title: Directed evolution of endoglucanase III (Cel12A) from Trichoderma reesei.
abstract: The stability and specific activity of endo-beta-1,4-glucanase III from Trichoderma reesei QM9414 was enhanced, and the expression efficiency of its encoding gene, egl3, was optimized by directed evolution using error-prone PCR and activity screening in Escherichia coli RosettaBlue (DE3) pLacI as a host. Relationship between increase in yield of active enzyme in the clones and improvement in its stability was observed among the mutants obtained in the present study. The clone harboring the best mutant 2R4 (G41E/T110P/K173M/Y195F/P201S/N218I) selected in via second-round mutagenesis after optimal recombinating of first-round mutations produced 130-fold higher amount of mutant enzyme than the transformant with wild-type EG III. Mutant 2R4 produced by the clone showed broad pH stability (4.4-8.8) and thermotolerance (entirely active at 55 degrees C for 30 min) compared with those of the wild-type EG III (pH stability, 4.4-5.2; thermostability, inactive at 55 degrees C for 30 min). k (cat) of 2R4 against carboxymethyl-cellulose was about 1.4-fold higher than that of the wild type, though the K (m) became twice of that of the wild type.
authors.affiliation: Department of Bioengineering, Nagaoka University of Technology, Japan.
authors.forename: Hikaru
authors.initials: H
authors.lastname: Nakazawa
authors.forename: Katsunori
authors.initials: K
authors.lastname: Okada
authors.forename: Tomoko
authors.initials: T
authors.lastname: Onodera
authors.forename: Wataru
authors.initials: W
authors.lastname: Ogasawara
authors.forename: Hirofumi
authors.initials: H
authors.lastname: Okada
authors.forename: Yasushi
authors.initials: Y
authors.lastname: Morikawa
mesh_terms: D000818:Animals; D002266:Carboxymethylcellulose Sodium / Q000378:metabolism; D002415:Cats; D002480:Cellulase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004271:DNA, Fungal / Q000235:genetics; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D016133:Polymerase Chain Reaction / Q000379:methods; D013379:Substrate Specificity; D013696:Temperature; D014242:Trichoderma / Q000201:enzymology*
chemical_list: D004271:DNA, Fungal; D005656:Fungal Proteins; D002480:Cellulase; D002266:Carboxymethylcellulose Sodium