title: Removal of the free cysteine residue reduces irreversible thermal inactivation of feruloyl esterase: evidence from circular dichroism and fluorescence spectra.
abstract: Feruloyl esterase A from Aspergillus niger (AnFaeA) contains three intramolecular disulfide bonds and one free cysteine at position 235. Saturated mutagenesis at Cys235 was carried out to produce five active mutants, all of which displayed unusual thermal inactivation patterns with the most residual activity achieved at 75°C, much higher than the parental AnFaeA. But their optimal reaction temperatures were lower than the parental AnFaeA. Extensive investigation into their free thiol and disulfide bond, circular dichroism spectra and fluorescence spectra revealed that the unfolding of the parental enzyme was irreversible on all the tested conditions, while that of the Cys235 mutants was reversible, and their ability to refold was highly dependent on the denaturing temperature. Mutants denatured at 75°C were able to efficiently reverse the unfolding to regain native structure during the cooling process. This study provided valid evidence that free cysteine substitutions can reduce irreversible thermal inactivation of proteins.
authors.affiliation: Key Laboratory of Environmental and Applied Microbiology, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China Environmental Microbiology Key Laboratory of Sichuan Province, Chengdu 610041, China University of Chinese Academy of Sciences, Beijing 10049, China.
authors.forename: Jingjing
authors.initials: J
authors.lastname: Li
authors.affiliation: College of Bioengineering, Henan University of Technology, Zhengzhou 450001, China.
authors.forename: Shuaibing
authors.initials: S
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Environmental and Applied Microbiology, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China.
authors.forename: Zhuolin
authors.initials: Z
authors.lastname: Yi
authors.affiliation: Key Laboratory of Environmental and Applied Microbiology, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China Environmental Microbiology Key Laboratory of Sichuan Province, Chengdu 610041, China University of Chinese Academy of Sciences, Beijing 10049, China.
authors.forename: Xiaoqiong
authors.initials: X
authors.lastname: Pei
authors.affiliation: Key Laboratory of Environmental and Applied Microbiology, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China Environmental Microbiology Key Laboratory of Sichuan Province, Chengdu 610041, China wuzhl@cib.ac.cn.
authors.forename: Zhongliu
authors.initials: Z
authors.lastname: Wu
mesh_terms: D019943:Amino Acid Substitution; D001234:Aspergillus niger / Q000201:enzymology*; D002265:Carboxylic Ester Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D003545:Cysteine; D004789:Enzyme Activation; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013696:Temperature
chemical_list: D005656:Fungal Proteins; D002265:Carboxylic Ester Hydrolases; C070196:feruloyl esterase; D003545:Cysteine
keywords: circular dichroism; disulfide bond; feruloyl esterase; free cysteine; intrinsic fluorescence