Open AccessStepwise error‐prone PCR and DNA shuffling changed the pH activity range and product specificity of the cyclodextrin glucanotransferase from an alkaliphilic Bacillus sp
Author(s) -
Melzer Susanne,
Sonnendecker Christian,
Föllner Christina,
Zimmermann Wolfgang
Publication year - 2015
Publication title -
febs open bio
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.718
H-Index - 31
ISSN - 2211-5463
DOI - 10.1016/j.fob.2015.06.002
Subject(s) - dna shuffling , cyclodextrin , enzyme , biochemistry , starch , chemistry , dna , biology , amino acid , gene , directed evolution , mutant
Cyclodextrin glucanotransferase (EC 2.4.1.19) from the alkaliphilic Bacillus sp. G‐825‐6 converts starch mainly to γ‐cyclodextrin (CD 8 ). A combination of error‐prone PCR and DNA shuffling was used to obtain variants of this enzyme with higher product specificity for CD 8 and a broad pH activity range. The variant S54 with seven amino acid substitutions showed a 1.2‐fold increase in CD 8 ‐synthesizing activity and the product ratio of CD 7 :CD 8 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 CD 8 ‐synthesizing activity at pH 4.0.