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MOLECULAR CLONING AND EXPRESSION OF A FAMILY 7 CELLOBIOHYDROLASE GENE CBH1 FROM PENICILLIUM FUNICULOSUM NCL1

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Author(s): VANITHA MC, MEERA B, RAMANI G, MALA RAO, SEETA LAXMAN AND GUNASEKARAN P

Journal: International Journal of Microbiology Research
ISSN 0975-5276

Volume: 3;
Issue: 2;
Start page: 97;
Date: 2011;
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Keywords: Penicillium funiculosum | cellulase | cellobiohydrolase | molecular cloning | affinity chromatography | molecular simulation

ABSTRACT
A cellobiohydrolase gene cbh1 was cloned from a cellulolytic fungus, Penicillum funiculosum NCL1. Nucleotidesequencing of cbh1 gene revealed that this gene was 1590 bp length encoding a putative protein consisting of 529 aminoacids. The deduced amino acid sequence showed that the predicted molecular mass of the CBHI was 54.9 kDa, andshowed significant homology to glycoside hydrolase family 7 cellobiohydrolases. The cbh1 gene was cloned using pET30band expressed in E. coli BL21 (DE3). The expression analysis of the recombinant E. coli BL21 (pETC7) revealed theproduction of cbh1 transcript; however, functional cellobiohydrolase could not be detected. Therefore, the cbh1 gene wassub-cloned into GST tagged expression vector pGEX4t-3. The GST tagged cellobiohydrolase was purified to homogeneityusing affinity chromatography. The recombinant enzyme exhibited optimum catalytic activity at pH 5.0 and 50 °Crespectively. It was thermostable at 50 °C and retained 70% of its original activity after 30 min at 60 °C
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