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Abstract
Overexpression of a Chitinase Gene from the Thermophilic Fungus, Thermomyces lanuginosus in Saccharomyces cerevisiae and Characterization of the Recombinant Chitinase
A chitinase gene from the thermophilic fungus, Thermomyces lanuginosus ATCC 44008 has been cloned and overexpressed in Saccharomyces cerevisiae SEY 2101. The recombinant chitinase was produced as soluble secreted protein. The enzyme activity was found to be maximum on fourth day at 30°C in the induction medium. The overexpressed chitinase displayed optimum activity at pH 6.5 and at 60°C. The recombinant chitinase attributed remarkable thermostability by holding more than 60% of the enzyme activity after 6 h at 50°C. The molecular mass of the overexpressed chitinase was 42 kDa as measured by SDS-PAGE. The kinetic parameters such as KM and Vmax of the enzyme were found to be 0.403 mM and 8.74 mmoles/min/mg protein, respectively. Synthesis of the recombinant chitinase was strongly repressed by glucose. The eukaryotic translational inhibitor, cycloheximide in the induction medium showed 10% of higher activity whereas 30% of the activity was inhibited by transcriptional inhibitors, viz. 8-azaguanine and 8-hydroxyquinoline. The overexpressed recombinant chitinase may find potential application in pharmaceutical industry to prepare chitooligosaccharides.