Molecular cloning and characterization of two major endoglucanases from Penicillium decumbens.
Two major endoglucanase genes (cel7B and cel5A) were cloned from Penicillium decumbens 114-2 using the method of modified thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR). The result of Southern blotting suggested that P. decumbens has a single copy of the cel5A gene and a single copy of cel7B gene in its chromosomal DNA. The expression level of cel5A and cel7B were determined by means of real-time quantitative PCR, suggesting that two genes were coordinately expressed and repressed by glucose and induced by cellulose. Both endoglucanase genes were expressed in Saccharomyces cerevisiae and the recombinant proteins were purified. The recombinant Cel7B and Cel5A were both optimal active at 60 C and pH 4.0. The recombinant Cel7B showed more than 8-fold, 30-fold, 5-fold higher enzyme activity towards carboxymethyl cellulose, barely beta-glucan and PASC respectively in comparison with that of Cel5A. However, their activities toward pNPC and Avicel were minor difference. The result suggested that Cel7B is a strict endoglucanase, while Cel5A show processivity because of its relative higher ability to hydrolyze the crystal cellulose.
Wei
Xiao-Min
XM
State Key Laboratory of Microbial Technology,Shandong University, 27 Shanda South Road, Jinan, Shandong 250100, P. R. China.
Qin
Yu-Qi
YQ
Qu
Yin-Bo
YB
D002480:Cellulase / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010407:Penicillium / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
D005656:Fungal Proteins; D002480:Cellulase