title: Engineering endoglucanase II from Trichoderma reesei to improve the catalytic efficiency at a higher pH optimum.
abstract: The catalytic efficiency and pH optimum of Trichoderma reesei endo-beta-1,4-glucanase II were improved by protein engineering. We subjected residue 342 to saturation mutagenesis, and further changed the enzyme by random mutagenesis and two rounds of DNA shuffling. Enzyme variants were purified and characterized. Variant N342V exhibited an optimal activity at pH 5.8, corresponding to a basic shift of 1 pH unit compared with the wild-type enzyme, and had improved catalytic efficiency (1.5-fold of k(cat)/K(m)) for the main substrates at pH 6.2. Variants N342R and N39R/L218H/W276R/N342T both had a pH optimum of 6.2 and the latter had improved catalytic efficiency (1.4-fold of k(cat)/K(m)) at pH 6.2. Variants L218H, Q139R/N342T and Q139R/L218H/W276R/N342T all had more than 4.5-fold higher activity in reactions compared with the wild-type at pH 7.0. The relationship between the structures and the activities of the variants were analyzed by modeling the structures of the endoglucanase II variants. More stable helixes and changed electrostatic interactions between the catalytic residues and substrates may explain the higher activities and higher pH optima of the variants.
authors.affiliation: State Key Laboratory of Microbial Technology, Shandong University, Shanda Nan Road 27, Jinan 250100, PR China.
authors.forename: Yuqi
authors.initials: Y
authors.lastname: Qin
authors.forename: Xiaomin
authors.initials: X
authors.lastname: Wei
authors.forename: Xin
authors.initials: X
authors.lastname: Song
authors.forename: Yinbo
authors.initials: Y
authors.lastname: Qu
mesh_terms: D019943:Amino Acid Substitution; D002384:Catalysis; D002480:Cellulase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019020:Directed Molecular Evolution; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D016296:Mutagenesis; D009154:Mutation; D013329:Structure-Activity Relationship; D014242:Trichoderma / Q000201:enzymology*
chemical_list: C056373:endoglucanase 2; D002480:Cellulase