Modification of the Catalytic Properties of β-cyclodextrin Glucosyltransferase through Error-prone PCR
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Abstract:
β-cyclodextrin is a cyclic polysaccharide that can be used to alter the physical and chemical properties of certain macromolecular compounds. It has remarkable industrial application prospects in the fields of food, biology, and medicine. β-cyclodextrin is obtained by converting polysaccharide compounds containing glucose groups, such as starch, by the action of β-cyclodextrin glucosyltransferase (β-CGTase). In the present study, β-CGTase derived from Paenibacillus campinasensis was subjected to directed evolution through error-prone PCR to obtain a mutant with potent enzymatic activity. The mutants were subjected to nickel column affinity purification and analyzed for their enzymatic properties. Finally, the mutant Q280L was obtained. Compared with that of the wildtype β-CGTase, the enzymatic activity of Q280L was increased by 42.10%, conversion rate of β-cyclodextrin was increased by 7.6%, its optimal reaction pH and pH stability were shifted, and its substrate affinity was increased by 46.13%. Sequence alignment and structural analysis revealed that compared with that in wildtype β-CGTase, the amino acid residue at position 280 and the hydrogen bonding interaction between this and its surrounding residues were altered in Q280L. These findings indicate that directed evolution of β-CGTase based on error-prone PCR could improve enzymatic activity and properties, thereby providing a reference for the industrial production of β-cyclodextrin.
