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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: Rational design of disulfide bonds for enhancing the thermostability of the 1,4-a-glucan branching enzyme from geobacillus thermoglucosidasius stb02

item LI, CAIMING - Jiangnan University
item BAN, XIAOFENG - Jiangnan University
item Zhang, Yuzhu
item GU, ZHENGBIAO - Jiangnan University
item HONG, YAN - Jiangnan University
item CHENG, LI - Jiangnan University
item LI, ZHAOFENG - Jiangnan University

Submitted to: Journal of Agricultural and Food Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/29/2020
Publication Date: 11/9/2020
Citation: Li, C., Ban, X., Zhang, Y., Gu, Z., Hong, Y., Cheng, L., Li, Z. 2020. Rational design of disulfide bonds for enhancing the thermostability of the 1,4-a-glucan branching enzyme from geobacillus thermoglucosidasius stb02. Journal of Agricultural and Food Chemistry. 68(47):13791-13797.

Interpretive Summary: There is a constant need to improve the stability and functionality of proteins with industrial use. However, the stability of proteins is poorly understood. Thus, proteins from thermophilic microbial organisms are a source of enzymes with potential widespread applications in research and the food industry. Geobacillus thermoglucosidasius STB02 is such an organism, from which a 1,4-a-glucan branching enzyme (GBE) has been isolated. GBE catalyzes the formation of branch points in liner polysaccharide chains. Branching of the polysaccharide can improve the physical and chemical properties of carbohydrates, including their solubility and degradation. To increase the utility of GBE from STB02, it is desirable to increase the stability of the enzyme. One of the natural attributes that help to stabilize proteins is the formation of disulfide bonds. In this study, we used bioinformatics based in silico design to introduced disulfide bonds in GBE. Recombinant expression was used to produce the mutants, and their effects on the specific enzyme activity and thermostability were tested experimentally. Three mutants were found to have better properties than the wild type enzyme. The results increased the applicability of the GBE in the food industry and suggested a possible strategy for introducing stabilizing disulfide bonds in proteins.

Technical Abstract: Disulfide bonds play crucial roles in thermostabilization, recognition, or activation of proteins. They are vital in holding respective conformations of globular structures, thereby enhancing thermostability. Bioinformatics approaches provide effective strategies to build disulfide bonds based on known structural information. We constructed nine mutants by rational analysis of 1,4-a-glucan branching enzyme (GBE, EC from Geobacillus thermoglucosidasius STB02 that catalyzes the synthesis of a-1,6-glucosidic bonds through acting upon a-(1,4) and/or a-(1,6) glucosidic linkages. Four of them enhanced thermostability, and five of them had adverse or negligible effects on stability. The circular dichroism (CD) spectra and intrinsic fluorescence analysis showed that introducing disulfide bonds might only affect secondary structures. The results also demonstrated that not only the distances of the Ca carbons and the thiol groups but also the sequence between the two cysteines need to be considered for designing disulfide bonds.