Contribution of Disulfide Bridges to the Thermostability of a Type A Feruloyl Esterase from Aspergillus usamii

Xin Yin; Die Hu; Jian-Fang Li; Yao He; Tian-Di Zhu; Min-Chen Wu

doi:10.1371/journal.pone.0126864

Contribution of Disulfide Bridges to the Thermostability of a Type A Feruloyl Esterase from Aspergillus usamii

PLoS One. 2015 May 13;10(5):e0126864. doi: 10.1371/journal.pone.0126864. eCollection 2015.

Authors

Xin Yin¹, Die Hu¹, Jian-Fang Li², Yao He¹, Tian-Di Zhu¹, Min-Chen Wu³

Affiliations

¹ Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.
² State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, China.
³ Wuxi Medical School, Jiangnan University, Wuxi, China.

Abstract

The contribution of disulfide bridges to the thermostability of a type A feruloyl esterase (AuFaeA) from Aspergillus usamii E001 was studied by introducing an extra disulfide bridge or eliminating a native one from the enzyme. MODIP and DbD, two computational tools that can predict the possible disulfide bridges in proteins for thermostability improvement, and molecular dynamics (MD) simulations were used to design the extra disulfide bridge. One residue pair A126-N152 was chosen, and the respective amino acid residues were mutated to cysteine. The wild-type AuFaeA and its variants were expressed in Pichia pastoris GS115. The temperature optimum of the recombinant (re-) AuFaeAA126C-N152C was increased by 6°C compared to that of re-AuFaeA. The thermal inactivation half-lives of re-AuFaeAA126C-N152C at 55 and 60°C were 188 and 40 min, which were 12.5- and 10-folds longer than those of re-AuFaeA. The catalytic efficiency (kcat/Km) of re-AuFaeAA126C-N152C was similar to that of re-AuFaeA. Additionally, after elimination of each native disulfide bridge in AuFaeA, a great decrease in expression level and at least 10°C decrease in thermal stability of recombinant AuEaeA variants were also observed.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Substitution
Aspergillus / enzymology*
Carboxylic Ester Hydrolases / chemistry*
Carboxylic Ester Hydrolases / genetics
Cystine / chemistry
Enzyme Stability
Fungal Proteins / chemistry*
Fungal Proteins / genetics
Half-Life
Hot Temperature
Kinetics
Mutagenesis, Site-Directed
Pichia

Substances

Fungal Proteins
Cystine
Carboxylic Ester Hydrolases
feruloyl esterase

Grants and funding

This work was financially supported by the Fundamental Research Fund for the Central Universities of China (No. JUDCF13011, JUSRP51412B) (http://yjsb.jiangnan.edu.cn/, http://www.moe.gov.cn/) and the Postgraduate Innovation Training Project of Jiangsu (No. CXZZ13_0757) (http://www.ec.js.edu.cn/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.