Cloning,Sequencing and Expression Analysis of the First Cellulase Gene Encoding Cellobiohydrolase 1 from a Cold-adaptive Penicillium chrysogenum FS010  

作　　者：Yunhua HOU Tianhong WANG Hao LONG Huiynan ZHU 

机构地区：[1]State Key Laboratory of Microbial Technology,Shandong University,Jinan 250100,China [2]Department of Food and Biologic Engineering,Shandong Institute of Light Industry,Jinan 250323,China

出　　处：《Acta Biochimica et Biophysica Sinica》2007年第2期101-107,共7页生物化学与生物物理学报（英文版）

基　　金：This work was supported by the grants from the Major State Basic Research Development Program （973） of China （N0. 2003CB716006 and 2004CB719702）;the Natural Science Foundation of Shandong Province （No. L2003D01） ; the 0pen Foundation of State Key laboratory of Microbial Technology, Shandong University

摘　　要：A cellobiohydrolase 1 gene （cbhl） was cloned from Penicillium chrysogenum FS010 by a modified thermal asymmetric interlaced polymerase chain reaction （TAIL-PCR）. DNA sequencing shows that cbhl has an open reading frame of 1590 bp, encoding a putative protein of 529 amino acid residues. The deduced amino acid sequence revealed that CBHI has a modular structure with a predicted molecular mass of 56 kDa and consists of a fungal type carbohydrate binding module separated from a catalytic domain by a threonine rich linker region. The putative gene product is homologous to fungal cellobiohydrolases in Family 7 of the glycosyl hydrolases. A novel cbhl promoter （1.3 kb） was also cloned and sequenced, which contains seven putative binding sites （5＇-SYGGRG-3＇） for the carbon catabolite repressor CRE1. Effect of various carbon sources to the cbhl transcription of P. chrysogenum was examined by Northern analysis, suggesting that the expression of cbhl is regulated at transcriptional level. The cbhl gene in cold-adaptive fungus P. chysogenum was expressed as an active enzyme in Saccharomyces cerevisiae H158. The recombinant CBHI accumulated intracellularly and could not be secreted into the medium.A cellobiohydrolase 1 gene （cbhl） was cloned from Penicillium chrysogenum FS010 by a modified thermal asymmetric interlaced polymerase chain reaction （TAIL-PCR）. DNA sequencing shows that cbhl has an open reading frame of 1590 bp, encoding a putative protein of 529 amino acid residues. The deduced amino acid sequence revealed that CBHI has a modular structure with a predicted molecular mass of 56 kDa and consists of a fungal type carbohydrate binding module separated from a catalytic domain by a threonine rich linker region. The putative gene product is homologous to fungal cellobiohydrolases in Family 7 of the glycosyl hydrolases. A novel cbhl promoter （1.3 kb） was also cloned and sequenced, which contains seven putative binding sites （5＇-SYGGRG-3＇） for the carbon catabolite repressor CRE1. Effect of various carbon sources to the cbhl transcription of P. chrysogenum was examined by Northern analysis, suggesting that the expression of cbhl is regulated at transcriptional level. The cbhl gene in cold-adaptive fungus P. chysogenum was expressed as an active enzyme in Saccharomyces cerevisiae H158. The recombinant CBHI accumulated intracellularly and could not be secreted into the medium.

关 键 词：Penicillium chrysogenum CELLOBIOHYDROLASE TAIL-PCR promoter of cbhl 

分 类 号：Q78[生物学—分子生物学]