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CN-121991925-A - Novel thermophilic beta-1, 4-endoxylanase Xyn413

CN121991925ACN 121991925 ACN121991925 ACN 121991925ACN-121991925-A

Abstract

The invention discloses a thermophilic beta-1, 4-endoxylanase Xyn413, the amino acid sequence of which is shown as SEQ ID No.1, and the enzyme is a novel beta-1, 4-endoxylanase which is derived from a metagenome of a hot spring of a hot water pond in Yuanjiang county of Yunnan province through amino acid sequence comparison. The thermophilic beta-1, 4-endoxylanase Xyn413 provided by the invention can hydrolyze beech xylan, oat beta-glucan and lichenin, has the activities of beta-1, 4-endoxylanase and beta-1, 3-1, 4-glucanase, wherein the activity of hydrolyzed xylan is the highest. The optimal reaction temperature of the enzyme is 100 ℃, which is far higher than that of the beta-1, 4-endoxylanase reported in the prior art, the residual enzyme activity is kept at 85.3% after the enzyme is placed at 80 ℃ for 24 h%, and 5% ethanol can enhance the activity of the enzyme, so that the enzyme is beneficial to the industrial application of lignocellulose biomass under the high-temperature condition.

Inventors

  • DU GANG
  • YANG HAIYING
  • ZHOU XINGKUI

Assignees

  • 云南民族大学

Dates

Publication Date
20260508
Application Date
20241101

Claims (14)

  1. 1. A novel thermophilic beta-1, 4-endoxylanase Xyn 413/beta-1, 3-1, 4-glucanase Xyn413 has an amino acid sequence shown as SEQ ID No. 1.
  2. 2. A novel thermophilic beta-1, 4-endoxylanase Xyn 413/beta-1, 3-1, 4-glucanase Xyn413 has an amino acid sequence shown as SEQ ID No. 1.
  3. 3. The enzyme or functional analogue thereof according to claim 1, which has an amino acid sequence having at least 82% identity, more preferably at least 85% identity, more preferably at least 90% identity, more preferably at least 95% identity, more preferably at least 98% identity, most preferably at least 99% identity to the polypeptide sequence shown in SEQ ID No. 2.
  4. 4. The enzyme or functional analogue thereof according to claim 1, which has an amino acid sequence having at least 92% identity, more preferably at least 95% identity, more preferably at least 98% identity, most preferably at least 99% identity to the polypeptide sequence shown in SEQ ID No. 3.
  5. 5. The enzyme or functional analogue thereof according to claim 1, which has an amino acid sequence having at least 88% identity, more preferably at least 90% identity, more preferably at least 95% identity, more preferably at least 98% identity, most preferably at least 99% identity to the polypeptide sequence as shown in SEQ ID No. 4.
  6. 6. The enzyme or functional analogue thereof according to claim 1, which has an amino acid sequence having at least 86% identity, more preferably at least 90% identity, more preferably at least 95% identity, more preferably at least 98% identity, most preferably at least 99% identity to the polypeptide sequence shown in SEQ ID No. 5.
  7. 7. The enzyme or functional analogue thereof according to claim 1, which has an amino acid sequence having at least 86% identity, more preferably at least 90% identity, more preferably at least 95% identity, more preferably at least 98% identity, most preferably at least 99% identity to the polypeptide sequence as shown in SEQ ID No. 6.
  8. 8. The enzyme or functional analogue thereof according to claim 1, which has an amino acid sequence having at least 88% identity, more preferably at least 90% identity, more preferably at least 95% identity, more preferably at least 98% identity, most preferably at least 99% identity to the polypeptide sequence shown in SEQ ID No. 7.
  9. 9. The enzyme or functional analogue thereof according to claim 1, which has an amino acid sequence having at least 86% identity, more preferably at least 90% identity, more preferably at least 95% identity, more preferably at least 98% identity, most preferably at least 99% identity to the polypeptide sequence shown in SEQ ID No. 8.
  10. 10. A DNA sequence encoding the beta-1, 4-endoxylanase or functional analogue of any one of claims 1-9.
  11. 11. A recombinant expression vector comprising the DNA sequence of claim 10.
  12. 12. A host cell transformed, transduced or transfected with the recombinant expression vector of claim 11.
  13. 13. Use of a DNA sequence according to any one of claims 10 to 12 for the preparation of β -1, 4-endo-xylanase and β -1,3-1, 4-glucanase.
  14. 14. Use of the enzyme or functional analogue according to claims 1-9 in the fields of textile, pulp production, paper making and lignocellulose hydrolysis, etc. of the enzyme prepared according to claim 13.

Description

Novel thermophilic beta-1, 4-endoxylanase Xyn413 Technical Field The invention belongs to the technical field of genetic engineering of enzymes and biochemical engineering of enzymes, and particularly relates to a novel thermophilic beta-1, 4-endoxylanase Xyn413. Background Xylan is about one third of all renewable organic carbon resources on earth, and is a polysaccharide which is second only to cellulose in nature (Dan Xiaoyu, etc. A strain of cultivated termite is used for separating and identifying intestinal xylan degrading bacteria, and microbiological report, 2016, 43 (3): 6). Xylan is the most important structural polysaccharide in plant cells, and forms an important component of plant cell walls together with lignin and cellulose, and also forms one of the main components of hemicellulose. Xylan is a complex polysaccharide whose sugar chain backbone is formed by the linkage of xylose residues via beta-1, 4 glycosidic linkages. The xylan backbone consists of xylopyranose residues, the side chains can be substituted with arabinose, methyl glucuronic acid, acetyl groups, etc., and xylans can be divided into four groups according to the type of side chains, homoxylan, arabinoxylan, glucuronoxylomannan and arabinoxylan (Naidu et al Carbohydrate Polymers, 2017, 179:28-41). Xylan is an important raw material in industrial production, such as energy, energy consumption, paper making, textile and the like, and is an emerging biological material due to the characteristics of bioactivity, biocompatibility, biodegradability, oxygen barrier and the like (Urtiga S et al Eur J Pharm Biopharm, 2020, 151:199-208). The hydrolysate of the xylan can produce by-products such as ethanol, xylitol, lactic acid and the like after fermentation, the xylan can selectively stimulate microbial flora in intestinal tracts, has beneficial effects on hosts, improves the health condition of the hosts, and is a common prebiotic (AKPINAR ET al Carbohydrate Research, 2009, 344 (5): 660-6). Depolymerization of xylan must rely on beta-1, 4-xylan hydrolase (EC 3.2.1.8) to produce xylooligosaccharides by cleavage of beta-1, 4 glycosidic linkages in the backbone of xylose residues. Degradation of the substrate by beta-1, 4-xylanases depends on the nature of the substrate molecule, such as chain length, branching degree and type of substituents, the initial hydrolysis product is beta-D-xylopyranose oligomer, and small molecular (Birsan et al. Biochem Soc Trans, 1998, 26(2): 156-60; Polizeli et al. Appl Microbiol Biotechnol, 2005, 67(5): 577-91.).β-1,4- xylanases which may produce beta-D-xylopyranosyl monosaccharides, disaccharides and trisaccharides in the late stage of hydrolysis are widely distributed in microorganisms including bacteria, yeasts, aspergillus, trichoderma and the like, and some fungi, plants, protozoa and crustaceans may also produce xylanases (Carvalho et al Food Research International, 2013, 51 (1): 75-85). Xylanases of different microbial origin can be divided into several glycoside hydrolase families (GH 5, 7, 8, 10, 11, 26, 30 and 43), and according to the amino acid sequence similarity of the catalytic domains of the xylanases, the xylanases mainly belong to GH10 and GH11(Tony et al. Fems Microbiology Reviews, 2010, 29(1): 3-23; Cheng et al. J Biol Chem, 2014, 289(16): 11020-8; Hong et al. Appl Environ Microbiol, 2014, 80(7): 2084-93)., which require that the xylanases can withstand severe conditions such as acid-base environment and high temperature in industrial applications, e.g. high temperature resistant xylanases are required during bleaching of pulp to achieve catalysis under hot alkaline conditions of high temperature, alkaline pretreatment, and the use of high temperature resistant beta-1, 4-xylanases during bleaching degumming of flax and ramie fibers can reduce costs and pollution, and the use of thermophilic beta-1, 4-xylanases during liquefaction and saccharification of malt can reduce wort viscosity and increase filtration rate. In view of the great value and the demand of thermophilic beta-1, 4-xylanases in industrial application, the search and development of thermophilic beta-1, 4-xylanases by means of pure culture microorganisms are greatly limited, and on the contrary, hot spring metagenomic samples clearly provide a rich resource treasury for the development and application of thermophilic beta-1, 4-xylanases. Disclosure of Invention The first aim of the invention is to provide a thermophilic beta-1, 4-endoxylanase Xyn413, the amino acid sequence of which is shown as SEQ ID No.1, which is derived from a hot spring metagenome of Yuanjiang county of Yunnan province, and the amino acid sequence similarity analysis shows that the enzyme is a novel beta-1, 4-endoxylanase Xyn413. It is a second object of the present invention to provide an amino acid sequence encoding said beta-1, 4-endoxylanase Xyn 413. It comprises an amino acid sequence having at least 89% identity to the amino acid sequence of SEQ ID