title: Enhanced activity toward PET by site-directed mutagenesis of Thermobifida fusca cutinase-CBM fusion protein.
abstract: In the present study, cutinase-CBMCenA fusion protein was genetically modified in the carbohydrate-binding module (CBM) binding sites, by site-directed mutagenesis, to enhance its activity toward polyethylene terephthalate (PET) fiber. The effects of tryptophan at particular positions of CBMCenA on the binding and hydrolysis of polyester substrate were investigated by replacing each of Trp14, Trp50 and Trp68 with leucine or tyrosine, respectively. All the mutants were expressed in Escherichia coli and purified to homogeneity. Enzyme characterization showed that the mutants displayed similar thermostability and pH stabilities in response to the native enzyme. Furthermore, W68L and W68Y, among all the mutants, exhibited significant improvement in binding and catalytic efficiency (1.4-1.5 fold) toward PET fiber when compared to that of the native enzyme. The enhanced binding and hydrolytic activity might be a result of creating new hydrogen bond or hydrophobic interaction between the enzyme and PET fiber.
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China.
authors.forename: Yao
authors.initials: Y
authors.lastname: Zhang
authors.forename: Lei
authors.initials: L
authors.lastname: Wang
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
authors.forename: Jing
authors.initials: J
authors.lastname: Wu
mesh_terms: D000192:Actinomycetales / Q000201:enzymology; D001665:Binding Sites; D055162:Biocatalysis; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D057927:Hydrophobic and Hydrophilic Interactions; D016297:Mutagenesis, Site-Directed; D011093:Polyethylene Terephthalates / Q000737:chemistry*; D055550:Protein Stability; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D011093:Polyethylene Terephthalates; D011993:Recombinant Fusion Proteins; D002265:Carboxylic Ester Hydrolases; C019952:cutinase