IMPROVEMENT OF ENDOGLUCANASE ENZYME OPERATING PROPERTIES BY MUTATION IN AMINO ACID

Ethanol produced from plant cellulose is called bioethanol and is recognized as a unique sustainable liquid fuel with powerful economic and environmental effects. In the present study we aimed at integrate a cellulase gene in to yeast genome to have the enzyme secreted out of the cell. Subsequently cellulose is depredated to glucose by the enzyme، and then it is ferment to ethanol. Using site directed mutagenesis، one of free cysteines was replaced by a valine residue. The gene with codon optimization in respect to pichia pastoris was sub-cloned in to cloning plasmid، then for amplification it was transformed into E. coli. The plasmid was extracted and excised by restriction enzyme and then was sub-cloned in to expression vector. In the next step using electroporation، gene was transferred in to yeast and recombinant protein was expressed. Enzyme properties like temperature and pH optimum، specific activity، specific constant، and thermostability survived were assessed. Applied mutation did not have any effect on features like pH and temperature optimum but it led to an improvement in catalytic efficiency and thermostability. The mutant enzyme has better properties for industrial application.