Change of the Product Specificity of a Cyclodextrin Glucanotransferase by Semi-Rational Mutagenesis to Synthesize Large-Ring Cyclodextrins
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...
Main Authors: | , |
---|---|
Format: | Article |
Language: | English |
Published: |
MDPI AG
2019-03-01
|
Series: | Catalysts |
Subjects: | |
Online Access: | http://www.mdpi.com/2073-4344/9/3/242 |
id |
doaj-7b36de7ab8c1418ca4707af17fb929f0 |
---|---|
record_format |
Article |
spelling |
doaj-7b36de7ab8c1418ca4707af17fb929f02020-11-25T02:16:31ZengMDPI AGCatalysts2073-43442019-03-019324210.3390/catal9030242catal9030242Change of the Product Specificity of a Cyclodextrin Glucanotransferase by Semi-Rational Mutagenesis to Synthesize Large-Ring CyclodextrinsChristian Sonnendecker0Wolfgang Zimmermann1Department of Microbiology and Bioprocess Technology, Institute of Biochemistry, Leipzig University, Johannisallee 23, 04103 Leipzig, GermanyDepartment of Microbiology and Bioprocess Technology, Institute of Biochemistry, Leipzig University, Johannisallee 23, 04103 Leipzig, GermanyCyclodextrin 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.http://www.mdpi.com/2073-4344/9/3/242cyclodextrin glucanotransferaseslarge-ring cyclodextrinssemi rational mutagenesis |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
author |
Christian Sonnendecker Wolfgang Zimmermann |
spellingShingle |
Christian Sonnendecker Wolfgang Zimmermann Change of the Product Specificity of a Cyclodextrin Glucanotransferase by Semi-Rational Mutagenesis to Synthesize Large-Ring Cyclodextrins Catalysts cyclodextrin glucanotransferases large-ring cyclodextrins semi rational mutagenesis |
author_facet |
Christian Sonnendecker Wolfgang Zimmermann |
author_sort |
Christian Sonnendecker |
title |
Change of the Product Specificity of a Cyclodextrin Glucanotransferase by Semi-Rational Mutagenesis to Synthesize Large-Ring Cyclodextrins |
title_short |
Change of the Product Specificity of a Cyclodextrin Glucanotransferase by Semi-Rational Mutagenesis to Synthesize Large-Ring Cyclodextrins |
title_full |
Change of the Product Specificity of a Cyclodextrin Glucanotransferase by Semi-Rational Mutagenesis to Synthesize Large-Ring Cyclodextrins |
title_fullStr |
Change of the Product Specificity of a Cyclodextrin Glucanotransferase by Semi-Rational Mutagenesis to Synthesize Large-Ring Cyclodextrins |
title_full_unstemmed |
Change of the Product Specificity of a Cyclodextrin Glucanotransferase by Semi-Rational Mutagenesis to Synthesize Large-Ring Cyclodextrins |
title_sort |
change of the product specificity of a cyclodextrin glucanotransferase by semi-rational mutagenesis to synthesize large-ring cyclodextrins |
publisher |
MDPI AG |
series |
Catalysts |
issn |
2073-4344 |
publishDate |
2019-03-01 |
description |
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. |
topic |
cyclodextrin glucanotransferases large-ring cyclodextrins semi rational mutagenesis |
url |
http://www.mdpi.com/2073-4344/9/3/242 |
work_keys_str_mv |
AT christiansonnendecker changeoftheproductspecificityofacyclodextringlucanotransferasebysemirationalmutagenesistosynthesizelargeringcyclodextrins AT wolfgangzimmermann changeoftheproductspecificityofacyclodextringlucanotransferasebysemirationalmutagenesistosynthesizelargeringcyclodextrins |
_version_ |
1724890792421490688 |