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Research Project: Defining, Measuring, and Mitigating Attributes that Adversely Impact the Quality and Marketability of Foods

Location: Healthy Processed Foods Research

Title: Maltose binding site 2 mutations affect product inhibition of bacilluscirculans stb01 cyclodextrin glycosyltransferase

Author
item LI, CAIMING - Jiangnan University
item YOU, YUXIAN - Jiangnan University
item Zhang, Yuzhu
item XIE, XIAOFANG - Jiangnan University
item XU, QI - Jiangnan University
item GU, ZHENGBIAO - Jiangnan University
item BAN, XIAOFENG - Jiangnan University
item TANG, XIAOSHU - Jiangnan University
item HONG, YAN - Jiangnan University
item CHENG, LI - Jiangnan University
item LI, ZHAOFENG - Jiangnan University

Submitted to: International Journal of Biological Macromolecules
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/4/2021
Publication Date: 4/6/2021
Citation: Li, C., You, Y., Zhang, Y., Xie, X., Xu, Q., Gu, Z., Ban, X., Tang, X., Hong, Y., Cheng, L., Li, Z. 2021. Maltose binding site 2 mutations affect product inhibition of Bacilluscirculans stb01 cyclodextrin glycosyltransferase. International Journal of Biological Macromolecules. 175:254-261. https://doi.org/10.1016/j.ijbiomac.2021.02.033.
DOI: https://doi.org/10.1016/j.ijbiomac.2021.02.033

Interpretive Summary: Cyclodextrins are a family of cyclic oligosaccharides. The most common members of this family are alpha-, beta- and gamma-cyclodextrin, which consist of 6, 7, and 8 glucose units, respectively. Cyclodextrins possess unique cavities that can accommodate a variety of hydrophobic molecules. Thus, cyclodextrins are used in food, pharmaceutical, cosmetics, and many other industries to improve the properties of small molecules. Cyclodextrin glycosyltransferases (CGTase)catalyze the conversion of starch or starch derivatives to alpha-, beta- and gamma- cyclodextrins. The enzymes are used to produce cyclodextrins. However, as the conversion progresses, product inhibition severely diminishes the cyclization activity to the proteins and decreases the reaction output. Thus limiting the industrial application of cyclodextrins. Therefore, there is a need to reduce product inhibition of CGTase for expanding the use of cyclodextrins. In this study, the maltose-binding site 2 of the ß-CGTase from Bacillus circulans STB01 was targeted for mutation. One of the mutants showed a 20% increase over the wildtype enzyme in cyclodextrin production. The results and information may serve as a new starting point for a better understanding of the structure, function, and industrial application of CGTases and relative enzymes.

Technical Abstract: In this study, the maltose binding site 2 (MBS2) of the ß-cyclodextrin glycosyltransferase (ß-CGTase) from bacillus circulans STB01 was modified to decrease product inhibition. First, two point mutants were prepared at position 599 (A599V and A599N). Then, the entire MBS2 region was replaced by that of the a-CGTase from Paenibacillus macerans JFB05-01 to form the mutant MBS2 ß'a. All three mutants exhibited mixed-type product inhibition, although both the competitive and uncompetitive components of this inhibition were decreased. The cyclization activities of A599N and A599V were 15.6% and 76.8% lower than that of the wild-type, respectively, while that of MBS2 ß'a was 4.3% higher. When allowed to incubate for 24 h, A599N and MBS2 ß'a produced cyclodextrin yields 13.1% and 19.7% greater than that of the wild-type, respectively. These results suggest that A599N and MBS2 ß'a may be more suitable than the wild-type for cyclodextrin production.