Cloning And Expression Of Xylanase Gene From Thermobifida Halotolerans YIM 90462~T

Thermophiles are becoming more and more abstractive because of their potential industrial applications. The more concerned about the thermophilic actinomycete, Thermobifida halotolerans YIM 90462'. was selected as target for its xylanase in this study, which was isolated from a salt mine sample collected from Heijing saline mine. Yunnan province.and identified by polyphasic taxonomy in 2008. and it is proposed that the organism be recognized as a novel species of Thermobifida. The members of all existed Thermobifida strains present an outstanding capability of xylanase decomposition. The xylanase of Thermobifida fusca YX has been thoroughly studied in many reports, while a xylanase XynⅠwas purified from the fermentation by Hu Songnan at Actinomycetal laboratory in Yunnan Institutue of Microbiology. Yunnan University in 2009. with which had suitable high temperature, alkali resistance and other special enzymatic properties, meanwhile the standards of the protein N/C terminal gene sequencing had not been done because of its purification and amount of enzymes at that time. Here, we use relevant molecular genetic manipulation including gene clone, express methods to obtain the xylanase gene sequences, and then study its enzymatic properties.Main results were mainly gained in this study as follows:1. The amplification method of T. halotolerans xylanase gene was studied and discussed. Using the online software CODEHOP designed degenerate primers,and the conserved region of T. halotolerans xylanase gene was amplified. The known flaking sequence was amplified by 5 different PCR. The 5 PCR for experimental principle, advantages and defects were explained.2. The gene sequence of T. halotolerans xylanase was amplified, as Xyn11-1. The gene consisted of 1008 bases, revealing a protein of 335 amino acid residues with the theoretical molecular mass of 35 kDa and the p I of 9.34. The three dimentional structure, conserved domain, multiple sequence alignement and phylogenetic tree of Xyn11-1 showed that it is suitable for high-temperature enzymes.3. The prokaryotic expression system (Xyn11-1-pET28a/BL21) was constructed and were overexpressed. Studies on properties of Xyn11-1 xylanase indicated that the optimum temperature and pH of the enzyme reaction was 70℃and pH 9.0. respectively. The activity of Xyn11-1 xylanase was dependent from mental ion. The active site of enzyme has serine residues and formed disulfide bond. The result provided valuable information in further study of this xylanase.In summary, the innovations of this study are presented as follows:1. Xylanase gene from the thermophilic actinomycete strain T. Halotolerans YIM 90462T was successfully amplified.2. A effective PCR technique for chromosome walking was applied, which was mainly based on chromosome-gene location.