Stepwise error-prone PCR and DNA shuffling changed the pH activity range and product specificity of the cyclodextrin glucanotransferase from an alkaliphilic Bacillus sp.

• Main Authors: Melzer, Susanne, Sonnendecker, Christian, Föllner, Christina, Zimmermann, Wolfgang
• Other Authors: Universität Leipzig, Institut für Biochemie
• Format: Article
• Language: English
• Published: Universitätsbibliothek Leipzig 2015
• Subjects: Cyclodextrin-Glucosyl-Transferase; Bacillus sp.; Gamma-Cyclodextrin; zufällige Mutagenese; DNA-Shuffling; Cyclodextrin glucanotransferase; Gamma-cyclodextrin; Random mutagenesis; DNA shuffling; ddc:572
• Online Access: http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-172353; http://nbn-resolving.de/urn:nbn:de:bsz:15-qucosa-172353; http://www.qucosa.de/fileadmin/data/qucosa/documents/17235/OAP-2015-104-Zimmermann_1-s2.0-S221154631500056X-main.pdf

Cyclodextrin glucanotransferase (EC 2.4.1.19) from the alkaliphilic Bacillus sp. G-825-6 converts starch mainly to c-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.