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Melzer S, Sonnendecker C, Föllner C, et al. Stepwise error-prone PCR and DNA shuffling changed the pH activity range and product specificity of the cyclodextrin glucanotransferase from an alkaliphilic Bacillus sp. FEBS Open Bio. 2015;5:528-34doi: 10.1016/j.fob.2015.06.002.
Melzer, S., Sonnendecker, C., Föllner, C., & Zimmermann, W. (2015). Stepwise error-prone PCR and DNA shuffling changed the pH activity range and product specificity of the cyclodextrin glucanotransferase from an alkaliphilic Bacillus sp. FEBS open bio, 5528-34. https://doi.org/10.1016/j.fob.2015.06.002
Melzer, Susanne, et al. "Stepwise error-prone PCR and DNA shuffling changed the pH activity range and product specificity of the cyclodextrin glucanotransferase from an alkaliphilic Bacillus sp." FEBS open bio vol. 5 (2015): 528-34. doi: https://doi.org/10.1016/j.fob.2015.06.002
Melzer S, Sonnendecker C, Föllner C, Zimmermann W. Stepwise error-prone PCR and DNA shuffling changed the pH activity range and product specificity of the cyclodextrin glucanotransferase from an alkaliphilic Bacillus sp. FEBS Open Bio. 2015 Jun 11;5:528-34. doi: 10.1016/j.fob.2015.06.002. eCollection 2015. PMID: 26155461; PMCID: PMC4491590.
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FEBS Open Bio. 2015 Jun 11;5:528-34. doi: 10.1016/j.fob.2015.06.002. eCollection 2015.

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

FEBS open bio

Susanne Melzer, Christian Sonnendecker, Christina Föllner, Wolfgang Zimmermann

Affiliations

  1. Institute of Biochemistry, Department of Microbiology and Bioprocess Technology, Leipzig University, Johannisallee 23, 04103 Leipzig, Germany.

PMID: 26155461 PMCID: PMC4491590 DOI: 10.1016/j.fob.2015.06.002

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.

Keywords: Bacillus sp.; CD, cyclodextrin; CGTase, cyclodextrin glucanotransferase; Cyclodextrin glucanotransferase; DNA shuffling; Gamma-cyclodextrin; Random mutagenesis

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