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CN-121991932-A - Beta-glucosidase mutant, immobilized enzyme thereof and application of immobilized enzyme in efficient synthesis of anoectochilus formosanus glycoside

CN121991932ACN 121991932 ACN121991932 ACN 121991932ACN-121991932-A

Abstract

The invention discloses a beta-glucosidase mutant, immobilized enzyme thereof and application thereof in high-efficiency synthesis of anoectochilus formosanus glycoside, belonging to the fields of enzyme engineering and natural product biosynthesis. The mutant introduces one or more mutations in W177Y, W179F, Q242A, F245G, R298S, R313T at key sites of the wild type beta-glucosidase amino acid sequence, so that the yield of the catalytic synthesis of anoectochilus formosanus is remarkably improved. Furthermore, the immobilized enzyme is immobilized by adopting a metal organic framework material UiO-66-NH 2 as an immobilization carrier and adopting a physical adsorption mode, and the obtained immobilized enzyme has excellent reusability. Under the same reaction conditions, the immobilized mutant enzyme is used for catalyzing the synthesis of anoectoin, and the yield of the immobilized mutant enzyme is 3 times that of the wild type enzyme. The invention provides a high-efficiency, green and sustainable anoectochilus formosanus glycoside biocatalysis synthesis scheme, and has good industrial application prospect.

Inventors

  • HU SONGQING
  • YUAN XIN
  • Hou Die

Assignees

  • 华南理工大学

Dates

Publication Date
20260508
Application Date
20250928

Claims (10)

  1. 1. The beta-glucosidase mutant is characterized in that the amino acid sequence of the beta-glucosidase mutant is shown as SEQ ID No.1, and the beta-glucosidase mutant is obtained by mutation through any one or more of the following combinations: W177Y, tryptophan at position 177 is mutated to tyrosine; W179F, tryptophan at position 179 to phenylalanine; mutation of glutamine 242 to alanine; F245G, mutating 245 th phenylalanine into glycine; 298S, the arginine at position 298 is mutated to serine; arginine at position 313 of R313T is mutated to threonine.
  2. 2. The beta-glucosidase mutant according to claim 1, wherein the amino acid sequence is SEQ ID No.1, and the mutant is obtained by the following mutation of W177Y/W179F, or W177Y/W179F/R313T, or Q242A/F245G/R298S, or W177Y/W179F/R313T/Q242A/F245G/R298S.
  3. 3. The biological material related to the beta-glucosidase mutant as claimed in claim 1 or 2, which is any one of the following biological materials: (1) A DNA molecule encoding the β -glucosidase mutant; (2) An expression cassette comprising the DNA molecule of (1); (3) A recombinant vector comprising the expression cassette of (2); (4) A recombinant host cell comprising the recombinant vector of (3).
  4. 4. The method for producing a beta-glucosidase mutant as claimed in claim 1 or 2, comprising the steps of: (1) Culturing the recombinant host cell of claim 3; (2) Inducing expression of the beta-glucosidase mutant; (3) Cell disruption and recovery of soluble proteins; (4) The enzyme was purified using Ni-NTA affinity chromatography.
  5. 5. An immobilized enzyme is characterized in that the beta-glucosidase mutant in the claim 1 or 2 is immobilized on a metal organic framework material in a physical adsorption mode, wherein the metal organic framework material is UiO-66-NH 2 .
  6. 6. The method for preparing immobilized enzyme according to claim 5, wherein a metal organic framework material is added into the solution of the beta-glucosidase mutant, the reaction is oscillated, the supernatant is removed by centrifugation of the reaction solution, and the immobilized enzyme is obtained by precipitation and washing; The concentration of the beta-glucosidase mutant is 0.1-1.2 mg/g, the ratio of the organic framework material to the solution is 10-30 mg/2 mL, and the oscillation reaction time is 1-6 h.
  7. 7. Use of the β -glucosidase mutant of claim 1 or 2 or the immobilized enzyme of claim 5 for catalyzing synthesis of anoectoin from 3 (R) -hydroxyγ -butyrolactone.
  8. 8. The method for synthesizing anoectoin by catalyzing 3 (R) -hydroxy gamma-butyrolactone according to claim 7, wherein the application is to dissolve beta-D-glucose and 3 (R) -hydroxy gamma-butyrolactone in water or a water-organic solvent mixed system, and add the beta-glucosidase mutant or immobilized enzyme to generate anoectoin.
  9. 9. The use of catalytic 3 (R) -hydroxygamma-butyrolactone in the synthesis of anoectochilus formosanus according to claim 8, characterized in that: The water-organic solvent mixed system is a sodium phosphate buffer solution-isoamyl acetate system, 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 beta-D-glucose to 3 (R) -hydroxygamma-butyrolactone is 1:2-1:12; The concentration of the beta-glucosidase mutant or immobilized enzyme is 25-150 mug/mL; the conditions of the reverse hydrolysis reaction are that the temperature is 50+/-10 ℃ and the time is 12-72 h.
  10. 10. A biocatalytic kit for synthesizing anoectochilus formosanus glycoside by inverse hydrolysis, which is characterized by comprising the beta-glucosidase mutant as claimed in claim 1 or 2 or the immobilized enzyme as claimed in claim 5, and glycosyl donor beta-D-glucose, glycosyl acceptor substrate 3 (R) -hydroxygamma-butyrolactone and reaction buffer solution.

Description

Beta-glucosidase mutant, immobilized enzyme thereof and application of immobilized enzyme in efficient synthesis of anoectochilus formosanus glycoside Technical Field The invention relates to the fields of enzyme engineering and natural product biosynthesis, in particular to a beta-glucosidase mutant, immobilized enzyme thereof and application thereof in high-efficiency synthesis of anoectochilus formosanus glycoside. Background Anoectochilus roxburghii (Anoectochilus roxburghii) is an orchid with important medicinal value, and is rich in various natural products including flavonoids, polysaccharides, steroids and glycoside compounds. Wherein, anoectochilus formosanus glycoside (CAS accession number: 151870-74-5) is taken as a representative glycoside compound, has the biological activities of antioxidation, immunoregulation, anti-tumor, blood sugar reduction and the like, and is widely applied to the fields of health-care food, chinese medicinal preparation and natural medicament research and development. At present, the main source of anoectochilus formosanus glycoside still depends on direct extraction of plants, so that the problems of resource shortage, low extraction efficiency, heavy environmental burden and the like exist, and chemical synthesis faces the challenges of poor region selectivity, complicated steps and unsustainable. Therefore, developing a highly efficient, green and sustainable anoectoin biocatalysis strategy has become a key direction of current research in this field. Beta-glucosidase (EC 3.2.1.21) is a class of enzymes capable of catalyzing the hydrolysis or synthesis of beta-glycosidic bonds, which have important biological functions in carbohydrate metabolism, biomass degradation and glycoside derivative synthesis. Notably, beta-glucosidase can reverse its hydrolysis reaction direction under the conditions of specific water activity and high concentration of donor substrate, catalyze the formation of glycosidic bond, and show "reverse hydrolysis" or "transglycosylation" activity. However, the enzyme of natural origin still faces various limitations in catalyzing and synthesizing glycoside, including problems of weak substrate recognition capability, low catalytic efficiency, poor thermal stability, unrepeatable use, etc., and is difficult to meet the requirements of industrial-scale synthesis. To solve the above problems, protein engineering is an effective means for improving enzyme performance. Through rationally designing an active center structure of the targeted regulatory enzyme, the catalytic efficiency and substrate adaptability of the glycoside synthesis direction are improved. Meanwhile, the enzyme immobilization technology can obviously enhance the thermal stability, the organic solvent tolerance and the recycling capability of the enzyme, and particularly, an immobilization mode taking metal organic framework Materials (MOFs) as carriers becomes a research hot spot of immobilization carriers in recent years due to the high specific surface area and the adjustable pore diameter and good biocompatibility. Therefore, the beta-glucosidase mutant with the multi-site fixed-point mutation and the high-catalytic activity are developed, and the stable and high-efficient biocatalysis system is built through MOFs immobilization, so that the beta-glucosidase mutant is used for catalyzing and synthesizing anoectochilus formosanus, has obvious technical innovation, has clear industrialization prospect, and is in line with the development trend of high-value utilization of the current green biological manufacturing and natural products. Disclosure of Invention The invention aims to solve the problems of low catalytic efficiency, poor substrate selectivity, poor recycling property and the like of the existing beta-glucosidase in anoectochilus formosanus glycoside biosynthesis, forms a beta-glucosidase mutant with high catalytic activity and an immobilized form thereof through molecular modification, and establishes an application system of the beta-glucosidase mutant in anoectochilus formosanus glycoside efficient synthesis so as to realize green, efficient and sustainable synthesis of glycoside natural products. The primary object of the present invention is to provide a mutant of beta-glucosidase. It is another object of the present invention to provide a beta-glucosidase immobilized enzyme. It is a further object of the present invention to provide the use of the above-mentioned β -glucosidase mutant or β -glucosidase immobilized enzyme. In order to achieve the above object, the present invention provides the following technical solutions: The first aspect of the invention provides a beta-glucosidase mutant, which is wild-type beta-glucosidase DtBGL from Thermococcus (Dictyoglomus thermophilum) with an amino acid sequence shown as SEQ ID No.1, wherein the amino acid sequence of the beta-glucosidase mutant is obtained by mutation of any one or more of the following combinations sho