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CN-121991918-A - Amylosucrase mutant with improved enzyme activity and application thereof

CN121991918ACN 121991918 ACN121991918 ACN 121991918ACN-121991918-A

Abstract

The invention relates to an amylase gene from microorganism Calidithermus timidus DSM 17022, which is used as a template, asparagine residues at 577 position on the interface between enzyme dimers are mutated into arginine and aspartic acid respectively to obtain mutants N577R and N577D, the aggregation state of the amylase gene is changed into a single polymer from a wild type tetramer state, and the total enzyme activity is respectively improved from 2.7+/-0.1U/mg to 2.9+/-0.2U/mg and 3.2+/-0.1U/mg of the wild type tetramer state to 1.07 and 1.19 times of the wild type.

Inventors

  • XU WEI
  • NI DAWEI
  • ZHANG WENLI
  • MU WANMENG

Assignees

  • 江南大学

Dates

Publication Date
20260508
Application Date
20260318

Claims (10)

  1. 1. An amylosucrase mutant with improved enzyme activity is characterized in that the amino acid sequence of the amylosucrase mutant is shown as SEQ ID NO.4 or SEQ ID NO. 6.
  2. 2. The mutant of amylosucrase with improved enzymatic activity according to claim 1, wherein the mutant of amylosucrase is obtained by site-directed mutagenesis of an asparagine residue at position 577 of an amylosucrase with the amino acid sequence shown in SEQ ID No.2 into arginine or aspartic acid.
  3. 3. An enzyme activity-enhanced amylosucrase mutant according to claim 2, wherein the amylosucrase as shown in SEQ ID No.2 is derived from Calidithermus timidus DSM 17022,17022.
  4. 4. A gene encoding the amylosucrase mutant according to claim 1.
  5. 5. A recombinant plasmid carrying the gene of claim 4.
  6. 6. The recombinant plasmid according to claim 5, characterized in that the expression vector of the recombinant plasmid is PET-22b (+).
  7. 7. A recombinant cell expressing the amylosucrase mutant according to claim 1 or the gene according to claim 4, characterized in that it is obtained by transforming the recombinant plasmid according to claim 5 into a host cell comprising e. coliBL21 (DE 3).
  8. 8. A method for preparing an amylosucrase mutant according to claim 1, wherein the recombinant cell according to claim 7 is subjected to induction culture to produce an amylosucrase mutant.
  9. 9. Use of an amylosucrase mutant according to claim 1 for catalyzing the production of dextran from sucrose.
  10. 10. Use according to claim 9, characterized in that the glucan is produced by an enzymatic reaction with sucrose as substrate under the action of an amylosucrase mutant, wherein the temperature of the enzymatic reaction is 50 ℃ and the reaction time of the enzymatic reaction is 30 min.

Description

Amylosucrase mutant with improved enzyme activity and application thereof Technical Field The invention belongs to the technical field of enzyme genetic engineering, and particularly relates to an amylosucrase mutant with improved enzyme activity and application thereof. Background Amylosucrase (ASase) belongs to the glycoside hydrolase 13 family and is the only functionally diverse sucrose utilization enzyme in this family having a polymeric activity. ASase can catalyze various reactions such as polymerization, isomerism, transglycosylation and the like by taking low-cost sucrose as a substrate, is used for producing functional sweeteners, dietary fibers, embedding materials based on carbohydrate, bioactive substances and the like, and has wide application potential in the food industry. The polymerization reaction synthesizes amylose (glucose units are connected only by alpha-1, 4 bonds) from cheap substrate sucrose, and can further synthesize starch-based immune particles and starch-based nanoparticle embedding systems by utilizing the self-assembly characteristic of the amylose of the product, or is coupled with other enzymes (such as cyclodextrin glycosyltransferase, maltooligosaccharide synthetase, glycogen branching enzyme and the like) to produce cyclodextrin, maltooligosaccharide, hyperbranched glucan and the like. The isomerization reaction produces the isomers melezitose and trehalulose of sucrose, both of which are the scarce functional disaccharides found in nature. When an additional glycosyl acceptor substrate is also present in the system besides sucrose, ASase has the glycosyl transferring capability, so that the compound is not only used as a glycosylation modification tool of bioactive substances, but also used for the synthesis of arbutin, flavone derivatives and the like, and also used for glycosylation protection, intermediate fragment synthesis and the like in the shigella antigen polysaccharide synthesis path. However, the problem of insufficient catalytic activity of the currently reported amylosucrases, especially the low overall catalytic activity at high temperatures, greatly limits the further development and use of the enzyme. The amylosucrase (CT-ASase) from the heat-resistant microorganism Calidithermus timidusDSM 17022 obtained by earlier successful excavation of the subject group is the only currently reported amylosucrase in solution in the form of tetramer, and in the structure, the enzyme consists of 5 residue regions (regions) widely involved in hydrophobic interaction, including amino acid residues Arg 368-Leu 372, ala 404-His 418, leu 573-Val 580, val 602-Val 609 and Glu 630-Val 634. The directional transformation is performed based on the amylosucrase at present so as to obtain an amylosucrase mutant with improved enzyme activity, and the mutant has important significance for industrial production and application of the amylosucrase. Disclosure of Invention The invention aims to overcome the defects in the prior art and provides an amylosucrase mutant with improved enzyme activity and application thereof, wherein the aggregation state of the amylosucrase mutant is changed from a wild tetramer state to a single polymer, and the enzyme activity is improved to a certain extent. In order to achieve the above purpose, the technical scheme adopted by the invention is as follows: An amylosucrase mutant with improved enzyme activity, wherein the amino acid sequence of the amylosucrase mutant is shown as SEQ ID NO.4 or SEQ ID NO. 6. As a further technical scheme, the amylosucrase mutant is obtained by site-directed mutagenesis of an asparagine residue at position 577 of the amylosucrase with the amino acid sequence shown in SEQ ID No.2 into arginine or aspartic acid. As a further technical scheme, the nucleotide sequence of the amylosucrase with the amino acid sequence shown as SEQ ID No.2 is shown as SEQ ID No. 1. As a further technical solution, the amylosucrase shown in SEQ ID No.2 is derived from Calidithermus timidusDSM.sup.17022. A gene encoding the amylosucrase mutant. As a further technical scheme, the nucleotide sequence of the gene of the amylosucrase mutant with the amino acid sequence shown as SEQ ID NO.4 is shown as SEQ ID NO. 3. As a further technical scheme, the nucleotide sequence of the gene of the amylosucrase mutant with the amino acid sequence shown as SEQ ID NO.6 is shown as SEQ ID NO. 5. A recombinant plasmid carrying the gene. As a further technical scheme, the expression vector of the recombinant plasmid is PET-22b (+). A recombinant cell expressing said amylosucrase mutant or said gene, said recombinant plasmid being obtained by transformation of a host cell comprising e. coliBL21 (DE 3). A method for preparing the amylosucrase mutant, and the recombinant cells are subjected to induction culture to produce the amylosucrase mutant. The method comprises the steps of adding seed liquid of the recombinant cells into liquid LB culture medium containing 50