- or endo-cut manner, thereby effectively and directionally hydrolyzing natural active substances containing glucosides, such as ginsenosides. At present, the ginsenoside components of plants belonging to the Araliaceae family, such as Panax notoginseng, ginseng, and American ginseng, generally show a high degree of glycosylation of monomer saponins, resulting in low oral bioavailability in humans. Modern studies have shown that compared with prototype saponins, deglycosylated rare saponins show higher biological and pharmacological activities. Therefore, finding monomer enzymes or combined enzymes that can specifically hydrolyze a variety of large polar saponins is the key to solving the problem of efficient production and high-value utilization in the actual industrial application process. However, there are still large technical barriers for β-glucosidase with specific hydrolysis of ginsenosides in large-scale applications. The activity of natural β-glucosidase is insufficient, and its reaction environment is often difficult to meet the optimal conditions of natural enzymes. Summary of the invention

[0004] In order to overcome the deficiencies of the prior art, the present invention provides a glucosyl hydrolase mutant capable of selectively converting ginsenoside Rg1 into ginsenoside F1, which can solve the problem of insufficient glucosidase activity.

[0005] In order to achieve the technical goal, the present invention is specifically implemented through the following technical solutions:

[0006] The first object of the present invention is to provide a glucosyl hydrolase mutant.

[0007] Through bioinformatics techniques such as molecular docking of proteins and substrates, the present invention designs a glucosyl hydrolase mutant, wherein the glucosyl hydrolase mutant is prepared by mutating asparagine at position 338 of the BglSK enzyme whose amino acid sequence is shown in SEQ ID NO:1 to alanine having a strong hydrophobic side chain and weak steric hindrance; or, valine at position 340 of the BglSK enzyme whose amino acid sequence is shown in SEQ ID NO:1 to alanine having a strong hydrophobic side chain and weak steric hindrance; or, leucine at position 508 of the BglSK enzyme whose amino acid sequence is shown in SEQ ID NO:1 to alanine having a strong hydrophobic side chain and weak steric hindrance.

[0008] Furthermore, the asparagine at position 338 of the BglSK enzyme mutated to alanine to obtain mutant N338A, whose amino acid sequence is shown in SEQ ID NO:3; the valine at position 340 of the BglSK enzyme mutated to alanine to obtain mutant V340A, whose amino acid sequence is shown in SEQ ID NO:5; the leucine at position 508 of the BglSK enzyme mutated to alanine to obtain mutant L508A, whose amino acid sequence is shown in SEQ ID NO:7.

[0009] Furthermore, the nucleotide sequence encoding the mutant N338A gene is shown in SEQ ID NO:4; the nucleotide sequence encoding the mutant V340A gene is shown in SEQ ID NO:6; and the nucleotide sequence encoding the mutant L508A gene is shown in SEQ ID NO:8.

[0010] Furthermore, the BglSK enzyme is derived from Sanguibacter keddieii, specifically, by codon optimizing the nucleotide sequence of the Sanguibacter keddieii β-glucosidase sequence D1BKC5 (sequence number ACZ20402 translated protein sequence; SEQ ID NO: 1), and the nucleotide sequence of the codon-optimized gene of the BglSK enzyme is shown in SEQ ID NO: 2.

[0011] The second object of the present invention is to provide a method for preparing the above-mentioned glucosyl hydrolase mutant, which can solve the problem of preparing high-activity glucosidase.

[0012] A method for preparing a glucosyl hydrolase mutant comprises the following steps:

[0013] Step 1: Connect the coding gene of the BglSK enzyme to be mutated into the plasmid to obtain a recombinant plasmid;

[0014] Step 2: designing and using site-directed mutagenesis primers, amplifying with the recombinant plasmid as a template, and obtaining a mutant product after enzyme digestion; the site-directed mutagenesis primers mutate the asparagine at position 338, the valine at position 340, or the leucine at position 508 of the BglSK enzyme;

[0015] Step 3: Transform the mutant product of step 2 into host cells, screen and induce the host cells to express and obtain the glucosyl hydrolase mutant.

[0016] Furthermore, the nucleotide sequence of the gene encoding the BglSK enzyme is shown in SEQ ID NO:2.

[0017] Further, in the step 2, the asparagine at position 338 of the BglSK enzyme is mutated to alanine by upstream primers N338A-F and downstream primers N338A-R, the nucleotide sequence of the upstream primer N338A-F is shown in SEQ ID NO: 15, and the nucleotide sequence of the downstream primer N338A-R is shown in SEQ ID NO: 16; the valine at position 340 of the BglSK enzyme is mutated to alanine by upstream primers V340A-F and downstream primers V340A-R, the nucleotide sequence of the upstream primer V340A-F is shown in SEQ ID NO: 17, and the nucleotide sequence of the downstream primer V340A-R is shown in SEQ ID NO: 18; the leucine at position 508 of the BglSK enzyme is mutated to alanine by upstream primers L508A-F and downstream primers L508A-R, and the nucleotide sequence of the upstream primer L508A-F is shown in SEQ ID NO: NO:21, and the nucleotide sequence of the downstream primer L508A-R is shown in SEQ ID NO:22.

[0018] Furthermore, the host cell of the recombinant plasmid includes any one of Escherichia coli, Pichia pastoris and Saccharomyces cerevisiae.

[0019] The third object of the present invention is to provide the application of the above-mentioned glucosyl hydrolase mutant, which can solve the problem of preparing ginsenoside F1.

[0020] An application of a glucosyl hydrolase mutant, wherein the glucosyl hydrolase mutant is used for preparing ginsenoside F1.

[0021] Furthermore, the application catalyzes the conversion of ginsenosides Rg1 and Re into F1 through a glucoside mutant.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. The glucosyl hydrolase mutant of the present invention has a short side chain with strong hydrophobicity, which does not affect the substrate binding to hydrophobic amino acids. Mutants N338A, V340A, and L508A can promote the hydrolysis of ginsenoside Rg1 and effectively improve the efficiency of converting ginsenoside Rg1 into ginsenoside F1. The Kcat / Km of the pure enzymes of mutants N338A, V340A, and L508A are 2.07 times, 2.11 times, and 7.04 times that of the wild type, respectively, and the catalytic efficiency is higher;

[0024] 2. The preparation method of the glucosyl hydrolase mutant of the present invention uses computer-aided protein engineering enzyme molecule design to analyze the active pocket region of the β-glucosidase BglSK from Sanguibacter keddieii for molecular docking, and then constructs a mutant library through site-directed mutagenesis technology. The preparation method is accurate and has good effect;

[0025] 3. The glucosyl hydrolase mutant of the present invention can catalyze the conversion of ginsenoside Rg1 to prepare compositions containing ginsenoside F1 in different content ranges, and has a wide range of applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is the legend for the conversion of ginsenoside Rg1 into ginsenoside F1;

[0027] Figure 2 This is the simulation diagram of the molecular docking between BglSK enzyme and ginsenoside Rg1;

[0028] Figure 3 Comparison of the catalytic efficiency of wild-type and mutant BglSK enzymes in converting ginsenoside Rg1 to ginsenoside F1;

[0029] Figure 4 This is the result of polyacrylamide gel electrophoresis analysis of BglSK and mutant pure enzyme after protein purification. DETAILED DESCRIPTION

[0030] Natural beta-glucosidase is not active enough, and its reaction environment is often difficult to meet the optimal conditions of natural enzymes. As related technologies such as molecular biology and genetic engineering mature, protein sequences are edited and modified, which can strengthen the interpretation of enzyme catalysis mechanism and the design of guiding enzymes. Therefore, the present invention constructs a mutant library of glucosyl hydrolase by bioinformatics techniques such as molecular docking of protein and substrate, and provides a highly active glucosyl hydrolase mutant to solve the problem of insufficient activity of beta-glucosidase.

[0031] The invention provides a glucosyl hydrolase mutant, wherein the glucosyl hydrolase mutant is prepared by mutating the asparagine at position 338 of the BglSK enzyme whose amino acid sequence is shown in SEQ ID NO:1 into alanine having a strong hydrophobic side chain and weak steric hindrance; or mutating the valine at position 340 of the BglSK enzyme whose amino acid sequence is shown in SEQ ID NO:1 into alanine having a strong hydrophobic side chain and weak steric hindrance; or mutating the leucine at position 508 of the BglSK enzyme whose amino acid sequence is shown in SEQ ID NO:1 into alanine having a strong hydrophobic side chain and weak steric hindrance.

[0032] Furthermore, the asparagine at position 338 of the BglSK enzyme mutated to alanine to obtain mutant N338A, the amino acid sequence of which is shown in SEQ ID NO: 3; the valine at position 340 of the BglSK enzyme mutated to alanine to obtain mutant V340A, the amino acid sequence of which is shown in SEQ ID NO: 5; the leucine at position 508 of the BglSK enzyme mutated to alanine to obtain mutant L508A, the amino acid sequence of which is shown in SEQ ID NO: 7. After the sites at positions 338, 340 and 508 of the BglSK enzyme were mutated respectively, the hydrolysis efficiency was improved compared with the wild-type β-glucosidase.

[0033] Furthermore, the nucleotide sequence encoding the mutant N338A gene is shown in SEQ ID NO:4; the nucleotide sequence encoding the mutant V340A gene is shown in SEQ ID NO:6; and the nucleotide sequence encoding the mutant L508A gene is shown in SEQ ID NO:8.

[0034] Furthermore, the BglSK enzyme is derived from Sanguibacter keddieii, specifically, the nucleotide sequence of the β-glucosidase sequence D1BKC5 (sequence number ACZ20402 translated protein sequence; SEQ ID NO: 1) of Sanguibacter keddieii is codon-optimized, and the nucleotide sequence of the codon-optimized gene of the BglSK enzyme is shown in SEQ ID NO: 2.

[0035] The present invention also provides a method for preparing the above-mentioned glucosyl hydrolase mutant, which can solve the problem of preparing high-activity glucosidase.

[0036] The method for preparing the glucosyl hydrolase mutant comprises the following steps:

[0037] Step 1. Connect the coding gene of the BglSK enzyme to be mutated into a plasmid to obtain a recombinant plasmid;

[0038] Step 2. Design and use site-directed mutagenesis primers, use the recombinant plasmid as a template for amplification, and obtain a mutant product after enzyme digestion; the site-directed mutagenesis primers mutate asparagine at position 338, valine at position 340, and leucine at position 508 of the BglSK enzyme;

[0039] Step 3. Transform the mutant product of step 2 into host cells, screen and induce the host cells to express and obtain the glucosyl hydrolase mutant.

[0040] Further, the nucleotide sequence of the gene encoding the BglSK enzyme is shown in SEQ ID NO: 2; in the step 2, the asparagine at position 338 of the BglSK enzyme is mutated to alanine by upstream primers N338A-F and downstream primers N338A-R, the nucleotide sequence of the upstream primer N338A-F is shown in SEQ ID NO: 17, and the nucleotide sequence of the downstream primer N338A-R is shown in SEQ ID NO: 18; the valine at position 340 of the BglSK enzyme is mutated to alanine by upstream primers V340A-F and downstream primers V340A-R, the nucleotide sequence of the upstream primer V340A-F is shown in SEQ ID NO: 19, and the nucleotide sequence of the downstream primer V340A-R is shown in SEQ ID NO: 20; the leucine at position 508 of the BglSK enzyme is mutated to alanine by upstream primers L508A-F and downstream primers L508A-R, and the nucleotide sequence of the upstream primer L508A-F is shown in SEQ ID NO: NO:21, and the nucleotide sequence of the downstream primer L508A-R is shown in SEQ ID NO:22.

[0041] Furthermore, the host cell of the recombinant plasmid includes any one of Escherichia coli, Pichia pastoris and Saccharomyces cerevisiae.

[0042] The third object of the present invention is to provide an application of a glucosyl hydrolase mutant, which can solve the problem of preparing ginsenoside F1.

[0043] An application of a glucosyl hydrolase mutant, wherein the glucosyl hydrolase mutant is used for preparing ginsenoside F1.

[0044] Furthermore, the application catalyzes the conversion of ginsenosides Rg1 and Re into F1 through a glucoside mutant.

[0045] Below, the present invention is further described in conjunction with the accompanying drawings and specific embodiments. It is necessary to point out that the embodiments are only used to further illustrate the present invention and cannot be understood as limiting the scope of protection of the present invention. In addition, it should be understood that after reading the content taught by the present invention, a person skilled in the art in this field can make some non-essential improvements and adjustments based on the content of the present invention described above, and these equivalent forms also fall within the scope defined by the claims attached to this application.

[0046] The following examples are experimental methods with specific conditions indicated. Conventional experimental conditions or experimental conditions recommended by the manufacturer are generally followed. Unless otherwise specified, the various reaction reagents involved in the examples can be purchased through commercial channels. For molecular biology experimental methods not specifically described in this example, please refer to the Molecular Cloning Experiment Guide.

[0047] Example 1 Molecular docking of the tertiary structure model of BglSK enzyme and ginsenoside Rg1

[0048] Combined with the deglycosylation mechanism of ginsenoside Rg1 into ginsenoside F1, as shown in Figure 1, the constructed three-dimensional structure model of BglSK enzyme was molecularly docked with the substrate ginsenoside Rg1. Figure 2 As shown. The glucose group connected to the C6 position of ginsenoside Rg1 is located in the active pocket of the BglSK enzyme, while the rest of the structure of ginsenoside Rg1 is located on the surface of the enzyme. The β-D-pyranose glucose group connected to the glucose group connected to the C6 position of the parent ring of ginsenoside Rg1 has an obvious steric hindrance relationship. It is preferred to mutate the five amino acids located on the surface of the active pocket, including isoleucine at position 75, threonine at position 137, asparagine at position 338, valine at position 340, and leucine at position 508. These five mutants are composed into a small mutation library and their amino acid residues are mutated to alanine, and their importance is explored by testing the activity of the mutant enzyme.

[0049] Example 2 BglSK enzyme to be mutated and plasmid

[0050] The gene of Sanguibacter keddieii ACZ20402 was optimized for codon preference in E. coli, and the steps were as follows: the β-glucosidase gene of the nucleotide sequence of SEQ ID NO: 2 was connected to the pET-28a plasmid to obtain the recombinant plasmid pET-28a-BglSK; the plasmid was transformed into E. coli BL21 (DE3), and the recombinant strain was named BL21 (DE3) / pET-28a-BglSK. The amino acid sequence of the BglSK enzyme expressed by the recombinant enzyme strain is shown in SEQ ID NO: 1.

[0051] Example 3 Obtaining BglSK mutant strain

[0052] 1. Construction of mutant enzyme vector by whole plasmid PCR

[0053] Step 1. Extract a small amount of the recombinant plasmid pET-28a-BglSK;

[0054] Step 2. Design mutation primers. The primers include a 15 bp overlap region and a 15 bp extension region, wherein the mutation site is designed in the overlap region. The mutation sites include single-site mutations of Ile75, Thr137, Asn338, Val340 and Leu508, and the mutated amino acid is alanine with a strong hydrophobic side chain and weak steric hindrance;

[0055] Step 3. Perform full plasmid PCR amplification using plasmid pET-28a-BglSK as a template. The PCR system is shown in Table 1:

[0056] Table 1. Full plasmid PCR amplification system

[0057]

[0058] The primers Primer-F and Primer-R are PCR upstream primers and downstream primers designed accordingly according to different mutation sites. The specific primer information is shown in Table 2.

[0059] Table 2 Primers for PCR amplification of single point mutations in whole plasmid

[0060]

[0061] Primer pair I75A-F and I75A-R are used to obtain mutant enzyme I75A, the amino acid sequence of mutant I75A is SEQ ID NO: 9, and the nucleotide sequence is SEQ ID NO: 10; primer pair T137A-F and T137A-R are used to obtain mutant T137A, the amino acid sequence of mutant T137A is SEQ ID NO: 11, and the nucleotide sequence is SEQ ID NO: 12; primer pair N338A-F and N338A-R are used to obtain mutant N338A, the amino acid sequence of mutant N338A is SEQ ID NO: 3, and the nucleotide sequence is SEQ ID NO: 4; primer pair V340A-F and V340A-R are used to obtain mutant enzyme V340A, the amino acid sequence of mutant V340A is SEQ ID NO: 5, and the nucleotide sequence is SEQ ID NO:6; primer pair L508A-F and L508A-R were used to obtain mutant L508A, the amino acid sequence of mutant L508A was SEQ ID NO:7, and the nucleotide sequence was SEQ ID NO:8.

[0062] PCR amplification program: pre-denaturation at 98°C for 5 min; cycle setting: denaturation at 98°C for 15 s, annealing at 62°C for 30 s, extension at 72°C for 1.5 min, 30 cycles; final extension at 72°C for 10 min; after the reaction, the PCR product was recovered using a kit.

[0063] Step 4. Remove the template DNA by enzyme digestion, and perform enzyme digestion on the PCR recovered product. The enzyme digestion system is shown in Table 3:

[0064] Table 3 Enzyme digestion system of whole plasmid PCR product

[0065] QuickCut DpnⅠ 1μL PCR products ≤1 μg 10×QuickCut Buffer 3μL <![CDATA[ddH2O]]> Make up to 30 μL

[0066] The above enzyme digestion system was placed in a 37°C metal bath for digestion for 1 hour. After the reaction was completed, the enzyme digestion product was recovered using a kit.

[0067] 2. Sequencing to verify whether the mutant enzyme strain is successfully constructed

[0068] The above enzyme digestion products were transformed into Escherichia coli BL21 (DE3) competent cells, inverted and cultured overnight at 37°C, and the pseudo-positive transformants were picked for sequencing verification, and mutant enzyme expression strains BL21 (DE3) / pET-28a-BglSK-I75A, BL21 (DE3) / pET-28a-BglSK-T137A, BL21 (DE3) / pET-28a-BglSK-N338A, BL21 (DE3) / pET-28a-BglSK-V340A, and BL21 (DE3) / pET-28a-BglSK-L508A strains were successfully obtained.

[0069] Example 4 Determination of the enzymatic reaction kinetic parameters of BglSK and its mutant enzymes

[0070] Enzyme protein induction of BglSK and its mutant enzyme recombinant strains BL21 (DE3) / pET-28a-BglSK-I75A, BL21 (DE3) / pET-28a-BglSK-T137A, BL21 (DE3) / pET-28a-BglSK, BL21 (DE3) / pET-28a-BglSK-N338A, BL21 (DE3) / pET-28a-BglSK-V340A, BL21 (DE3) / pET-28a-BglSK-L508A strains were streaked on LB plates containing Kan (100 μg / mL). After inverted culture at 37 ° C overnight, a single colony was picked and inoculated into 1 mL of LB liquid culture medium containing Kan, and cultured at 37 ° C, 200 r / min shaking for 12-16 hours. Inoculate the overnight cultured seed solution into 100 mL of fresh LB liquid medium containing Kan at a 1% inoculation rate, culture at 37°C, 200 r / min with shaking for 2-3 h until OD600 is 0.6-0.8, add IPTG with a final concentration of 0.1 mM, place the culture solution at 20°C, and culture at 150 r / min with shaking for 18 h to induce protein expression.

[0071] 2. Isolation and purification of BglSK and its mutant enzyme proteins

[0072] After protein induction, the cells were collected by centrifugation at 4°C and 8000rpm for 5min, and the cells were resuspended and washed with Lysis buffer. The cells were collected by centrifugation again and resuspended with Lysis buffer. The obtained cell suspension was subjected to cell disruption treatment with an ultrasonic disruptor under ice bath conditions for 10min, and centrifuged at 4°C and 8000rpm for 5min. The supernatant was taken to obtain a crude enzyme solution. The supernatant was filtered into an ultrafiltration tube with a 0.45μm sterile filter membrane, and ultrafiltered at 4°C and 8000rpm for 10min. The concentrated supernatant was taken out, filtered through a 0.45μm organic filter head, and poured into a Ni-NTA agarose gel purification column. After secondary flow-through, 4mL of Washbuffer with an imidazole concentration of 20mM was added to wash 4 times, and finally a total volume of 7mL of Elution buffer with an imidazole concentration of 250mM was added and the column filtrate was collected to obtain a protein purification solution of the BglSK mutant. The protein concentration was determined using a BCA protein concentration kit.

[0073] 3. Protein Expression

[0074] The obtained pure enzyme solution was mixed with 5× Loading Buffer and denatured at 95°C for 10 min. The mixture was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to verify whether the protein was successfully expressed. The SDS-PAGE gel preparation system is shown in Table 4.

[0075] Table 4 SDS-PAGE gel preparation system

[0076]

[0077] 4. Determination of Kinetic Parameters of Protease-Catalyzed Reaction

[0078] Take the pure enzyme solution diluted a certain number of times, add the ginsenoside Rg1 solution with a final concentration of 10, 30, 50, 70, 100, 150, 200, 300, 400 and 500 μM, respectively, and react for 30 minutes under the optimal conditions. After the reaction, add 1 volume of methanol to terminate the reaction, and the reaction product is filtered through a 0.45 μm organic filter head and then the product ginsenoside F1 is detected by HPLC. The experiment is repeated three times.

[0079] 5. Detection method of ginsenoside F1

[0080] HPLC was used for quantitative analysis of ginsenoside F1, and the chromatographic conditions were as follows:

[0081] High performance liquid chromatograph: Agilent 1100 Series

[0082] Column: Diamonsil® 5 μm C18 (250 mm x 4.6 mm x 5 μm)

[0083] Detector: VWD detector, detection wavelength 208nm

[0084] Mobile phase ratio and elution conditions: flow rate 1mL / min; column temperature 30°C; injection volume 10μL; gradient elution system as shown in Table 5:

[0085] Table 5 HPLC gradient elution system

[0086]

[0087] 6. Results Analysis

[0088] The obtained pure enzyme solution was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Figure 4The results showed that there was an obvious band at 70KDa, proving that the wild-type WT and its mutants I75A, T137A, N338A, V340A and L508A of β-glucosidase BglSK were expressed.

[0089] Table 6 Determination of kinetic parameters of enzymatic reaction of BglSK mutation

[0090]

[0091] When ginsenoside Rg1 is used as substrate, Figure 3 As shown in Table 6, the K of the wild-type WT BglSK enzyme cat / K m 0.297 μM -1 min -1 , K of mutant I75A cat / K m 0.094 μM -1 min -1 , its catalytic efficiency is only 0.32 times that of WT, and the K cat / K m 0.3066μM -1 min -1 , and their catalytic efficiency was not significantly different from that of WT, while the K cat / K m 0.6157 μM respectively -1 min -1 , 0.6267μM -1 min -1 and 2.092 μM -1 min -1 The catalytic efficiency is 2.07 times, 2.11 times and 7.04 times that of the wild type WT. According to the test results, the mutants N338A, V340A and L508A are more affinity to ginsenoside Rg1 and can effectively bind to the substrate for catalysis, thereby improving the catalytic efficiency of the specific conversion of ginsenoside Rg1 to ginsenoside F1.

[0092] In summary, it can be seen that the present invention improves the activity of β-glucosidase based on steric hindrance, constructs a mutant library of glucosyl hydrolase, simulates the three-dimensional structure of wild-type β-glucosidase through computer-assisted protein and molecular visualization, and performs molecular docking with the substrate. Through molecular docking, the conformational relationship between the enzyme and the substrate is accurately predicted, and the relevant target is efficiently obtained, thereby reducing the capacity of the mutant library. Through this site-directed mutagenesis technology, based on the understanding of the relationship between protein structure and function, the protein is purposefully designed and modified, and a possible mutant structure of glucosyl hydrolase is proposed, and related mutants are synthesized through genetic engineering, and then experimentally verified that the mutants N338A, V340A and L508A are more affinity to ginsenoside Rg1, can effectively bind to the substrate for catalysis, and improve the catalytic efficiency of the specific conversion of ginsenoside Rg1 into ginsenoside F1, which can be used for the preparation of ginsenoside F1.

[0093] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0094] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A glucosyl hydrolase mutant, characterized in that: The glucosyl hydrolase mutant is a mutant in which the asparagine at position 338 of the BglSK enzyme with an amino acid sequence as shown in SEQ ID NO: 1 is mutated to alanine having a strongly hydrophobic side chain and weak steric hindrance; Or, the valine at position 340 of the BglSK enzyme whose amino acid sequence is shown in SEQ ID NO: 1 is mutated to alanine having a strongly hydrophobic side chain and weak steric hindrance; Alternatively, the leucine at position 508 of the BglSK enzyme whose amino acid sequence is shown in SEQ ID NO: 1 is mutated to alanine having a strongly hydrophobic side chain and weak steric hindrance.

2. The method for preparing the glucosyl hydrolase mutant according to claim 1, characterized in that: The following steps are involved: Step 1: Connect the coding gene of the BglSK enzyme to be mutated into the plasmid to obtain a recombinant plasmid; Step 2: Design and use site-directed mutagenesis primers, use the recombinant plasmid as a template for amplification, and obtain a mutant product by enzyme digestion; wherein the site-directed mutagenesis primers mutate the asparagine at position 338, the valine at position 340, and the leucine at position 508 of the BglSK enzyme, respectively; Step 3: Transform the mutant product into host cells, screen and induce the host cells to express the glucosyl hydrolase mutant.

3. The method for preparing a glucosyl hydrolase mutant according to claim 2, characterized in that: The nucleotide sequence of the gene encoding the BglSK enzyme is shown in SEQ ID NO:

2.

4. The method for preparing a glucosyl hydrolase mutant according to claim 2, characterized in that: In the step 2, The asparagine at position 338 of the BglSK enzyme was mutated to alanine by upstream primers N338A-F and downstream primers N338A-R. The nucleotide sequence of the upstream primer N338A-F is shown in SEQ ID NO: 15, and the nucleotide sequence of the downstream primer N338A-R is shown in SEQ ID NO: 16; The valine at position 340 of the BglSK enzyme was mutated to alanine by upstream primers V340A-F and downstream primers V340A-R. The nucleotide sequence of the upstream primer V340A-F is shown in SEQ ID NO: 17, and the nucleotide sequence of the downstream primer V340A-R is shown in SEQ ID NO: 18; The leucine at position 508 of the BglSK enzyme was mutated to alanine by upstream primers L508A-F and downstream primers L508A-R. The nucleotide sequence of the upstream primer L508A-F is shown in SEQ ID NO: 21, and the nucleotide sequence of the downstream primer L508A-R is shown in SEQ ID NO:

22.

5. The method for preparing a glucosyl hydrolase mutant according to claim 2, characterized in that: The host cell is any one of Escherichia coli, Pichia pastoris and Saccharomyces cerevisiae.

6. The use of the glucosyl hydrolase mutant according to claim 1, characterized in that: The glucosyl hydrolase mutant is used to catalyze ginsenoside Rg1 to produce ginsenoside F1.

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