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CN-122012468-A - Beta-glucosidase mutant and application thereof in catalyzing flavonoid glycoside hydrolysis

CN122012468ACN 122012468 ACN122012468 ACN 122012468ACN-122012468-A

Abstract

The invention relates to the technical field of enzyme catalysis, and discloses a beta-glucosidase mutant and application thereof in catalyzing flavonoid glycoside hydrolysis. In order to solve the problem of low catalytic activity of beta-glucosidase at present, mutants are obtained by single-point or multi-point combined substitution of amino acids at 49 th, 282 th, 306 th and/or 445 th of beta-glucosidase from pyrococcus (Pyrococcus furiosus), wherein the mutants comprise single mutant V46A, D49E, I282V, Y306H, L445K, double-point combined mutant V46A-L445K, triple-point combined mutant V46A‑Y306H‑L445K, V46A‑D49E‑L445K, D49E‑Y306H‑L445K, D49E‑I282V‑Y306H, I282V‑Y306H‑L445K and four-point mutant D49E‑I282V‑Y306H‑L445K, V46A‑D49E‑I282V‑Y306H, V46A‑D49E‑Y306H‑L445K. Experiments prove that the enzyme activity of the mutant has a larger improvement on the catalytic activity of the substrate naringin compared with a wild type, and has better tolerance on high-concentration substrates.

Inventors

  • CHEN HANCHI
  • Gao Shenze
  • LU YUELE
  • ZHU LINJIANG
  • CHEN XIAOLONG

Assignees

  • 浙江工业大学

Dates

Publication Date
20260512
Application Date
20260209

Claims (10)

  1. 1. A beta-glucosidase mutant is characterized in that the beta-glucosidase mutant is obtained by single-point mutation or multi-point combination mutation of the following sites from an amino acid sequence shown as SEQ ID NO. 1: (a) Valine at position 46 to alanine; (b) Aspartic acid at position 49 to glutamic acid; (c) Isoleucine 282 to valine; (d) Tyrosine at position 306 is mutated to histidine; (e) Leucine 445 to lysine.
  2. 2. A mutant beta-glucosidase according to claim 1, characterized in that it is obtained by the following mutation of the amino acid sequence shown in SEQ ID NO. 1: aspartic acid at position 49 is mutated to glutamic acid, while isoleucine at position 282 is mutated to valine, tyrosine at position 306 is mutated to histidine, and leucine at position 445 is mutated to lysine.
  3. 3. The gene encoding a β -glucosidase mutant according to any one of claims 1 to 2.
  4. 4. A recombinant vector comprising the coding gene according to claim 3.
  5. 5. The recombinant vector according to claim 4, wherein the vector is pET-28a.
  6. 6. A genetically engineered bacterium comprising the coding gene of claim 3 or the recombinant vector of claim 4.
  7. 7. The genetically engineered bacterium of claim 6, wherein the genetically engineered bacterium is E.coli.
  8. 8. Use of a beta-glucosidase mutant according to claim 1 for catalyzing a flavonoid glycoside hydrolysis reaction.
  9. 9. The method according to claim 8, wherein the flavonoid glycoside is naringin.
  10. 10. The method according to claim 8, wherein the reaction comprises the step of catalyzing the reaction at a temperature of no more than 75 ℃ with naringin as a substrate and a beta-glucosidase mutant as a catalyst.

Description

Beta-glucosidase mutant and application thereof in catalyzing flavonoid glycoside hydrolysis Technical Field The invention relates to the technical field of enzyme catalysis, in particular to a beta-glucosidase mutant and application thereof in catalyzing and hydrolyzing flavone glycoside. Background The citrus flavone has the effects of resisting oxidation, strengthening capillary vessel strength, improving vascular permeability, regulating cholesterol and the like, and is a natural active substance widely existing in citrus fruits. Natural citrus flavones generally exist in a glycoside form, and are required to be degraded into aglycone under the action of intestinal microorganisms so as to be absorbed and utilized by organisms. However, the hydrolysis of intestinal microorganisms is weak, so that the natural citrus flavones are difficult to effectively utilize by organisms, and the application range and the efficacy of the natural citrus flavones are limited. At present, the method for converting the citrus flavonoid glycoside into aglycone mainly comprises an acid hydrolysis method and an enzymolysis method. The hydrolysis method has the advantages of utilizing the specific catalytic action of enzyme, being mild in reaction condition, being capable of better protecting the acid-sensitive aglycone structure and catalyzing the hydrolysis of glycosyl, having unique advantages and being a green and environment-friendly process. Beta-glucosidase (beta-D-Glucosidase, bgl, EC 3.2.1.21) is a class of glycoside hydrolases which liberate beta-D-glucose and the corresponding ligands by hydrolyzing the non-reducing beta-D-glucosidic bonds at the end of the substrate. Beta-glucosidase is widely accepted as an important biocatalyst for use in the production of flavonoid glycosylated substances. For example, shin et al hydrolyse hesperidin from orange peel extract with beta-glucosidase from Pyrococcus furiosus, converting 18.2 g/L hesperidin to 9.0 g/L hesperetin completely within 9 hours, with a productivity of 1.00 g L-1 h-1. After 24h of Lu et al using an enzymatic cascade between alpha-rhamnosidase and beta-glucosidase, the conversion of 60 mM naringin was 70%. However, when enzymatic hydrolysis of flavone glycosides is used, although this approach has shown potential in production, some challenges remain. At present, enzymes mainly used for naringin hydrolysis comprise naringinase, alpha-rhamnosidase and beta-glucosidase, but the naringinase has the defects of inconsistent activity, insufficient stability and the like, so that the process of naringin hydrolysis into naringenin is hindered. Whereas α -rhamnosidase and β -glucosidase are commonly cascade-connected for hydrolysis of naringin, which results in increased cost of the enzymes and difficulty in unified coordination of reaction conditions of optimal temperature, pH, substrate concentration, etc. of the two enzymes. The current naringin hydrolysis has the problems that naringin yield and conversion rate are still low, and the naringin hydrolysis is difficult to be applied to industry. Disclosure of Invention In order to solve the technical problems of yield and conversion rate of catalyzing naringin to naringin at present, and more particularly to solve the technical problem of low activity of beta-glucosidase in catalyzing naringin conversion, the invention provides a beta-glucosidase mutant and application thereof in catalyzing flavone glycoside hydrolysis. The specific technical scheme of the invention is as follows: the invention provides a kind of In a first aspect, there is provided a β -glucosidase mutant obtained by single point mutation or multiple point combination mutation of the following sites from the amino acid sequence shown in SEQ ID No. 1: (a) Valine (V) at position 46 to alanine (a); (b) Aspartic acid (D) at position 49 to glutamic acid (E); (c) Isoleucine (I) at position 282 to valine (V); (d) Tyrosine (Y) at position 306 is mutated to histidine (H); (e) Leucine (L) at position 445 is mutated to lysine (K). In order to solve the problems of low catalytic activity and low catalytic efficiency of the existing beta-glucosidase, the invention obtains a mutant by carrying out single-point or multi-point combined substitution on amino acids 49, 282, 306 and/or 445 of the wild type beta-glucosidase shown in SEQ ID NO.1, wherein the substitution is V46A, D, E, I, 282V, Y, 306 and H, L K. Particularly preferred mutants are the single mutant V46E A, D49E, I282V, Y306H, L445K, the double site combined mutant V46A-L445K, the triple site combined mutant V46A-Y306H-L445K, V A-D49E-L445K, D E-Y306H-L445K, D E-I282V-Y306H, I282V-Y306H-L445K, and the four site mutant D49E-I282V-Y306H-L445 3446A-D49E-I282V-Y306H, V A-D49E-Y306H-L445K. The enzyme activity of the preferred mutants is greatly improved relative to the wild type catalytic activity of naringin as a substrate. Compared with the wild type, the relative enzyme activity of the beta-glucosidase mut