Beta-glucosidase mutant, immobilized enzyme of beta-glucosidase mutant and application of beta-glucosidase mutant in efficient synthesis of kinsenoside

Abstract

The invention discloses a beta-glucosidase mutant, an immobilized enzyme of the beta-glucosidase mutant and application of the beta-glucosidase mutant in efficient synthesis of kinsenoside, and belongs to the field of enzyme engineering and natural product biosynthesis. According to the mutant, one or more mutations of W177Y, W179F, Q242A, F245G, R298S and R313T are introduced at key sites of an amino acid sequence of wild type beta-glucosidase, so that the yield of catalytic synthesis of kinsenoside is remarkably improved. Furthermore, a metal organic framework material UiO-66-NH2 is adopted as an immobilization carrier, enzyme immobilization is achieved in a physical adsorption mode, and the obtained immobilized enzyme is excellent in reusability. Under the same reaction conditions, the immobilized mutant enzyme is used for catalyzing synthesis of kinsenoside, and the yield of the immobilized mutant enzyme is 3 times that of a wild enzyme. The invention provides an efficient, green and sustainable kinsenoside biocatalytic synthesis scheme, and the kinsenoside biocatalytic synthesis method has a good industrial application prospect.

Description

Technical Field

[0001] This invention relates to the fields of enzyme engineering and natural product biosynthesis, specifically to a β-glucosidase mutant, its immobilized enzyme, and its application in the efficient synthesis of Anoectochilus roxburghii glycosides. Background Technology

[0002] Anoectochilus roxburghii is an orchid with significant medicinal value, rich in various natural products, including flavonoids, polysaccharides, steroids, and glycosides. Among them, anoectochilosin (CAS Registry No.: 151870-74-5), as a representative glycoside, has been proven to possess antioxidant, immunomodulatory, antitumor, and hypoglycemic biological activities, and is widely used in the research and development of health foods, traditional Chinese medicine preparations, and natural drugs.

[0003] Currently, the main source of Anoectochilus glycosides still relies on direct plant extraction, which suffers from problems such as resource scarcity, low extraction efficiency, and heavy environmental burden. Chemical synthesis, on the other hand, faces challenges such as poor regional selectivity, cumbersome procedures, and unsustainability. Therefore, developing an efficient, green, and sustainable biocatalytic strategy for Anoectochilus glycosides has become a key research direction in this field.

[0004] β-glucosidase (EC 3.2.1.21) is a class of enzymes that catalyze the hydrolysis or synthesis of β-glycosidic bonds, playing important biological functions in carbohydrate metabolism, biomass degradation, and glycoside derivative synthesis. Notably, under specific water activity and high-concentration donor substrate conditions, β-glucosidase can reverse the direction of its hydrolysis reaction, catalyzing the formation of glycosidic bonds, exhibiting "reverse hydrolysis" or "transglycosylation" activity. However, naturally derived enzymes still face several limitations in catalyzing glycoside synthesis, including weak substrate recognition, low catalytic efficiency, poor thermal stability, and non-reusability, making it difficult to meet the needs of industrial-scale synthesis.

[0005] To address these challenges, protein engineering has become an effective means of improving enzyme performance. By rationally designing and targeting the structure of the enzyme's active site, its catalytic efficiency and substrate adaptability in glycoside synthesis can be enhanced. Meanwhile, enzyme immobilization technology can significantly enhance the enzyme's thermal stability, organic solvent tolerance, and recyclability. In particular, immobilization using metal-organic frameworks (MOFs) as carriers has become a research hotspot in recent years due to its high specific surface area, tunable pore size, and good biocompatibility.

[0006] Therefore, developing a highly catalytically active β-glucosidase mutant with multi-site site-directed mutations and immobilizing it with MOFs to construct a stable and efficient biocatalytic system for catalytic synthesis of Anoectochilus roxburghii glycosides not only has significant technological innovation but also clear industrialization prospects, aligning with the current development trend of green biomanufacturing and high-value utilization of natural products. Summary of the Invention

[0007] This invention aims to overcome the problems of low catalytic efficiency, poor substrate selectivity, and poor reusability of existing β-glucosidases in the biosynthesis of Anoectochilus roxburghii glycosides. Through molecular modification, a β-glucosidase mutant with high catalytic activity and its immobilized form are formed, and an application system for its high-efficiency synthesis of Anoectochilus roxburghii glycosides is established to achieve green, efficient, and sustainable synthesis of glycoside natural products.

[0008] The primary objective of this invention is to provide a β-glucosidase mutant.

[0009] Another object of the present invention is to provide a β-glucosidase immobilized enzyme.

[0010] Another object of the present invention is to provide the application of the above-mentioned β-glucosidase mutant or β-glucosidase immobilized enzyme.

[0011] To achieve the above objectives, the present invention provides the following technical solution:

[0012] The first aspect of the present invention provides a β-glucosidase mutant, wherein the β-glucosidase is a wild-type β-glucosidase DtBGL derived from *Dictyoglomus thermophilum*, the amino acid sequence of which is shown in SEQ ID No. 1; the β-glucosidase mutant is obtained by any one or more combinations of mutations of the amino acid sequence of SEQ ID No. 1 as follows:

[0013] W177Y: The 177th tryptophan (W177) is mutated to tyrosine (Y);

[0014] W179F: The 179th tryptophan (W179) is mutated to phenylalanine (F);

[0015] Q242A: Glutamine (Q242) at position 242 is mutated to alanine (A);

[0016] F245G: The phenylalanine at position 245 (F245) is mutated to glycine (G);

[0017] R298S: Arginine at position 298 (R298) is mutated to serine (S);

[0018] R313T: Arginine at position 313 (R313) is mutated to threonine (T).

[0019] Preferably, the β-glucosidase mutant has the amino acid sequence of SEQ ID No. 1 obtained by the following mutations: W177Y / W179F, or W177Y / W179F / R313T, or Q242A / F245G, or Q242A / F245G / R298S, or W177Y / W179F / R313T / Q242A / F245G, or W177Y / W179F / R313T / Q242A / F245G / R298S.

[0020] The second aspect of the present invention provides biological materials related to the above-mentioned β-glucosidase mutant, which are any one of the following biological materials:

[0021] (1) The DNA molecule encoding the β-glucosidase mutant;

[0022] (2) An expression cassette containing the DNA molecule described in (1);

[0023] (3) A recombinant vector containing the expression cassette described in (2);

[0024] (4) Recombinant host cells containing the recombinant vector described in (3).

[0025] Preferably, the recombinant vector used is pMAL-c6T.

[0026] Preferably, the recombinant host cell used is Escherichia coli BL21(DE3).

[0027] A third aspect of the present invention provides a method for preparing the above-mentioned β-glucosidase mutant, comprising the following steps: (1) culturing the recombinant host cells; (2) inducing expression of the β-glucosidase mutant; (3) cell disruption and recovery of soluble proteins; and (4) purifying the enzyme using Ni-NTA affinity chromatography.

[0028] A fourth aspect of the present invention provides an immobilized enzyme obtained by physically adsorbing the above-mentioned β-glucosidase mutant onto a metal-organic framework material. The immobilized enzyme retains ≥50% of its initial activity after at least three catalytic cycles.

[0029] Preferably, the metal-organic framework material is UiO-66-NH2.

[0030] The fifth aspect of the present invention provides a method for preparing the above-mentioned immobilized enzyme, which involves adding a metal-organic framework material to a solution of the β-glucosidase mutant, shaking the reaction mixture, centrifuging the reaction solution to discard the supernatant, washing the precipitate, and obtaining the immobilized enzyme.

[0031] Preferably, the concentration of the β-glucosidase mutant is 0.1–1.2 mg / g, the ratio of organic framework material to solution is 10–30 mg: 2 mL, and the shaking reaction time is 1–6 h.

[0032] A sixth aspect of the present invention provides the application of the above-mentioned β-glucosidase mutant or immobilized enzyme in the catalytic synthesis of romosiderin from 3(R)-hydroxyγ-butyrolactone. The yield is increased by at least 1.5 times, and even more than 2 times, compared to wild-type β-glucosidase.

[0033] Preferably, the application involves dissolving β-D-glucose (as a glycosyl donor) and 3(R)-hydroxyγ-butyrolactone (as a glycosyl acceptor substrate) in water or a water-organic solvent mixture, and then adding the β-glucosidase mutant or immobilized enzyme to generate Anoectochilin.

[0034] Preferably, the water-organic solvent mixture is a sodium phosphate buffer solution-isoamyl acetate system, wherein the concentration of the sodium phosphate buffer solution is 30-70 mM, the volume fraction of the organic solvent is 70%-95%, and the molar ratio of β-D-glucose to 3(R)-hydroxyγ-butyrolactone is 1:2-1:12.

[0035] Preferably, the concentration of the β-glucosidase mutant or immobilized enzyme is 25–150 μg / mL.

[0036] Preferably, the conditions for the reverse hydrolysis reaction are: temperature 50±10℃, time 12~72h.

[0037] A seventh aspect of the present invention provides a biocatalytic kit for the reverse hydrolysis synthesis of anolysin, comprising the above-mentioned β-glucosidase mutant or immobilized enzyme, as well as the glycosyl donor β-D-glucose, the glycosyl acceptor substrate 3(R)-hydroxyγ-butyrolactone, and a reaction buffer.

[0038] Compared with the prior art, the present invention has the following advantages and positive effects:

[0039] 1. By improving the catalytic activity of β-glucosidase in the synthesis of Anoectochilus roxburghii glycosides through molecular modification, the problem of poor synthetic activity of natural enzymes was solved;

[0040] 2. The immobilization method is mild and simple. UiO-66-NH2 material has excellent biocompatibility and enzyme carrying capacity. The immobilized enzyme has high stability and strong reusability.

[0041] 3. The established enzyme-catalyzed synthesis system is characterized by being green and environmentally friendly, easy to operate, and having mild reaction conditions, making it suitable for the efficient synthesis and industrial scale-up of Anoectochilus roxburghii glycosides.

[0042] 4. This invention provides a novel enzyme engineering and immobilization combination strategy with universal applicability in the field of glycoside natural product synthesis, and also has reference and promotion value for the synthesis of other functional glycosides. Attached Figure Description

[0043] Figure 1 The plasmid map of the recombinant expression plasmid pMAL-c6T-DtBGL.

[0044] Figure 2 The image shows the SDS-PAGE analysis results for W177Y. Lane descriptions are as follows: M - Protein molecular weight standard (Marker); B - Total protein before induction; T - Total protein after induction; S - Supernatant after induction; P - Precipitate after induction; F - Ni-NTA affinity chromatography breakthrough buffer; W - Elution fraction from 15% B elution buffer; E - Elution fraction from 60% B elution buffer.

[0045] Figure 3 The graph shows the yield determination results of β-glucosidase mutant catalyzing the production of roxithrin.

[0046] Figure 4 The figure shows the results of the determination of the effect of reaction time on the yield of anolysin synthesized by immobilized enzyme M6@UiO-66-NH2.

[0047] Figure 5 The figure shows the reusability of M6@UiO-66-NH2 in the catalytic synthesis of Anoectochilus roxburghii glycosides. Figure 6 The graph shows the results of the ability of each mutant to catalyze the synthesis of Anoectochilin. Detailed Implementation

[0048] The following embodiments of the present invention are intended to provide a detailed description of the construction, expression, purification, immobilization, and application of the β-glucosidase mutant in the catalytic synthesis of Anoectochilus roxburghii glycoside, so that those skilled in the art can understand and implement the present invention, but are not intended to limit the scope of protection of the present invention.

[0049] The main reagents used in the embodiments of this invention are all analytical grade or biochemical reagent grade, purchased from Shanghai Yuanye Biotechnology Co., Ltd., Sinopharm Chemical Reagent Co., Ltd., Aladdin Reagent (Shanghai) Co., Ltd., etc., and mainly include: LB medium powder, isopropyl-β-D-thiogalactoside (IPTG), ampicillin (Amp), β-D-glucose, 3(R)-hydroxyγ-butyrolactone (3HBL), isoamyl acetate, p-nitrobenzene-β-D-glucopyranoside (pNPG), zirconium tetrachloride (ZrCl4), 2-aminoterephthalic acid, N,N-dimethylformamide (DMF), anhydrous ethanol, imidazole, Tris, NaCl, etc.

[0050] The culture media involved in the embodiments of the present invention are as follows:

[0051] LB liquid medium: Weigh 25.0g of LB medium powder, add distilled water to completely dissolve and bring the volume to 1L, autoclave at 121℃ for 20min, and store at 4℃ for later use.

[0052] LB liquid medium containing Amp: After the sterilized LB liquid medium has cooled to about 50°C, add Amp to a final concentration of 100 μg / mL in a sterile operating table. Prepare and use immediately.

[0053] LB solid medium containing Amp: Weigh 2.5g LB medium powder and 1.5g agar powder, add distilled water to a final volume of 100mL, and autoclave at 121℃ for 20min. When the medium cools to about 50℃, add Amp to a final concentration of 100μg / mL in a sterile operating table, gently shake to mix, pour the medium to a thickness of about 3mm on each plate, seal with sealing film after solidification, and store at 4℃ for later use.

[0054] The buffer solutions involved in the embodiments of the present invention are as follows:

[0055] Ni-NTA affinity chromatography equilibration buffer: Weigh 6.1g Tris and 17.5g NaCl and dissolve them completely in 900mL of ultrapure water. Adjust the pH of the solution to 8.0 with concentrated hydrochloric acid, then bring the volume to 1L with ultrapure water. Filter the solution through a 0.45μm aqueous filter membrane and store at 4℃ for later use.

[0056] Ni-NTA affinity chromatography elution buffer: Weigh 6.1g Tris, 34.0g imidazole and 17.5g NaCl and dissolve them completely in 900mL ultrapure water. Adjust the pH of the solution to 8.0 with concentrated hydrochloric acid and then bring the volume to 1L with ultrapure water. Filter through a 0.45μm aqueous filter membrane and store at 4℃ for later use.

[0057] Protein dialysis buffer: Weigh 3.0g NaH2PO4·2H2O, 10.9g Na2HPO4·12H2O and 2.9g NaCl and dissolve them completely in 700mL ultrapure water. Add 200mL glycerol and stir well. Adjust the pH of the solution to 7.0 and then bring the volume up to 1L with ultrapure water. Store at 4℃ for later use.

[0058] Example 1: Construction of β-glucosidase mutant

[0059] This embodiment employs a semi-rational design strategy to molecularly modify the wild-type β-glucosidase DtBGL derived from *Tetracentron sinense*. Through molecular docking, substrate binding pocket analysis, and sequence conservation comparison, key amino acid residues that may affect substrate affinity and steric hindrance were screened as mutation sites. A total of six single-point mutants were designed: W177Y, W179F, Q242A, F245G, R298S, and R313T. Based on this, six combinatorial mutants were further constructed, namely: M1(W177Y / W179F), M2(W177Y / W179F / R313T), M3(Q242A / F245G), M4(Q242A / F245G / R298S), M5(W177Y / W179F / R313T / Q242A / F245G) and M6(W177Y / W179F / R313T / Q242A / F245G / R298S). The primer sequences used are shown in Table 1.

[0060] The amino acid sequence of DtBGL is shown in SEQ ID NO:1. The coding sequence obtained by reverse engineering from this amino acid sequence is shown in SEQ ID NO:2. It was synthesized by Guangzhou Aiji Biotechnology Co., Ltd. based on the codon preference of the *E. coli* expression system and cloned into the expression vector pMAL-c6T using the BamHI and EcoRI restriction sites. The resulting recombinant plasmid was named pMAL-c6T-DtBGL, and its plasmid map is shown below. Figure 1 As shown, it serves as a template for subsequent mutational responses.

[0061] The mutant was constructed using whole-plasmid PCR. Using pMAL-c6T-DtBGL plasmid as a template, the total reaction volume was 20 μL, containing the following components: 0.2 μL template plasmid (1 ng / μL), 0.4 μL each of forward and reverse primers (10 μM), 10 μL PfuMax HiFi PCR ProMix (Guangzhou Yingzan Biotechnology Co., Ltd.), and ddH2O to a final volume of 20 μL.

[0062] The PCR amplification program was as follows: pre-denaturation at 98℃ for 2 min; followed by 30 cycles, each cycle consisting of denaturation at 98℃ for 10 s, annealing at (Tm-3)℃ for 30 s, and extension at 68℃ for 3 min; finally, extension at 68℃ for 5 min. After amplification, the amplified products were detected by agarose gel electrophoresis. PCR products with correct bands were digested with DpnI rapid digestion enzyme (Takara Corporation, Japan) to remove methylated template plasmids. The DpnI digestion system and conditions were set according to the product instructions.

[0063] The digestion products were transformed into *E. coli* DH5α competent cells and plated on LB agar plates containing ampicillin. Single colonies were picked and sent to Guangzhou Aiji Biotechnology Co., Ltd. for sequencing verification. Verified plasmids were extracted using the Tiangen Rapid Plasmid Mini-Prep Kit and stored at -20℃.

[0064] Combinatorial mutants were constructed using plasmids of each single-point mutant as templates, following the PCR amplification and cloning methods described above, to obtain target mutants M1 to M6.

[0065] Table 1 Primer sequences for constructing mutants

[0066]

[0067]

[0068]

[0069] Note: Lowercase letters indicate mutation sites.

[0070] Example 2: Expression and purification of mutant enzymes

[0071] The mutant plasmids constructed in Example 1 were transformed into the *E. coli* expression host BL21(DE3). Single colonies of the transformed bacteria were picked and inoculated into LB liquid medium containing 100 μg / mL Amp, and cultured overnight at 37°C with shaking at 180 rpm to prepare a seed culture. The seed culture was then inoculated into fresh LB medium at a ratio of 1% (v / v) and cultured until the optical density (OD) of the bacterial culture at 600 nm was measured. 600 The expression level reached 0.6–0.8. At this point, IPTG was added at a final concentration of 0.2 mM to induce the expression of the target protein. The induction time was 8 h, and the temperature was maintained at 37 °C.

[0072] After induction, bacterial cells were collected by centrifugation at 8000g, 4℃ for 15 min. Cells were resuspended in 15 mL of Ni-NTA affinity chromatography equilibration buffer per gram of wet bacterial cell weight and lysed using ultrasound under ice bath conditions (parameters: power 200W, 3s interval, 3s incubation, total duration 20 min). The lysate was centrifuged at 12000g, 4℃ for 20 min, and the supernatant was collected and filtered through a 0.22 μm microporous membrane for later use.

[0073] The obtained filtrate was purified into the target protein using a Ni-NTA affinity chromatography column (Changzhou Tiandi Renhe Biotechnology Co., Ltd.). The specific procedure is as follows: the chromatography column was pretreated with 5 column volumes of equilibration buffer; after loading the sample, gradient elution was performed sequentially with 15%, 60%, and 100% Ni-NTA elution buffer to remove contaminating proteins, elute the target protein, and remove washing residues, respectively. The purification effect was analyzed by SDS-PAGE of each eluted fraction. Figure 2 As shown, taking the W177Y mutant enzyme as an example, a clear single band is visible at approximately 97 kDa, indicating that high-purity recombinant protein can be obtained after a single Ni-NTA chromatography step. The purification results for other mutant enzymes are similar.

[0074] After collecting 60% of the elution fraction, dialyze overnight in dialysis buffer at 4°C to remove low molecular weight impurities.

[0075] Example 3: Synthesis of Anoectochilin Catalyzed by Mutant Enzyme

[0076] Using the mutant enzyme obtained in Example 2, a reverse hydrolysis reaction was carried out under enzymatic catalysis with 3HBL and β-D-glucose as substrates to synthesize roximate. The reaction was carried out in a brown sealed flask with a total volume of 0.5 mL, specifically consisting of: 90% (v / v) isoamyl acetate, 50 mM sodium phosphate buffer (pH 6.0), 0.75 M 3HBL, 0.125 M β-D-glucose, and 125 μg / mL mutant enzyme. The reaction was carried out at 50 °C and 180 rpm with shaking for 48 h. After the reaction was completed, the reaction was terminated by heating at 90 °C for 10 min, followed by centrifugation at 8000 g and 25 °C for 5 min to collect the supernatant. The supernatant was vacuum dried at 50 °C to remove organic solvents, and the residues were dissolved in ultrapure water for the determination of roximate content.

[0077] The content of roximate in *Anoectochilus roxburghii* was analyzed by high-performance liquid chromatography-evaporative light detection (HPLC-ELSD). An Agilent 1260 system was used, with a Dikma Diamonsil Plus C18 column (250 × 4.6 mm, 5 μm), a column temperature of 25 °C, a mobile phase of methanol:water = 2:98 (v / v), and a flow rate of 0.6 mL / min. The sample was filtered through a 0.22 μm filter, and the injection volume was 10 μL. ELSD detection conditions were: nitrogen carrier gas, flow rate 1.7 L / min, and nebulizer and drift tube temperatures of 60 °C. A standard curve was established by comparing standard concentration with the logarithm of peak area, and the concentration of roximate in the sample was calculated.

[0078] The yield of glycosides from *Anoectochilus roxburghii* was calculated according to Formula 1:

[0079] Formula 1: Yield of Anoectochilus roxburghii glycosides (%) = Actual yield of Anoectochilus roxburghii glycosides / Theoretical yield of Anoectochilus roxburghii glycosides × 100, where the theoretical yield of Anoectochilus roxburghii glycosides refers to the amount of Anoectochilus roxburghii glycosides produced when β-D-glucose is completely converted in the reverse hydrolysis reaction.

[0080] The results are as follows Figure 3 As shown in the figure, compared with the wild-type enzyme, mutants W177Y, W179F, Q242A, F245G, R298S, and R313T significantly increased the yield of anolyte glycosides, in the following order: W179F (32.23%) > R313T (31.92%) > W177Y (31.39%) > R298S (26.06%) > Q242A (23.17%) > F245G (22.20%). Furthermore, all combined mutants (M1 to M6) exhibited a positive additive effect, with yields 1.94-fold, 2.05-fold, 1.68-fold, 1.89-fold, 2.12-fold, and 2.35-fold higher than the wild-type, respectively, indicating that multi-site combined mutations significantly enhance catalytic performance.

[0081] Example 4: Immobilization of β-glucosidase mutant M6

[0082] The β-glucosidase mutant M6 was immobilized using the metal-organic framework material UiO-66-NH2 as an immobilization carrier.

[0083] Preparation method of UiO-66-NH2: Weigh 0.81 g of 2-aminoterephthalic acid and dissolve it in 40 mL of DMF (2%, v / v). Sonicate the solution for 20 min, then add ZrCl4 (1.05 g) and hydrochloric acid (17 mL) sequentially. After mixing thoroughly, transfer the mixture to a Teflon-lined stainless steel autoclave and heat at 120 °C for 24 h. After the reaction is complete, cool to room temperature. Centrifuge the product at 6000 g for 30 min to collect the precipitate, and wash it three times with DMF. Dry the precipitate under vacuum at 80 °C for 12 h to obtain UiO-66-NH2.

[0084] The enzyme immobilization procedure was as follows: Based on the purified β-glucosidase mutant M6 obtained in Examples 1 and 2, 20 mg of UiO-66-NH2 was added to 2 mL of 0.7 mg / mL M6 purified enzyme solution, and the mixture was shaken and adsorbed at 25 °C for 3 h. After adsorption, the mixture was centrifuged at 6000 g for 15 min, the supernatant was discarded, and the precipitate was washed three times with 50 mM, pH 6.0 sodium phosphate buffer to finally obtain the immobilized enzyme M6@UiO-66-NH2.

[0085] Example 5: Synthesis of Anoectochilus roxburghii glycosides catalyzed by immobilized enzyme M6@UiO-66-NH2

[0086] Using the immobilized enzyme M6@UiO-66-NH2 prepared in Example 4, and with 3HBL and β-D-glucose as substrates, a reverse hydrolysis reaction was carried out under enzyme catalysis to synthesize roximate, and its yield was determined. The reaction system, reaction conditions, and methods for detecting and calculating roximate were all the same as in Example 3.

[0087] To investigate the effect of reaction time on the efficiency of *Anoectochilus roxburghii* glycoside formation, samples were taken and analyzed at 12, 24, 36, 48, 60, and 72 hours after the reaction. Parallel experiments were conducted using the following control groups: free wild-type β-glucosidase DtBGL (prepared using the method described in Example 2), immobilized wild-type β-glucosidase DtBGL@UiO-66-NH2 (prepared using the method described in Example 4), and free β-glucosidase mutant M6, to evaluate the effects of immobilization and mutation on catalytic performance.

[0088] Experimental results are as follows Figure 4 As shown, within the range of 12-72 h, the yields of anoectochilin in all reaction systems exhibited similar trends: the yield continuously increased with increasing reaction time up to 48 h; however, after 48 h, the yield essentially stabilized, indicating that the reaction was close to thermodynamic equilibrium. Therefore, the optimal reaction time for the reverse hydrolysis reaction to synthesize anoectochilin is 48 h.

[0089] Under these conditions, the yields of roximate synthesis in each reaction system were as follows: 17.90% for free DtBGL, 42.07% for free M6, 34.12% for DtBGL@UiO-66-NH2, and 54.51% for M6@UiO-66-NH2. Compared with the free enzyme, the immobilized enzymes all exhibited higher catalytic efficiency. Among them, the yield increase of M6@UiO-66-NH2 was the most significant, increasing by 29.47% compared to its free form and by 20.39% compared to the immobilized wild-type enzyme. This indicates that enzyme mutation and immobilization have a synergistic enhancing effect in improving the roximate synthesis yield.

[0090] Example 6: Reusability of immobilized enzyme M6@UiO-66-NH2 in the catalytic synthesis of roximate glycosides

[0091] Following the method described in Example 5, immobilized enzyme M6@UiO-66-NH2 was used to catalyze the synthesis of roximate glycosides. After each reaction, the immobilized enzyme M6@UiO-66-NH2 was recovered by centrifugation, and the supernatant was collected and the yield of roximate glycosides was determined by HPLC-ELSD. The recovered immobilized enzyme was washed three times with 50 mM, pH 6.0 PBS buffer, and then added back to a fresh reaction system for the next reaction. The above process was repeated continuously to examine the reusability of the immobilized enzyme in multiple catalytic reactions. The experimental results are as follows: Figure 5 As shown, the yield of roximate gradually decreased with increasing reaction cycles. After three repeated uses, the relative yield of roximate remained above 50%, indicating that the immobilized enzyme M6@UiO-66-NH has certain recycling potential and exhibits good stability and reproducibility in practical applications.

[0092] Comparative Example 1

[0093] Following the method in Example 1, 20 single-point mutants were designed and constructed around 16 key amino acid sites in the DtBGL substrate binding pocket. These mutants were W177F, W179V, N222F, V223G, V223N, Q242R, F245L, G246N, N247F, N247Y, Y295H, T297S, R298G, N299F, N299Y, R313F, R313Y, W325F, E404Q, and F413Y. The primer sequences used are shown in Table 2. Each mutant was heterologously expressed and purified according to the method in Example 2. Subsequently, their ability to catalyze the synthesis of roximate was determined according to Example 3. The results are as follows: Figure 6As shown, mutants N222F, F245L, G246N, T297S, N299F, R313F, and E404Q almost completely lost the ability to synthesize roxithrin, with roxithrin yields below 1.5%. Mutants W179V, V223G, N247F, N247Y, R298G, and N299Y showed significantly lower roxithrin yields compared to the wild type, at 8.08%, 11.75%, 14.21%, 4.34%, 3.46%, and 4.81%, respectively. Mutants W177F, V223N, Q242R, Y295H, W325F, and F413Y showed no significant difference compared to the wild type. These results validate the rationality of the present invention and the effectiveness of the mutation strategy.

[0094] Table 2 Primer sequences for constructing mutants

[0095]

[0096]

[0097] Note: Lowercase letters indicate mutation sites.

[0098] It should be noted that the above-described embodiments are merely preferred embodiments of the present invention, intended to more clearly illustrate the technical solution of the present invention and facilitate understanding and implementation by those skilled in the art, and do not constitute a limitation on the scope of protection of the present invention. Any equivalent transformations, substitutions, modifications, or improvements made based on the core ideas and technical concepts of the present invention and in accordance with the prior art should be considered to fall within the scope of protection of the present invention.

Claims

1. A β-glucosidase mutant, characterized in that: Its amino acid sequence is as shown in SEQ ID No. 1, obtained by any one or more of the following combinations of mutations: W177Y: Tryptophan at position 177 is mutated to tyrosine; W179F: Tryptophan at position 179 is mutated to phenylalanine; Q242A: Glutamine at position 242 is mutated to alanine; F245G: The phenylalanine at position 245 is mutated to glycine; R298S: Arginine at position 298 is mutated to serine; R313T: Arginine at position 313 is mutated to threonine.

2. The β-glucosidase mutant according to claim 1, characterized in that: Its amino acid sequence is obtained by the following mutations of SEQ ID No. 1: W177Y / W179F, or W177Y / W179F / R313T, or Q242A / F245G, or Q242A / F245G / R298S, or W177Y / W179F / R313T / Q242A / F245G, or W177Y / W179F / R313T / Q242A / F245G / R298S.

3. The biomaterials related to the β-glucosidase mutant as described in claim 1 or 2, characterized in that: It can be any one of the following biological materials: (1) The DNA molecule encoding the β-glucosidase mutant; (2) An expression cassette containing the DNA molecule described in (1); (3) A recombinant vector containing the expression cassette described in (2); (4) Recombinant host cells containing the recombinant vector described in (3).

4. The method for preparing the β-glucosidase mutant according to claim 1 or 2, characterized in that: Includes the following steps: (1) Culturing the recombinant host cells as described in claim 3; (2) Inducing expression of the β-glucosidase mutant; (3) Cell disruption and recovery of soluble proteins; (4) The enzyme was purified by Ni-NTA affinity chromatography.

5. An immobilized enzyme, characterized in that: It is obtained by immobilizing the β-glucosidase mutant described in claim 1 or 2 on a metal-organic framework material by physical adsorption; the metal-organic framework material is UiO-66-NH2.

6. The method for preparing the immobilized enzyme as described in claim 5, characterized in that: The metal-organic framework material was added to the solution of the β-glucosidase mutant, the reaction was shaken, the reaction solution was centrifuged and the supernatant was discarded, the precipitate was washed, and the immobilized enzyme was obtained. The concentration of the β-glucosidase mutant was 0.1–1.2 mg / g, the ratio of organic framework material to solution was 10–30 mg: 2 mL, and the shaking reaction time was 1–6 h.

7. The use of the β-glucosidase mutant of claim 1 or 2 or the immobilized enzyme of claim 5 in the catalytic synthesis of romosiderin from 3(R)-hydroxyγ-butyrolactone.

8. The application of the method described in claim 7 in the synthesis of romosiderin from 3(R)-hydroxyγ-butyrolactone, characterized in that: The application is as follows: β-D-glucose and 3(R)-hydroxyγ-butyrolactone are dissolved in water or a water-organic solvent mixture, and the β-glucosidase mutant or immobilized enzyme is added to generate Anoectochilin.

9. The application of the method described in claim 8 in the synthesis of romosiderin from 3(R)-hydroxyγ-butyrolactone, characterized in that: The water-organic solvent mixture is a sodium phosphate buffer solution-isoamyl acetate system, with a sodium phosphate buffer solution concentration of 30-70 mM, an organic solvent volume fraction of 70%-95%, and a molar ratio of β-D-glucose to 3(R)-hydroxyγ-butyrolactone of 1:2-1:

12. The concentration of the β-glucosidase mutant or immobilized enzyme is 25–150 μg / mL; The conditions for the reverse hydrolysis reaction are: temperature 50±10℃, time 12~72h.

10. A biocatalytic kit for the reverse hydrolysis synthesis of Anoectochilus roxburghii glycosides, characterized in that: It comprises the β-glucosidase mutant of claim 1 or 2 or the immobilized enzyme of claim 5, as well as the glycosyl donor β-D-glucose, the glycosyl acceptor substrate 3(R)-hydroxyγ-butyrolactone, and a reaction buffer.

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