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Báo cáo Y học: Expression and characterization of active site mutants of hevamine, a chitinase from the rubber tree Hevea brasiliensis

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Hevamine is a chitinase from the rubber tree Hevea brasiliensis. Its active site contains Asp125, Glu127, and Tyr183, which interact with the )1 sugar residue of the substrate. To investigate their role in catalysis, we have successfully expressed wild-type enzyme and mutants of these residues as inclusion bodies in Escherichia coli. After refolding and purification they were characterized by both structural and enzyme kinetic studies. Mutation of Tyr183 to phenylalanine produced an enzyme with a lower kcat and a slightly higher Km than the wild-type enzyme. Mutating Asp125 and Glu127 to alanine gave mutants with 2% residual activity

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