Gene cloning, overexpression, and characterization of a xylanase from Penicillium sp. CGMCC 1669.
A xylanase-encoding gene, xyn11F63, was isolated from Penicillium sp. F63 CGMCC1669 using degenerated polymerase chain reaction (PCR) and thermal asymmetric interlaced (TAIL)-PCR techniques. The full-length chromosomal gene consists of 724 bp, including a 73-bp intron, and encodes a 217 amino acid polypeptide. The deduced amino acid sequence of xyn11F63 shows the highest identity of 70% to the xylanase from Penicillium sp. strain 40, which belongs to glycosyl hydrolases family 11. The gene was overexpressed in Pichia pastoris, and its activity in the culture medium reached 516 U ml(-1). After purification to electrophoretic homogeneity, the enzyme showed maximal activity at pH 4.5 and 40 degrees C, was stable at acidic buffers of pH 4.5-9.0, and was resistant to proteases (proteinase K, trypsin, subtilisin A, and alpha-chymotrypsin). The specific activity, K (m), and V (max) for oat spelt xylan substrate was 7,988 U mg(-1), 22.2 mg ml(-1), and 15,105.7 micromol min(-1) mg(-1), respectively. These properties make XYN11F63 a potential economical candidate for use in feed and food industrial applications.
Liu
Wanli
W
Key Laboratory of Feed Biotechnology of Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing 100081, People's Republic of China.
Shi
Pengjun
P
Chen
Qiang
Q
Yang
Peilong
P
Wang
Guozeng
G
Wang
Yaru
Y
Luo
Huiying
H
Yao
Bin
B
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D043364:Endo-1,4-beta Xylanases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D015870:Gene Expression; D006868:Hydrolysis; D007221:Industry; D007700:Kinetics; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010407:Penicillium / Q000201:enzymology / Q000235:genetics* / Q000302:isolation & purification; D010447:Peptide Hydrolases / Q000378:metabolism; D010843:Pichia / Q000235:genetics; D013379:Substrate Specificity
D043364:Endo-1,4-beta Xylanases; D010447:Peptide Hydrolases