title: Gene cloning of endoglucanase Cel5A from cellulose-degrading Paenibacillus xylanilyticus KJ-03 and purification and characterization of the recombinant enzyme.
abstract: The bacterial strain Paenibacillus xylanilyticus KJ-03 was isolated from a sample of soil used for cultivating Amorphophallus konjac. The cellulase gene, cel5A was cloned using fosmid library and expressed in Escherichia coli BL21 (trxB). The cel5A gene consists of a 1,743 bp open reading frame and encodes 581 amino acids of a protein. Cel5A contains N-terminal signal peptide, a catalytic domain of glycosyl hydrolase family 5, and DUF291 domain with unknown function. The recombinant cellulase was purified by Ni-affinity chromatography. The cellulase activity of Cel5A was detected in clear band with a molecular weight of 64 kDa by zymogram active staining. The maximum activity of the purified enzyme was displayed at a temperature of 40 °C and pH 6.0 when carboxymethyl cellulose was used as a substrate. It has 44% of its maximum activity at 70 °C and retained 66% of its original activity at 45 °C for 1 h. The purified cellulase hydrolyzed avicel, CMC, filter paper, xylan, and 4-methylumbelliferyl β-D-cellobiose, but no activity was detected against p-nitrophenyl β-D-glucoside. The end products of the hydrolysis of cellotetraose and cellopentaose by Cel5A were detected by thin layer chromatography, while enzyme did not hydrolyze cellobiose and cellotriose.
authors.affiliation: Department of Biotechnology, College of Natural Resources and Life Science, Dong-A University, Pusan 604-714, South Korea.
authors.forename: In-Hye
authors.initials: IH
authors.lastname: Park
authors.forename: Jie
authors.initials: J
authors.lastname: Chang
authors.forename: Yong-Seok
authors.initials: YS
authors.lastname: Lee
authors.forename: Shu-Jun
authors.initials: SJ
authors.lastname: Fang
authors.forename: Yong-Lark
authors.initials: YL
authors.lastname: Choi
mesh_terms: D002480:Cellulase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D002846:Chromatography, Affinity; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D056507:Paenibacillus / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D011994:Recombinant Proteins; C407024:endoglucanase Cel5A; D002480:Cellulase