Cloning, expression, and characterization of protease-resistant xylanase from Streptomyces fradiae var. k11.
The gene SfXyn10, which encodes a protease-resistant xylanase, was isolated using colony PCR screening from a genomic library of a feather-degrading bacterial strain Streptomyces fradiae var. k11. The full-length gene consists of 1,437 bp and encodes 479 amino acids, which includes 41 residues of a putative signal peptide at its N terminus. The amino acid sequence shares the highest similarity (80%) to the endo-1,4-beta-xylanase from Streptomyces coelicolor A3, which belongs to the glycoside hydrolase family 10. The gene fragment encoding the mature xylanase was expressed in Escherichia coli BL21 (DE3). The recombinant protein was purified to homogeneity by acetone precipitation and anion-exchange chromatography, and subsequently characterized. The optimal pH and temperature for the purified recombinant enzyme were 7.8 and 60 degrees , respectively. The enzyme showed stability over a pH range of 4-10. The kinetic values on oat spelt xylan and birchwood xylan substrates were also determined. The enzyme activity was enhanced by Fe2+ and strongly inhibited by Hg2+ and SDS. The enzyme also showed resistance to neutral and alkaline proteases. Therefore, these characteristics suggest that SfXyn10 could be an important candidate for protease-resistant mechanistic research and has potential applications in the food industry, cotton scouring, and improving animal nutrition.
Li
Ning
N
Microbial Engineering Department, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
Yang
Peilong
P
Wang
Yaru
Y
Luo
Huiying
H
Meng
Kun
K
Wu
Ningfeng
N
Fan
Yunliu
Y
Yao
Bin
B
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism*; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D017421:Sequence Analysis; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D013696:Temperature
D001426:Bacterial Proteins; D043364:Endo-1,4-beta Xylanases; D010450:Endopeptidases; C087822:alkaline protease