Cloning of a new xylanase gene from Streptomyces sp. TN119 using a modified thermal asymmetric interlaced-PCR specific for GC-rich genes and biochemical characterization.
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 degrees Celsius, was stable at pH 4.0 to 10.0 and 50 degrees Celsius, was resistant to most chemicals (except for Cu(2+), Mn(2+), Ag(+), Hg(2+) 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 micromol min(-1) mg(-1), and 49.2 s(-1), respectively.
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing 100081, People's Republic of China.
Junpei
J
Zhou
Huoqing
H
Huang
Kun
K
Meng
Pengjun
P
Shi
Yaru
Y
Wang
Huiying
H
Luo
Peilong
P
Yang
Yingguo
Y
Bai
Bin
B
Yao
D001482:Base Composition / Q000235:genetics*; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D043364:Endo-1,4-beta Xylanases / Q000235:genetics* / Q000378:metabolism; D005798:Genes, Bacterial / Q000235:genetics*; D006868:Hydrolysis / Q000187:drug effects; D007477:Ions; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D011994:Recombinant Proteins / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D013302:Streptomyces / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics*; D013696:Temperature
D007477:Ions; D008670:Metals; D011994:Recombinant Proteins; D043364:Endo-1,4-beta Xylanases