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CN-116121226-B - Rhamnosyl hydrolase mutant and preparation method and application thereof

CN116121226BCN 116121226 BCN116121226 BCN 116121226BCN-116121226-B

Abstract

The invention discloses a rhamnosyl hydrolase mutant, which is characterized in that lysine of 513 st part of rhamnosidase TpeRha is mutated, the site mutation is amino acid with a hydrophobic side chain, the hydrolysis efficiency of the mutant K513A and the mutant K513V is respectively improved by 2.39 times and 6.27 times compared with that of a wild type, the invention also discloses a preparation method of the rhamnosyl hydrolase mutant, and the invention also discloses application of the rhamnosyl hydrolase mutant, wherein the rhamnosyl hydrolase mutant is used for preparing hesperetin-7-O-glucoside, hesperetin or a composition of the two.

Inventors

  • HUANG JIAJUN
  • LI HUILING
  • Hu Haoxuan
  • ZHOU JINLIN
  • LU YUJING
  • BAO YADAN

Assignees

  • 广东金骏康生物技术有限公司

Dates

Publication Date
20260508
Application Date
20221129

Claims (9)

  1. 1. A rhamnosyl hydrolase mutant is characterized in that the rhamnosyl hydrolase mutant is obtained by mutating the 513 st lysine of TpeRha th enzyme with an amino acid sequence shown as SEQ ID NO.2 into any one of tryptophan, phenylalanine or valine.
  2. 2. The rhamnosyl hydrolase mutant according to claim 1, wherein the TpeRha th lysine is mutated to valine (K513V) with the amino acid sequence shown in SEQ ID No. 6.
  3. 3. The rhamnosyl hydrolase mutant according to claim 2, wherein the nucleotide sequence of the gene encoding K513V is shown in SEQ ID No. 5.
  4. 4. The rhamnosyl hydrolase mutant according to any of claims 1-3, wherein the TpeRha enzyme is derived from thermotoga petroleum (Thermotoga petrophila) DSM 13995.
  5. 5. A method for preparing the rhamnosyl hydrolase mutant according to any of claims 1 to 4, comprising the steps of: (1) Connecting TpeRha enzyme coding genes to be mutated to a plasmid vector to obtain a recombinant plasmid; (2) Designing a site-directed mutagenesis primer, amplifying by taking the recombinant plasmid as a template, and performing enzyme digestion treatment to obtain a mutation product containing a mutation site, wherein the site-directed mutagenesis primer is used for mutating the 513 st lysine of TpeRha enzyme; (3) And transforming the mutant product into a host cell, and screening and inducing expression to obtain the rhamnosyl hydrolase mutant.
  6. 6. The preparation method of claim 5, wherein the nucleotide sequence of the TpeRha enzyme coding gene is shown as SEQ ID NO. 1, and in the step (2), the 513 st lysine of the TpeRha enzyme is mutated into valine by an upstream primer K513V-F and a downstream primer K513V-R, wherein the nucleotide sequence of the upstream primer K513V-F is shown as SEQ ID NO. 17, and the nucleotide sequence of the downstream primer K513V-R is shown as SEQ ID NO. 18.
  7. 7. The method of claim 5 or 6, wherein the host cell is any one of E.coli, saccharomyces cerevisiae or Pichia pastoris.
  8. 8. Use of the rhamnosyl hydrolase mutant according to any of claims 1-4 for the preparation of a composition comprising hesperetin-7-O-glucoside and/or hesperetin.
  9. 9. The use according to claim 8, wherein the use is the catalytic conversion of hesperidin to hesperetin-7-O-glucoside and/or hesperetin using the rhamnosyl hydrolase mutant.

Description

Rhamnosyl hydrolase mutant and preparation method and application thereof Technical Field The invention relates to the technical field of biological medicine, in particular to a rhamnosyl hydrolase mutant and a preparation method and application thereof. Background Hesperetin-7-O-glucoside with molecular formula of C 22H24O11 is one of the components of citrus flavonoid. In recent years, the study on the hesperetin-7-O-glucoside is deeper, and the study result shows that the absorption time of the hesperetin-7-O-glucoside is shorter than that of hesperidin and neohesperidin, the bioavailability is higher, and the hesperetin-7-O-glucoside also has the characteristic of prebiotics after entering the animal body, can promote the growth of intestinal probiotics, increase the content of flora metabolite short chain fatty acid, play a practical effect of resisting enteritis, and play a role in solving the intestinal problem. Hesperetin-7-O-glucoside is usually formed from hesperidin or neohesperidin, after removal of a portion of rhamnose by alpha-L-rhamnosidase. The alpha-L-rhamnosidase can cleave the glycosidic bond formed by reacting an alcoholic hydroxyl group with a hemiacetal in an exo-or endo-manner, thereby efficiently directing the hydrolysis of natural active substances containing rhamnoside, such as hesperidin. However, the alpha-L-rhamnosidase still has a large technical barrier when being applied in large scale, the activity of the natural alpha-L-rhamnosidase is insufficient, and the reaction environment of the natural alpha-L-rhamnosidase is difficult to meet the optimal condition of the natural enzyme. Disclosure of Invention To overcome the defects in the prior art, a first object of the invention is to provide a rhamnosyl hydrolase mutant which can solve the problem of insufficient activity of rhamnosidase. A rhamnosyl hydrolase mutant is obtained by mutating lysine at 513 st position of TpeRha enzyme with an amino acid sequence shown as SEQ ID NO. 2 into amino acid with a hydrophobic side chain. Further, the amino acid having a hydrophobic side chain is one of alanine, tryptophan, phenylalanine or valine. Further, the lysine at 513 st position of TpeRha enzyme is mutated into alanine to obtain mutant K513A, the amino acid sequence is shown as SEQ ID NO. 4, and the lysine at 513 st position of TpeRha enzyme is mutated into valine to obtain mutant K513V, the amino acid sequence is shown as SEQ ID NO. 6. Further, the nucleotide sequence of the mutant K513A gene is shown as SEQ ID NO.3, and the nucleotide sequence of the mutant K513V gene is shown as SEQ ID NO. 5. Further, the TpeRha enzyme is derived from Thermotoga petroleum DSM 13995. A second object of the present invention is to provide a method for preparing a rhamnosyl hydrolase mutant, which can solve the problem of preparing rhamnosidase with high activity. A method for preparing a rhamnosyl hydrolase mutant, comprising the following steps: Step 1, connecting a coding gene of TpeRha enzyme to be mutated into a plasmid to obtain a recombinant plasmid; Step 2, designing and utilizing a site-directed mutagenesis primer, and amplifying by taking a recombinant plasmid as a template, and performing enzyme digestion to obtain a mutation product, wherein the site-directed mutagenesis primer mutates lysine at 513 th site of TpeRha enzyme; And step 3, transforming the mutation product into a host cell, and screening and inducing the host cell to express to obtain the rhamnosyl hydrolase mutant. Further, the nucleotide sequence of the coding gene of TpeRha enzyme is shown as SEQ ID NO. 1, in the step 2, the 513 st lysine of TpeRha enzyme is mutated into alanine through an upstream primer K513A-F and a downstream primer K513A-R, the nucleotide sequence of the upstream primer K513A-F is shown as SEQ ID NO. 9, the nucleotide sequence of the downstream primer K513A-R is shown as SEQ ID NO. 10, the 513 st lysine of TpeRha enzyme is mutated into valine through an upstream primer K513V-F and a downstream primer K513V-R, the nucleotide sequence of the upstream primer K513V-F is shown as SEQ ID NO. 17, and the nucleotide sequence of the downstream primer K513V-R is shown as SEQ ID NO. 18. Further, the host cell is any one of escherichia coli, saccharomyces cerevisiae and pichia pastoris. The third object of the invention is to provide an application of a rhamnosyl hydrolase mutant, which can solve the problem of hesperetin-7-O-glucoside preparation. Use of a rhamnosyl hydrolase mutant for the preparation of one or both of hesperetin-7-O-glucoside or hesperetin. Further, the application is to catalyze and convert hesperidin into one or two of hesperetin-7-O-glucoside or hesperetin by using a rhamnoside mutant. Compared with the prior art, the invention has the beneficial effects that: 1. the rhamnosyl hydrolase mutants K513A and K513V can promote the hydrolysis of hesperidin, and effectively improve the efficiency of converting hesperidin into hesperetin-7-O-g