title: Expression, purification and characterization of two thermostable endoglucanases cloned from a lignocellulosic decomposing fungi Aspergillus fumigatus Z5 isolated from compost.
abstract: Two genes encoding endoglucanase, designated as egl2 and egl3, were cloned from a lignocellulosic decomposing fungus Aspergillus fumigatus Z5 and were successfully expressed in Pichia pastoris X33. The deduced amino acid sequences encoded by egl2 and egl3 showed strong similarity with the sequence of glycoside hydrolase family 5. SDS-PAGE and western blot assays indicated that the recombinant enzymes were secreted into the culture medium and the zymogram analysis confirmed that both recombinant enzymes had endoglucanase activity. Several biochemical properties of the two recombinant enzymes were studied: Egl2 and Egl3 showed optimal activity at pH 5.0 and 4.0, respectively, and at 50 and 60°C, respectively. Egl2 and Egl3 showed good pH stability in the range of 4-7, and both enzymes demonstrated good thermostability ranging from 30 to 60°C. The K(m) and V(max) values using carboxymethyl cellulose (CMC, soluble cellulose, polymerized by β-1, 4-linked glucose residues) as the substrate at optimal conditions were determined. The activities of the enzymes on a variety of cello-oligosaccharide substrates were investigated, and Egl2 can hydrolyze cellotetraose and cellopentaose but not cellobiose and cellotriose, whereas Egl3 can hydrolyze all cello-oligosaccharides, except cellobiose.
authors.affiliation: Jiangsu Key Laboratory for Organic Solid Waste Utilization, Nanjing Agricultural University, Nanjing 210095, China.
authors.forename: Dongyang
authors.initials: D
authors.lastname: Liu
authors.forename: Ruifu
authors.initials: R
authors.lastname: Zhang
authors.forename: Xingming
authors.initials: X
authors.lastname: Yang
authors.forename: Yangchun
authors.initials: Y
authors.lastname: Xu
authors.forename: Zhu
authors.initials: Z
authors.lastname: Tang
authors.forename: Wei
authors.initials: W
authors.lastname: Tian
authors.forename: Qirong
authors.initials: Q
authors.lastname: Shen
mesh_terms: D000595:Amino Acid Sequence; D001232:Aspergillus fumigatus / Q000201:enzymology / Q000235:genetics; D015153:Blotting, Western; D002480:Cellulase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002482:Cellulose / Q000031:analogs & derivatives / Q000378:metabolism; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008031:Lignin / Q000378:metabolism*; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D010843:Pichia; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D016415:Sequence Alignment; D012987:Soil / Q000737:chemistry; D013379:Substrate Specificity; D013696:Temperature; D013780:Tetroses / Q000378:metabolism; D014170:Transformation, Genetic
chemical_list: D007527:Isoenzymes; D009844:Oligosaccharides; D011994:Recombinant Proteins; D012987:Soil; D013780:Tetroses; C036909:lignocellulose; C019193:maltopentaose; C048468:cellotetraose; D002482:Cellulose; D008031:Lignin; D002480:Cellulase