Improving the thermostability of methyl parathion hydrolase from Ochrobactrum sp. M231 using a computationally aided met
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APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY
Improving the thermostability of methyl parathion hydrolase from Ochrobactrum sp. M231 using a computationally aided method Jian Tian & Ping Wang & Lu Huang & Xiaoyu Chu & Ningfeng Wu & Yunliu Fan
Received: 22 June 2012 / Revised: 28 August 2012 / Accepted: 3 September 2012 / Published online: 22 September 2012 # Springer-Verlag Berlin Heidelberg 2012
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 wild-type (WT) methyl parathion hydrolase enzyme from Ochrobactrum sp. M231 [Ochr-methyl parathion hydrolase (MPH)]. As a result, seven amino acid sites within Ochr-MPH 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 Tm and T50 of the mutant enzyme were 11.7 and 10.2 °C higher, respectively, than that of the WT enzyme. The success of this design methodology for Ochr-MPH suggests that it was an efficient strategy for enhancing protein thermostability and suitable for protein engineering. Keywords Methyl parathion hydrolase . Site evolutionary entropy . Thermostability . Prethermut . Unfolding free energy
Electronic supplementary material The online version of this article (doi:10.1007/s00253-012-4411-7) contains supplementary material, which is available to authorized users. J. Tian : P. Wang : L. Huang : X. Chu : N. Wu (*) : Y. Fan Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, 12 Zhongguancun South Street, Beijing 100081, China e-mail: [email protected]
Introduction As natural biological catalysts, enzymes usually have high catalytic efficiency and substrate specificity. Despite their great potential for industrial applications, many enzymes have poor thermal stability, which greatly restricts their use in industrial production (Matsui and Harata 2007; Radestock and Gohlke 2011; Vieille and Zeikus 2001). For this reason, research on the mechanisms and rational design methods for improving protein thermal stability has become a hot spot in the fields of computational biology and protein engineering (Benedix et al. 2009; Capriotti et al. 2005; Heinzelman et al. 2009; Jeong et al. 2007; Schymkowitz et al. 2005; Yin et al. 2007). This will not only expand the number of applications involving enzymes but also help us to understand the relationship between protein structure and function. The two main
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