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Denaturing Effects of Urea and Guanidine Hydrochloride on hyperthermophilic esterase from Aeropyrum pernix K1

Chemical Research in Chinese Universities 2006年第2期22卷 168-172页

作者： GAO Ren-jun XIE Gui-qiu ZHOU Jun FENG Yan CAO Shu-gui Key Laboratory for Molecular Enzymology and Engineering the Ministry of Education School of Pharmacy Jilin University Changchun 130021 P. R. China

The changes in the activity and the conformation of the hyperthermophilic esterase derived from aerobic thermophilic Aeropyrum pernix K1 （ APE1547 ） were studied during denaturation by guanidine hydrochlofide （ GdnHC1 ） and urea. The denaturation course of APE1547 was followed by the steady-state and time resolved fluorescence methods. An increase in the denaturant concentration in the denatured system can significantly enhance the inactivation and unfolding of APE1547. The enzyme can be completely inactivated with a urea concentration of 2.7 mol/L or a GdnHCl concentration of 7.5 mol/L. The fluorescence emission maximum of the enzyme protein red shifts in magnitude to a maximum value（355 nm） when the concentration of GdnHCl is 5.1 mol/L. The experimental results indicate that APE1547 has a high resistance to urea. Unfolding of APE1547 in GdnHCI（4. 2-6.0 mol/L） was shown to be an irreversible process. The present results indicate that the ion pairs in this protein may be a key factor for the stability of this esterase.

关键词： Stability hyperthermophilic esterase Urea Guanidine hydrochloride

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High-throughput Screening of the Enantioselectivity of hyperthermophilic Mutant esterases from Archaeon Aeropyrum pernix K1 for Resolution of (R,S)-2-Octanol Acetate

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Chemical Research in Chinese Universities 2007年第3期23卷 319-324页

作者： ZHANG Gui-rong GAO Ren-jun ZHANG Ai-jun RAO Lang CAO Shu-gui Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education Jilin University Changchun 130021 P. R. China

To identify the desired hypertherrnophilic variants within a mutant esterase library for the resolution of （R, S）-2- octanol acetate, a simple, reliable, and versatile method was developed in this study. We built a screening strategy including two steps, first we selected agar plate with substrate to screen the enzymatic activity; secondly we used a pH indicator to screen the enantioselectivity. This method could rapidly detect favorable mutants with high activity and enantioselectivity. A total of 96. 2% of tedious screening work can be precluded using this screening strategy. It is an effective screening for alkyl ester and can be applied to relative screening researches. The four improved mutants were screened from the mutant esterase library. Their enantioselectivities, activities, and structures were investigated at different temperatures.

关键词： High-throughput screening Enantioselectivity hyperthermophilic esterase Directed evolution

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