Authors: Jian Tian Ping Wang Lu Huang Xiaoyu Chu Ningfeng Wu Yunliu Fan
Publish Date: 2012/09/22
Volume: 97, Issue: 7, Pages: 2997-3006
Abstract
Good protein thermostability is very important for the protein application In this report we propose a strategy which contained a prediction method to select residues related to protein thermal stability but not related to protein function and an experiment method to screen the mutants with enhanced thermostability The prediction strategy was based on the calculated site evolutionary entropy and unfolding free energy difference between the mutant and wildtype WT methyl parathion hydrolase enzyme from Ochrobactrum sp M231 Ochrmethyl parathion hydrolase MPH As a result seven amino acid sites within OchrMPH were selected and used to construct seven saturation mutagenesis libraries The results of screening these libraries indicated that six sites could result in mutated enzymes exhibiting better thermal stability than the WT enzyme A stepwise evolutionary approach was designed to combine these selected mutants and a mutant with four point mutations S274Q/T183E/K197L/S192M was selected The T m and T 50 of the mutant enzyme were 117 and 102 °C higher respectively than that of the WT enzyme The success of this design methodology for OchrMPH suggests that it was an efficient strategy for enhancing protein thermostability and suitable for protein engineering
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