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逐步易错PCR和DNA改组改变了嗜碱芽孢杆菌环糊精葡糖基转移酶的pH活性范围和产物特异性。

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

作者信息

Melzer Susanne, Sonnendecker Christian, Föllner Christina, Zimmermann Wolfgang

机构信息

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

出版信息

FEBS Open Bio. 2015 Jun 11;5:528-34. doi: 10.1016/j.fob.2015.06.002. eCollection 2015.

DOI:10.1016/j.fob.2015.06.002
PMID:26155461
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC4491590/
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.

摘要

来自嗜碱芽孢杆菌G-825-6的环糊精葡糖基转移酶（EC 2.4.1.19）主要将淀粉转化为γ-环糊精（CD8）。采用易错PCR和DNA改组相结合的方法，获得了对CD8具有更高产物特异性和更宽pH活性范围的该酶变体。具有七个氨基酸取代的变体S54的CD8合成活性提高了1.2倍，与野生型酶的1:3相比，CD7:CD8的产物比例变为1:7。对环糊精葡糖基转移酶进行了九个氨基酸取代，以产生在pH 4.0-10.0范围内具有活性的变体S35。与在pH 6.0以下无活性的野生型酶相比，S35在pH 4.0时保留了其70%的CD8合成活性。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7605/4491590/d5ef3a41cb6e/gr2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7605/4491590/5384d0e52240/fx1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7605/4491590/81bff10b32e5/gr1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7605/4491590/d5ef3a41cb6e/gr2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7605/4491590/5384d0e52240/fx1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7605/4491590/81bff10b32e5/gr1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/7605/4491590/d5ef3a41cb6e/gr2.jpg

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