Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Molecular Cloning and Characterization of Two Major Endoglucanases from Penicillium decumbens

  • Wei, Xiao-Min (State Key Laboratory of Microbial Technology) ;
  • Qin, Yu-Qi (School of Pharmaceutical Science Shandong University) ;
  • Qu, Yin-Bo (State Key Laboratory of Microbial Technology)
  • Published : 2010.02.28
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Abstract

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 the cel7B gene in its chromosomal DNA. The expression levels of cel5A and cel7B were determined by means of real-time quantitative PCR, suggesting that the 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 optimally active at $60^{\circ}C$ and pH 4.0. The recombinant Cel7B showed more than 8-fold, 30-fold, and 5-fold higher enzyme activities toward carboxymethyl cellulose, barley $\beta$-glucan, and PASC, respectively, in comparison with that of Cel5A. However, their activities toward pNPC and Avicel showed minor differences. The results suggested that Cel7B is a strict endoglucanase, whereas Cel5A showed processivity because of its relative higher ability to hydrolyze the crystal cellulose.

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