CN-121991926-A - Glucanase mutant with high antibacterial property and high specific activity heat resistance and application thereof

Abstract

The invention relates to the technical fields of genetic engineering and protein engineering, in particular to a glucanase mutant with high bacteriostasis and high specific activity heat resistance and application thereof. The invention takes 1,3-1, 4-beta-glucanase Feglc A as a female parent and screens out a combined mutant M4 by a directed evolution technology. In terms of heat stability, the T 50 value and the half-life period of the mutant M4 at 60 ℃ are respectively improved by 9 ℃ and 62 min compared with that of a wild enzyme, in terms of enzyme activity, the specific activity and the catalytic efficiency of the mutant M4 are respectively improved by 54% and 142% compared with that of the wild enzyme, and in terms of antibacterial performance, the antibacterial rate of the mutant M4 on aspergillus niger and aspergillus flavus is respectively improved by 44% and 53%. The acquisition of the high-catalytic-efficiency heat-resistant glucanase mutant M4 provides a reference for improving the catalytic performance of glucanase, and the mutant M4 with excellent antibacterial performance has a huge application prospect in the production of green antibacterial agents.

Inventors

• LI JING
• HE JIANQIANG
• WANG SHENGJUN
• GONG HUIFANG

Assignees

• 江苏大学附属医院

Dates

Publication Date

20260508

Application Date

20260212

Claims (10)

1. 1. A glucanase mutant with high bacteriostasis and high specific activity heat resistance is characterized in that the amino acid sequence of the glucanase mutant is shown as SEQ ID NO. 2.
2. 2. A nucleic acid molecule encoding the glucanase mutant of claim 1, characterized in that the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID No. 4.
3. 3. A recombinant vector comprising the nucleic acid molecule of claim 2.
4. 4. A recombinant bacterium is characterized in that, the recombinant bacterium comprising the recombinant vector of claim 3.
5. 5. The recombinant bacterium according to claim 4, wherein the base strain of the recombinant bacterium is pichia pastoris GS115.
6. 6. Use of the glucanase mutant of claim 1, the nucleic acid molecule of claim 2, the recombinant vector of claim 3 or the recombinant bacterium of claim 4 or 5 for the preparation of an antibacterial agent.
7. 7. The use according to claim 6, wherein the antibacterial agent comprises an aspergillus inhibitor.
8. 8. The use according to claim 7, wherein the aspergillus comprises aspergillus niger and/or aspergillus flavus.
9. 9. An antibacterial agent, which is characterized in that, the antibacterial agent comprising the glucanase mutant of claim 1.
10. 10. The antimicrobial agent of claim 9, wherein the antimicrobial agent is used to inhibit aspergillus; the aspergillus comprises aspergillus niger and/or aspergillus flavus.

Description

Glucanase mutant with high antibacterial property and high specific activity heat resistance and application thereof Technical Field The invention relates to the technical fields of genetic engineering and protein engineering, in particular to a glucanase mutant with high bacteriostasis and high specific activity heat resistance and application thereof. Background 1,3-1, 4-Beta-glucanase (EC 3.2.1.73) is a class of glycoside hydrolase capable of specifically hydrolyzing 1,3-1, 4-beta-glycosidic bonds in beta-glucan, and is widely found in microorganisms and plants. The enzyme has important application value in the fields of food, feed, brewing, biological energy sources, antibiosis and the like, particularly in the fields of feed and antibiosis, can degrade beta-glucan in cell walls, reduce chyme viscosity, improve feed utilization rate, promote fungus dissolution and realize biological antibiosis effect. However, the natural 1,3-1, 4-beta-glucanase has the problems of low catalytic activity, insufficient thermal stability and the like, and the wide application of the natural 1,3-1, 4-beta-glucanase in industry is restricted. The catalytic activity of the enzyme directly determines the substrate conversion efficiency and economy, and the thermal stability is a key factor for keeping the activity of the enzyme in the industrial high-temperature processing or storage process. Traditional enzyme modification strategies focus on the improvement of single performance, but catalytic activity and thermal stability are often in a mutually restricted relationship, so that one side performance is improved while the other side performance is weakened. Therefore, how to realize synchronous improvement of catalytic activity and thermal stability becomes a long-standing challenge in the field of enzyme engineering. In addition, as the problem of resistance to chemical antibiotics by abuse becomes more and more severe, development of new, green, efficient antibiotics is becoming an urgent need. The 1,3-1, 4-beta-glucanase can inhibit bacterial growth by hydrolyzing beta-glucan components in bacterial cell walls to destroy the structural integrity, and particularly has potential antibacterial effect on harmful fungi, but the low activity and heat sensitivity of the natural enzyme limit the application effect of the natural enzyme in the antibacterial field. Therefore, the development of the excellent 1,3-1, 4-beta-glucanase mutant with high catalytic activity and high temperature resistance is beneficial to expanding the application range of the mutant in the traditional industry, and can provide a new idea for the development of novel enzyme-based antibacterial agents. Through enzyme molecular transformation, the performance cooperative promotion is realized, the functional durability of the enzyme in a complex environment can be enhanced, a foundation is laid for exploring the application of the enzyme in the emerging fields of biological antibiosis, medical protection and the like, and the enzyme has important scientific significance and application value for promoting the development of green biological manufacturing and anti-infection strategies. Disclosure of Invention The invention aims to provide a glucanase mutant with high bacteriostasis and high specific activity heat resistance and application thereof, so as to solve the problems in the prior art. The glucanase mutant provided by the invention is obtained by simultaneously carrying out site-directed mutagenesis on key amino acid loci Thr27, ser49, gly92 and Ser258 of Feglc A from Friedemann bacteria (Friedmanniomyces endolithicus), and has high specific activity, heat resistance and strong antibacterial property. In order to achieve the above object, the present invention provides the following solutions: The invention provides a glucanase mutant with high bacteriostasis and high specific activity heat resistance, and the amino acid sequence of the glucanase mutant is shown as SEQ ID NO. 2. The invention provides a nucleic acid molecule for encoding the dextranase mutant, and the nucleotide sequence of the nucleic acid molecule is shown as SEQ ID NO. 4. The invention provides a recombinant vector comprising the nucleic acid molecule described above. The invention provides a recombinant bacterium, which comprises the recombinant vector. Optionally, the basic strain of the recombinant bacterium is pichia pastoris GS115. The invention provides application of the glucanase mutant, the nucleic acid molecule, the recombinant vector or the recombinant bacterium in preparation of antibacterial agents. Optionally, the antimicrobial agent comprises an aspergillus inhibitor. Optionally, the aspergillus comprises aspergillus niger and/or aspergillus flavus. The invention provides an antibacterial agent, which comprises the glucanase mutant. Optionally, the antimicrobial agent is used to inhibit aspergillus; the aspergillus comprises aspergillus niger and/or aspergillus fla