CN-121991933-A - Thermophilic xylanase LD3-Xyn6 and application thereof

CN121991933ACN 121991933 ACN121991933 ACN 121991933ACN-121991933-A

Abstract

The application discloses thermophilic xylanase LD3-Xyn6 and application thereof. Belongs to the technical field of genetic engineering. The application identifies a brand new xylanase gene from a metagenome of the Yunnan Dajingdu hot spring, and obtains the recombinase through heterologous expression and purification. The characterization result of the enzymatic property shows that the optimal temperature of the enzyme is 85 ℃, the optimal pH value is 5.6, and the half-lives of the enzyme at 85 ℃ and 90 ℃ are 15min and 10 min respectively, so that the enzyme has excellent heat-resisting property and excellent temperature and pH stability. In addition, K + 、Ca 2+ and Mg 2+ have an activating effect on their enzymatic activities. The enzyme can effectively degrade various natural xylans, and the hydrolysate thereof is mainly composed of xylobiose and xylotetraose, and has been proved to be capable of remarkably promoting the growth of lactococcus lactis. Therefore, the xylanase LD3-Xyn6 has wide application prospect in the preparation of high-temperature resistant feed additives and prebiotics.

Inventors

Yin Yirui
Pu Lanhua
LI JIANLING
Shao Zongjin
LIU YANMI
MA WEN
Shao Qirong
LI MAOSONG
LI CHANJIN
CHEN YAN

Assignees

大理大学

Dates

Publication Date: 20260508
Application Date: 20260202

Claims (10)

1. The thermophilic xylanase LD3-Xyn6 is characterized in that the amino acid sequence of the thermophilic xylanase LD3-Xyn6 is shown as SEQ ID NO. 4.
2. A DNA molecule, wherein the DNA molecule encodes the thermophilic xylanase LD3-Xyn6 as defined in claim 1, and the nucleotide sequence of the DNA molecule is shown as SEQ ID NO. 3.
3. A biomaterial characterized in that it is any one of the following: 1) An expression cassette comprising the DNA molecule of claim 2; 2) A recombinant vector comprising the DNA molecule of claim 2, or a recombinant vector comprising 1) the expression cassette; 3) A recombinant comprising the DNA molecule of claim 2, or a recombinant comprising 1) the expression cassette, or a recombinant comprising 2) the recombinant vector.
4. Use of a DNA molecule according to claim 2 or a biomaterial according to claim 3 for the preparation of the thermophilic xylanase LD3-Xyn 6.
5. Use of the thermophilic xylanase LD3-Xyn6 as defined in claim 1 for the preparation of xylo-oligosaccharides.
6. Use of the thermophilic xylanase LD3-Xyn6 according to claim 1 for degrading xylan, characterized in that the xylan is derived from beech, corncob and/or bagasse.
7. Use of the thermophilic xylanase LD3-Xyn6 of claim 1 for promoting the growth of lactococcus lactis.
8. A method of degrading xylan comprising contacting a xylan substrate with a thermophilic xylanase LD3-Xyn6 according to claim 1 under reaction conditions of 85° C, pH 5.6.6.
9. The method of claim 8, wherein the xylan substrate is high temperature alkali treated corncob xylan or high temperature water treated wheat bran xylan.
10. A feed additive comprising the thermophilic xylanase LD3-Xyn6 of claim 1.

Description

Thermophilic xylanase LD3-Xyn6 and application thereof Technical Field The invention relates to the technical field of genetic engineering, in particular to thermophilic xylanase LD3-Xyn6 and application thereof. Background The cost of the feed accounts for 60 to 80 percent of the total cost of animal husbandry production, and is a core factor for restricting the economic benefit of the industry. Therefore, improving the feed utilization efficiency is always a key target of feed science research. In the modern feed processing technology, the granulation technology can obviously improve the palatability of the feed, improve the digestibility of nutrient substances and promote the growth performance of animals by compressing the powdery feed into granules and combining with adding external enzyme preparations such as xylanase and the like. However, the high temperature conditioning step of the process (which is typically required to be carried out at 70 ℃ to 95 ℃) presents a significant challenge to the use of enzyme preparations while optimizing feed quality. At present, commercial xylanase widely applied in the feed industry mainly comes from mesophilic microorganisms, and the optimum acting temperature is generally 50-65 ℃, so that the heat stability is poor. When subjected to high temperature conditioning in excess of 75 ℃ during pelleting, these enzyme molecules undergo irreversible denaturation and inactivation, resulting in very low residual rates of enzyme activity in the final feed product and failure to function effectively in the animal digestive tract. The technical bottleneck severely restricts the application mode of adding the enzyme preparation before granulating, and forces the production enterprises to adopt a post-spraying process with higher cost and uniformity problem, thereby not only increasing the production cost, but also affecting the stability of the product quality. In addition to insufficient heat resistance, the existing xylanases also face other prominent problems in practical application environments. First, it has poor stability under complex feed conditions. The animal digestive tract environment is complex, the pH value change range is large from the acidic environment of the stomach to the neutral environment of the intestinal tract, and the enzyme activity of many existing xylanases is drastically reduced after the xylanase deviates from the optimal pH value, so that the xylanase is difficult to continuously exert efficacy in the whole digestive tract. Secondly, various metal ions and chemicals contained in the feed may have an inhibitory effect on the enzyme activity, and the existing enzyme preparations often lack tolerance or activation effects on common ions (such as K +、Ca2+、Mg2+), so that the effects of the existing enzyme preparations in practical application are unstable. In addition, there is room for optimization of the catalytic product spectrum of existing xylanases. Xylo-oligosaccharide (XOS) which is a xylanase degradation product is taken as a high-quality prebiotic, can selectively promote the proliferation of beneficial intestinal flora, but the ratio of the XOS components generated by different xylanases from different sources is different due to the hydrolysis characteristic difference of the xylanases. Studies have shown that certain XOS components (e.g., xylobiose and xylotetraose) have superior prebiotic activity, and that xylanases currently capable of efficiently hydrolyzing xylans in a variety of feed materials and producing predominantly these high value XOS components are relatively lacking. Therefore, there is an urgent need in the feed industry to develop a novel xylanase with excellent thermostability, wide pH adaptability, good tolerance to common metal ions, and capable of efficiently producing XOS with high prebiotic value, which is a technical problem to be solved by those skilled in the art. Disclosure of Invention In view of the above, the present invention provides a thermophilic xylanase LD3-Xyn6 and its use, which has excellent heat resistance, stability, and in addition, its catalytic product has excellent prebiotic properties. In order to solve the technical problems, the application adopts the following technical scheme: The first aim of the application is to provide thermophilic xylanase LD3-Xyn6, wherein the amino acid sequence of the thermophilic xylanase LD3-Xyn6 is shown as SEQ ID NO. 4. It is still another object of the present application to provide a DNA molecule encoding the above thermophilic xylanase LD3-Xyn6, the nucleotide sequence of which is shown as SEQ ID NO. 3. It is still another object of the present application to provide a biomaterial, which is any one of the following: 1) An expression cassette comprising the above DNA molecule; 2) A recombinant vector comprising the above DNA molecule or a recombinant vector comprising 1) the expression cassette; 3) Recombinant bacteria comprising the above DNA molecule, or