title: Stepwise error-prone PCR and DNA shuffling changed the pH activity range and product specificity of the cyclodextrin glucanotransferase from an alkaliphilic Bacillus sp.
abstract: Cyclodextrin glucanotransferase (EC 2.4.1.19) from the alkaliphilic Bacillus sp. G-825-6 converts starch mainly to γ-cyclodextrin (CD8). A combination of error-prone PCR and DNA shuffling was used to obtain variants of this enzyme with higher product specificity for CD8 and a broad pH activity range. The variant S54 with seven amino acid substitutions showed a 1.2-fold increase in CD8-synthesizing activity and the product ratio of CD7:CD8 was shifted to 1:7 compared to 1:3 of the wild-type enzyme. Nine amino acid substitutions of the cyclodextrin glucanotransferase were performed to generate the variant S35 active in a pH range 4.0-10.0. Compared to the wild-type enzyme which is inactive below pH 6.0, S35 retained 70% of its CD8-synthesizing activity at pH 4.0.
authors.affiliation: Institute of Biochemistry, Department of Microbiology and Bioprocess Technology, Leipzig University, Johannisallee 23, 04103 Leipzig, Germany.
authors.forename: Susanne
authors.initials: S
authors.lastname: Melzer
authors.affiliation: Institute of Biochemistry, Department of Microbiology and Bioprocess Technology, Leipzig University, Johannisallee 23, 04103 Leipzig, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Sonnendecker
authors.affiliation: Institute of Biochemistry, Department of Microbiology and Bioprocess Technology, Leipzig University, Johannisallee 23, 04103 Leipzig, Germany.
authors.forename: Christina
authors.initials: C
authors.lastname: Föllner
authors.affiliation: Institute of Biochemistry, Department of Microbiology and Bioprocess Technology, Leipzig University, Johannisallee 23, 04103 Leipzig, Germany.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Zimmermann
keywords: Bacillus sp.; CD, cyclodextrin; CGTase, cyclodextrin glucanotransferase; Cyclodextrin glucanotransferase; DNA shuffling; Gamma-cyclodextrin; Random mutagenesis