期刊
APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
卷 160, 期 5, 页码 1277-1292出版社
SPRINGER
DOI: 10.1007/s12010-009-8642-8
关键词
Xylanase; Batocera horsfieldi; Gut; Streptomyces sp.; TN119; Thermal asymmetric interlaced (TAIL)-PCR; High-GC genes
资金
- National High Technology Research and Development Program of China (863 Program) [2007AA100601]
- National Key Technology R&D Program of China [2006BAD12B05-03]
- Chinese Program on Research for Public Good [2005DIB4J038]
A bacterial strain, Streptomyces sp. TN119, was isolated from the gut of Batocera horsfieldi larvae and showed xylanolytic activity. A degenerate primer set was designed based on the base usage of G and C in Actinobacteria xylanase-coding sequences belonging to the glycosyl hydrolases family 10 (GH 10), and used to clone the partial xylanase gene from Streptomyces sp. TN119. A modified thermal asymmetric interlaced (TAIL)-PCR specific for high-GC genes, named GC TAIL-PCR, was developed to obtain the full-length xylanase gene (xynA119; 1089 bp). Rich in GC content (67.8%), xynA119 encodes a new GH 10 xylanase (XynA119), which shares highest identity (48.8%) with an endo-1,4-beta-xylanase from Cellulosimicrobium sp. HY-12. Recombinant XynA119 was expressed in Escherichia coli BL21 (DE3) and purified to electrophoretic homogeneity. The enzyme showed maximal activity at pH 6.5 and 60 A degrees C, was stable at pH 4.0 to 10.0 and 50 A degrees C, was resistant to most chemicals (except for Cu2+, Mn2+, Ag+, Hg2+ and SDS) and trypsin, and produced simple products. The specific activity, K (m), V (max), and k (cat) using oat-spelt xylan as substrate were 57.9 U mg(-1), 1.0 mg ml(-1), 74.8 mu mol min(-1) mg(-1), and 49.2 s(-1), respectively.
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