Improving the thermostability of a GH97 dextran glucosidase by rational design
- PDF / 1,334,921 Bytes
- 11 Pages / 547.087 x 737.008 pts Page_size
- 48 Downloads / 189 Views
(0123456789().,-volV) ( 01234567 89().,-volV)
ORIGINAL RESEARCH PAPER
Improving the thermostability of a GH97 dextran glucosidase by rational design Xiaomin Zhang . Feiyun Chen . Chao He . Wei Fang . Zemin Fang . Xuecheng Zhang . Xiaotang Wang . Yazhong Xiao
Received: 11 December 2019 / Accepted: 29 May 2020 Ó Springer Nature B.V. 2020
Abstract This study was aimed at improving the thermostability of dextran glucosidase PspAG97A, a member of the glycoside hydrolase family 97, from Pseudoalteromonas sp. K8. A total of 9 lysine residues were chosen using the TKSA-MC program based on the optimization of surface charge-charge interactions and were mutated to glutamate for shifting the enzyme’s isoelectric point off its optimum pH value. Three mutants K75E, K363E and K420E showed enhanced thermostability. The triple mutant, K75E/ K363E/K420E, was found to be the best with a 7.3fold increase in half-life (t1/2) at 33 °C compared to
that of the wild-type (WT). Most importantly, this mutant showed comparable enzymatic activity to that of the WT protein. Structural modelling demonstrated that increased surface charge-charge interactions and optimization of surface hydrophobic and electrostatic contacts contributed to the improved thermostability displayed by K75E/K363E/K420E.
Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10529-020-02928-8) contains supplementary material, which is available to authorized users.
Introduction
X. Zhang F. Chen C. He (&) W. Fang Z. Fang X. Zhang Y. Xiao (&) School of Life Sciences, Anhui University, Hefei 230601, Anhui, China e-mail: [email protected] Y. Xiao e-mail: [email protected] X. Zhang F. Chen C. He W. Fang Z. Fang X. Zhang Y. Xiao Anhui Key Laboratory of Modern Biomanufacturing, Hefei 230601, Anhui, China X. Wang Department of Chemistry and Biochemistry, Florida International University, Miami, FL 33199, USA
Keywords Dextran glucosidase GH97 Surface charge–charge interactions Isoelectric point Thermostability improving Structural modelling
Microbial dextran is a homoglycan of a-D-glucopyranose molecule connected primarily by a-1,6 linkages, with various extents of branches via -1,2, -1,3, or -1,4 linkages to the main chain. Dextranases hydrolyze dextran to oligosaccharides and are widely used in pharmaceutical, sugar, and dental industries (Khalikova et al. 2005). In sugar industry, dextranase is used to reduce the viscosity and to increase sucrose recovery of juices (Jime´nez 2009). In pharmaceutical industry, dextranase can be used with commercial dextran to produce low molecular weight clinical grade dextran as plasma substitutes (Wu et al. 2011) or isomaltooligosaccharides as prebiotics (Gan et al. 2014; Goulas et al. 2004). In dental industry,
123
Biotechnol Lett
dextranase is added to oral care products, such as toothpaste and mouthwash, for degrading dextran in dental plaques and dislodging biofilm produced by Streptococcus mutans, a primary pathogen of dental caries (Pleszczynska et
Data Loading...
