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Catalysts 2019, 9(3), 242; https://doi.org/10.3390/catal9030242

Change of the Product Specificity of a Cyclodextrin Glucanotransferase by Semi-Rational Mutagenesis to Synthesize Large-Ring Cyclodextrins

Department of Microbiology and Bioprocess Technology, Institute of Biochemistry, Leipzig University, Johannisallee 23, 04103 Leipzig, Germany
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Received: 11 February 2019 / Revised: 28 February 2019 / Accepted: 1 March 2019 / Published: 6 March 2019
(This article belongs to the Special Issue Novel Enzyme and Whole-Cell Biocatalysts)
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Abstract

Cyclodextrin glucanotransferases (CGTases) convert starch to cyclodextrins (CD) of various sizes. To engineer a CGTase for the synthesis of large-ring CD composed of 9 to 12 glucose units, a loop structure of the protein involved in substrate binding was targeted for semi-rational mutagenesis. Based on multiple protein alignments and protein structure information, a mutagenic megaprimer was designed to encode a partial randomization of eight amino acid residues within the loop region. The library obtained encoding amino acid sequences occurring in wild type CGTases in combination with a screening procedure yielded sequences displaying a changed CD product specificity. As a result, variants of the CGTase from the alkaliphilic Bacillus sp. G825-6 synthesizing mainly CD9 to CD12 could be obtained. When the mutagenesis experiment was performed with the CGTase G825-6 variant Y183R, the same loop alterations that increased the total CD synthesis activity resulted in lower activities of the variant enzymes created. In the presence of the amino acid residue R183, the synthesis of CD8 was suppressed and larger CD were obtained as the main products. The alterations not only affected the product specificity, but also influenced the thermal stability of some of the CGTase variants indicating the importance of the loop structure for the stability of the CGTase. View Full-Text
Keywords: cyclodextrin glucanotransferases; large-ring cyclodextrins; semi rational mutagenesis cyclodextrin glucanotransferases; large-ring cyclodextrins; semi rational mutagenesis
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited (CC BY 4.0).

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Sonnendecker, C.; Zimmermann, W. Change of the Product Specificity of a Cyclodextrin Glucanotransferase by Semi-Rational Mutagenesis to Synthesize Large-Ring Cyclodextrins. Catalysts 2019, 9, 242.

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