title: Contribution of Disulfide Bridges to the Thermostability of a Type A Feruloyl Esterase from Aspergillus usamii.
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.
authors.affiliation: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.
authors.forename: Xin
authors.initials: X
authors.lastname: Yin
authors.affiliation: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.
authors.forename: Die
authors.initials: D
authors.lastname: Hu
authors.affiliation: State Key Laboratory of Food Science and Technology, School of Food Science and Technology, Jiangnan University, Wuxi, China.
authors.forename: Jian-Fang
authors.initials: JF
authors.lastname: Li
authors.affiliation: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.
authors.forename: Yao
authors.initials: Y
authors.lastname: He
authors.affiliation: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.
authors.forename: Tian-Di
authors.initials: TD
authors.lastname: Zhu
authors.affiliation: Wuxi Medical School, Jiangnan University, Wuxi, China.
authors.forename: Min-Chen
authors.initials: MC
authors.lastname: Wu
mesh_terms: D019943:Amino Acid Substitution; D001230:Aspergillus / Q000201:enzymology*; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics; D003553:Cystine / Q000737:chemistry; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics; D006207:Half-Life; D006358:Hot Temperature; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D010843:Pichia
chemical_list: D005656:Fungal Proteins; D003553:Cystine; D002265:Carboxylic Ester Hydrolases; C070196:feruloyl esterase