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Cloning of Aspergillus Niger BglA and expression of recombinant β-glucosidase in methylotrophic yeast Pichia Pastoris

Kamaruddin, Shazilah and Abu Bakar, Farah Diba and Md Illias, Rosli and Rabu, Amir and Said, Mamot and Hassan, Osman and Abdul Murad, Abdul Munir (2008) Cloning of Aspergillus Niger BglA and expression of recombinant β-glucosidase in methylotrophic yeast Pichia Pastoris. Jurnal Teknologi (49F). pp. 367-381. ISSN 0127-9696

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Abstract

Full length cDNA of bglA gene encoding Aspergillus niger ATCC10574 β-glucosidase was isolated and sequenced. The cDNA has a length of 2583 bp which encodes a polypeptide of 860 amino acid residues with predicted pI value of 4.6 and molecular weight of 93 kDa. Amino acid analysis of BGLA from four different isolates of A. niger, isolates ATCC10574, ATCC1015, B1 and CBS513.88, detected a total of 29 amino acids differences. The degree of differences varies between different variants, from 0.46% up to 2.9%. Around 34% of these differences were located in β-glucosidase two conserved domains, the glycosyl hydrolase family 3 N-terminal and the C-terminal domains. Both of the domains are important for the catalytic activity of the enzyme and these differences might contribute to different biophysical and biochemical enzyme properties. Heterologous expression of BGLA in methylotrophic yeast, Pichia pastoris has been carried out using methanol as inducer resulting in the production of recombinant protein with molecular weight around 90 kDa. β-glucosidase activity was detected from the culture filtrate using UVstimulated fluorescence of cleaved fluorescence substrate, 4-methylumbelliferyl-β-D-glucopyranoside (MUGlc). The specific activity of the crude recombinant enzyme for cellobiose hydrolysis was 18 U/mg.

Item Type:Article
Uncontrolled Keywords:Aspergillus niger; β-glucosidase; Pichia pastoris; heterologous expression; glycosyl hydrolase
Subjects:Q Science > QH Natural history > QH301 Biology
Divisions:Chemical and Natural Resources Engineering (Formerly known)
ID Code:8836
Deposited By: Pn Norhayati Abu Ruddin
Deposited On:15 May 2009 03:32
Last Modified:14 Oct 2010 04:49

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