A glucosidase mutant and its application in ginsenoside production

Abstract

This invention, entitled "A Glucosidase Mutant and Its Application in Ginsenoside Production," belongs to the fields of genetic engineering, enzyme engineering, and natural product biotransformation technology. The technical problem to be solved is to provide a glucosidase mutant capable of effectively catalyzing the conversion of ginsenosides Rb1, Rb2, Rb3, and Rc in Panax notoginseng stems and leaves into the rare ginsenoside CK. The key technical solution is to provide a glucosidase mutant that, with… Caldicellulosiruptor bescii Using β-glucosidase Cb-glu as the parent enzyme, the leucine at position 220 was mutated to alanine to obtain the mutant Cb-glu-L220A. The amino acid sequence of Cb-glu-L220A is shown in SEQ ID NO:1.

Description

Technical Field

[0001] This invention belongs to the fields of genetic engineering, enzyme engineering and natural product biotransformation technology, and specifically relates to a glucosidase mutant and its application in the production of ginsenosides. Background Technology

[0002] Sanqi ( Panax notoginseng Panax notoginseng is a traditional and precious Chinese medicinal herb, rich in various dammarane-type saponins in its roots, stems, and leaves. Besides the main root, the stems and leaves, as important byproducts of Panax notoginseng cultivation and processing, also contain large amounts of protopanaxadiol-type saponins such as Rb1, Rb2, Rb3, and Rc. However, these naturally abundant saponins have complex molecular structures and a large number of sugar groups, resulting in poor lipid solubility and cell membrane permeability, limiting their direct bioavailability. Therefore, converting the abundant high-glycosyl saponins in Panax notoginseng stems and leaves into low-glycosyl, highly active rare ginsenosides is an important direction for realizing the high-value utilization of Panax notoginseng byproduct resources.

[0003] Rare ginsenoside Compound K (CK) is an important bioactive product formed during the in vivo metabolism or in vitro deglycosylation of protopanaxadiol-type saponins. Due to its good absorption properties and various potential pharmacological activities, CK has significant application value in the pharmaceutical, functional food, health product, and cosmetic fields. However, the content of CK in natural plants is extremely low, and direct extraction is costly, making it difficult to meet the needs of large-scale applications. Therefore, converting naturally abundant ginsenosides such as Rb1, Rb2, Rb3, and Rc into CK is currently a promising green preparation route.

[0004] Existing methods for preparing rare ginsenosides (CK) mainly include acid hydrolysis, alkaline hydrolysis, microbial transformation, and enzymatic transformation. Among these, acid-alkaline hydrolysis typically suffers from poor reaction selectivity, numerous byproducts, and significant environmental pollution. While microbial transformation systems are lower in cost, their complex metabolic background makes product separation difficult, resulting in poor batch-to-batch stability. In contrast, enzymatic transformation offers advantages such as mild reaction conditions, high regioselectivity, fewer byproducts, and environmental friendliness, making it an important technological means for preparing rare ginsenoside CK.

[0005] β-glucosidases hydrolyze the β-glucosidic bonds in saponin molecules, playing a crucial role in the stepwise deglycosylation conversion of ginsenosides. However, ginsenosides Rb1, Rb2, Rb3, and Rc molecules are large in size and have significant steric hindrance of their glycosyl groups, resulting in low catalytic efficiency for many wild-type β-glucosidases on these large molecular substrates, making efficient conversion difficult. This is especially true in the complex substrate system of total saponins from Panax notoginseng stems and leaves, where diverse substrate compositions, limited solubility, and different glycosyl linkages place higher demands on the enzyme's substrate recognition ability, active pocket spatial adaptability, and catalytic stability.

[0006] Caldicellulosiruptor bescii This is a thermophilic microorganism, and its derived enzymes typically exhibit good thermal stability and industrial application potential. The β-glucosidase Cb-glu derived from this bacterium can serve as a candidate enzyme for the biotransformation of ginsenosides. However, there is still room for improvement in the conversion activity of wild-type Cb-glu for the main protopanaxadiol-type saponins in Panax notoginseng stems and leaves. Therefore, rationally modifying Cb-glu through protein engineering to obtain mutants with higher catalytic efficiency for sterically hindered saponin substrates is of great significance for improving the conversion efficiency of total saponins from Panax notoginseng stems and leaves and realizing the green preparation of rare ginsenoside CK.

[0007] Relevant patent documents retrieved: The document, published in China (CN121204026A) on December 26, 2025, discloses a highly catalytically active β-glucosidase mutant. The β-glucosidase mutant sequence is obtained by mutating the amino acid sequence shown in SEQ ID No. 1 through any of the following mutations: mutating proline at position 95 and arginine at position 178 to cysteine, respectively; mutating phenylalanine at position 98 and arginine at position 178 to cysteine, respectively.

[0008] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: The β-glucosidase mutant disclosed in patent CN121204026A has low catalytic activity and poor ability to convert ginsenoside Rb1 into ginsenoside CK. Summary of the Invention

[0009] The purpose of this invention is to provide: A glucosidase mutant and its application in ginsenoside production, and related technologies, to solve technical problems such as providing a glucosidase mutant or a combination thereof that can effectively catalyze the conversion of ginsenosides Rb1, Rb2, Rb3 and Rc in Panax notoginseng stems and leaves into rare ginsenoside CK.

[0010] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0011] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0012] The definition of the standard chemical term can be found in the reference "Introduction to Enzymology and Enzyme Engineering": Tsinghua University Press, 2021-08-01.

[0013] Unless otherwise specified, conventional methods within the scope of the art, such as enzyme activity detection and thin-layer chromatography, shall be used.

[0014] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0015] As used in this article, the term "amino acid" refers to one of the 20 naturally occurring amino acids encoded by DNA and RNA, or one of the artificially synthesized amino acids.

[0016] The term "amino acid substitution" as used in this article refers to replacing an amino acid at a specific position in a polypeptide sequence with a different amino acid.

[0017] As used herein, the term "vector" refers to a vector that can autonomously replicate within a host cell, preferably a multi-copy vector. Furthermore, vectors typically possess markers such as antibiotic resistance genes for selecting transformants. Additionally, vectors may have promoters and / or terminators for expressing the introduced gene. Vectors can be, for example, viral vectors, vectors derived from bacterial plasmids, vectors derived from yeast plasmids, vectors derived from bacteriophages, granules, phage particles, etc. Genetically engineered vectors are vectors that enable the expression of a target gene in cells, and are typically linear or circular DNA molecules comprising polynucleotides encoding proteins or polypeptides and operatively linked to an expression control sequence.

[0018] As used herein, the term "expression cassette" refers to DNA capable of expressing the protein in a host cell (such as a microbial cell or plant cell), which may include not only a promoter to initiate transcription of the protein gene but also a terminator to terminate transcription. Furthermore, the expression cassette may also include an enhancer sequence.

[0019] The term "substrate" as used in this article refers to a compound catalyzed by an enzyme. An enzyme acts on only one class of compounds or certain chemical bonds to promote a specific chemical change and produce a specific product.

[0020] In a first aspect, the present invention provides a glucosidase mutant, which, in order to Caldicellulosiruptor bescii Using β-glucosidase Cb-glu as the parent enzyme, the leucine at position 220 was mutated to alanine to obtain the mutant Cb-glu-L220A. The amino acid sequence of Cb-glu-L220A is shown in SEQ ID NO:1.

[0021] Among them are technical features such as the amino acid sequence of Cb-glu-L220A.

[0022] According to some embodiments of the present invention, the amino acid sequence of Cb-glu-L220A may be selected from the sequence that has at least 85% sequence identity with the sequence shown in SEQ ID NO:1.

[0023] According to some embodiments of the present invention, the amino acid sequence of Cb-glu-L220A may be selected from the sequence that has at least 90% sequence identity with the sequence shown in SEQ ID NO:1.

[0024] According to some embodiments of the present invention, the amino acid sequence of Cb-glu-L220A may be selected from the sequence that has at least 95% sequence identity with the sequence shown in SEQ ID NO:1.

[0025] According to some embodiments of the present invention, the amino acid sequence of Cb-glu-L220A may be selected from the sequence that has at least 99% sequence identity with the sequence shown in SEQ ID NO:1.

[0026] According to some embodiments of the present invention, the amino acid sequence of Cb-glu-L220A may be selected from those having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the sequence shown in SEQ ID NO:1.

[0027] SEQ ID NO:1 MSLPKGFLWGAATASYQIEGAWNEDGKGESIWDRFTHQKGNILYGHNGDVACDHYHRFEEDVSLMKELGLKAYRFSIAWARIFPDGFGTVNQKGLEFYDRLINKLVENGIEPV VTIYHWDLPQKLQDIGGWANPEIVNYYFEYAMLIVNRYKDKVKKWITFNEPYCIAFLGHFYGVHAPGIKDFKVAMDVVHNIMLSHFKVVKAVKENNIDVEVGITLNATPVYFQ TERLGYKVSEIEREMVNLSSQLDNELFLDPVLKGSYPQKLFDYLVQKDLLETQKVLSMQQEVKENFVFPDFLGINYYTRAVRLYDENSNWIFPIRWEHPAGEYTEMGWEVFPQ GLYDLLIWIKESYPQIPIYITENGAAYNDKVEDGRVHDQKRVEYLKQHFEAARKAIENGVDLRGYFVWSLLDNLEWAMGYTKRFGVIYVDYETQKRIKKDSFYFYQQYIKENS.

[0028] Specifically, the mutant Cb-glu-L220A can be heterologously expressed using an E. coli expression system.

[0029] Furthermore, the Cb-glu-L220A encoding gene was ligated into the pET-28a expression vector and transformed into Escherichia coli BL21(DE3). After IPTG induction, the purified enzyme protein was obtained through centrifugation, disruption, affinity chromatography, and other steps.

[0030] Secondly, a nucleic acid that encodes the aforementioned glucosidase mutant.

[0031] Specifically, the nucleic acid sequence is shown in SEQ ID NO:2.

[0032] SEQ ID NO:2

[0033] Thirdly, the present invention provides an expression cassette comprising the aforementioned nucleic acid.

[0034] Specifically, the expression box includes a promoter and a terminator.

[0035] Furthermore, the promoter is operatively linked to the 5' end of the nucleic acid to initiate nucleic acid transcription; the terminator is operatively linked to the 3' end of the nucleic acid to terminate gene transcription.

[0036] Fourthly, the present invention provides a gene engineering vector comprising the above-mentioned nucleic acid and / or the above-mentioned expression cassette.

[0037] Specifically, the gene engineering vector is selected from prokaryotic expression vectors and yeast expression vectors.

[0038] Furthermore, the prokaryotic expression vectors include the pET series, pGEX series, and pUC series vectors.

[0039] Furthermore, the yeast expression vectors include the pPIC9K, pAO815, and pYES2 series vectors.

[0040] Fifthly, the present invention provides a recombinant strain comprising the above-mentioned nucleic acid, expression cassette and / or gene engineering vector.

[0041] In a sixth aspect, the present invention provides biological materials comprising the above-mentioned nucleic acids, expression cassettes and / or gene engineering vectors, wherein the biological materials are selected from: plant cell lines, plant tissues, plant organs, animal cell lines, animal tissues or animal organs.

[0042] In a seventh aspect, the present invention provides the application of the above-mentioned glucosidase mutant or the above-mentioned nucleic acid, expression cassette or genetic engineering vector in the production of ginsenosides.

[0043] Specifically, the aforementioned glucosidase mutant or the aforementioned nucleic acid, expression cassette or genetic engineering vector can effectively catalyze the deglycosylation conversion of ginsenosides Rb1, Rb2, Rb3 and Rc substrates in Panax notoginseng stems and leaves to generate rare ginsenoside CK.

[0044] Specifically, ginsenosides Rb1, Rb2, Rb3, and Rc were used as substrates.

[0045] Furthermore, the application has at least one of the following functions: (1) Catalyzes the conversion of ginsenoside Rb1 to CK; (2) Catalyzes the conversion of ginsenoside Rb2 to CK; (3) Catalyzes the conversion of ginsenoside Rb3 to CK; (4) Catalyzes the conversion of ginsenoside Rc to CK.

[0046] The present invention has at least the following beneficial effects: Compared with existing technologies, the present invention has better technical effects in improving the catalytic activity of β-glucosidase.

[0047] According to experimental tests, the present invention increases the catalytic activity of β-glucosidase from 255.6-312.8 U / mg in the prior art to over 431.6 U / mg.

[0048] 1. This invention obtains Cb-glu-L220A through single-point mutation. Compared with wild-type Cb-glu, this mutant has significantly improved conversion activity for ginsenosides Rb1, Rb2, Rb3 and Rc.

[0049] 2. Applicable to the high-value utilization of total saponins in Panax notoginseng stems and leaves: Panax notoginseng stems and leaves are rich in protopanaxadiol-type saponins such as Rb1, Rb2, Rb3, and Rc. The mutant of this invention can efficiently convert them into rare ginsenoside CK, which is beneficial to improving the utilization value of Panax notoginseng agricultural by-products.

[0050] 3. The reaction is green and mild, and the product selectivity is high: Enzymatic conversion avoids the problems of many by-products and heavy pollution caused by strong acid and strong base hydrolysis. It has the advantages of mild reaction conditions, high selectivity and environmental friendliness.

[0051] 4. Possesses excellent potential for industrial application: Cb-glu originates from thermophilic microorganisms. Caldicellulosiruptor bescii Its mutant can catalyze reactions at higher temperatures, which is beneficial for improving substrate solubility, reducing pollution risk, and is suitable for the large-scale preparation of rare ginsenoside CK. Attached Figure Description

[0052] Figure 1 The nucleic acid electrophoresis diagram constructed for the mutant Cb-glu-L220A.

[0053] Figure 2 SDS-PAGE gel electrophoresis image of recombinant bacteria expressing Cb-glu-L220A.

[0054] Figure 3 A comparison of the optimal pH and temperature for wild-type Cb-glu and mutant Cb-glu-L220A.

[0055] Figure 4 A comparison of pH and temperature stability between wild-type Cb-glu and mutant Cb-glu-L220A.

[0056] Figure 5 TLC analysis of the conversion of ginsenosides Rb1 and Rb2 to CK catalyzed by Cb-glu and Cb-glu-L220A.

[0057] Figure 6 TLC analysis of the conversion of ginsenoside Rb3 to CK catalyzed by Cb-glu and Cb-glu-L220A.

[0058] Figure 7 TLC analysis of the conversion of ginsenoside Rc to CK catalyzed by Cb-glu and Cb-glu-L220A. Detailed Implementation

[0059] Unless otherwise specified, all raw materials and reagents used in this invention were purchased from commercial suppliers, and experiments were conducted in accordance with the operating instructions. Unless otherwise specified, all instruments, equipment, and apparatus used in this invention are conventional instruments, equipment, and apparatus, and experiments were conducted in accordance with the operating instructions and the accompanying reagents.

[0060] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. Unless otherwise specified in the embodiments, conditions are performed under conventional conditions or conditions recommended by the manufacturer. All reagents or instruments without specified manufacturers are commercially available conventional products. Numerous specific details are provided in the following detailed embodiments to better illustrate the invention. The specific embodiments described herein are for illustrative purposes only and are not intended to constitute any limitation on the invention.

[0061] Source of experimental materials: Rb1 standard was purchased from Sichuan Weikeqi Biotechnology Co., Ltd., product number 41753-43-9; Rb2 standard was purchased from Sichuan Weikeqi Biotechnology Co., Ltd., product number 11021-13-9; Rb3 standard was purchased from Sichuan Weikeqi Biotechnology Co., Ltd., product number 68406-26-8; Rd standard was purchased from Sichuan Weikeqi Biotechnology Co., Ltd., product number 52705-93-8; Rc standard was purchased from Sichuan Weikeqi Biotechnology Co., Ltd., product number 11021-14-0; The CK standard product was purchased from Sichuan Weikeqi Biotechnology Co., Ltd., product number 39262-14-1.

[0062] Example 1: Design and Construction of the Cb-glu-L220A Mutant From Caldicellulosiruptor besciiThe β-glucosidase Cb-glu is the parent enzyme. In this invention, leucine at position 220 is mutated to alanine, which has a smaller side chain, to obtain the mutant Cb-glu-L220A.

[0063] Site-directed mutagenesis primers were designed based on the Cb-glu encoding gene sequence: upstream primer L220A-F: 5'-CACTAAATGCAACTCCAGTTTACTTTCAAACAGAGC-3', as shown in SEQ ID NO:3; downstream primer L220A-R: 5'-CTGGAGTTGCATTTAGTGTAATTCCTACCTCAACATCAA-3', as shown in SEQ ID NO:4.

[0064] Using the recombinant plasmid Cb-glu-pET containing the wild-type Cb-glu gene as a template, the L220A mutation was introduced via site-directed mutagenesis PCR amplification. The PCR reaction system included the template plasmid, forward and reverse mutagenesis primers, high-fidelity DNA polymerase, dNTPs, and reaction buffer. The electrophoresis image of the Cb-glu-L220A mutant is shown below. Figure 1 As shown, after PCR amplification, the template plasmid was digested with DpnI and then the amplification product was transformed into E. coli competent cells.

[0065] Positive clones were selected and sent to bioengineering lab for colony PCR and sequencing verification. Sequencing results showed that the codon corresponding to leucine at position 220 had been successfully mutated to the codon corresponding to alanine, and no unexpected mutations were found in the remaining sequences. The resulting recombinant plasmid was named Cb-glu-L220A-pET.

[0066] Example 2: Induction and purification of Cb-glu-L220A mutant The recombinant plasmid Cb-glu-L220A-pET, which was verified to be correct by sequencing, was transformed into Escherichia coli BL21(DE3) competent cells, plated on LB solid medium plates containing the corresponding antibiotics, and incubated overnight at 37°C with the plates inverted.

[0067] Single clones were picked and inoculated into LB liquid medium containing antibiotics, and cultured overnight at 37°C with shaking. Then, 1% of the inoculum was transferred to fresh LB medium and cultured at 37°C with shaking until the OD600 reached 0.6–0.8. IPTG was then added to a final concentration of 1.0 mmol / L to induce expression. The induction temperature was 17°C, and the induction time was 13 h.

[0068] After induction, bacterial cells were collected by centrifugation at 8000 rpm for 5–10 min, resuspended in an appropriate amount of buffer, and sonicated under ice bath conditions. The lysate was centrifuged at 12000 rpm for 10–30 min, and the supernatant was collected. The expression vector contained a His tag and was purified using a Ni-NTA affinity chromatography column, with the target protein eluted using imidazole buffer of different concentrations.

[0069] SDS-PAGE analysis showed that the mutant Cb-glu-L220A could be successfully expressed in E. coli, and the target protein was obtained with high purity after purification. Figure 2 ).

[0070] Comparative Example 1 From Caldicellulosiruptor bescii The β-glucosidase Cb-glu was used as the parent enzyme, and in Comparative Example 1, the 174th amino acid was mutated to alanine.

[0071] Comparative Example 2 From Caldicellulosiruptor bescii The β-glucosidase Cb-glu was the parent enzyme, and Comparative Example 2 involved mutating the 220th amino acid to cysteine.

[0072] Comparative Example 3 From Caldicellulosiruptor bescii The β-glucosidase Cb-glu was used as the parent enzyme, and in Comparative Example 2, the 98th and 178th positions were both mutated to cysteine.

[0073] Comparative Example 4 From Caldicellulosiruptor bescii The β-glucosidase Cb-glu was used as the parent enzyme, and in Comparative Example 2, positions 95 and 178 were both mutated to cysteine.

[0074] Example 3 Enzyme activity assay of Cb-glu-L220A mutant p-nitrophenyl-β-D-glucopyranoside (pNPG) was used as a substrate to determine β-glucosidase activity.

[0075] The reaction system was as follows: 450 μL of 2 mM pNPG substrate solution; 50 μL of 0.1 mg / mL enzyme solution; appropriate amount of reaction buffer; total reaction volume 500 μL. The substrate solution was preheated at the set temperature for 5 min, then the enzyme solution was added to initiate the reaction. After 10 min, 1 mol / L Na₂CO₃ was added to terminate the reaction. The absorbance of the released p-nitrophenol was measured at 405 nm. Under the enzyme activity assay conditions, the amount of enzyme required to generate 1 μmol of pNP per minute was defined as 1 U of enzyme activity, and the enzyme activity corresponding to a unit mass of enzyme was defined as specific enzyme activity U / mg.

[0076] The results (Table 1) show that, compared with the wild-type Cb-glu, the mutant Cb-glu-L220A has significantly increased hydrolytic activity on pNPG substrates, while the hydrolytic activity of comparative examples 1-4 is lower, indicating that the L220A mutation is beneficial to improving the catalytic activity of β-glucosidase.

[0077] Table 1

[0078] Example 4: Determination of the optimal pH and optimal temperature for the Cb-glu-L220A mutant To determine the optimal pH for the Cb-glu-L220A mutant, its relative enzyme activity was measured in buffer systems with different pH values. The buffer system was citrate-phosphate buffer with a pH range of 4.5–7.0. The assay method was the same as for enzyme activity assay: 450 μL of 2 mM pNPG substrate solution and 50 μL of 0.1 mg / mL enzyme solution were used, and the reaction was carried out at 80 °C.

[0079] To determine the optimal temperature, the relative enzyme activity of the Cb-glu-L220A mutant was measured at different temperatures under optimal pH conditions, ranging from 65 to 85 °C. The measurement method was the same as that used for enzyme activity determination.

[0080] The results showed that the Cb-glu-L220A mutant exhibited catalytic activity over a wide pH range (5.0-6.5) and a temperature range (80-85℃), with optimal pH and optimal temperature at 5.5 and 80℃, respectively. Figure 3 ).

[0081] The residual activity of the enzyme was measured after treatment at different temperatures (75, 80, and 85 °C) for different times (30, 60, 90, and 120 min) at the optimal pH and temperature to assess the temperature stability of the enzyme. The assay method was the same as that for enzyme activity assay.

[0082] Example 5: Cb-glu-L220A catalyzes the conversion of ginsenoside Rb1 to CK Ginsenoside Rb1 was used as a substrate, and a solution of Rb1 at a certain concentration was prepared. The reaction system included 225 μL of 2 mM Rb1 substrate, 225 μL of McIlvaine buffer at the optimal pH, and 50 μL of Cb-glu-L220A enzyme solution. The reaction conditions were pH 5.5, temperature 80℃, and reaction time 2 h.

[0083] After the reaction was completed, an equal volume of methanol was added to terminate the reaction. The protein precipitate was removed by centrifugation, and the supernatant was analyzed by thin-layer chromatography (TLC). Rb1, Rd, CK standards and a control group were used as controls (50 μL of the same concentration of inactivated enzyme was added).

[0084] The specific TLC method is as follows: Prepare the developing solvent by mixing 700 μL of chloroform, 300 μL of methanol, and 50 μL of double-distilled water. Pour an appropriate amount into the developing tank and let it stand for 30 min to saturate the system. Activate the high-performance thin-layer chromatography silica gel G plate in a 110℃ oven for 30 min. After cooling to room temperature, draw a baseline 2 cm below the bottom of the silica gel plate. Spot the sample using a capillary tube, 0.5 μL each time. After drying, repeat the spotting process three times. Simultaneously spot the substrate and product standards as controls. Place the spotted silica gel plate, bottom down, into the saturated developing tank, ensuring that the spotted sample does not submerge in the developing solvent. Develop the sample until the leading edge of the developing solvent is 1.5 cm from the top of the silica gel plate. Remove the silica gel plate, dry it, and repeat the development process twice. After the second development, spray a 10% sulfuric acid ethanol color developer evenly onto the silica gel plate (slowly add 90 mL of anhydrous ethanol to 10 mL of concentrated sulfuric acid while stirring, and prepare immediately before use) until the surface of the silica gel plate is evenly wetted without any liquid accumulation. After standing at room temperature for 30 seconds, blow dry and place in a 105℃ oven for 15 minutes to allow the spots to develop clearly.

[0085] The results showed that Cb-glu-L220A could catalyze a stepwise deglycosylation reaction of Rb1 to generate rare ginsenoside CK. Compared with wild-type Cb-glu, the mutant Cb-glu-L220A catalytic system had lower Rb1 residue and higher CK production, indicating that the L220A mutation improved the enzyme's ability to convert Rb1 substrates. Figure 5 (a) in the middle.

[0086] Example 6: Cb-glu-L220A catalyzes the conversion of ginsenoside Rb2 to CK Ginsenoside Rb2 was used as a substrate, and an enzyme-catalyzed reaction system was established according to the method described in Example 5 (substrate concentration and enzyme concentration were the same). After the reaction was terminated with methanol, centrifuged, and filtered, the changes in substrate and product were analyzed by TLC.

[0087] The results showed that Cb-glu-L220A could effectively catalyze the deglycosylation conversion of Rb2, ultimately generating CK. Compared with wild-type Cb-glu, the mutant Cb-glu-L220A had a higher conversion efficiency for Rb2, indicating that this mutant has good substrate adaptability to protopanaxadiol-type saponins with different glycosyl modifications. Figure 5 (b) in the middle.

[0088] Example 7: Cb-glu-L220A catalyzes the conversion of ginsenoside Rb3 to CK Ginsenoside Rb3 was used as a substrate, and Cb-glu-L220A was added to catalyze the reaction at pH 5.5 and 80℃ for 2 h. The reaction was analyzed by TLC after completion.

[0089] The results showed that Cb-glu-L220A could catalyze the conversion of Rb3 to CK. Compared with wild-type Cb-glu, the peak area of ​​the CK product in the Cb-glu-L220A mutant reaction system was significantly increased, indicating that the L220A mutation improved the enzyme's catalytic conversion ability of Rb3 substrate. Figure 6 ).

[0090] Example 8: Cb-glu-L220A catalyzes the conversion of ginsenoside Rc to CK Using ginsenoside Rc as a substrate, an enzyme-catalyzed reaction system identical to that in Example 5 was established. The reaction was detected by TLC after completion.

[0091] The results showed that Cb-glu-L220A could catalyze the deglycosylation of Rc to generate CK. Although its spatial structure differs from Rb1, Rb2, and Rb3 due to different glycosyl substitutions in the Rc structure, Cb-glu-L220A still exhibited good catalytic activity, indicating that this mutant has broad substrate adaptability. Figure 7 ).

[0092] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A glucosidase mutant, characterized in that, Its Caldicellulosiruptor bescii Using β-glucosidase Cb-glu as the parent enzyme, the leucine at position 220 was mutated to alanine to obtain the mutant Cb-glu-L220A. The amino acid sequence of Cb-glu-L220A is shown in SEQ ID NO:

1.

2. A nucleic acid, characterized in that, The nucleic acid encodes the glucosidase mutant of claim 1.

3. The nucleic acid according to claim 2, characterized in that, The nucleic acid sequence is shown in SEQ ID NO:

2.

4. An expression box, characterized in that, The expression cassette comprises the nucleic acid as described in any one of claims 2-3.

5. A gene engineering vector, characterized in that, The genetic engineering vector comprises the nucleic acid as described in any one of claims 2-3 and / or the expression cassette as described in claim 4.

6. A recombinant bacterial strain, characterized in that, The recombinant strain comprises the nucleic acid of any one of claims 2-3, the expression cassette of claim 4, and / or the gene engineering vector of claim 5.

7. A biomaterial comprising the nucleic acid according to any one of claims 2-3, the expression cassette according to claim 4, and / or the gene engineering vector according to claim 5, characterized in that, The biological materials are selected from: plant cell lines, plant tissues, plant organs, animal cell lines, animal tissues, or animal organs.

8. The use of the glucosidase mutant of claim 1, the nucleic acid of any one of claims 2-3, the expression cassette of claim 4, and / or the gene engineering vector of claim 5 in the production of ginsenosides.

9. The application according to claim 8, characterized in that, Ginsenosides Rb1, Rb2, Rb3, and Rc were used as substrates.

10. The application according to claim 8, characterized in that, The application described has at least one of the following functions: (1) Catalyzes the conversion of ginsenoside Rb1 to CK; (2) Catalyzes the conversion of ginsenoside Rb2 to CK; (3) Catalyzes the conversion of ginsenoside Rb3 to CK; (4) Catalyzes the conversion of ginsenoside Rc to CK.

Images