4.7 Article

Increasing γ-CD conversion rates by improving thermostability of Bacillus sp. FJAT-44876 γ-CGTase

Journal

FOOD BIOSCIENCE
Volume 51, Issue -, Pages -

Publisher

ELSEVIER
DOI: 10.1016/j.fbio.2022.102204

Keywords

Starch; Protein engineering; Three-dimensional structure

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Poor thermostability of gamma-CGTase restricts its application in starch utilization and gamma-CD yield. By adding Ca2+ and conducting site-directed mutagenesis, the thermostability of Bacillus sp. FJAT-44876 gamma-CGTase (BF gamma-CGTase) was improved. The addition of 10 mM Ca2+ increased the half-life (t1/2) at 55 and 60 degrees C from 3.0 to 0.3 h to 17.4 and 2.0 h, respectively. Ca2+ stabilized the tertiary structure of BF gamma-CGTase, leading to improved thermostability. A mutation to serine in an alpha-helix glycine residue also enhanced stability, especially at higher temperatures. Furthermore, the G208S mutant in the presence of 10 mM Ca2+ exhibited increased t1/2 and yield of gamma-CD production from tapioca starch. This study highlights the importance of rational protein engineering in enhancing enzymatic gamma-CD production.
Poor thermostability is a limiting factor for applications of gamma-CGTase that affects starch utilization and yield of gamma-CD. Here thermostability of Bacillus sp. FJAT-44876 gamma-CGTase (BF gamma-CGTase) was improved by addition of Ca2+ and site-directed mutagenesis. Thus, 10 mM Ca2+ increased the half-life (t1/2) at 55 and 60 degrees C from 3.0 to 0.3 h to 17.4 and 2.0 h, respectively. Fluorescence spectra indicated that Ca2+ stabilized the tertiary structure of the BF gamma-CGTase and hence improved the thermostability. Mutation to serine of a glycine residue in an alpha-helix related to thermostability also improved the stability especially at the higher temperature. Importantly, in 10 mM Ca2+ the G208S mutant further increased t1/2 to 20.0 h and 4.0 h at 55 and 60 degrees C, respectively. The G208S mutant in 10 mM Ca2+ produced gamma-CD from tapioca starch with 40% increased yield of that from BF gamma-CGTase. This work involving rational protein engineering provided a new tool for enzymatic gamma-CD production.

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