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 ( Gdn...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.展开更多
The optimization of nutrient levels for the production of recombinant hyperthermophilie esterase by E. coli was carried out with response surface methodology(RSM) based on the central composite rotatable design(CCR...The optimization of nutrient levels for the production of recombinant hyperthermophilie esterase by E. coli was carried out with response surface methodology(RSM) based on the central composite rotatable design(CCRD). A 24 central composite rotatable design was used to study the combined effect of the nutritional constituents like yeast extract, peptone, mineral salt and trace metals. The P-value of the coefficient for the linear effect of peptone concentration was 0. 0081 and trace metals solution was less than 0. 0001, suggesting that these were the principal variables with significant effect on the hyperthermophilic esterase production. The predicted optimal hyperthermophilie esterase yield was 269. 17 U/mL, whereas an actual experimental value of 284. 58 U/mL was obtained.展开更多
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 ...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.展开更多
The stereospecific hydrolysis of mandelate can be effectively catalyzed by hyperthermophilic acylpeptide esterase APE 1547(Aeropyrum pernix esterase 1547). APE 1547 used in this reaction showed a remarkable stereodi...The stereospecific hydrolysis of mandelate can be effectively catalyzed by hyperthermophilic acylpeptide esterase APE 1547(Aeropyrum pernix esterase 1547). APE 1547 used in this reaction showed a remarkable stereodi-scrimination in favour of R-mandelic acid(99% e.e.) with an enantiomeric ratio E〉200. The results of computer simulation are consistent with the experimental results. It can be inferred that the R-substrate adopted a binding mode productive of the reaction due to the formation of the hydrogen bond at the active site of APE 1547.展开更多
文摘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.
文摘The optimization of nutrient levels for the production of recombinant hyperthermophilie esterase by E. coli was carried out with response surface methodology(RSM) based on the central composite rotatable design(CCRD). A 24 central composite rotatable design was used to study the combined effect of the nutritional constituents like yeast extract, peptone, mineral salt and trace metals. The P-value of the coefficient for the linear effect of peptone concentration was 0. 0081 and trace metals solution was less than 0. 0001, suggesting that these were the principal variables with significant effect on the hyperthermophilic esterase production. The predicted optimal hyperthermophilie esterase yield was 269. 17 U/mL, whereas an actual experimental value of 284. 58 U/mL was obtained.
基金Supported by the National Natural Science Foundation of China(Nos30400081, 30570405 and 20672045)the Key Tech-nology Research and Development Program of China(No2004BA713D03-04)
文摘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.
基金Supported by the National Natural Science Foundation of China(Nos.30870539, 21072075)the 38th Postdoctoral Foundation of China(No.801050321413)
文摘The stereospecific hydrolysis of mandelate can be effectively catalyzed by hyperthermophilic acylpeptide esterase APE 1547(Aeropyrum pernix esterase 1547). APE 1547 used in this reaction showed a remarkable stereodi-scrimination in favour of R-mandelic acid(99% e.e.) with an enantiomeric ratio E〉200. The results of computer simulation are consistent with the experimental results. It can be inferred that the R-substrate adopted a binding mode productive of the reaction due to the formation of the hydrogen bond at the active site of APE 1547.