Malaysian Applied Biology Journal

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44_4_03

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Malays. Appl. Biol. (2015) 44(4): 19-26

CLONING, HETEROLOGOUS EXPRESSION AND  CHARACTERISATION OF A RECOMBINANT CELLOBIOHYDROLASE FROM Humicola insolens ATCC16454 IN Pichia pastoris


AMATUL SAMAHAH MD ALI1, FARAH DIBA ABU BAKAR1, ROSLI MD ILLIAS2, OSMAN HASSAN3 AND ABDUL MUNIR ABDUL MURAD1,*


1 School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia

2 Department of Bioprocess Engineering, Faculty of Chemical and Natural Resources Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia

3 School of Chemical Sciences and Food Technology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia

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ABSTRACT

 

A cellobiohydrolase gene from the thermophilic fungus Humicola insolens ATCC 16454 was expressed in the methylotrophic yeast Pichia pastoris X-33, and the biochemical properties of the recombinant protein were characterised. The full-length cDNA of the cellobiohydrolase gene avi2 was cloned into the P. pastoris expression vector pPICZ?C and expressed extracellularly as a recombinant cellobiohydrolase protein with a molecular weight of approximately 52.3 kDa. The purified recombinant Avi2 enzyme displayed an optimal activity at 50°C and was found stable between temperatures of 30°C and 60°C. The optimal pH of the enzyme was pH 5.0. More than 80% of the enzyme activity was retained at pH values ranging from pH3.0 to pH9.0. Recombinant Avi2 enzyme showed its highest activity towards the substrates Avicel (0.075 U mg-1) and Sigmacell-cellulose (0.018 U mg-1). Very low or undetectable hydrolysis was observed with cellobiose and filter paper. Metal ions, such as Mn2+, Co2+, and Ba2+, increased the activity of the recombinant enzyme. Manganese ions caused the highest increase in activity of approximately 1.38-fold compared to the control assay. Other ions such as Pd2+, Cu2+, Zn2+, Fe2+, and SDS, however, inhibited Avi2 enzyme activity. Interestingly, this recombinant enzyme showed high pH stability when it was incubated in either acidic or basic solutions.

Keywords: Cellobiohydrolase, Humicola insolens, Pichia pastoris, recombinant enzyme

 

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