Thermophilic esterase APE1547 from Aeropyrum pernix K1 contained a β-propeller with seven blades. There are three hydrogen bonding interactions(Thr127-Gly154,Leu182- Arg145-Glu122) between blades 3 and 4 in the β-pr...Thermophilic esterase APE1547 from Aeropyrum pernix K1 contained a β-propeller with seven blades. There are three hydrogen bonding interactions(Thr127-Gly154,Leu182- Arg145-Glu122) between blades 3 and 4 in the β-propeller domain of the APE1547. To examine the role of these hydrogen bonds, we eliminated these three hydrogen bonds between blades 3 and 4, by site-directed mutagenesis. The analysis results of kinetics, thermostability and differential scanning calorimetry(DSC) of the mutants show that Kcat/Km value of each mutation increased, and stability decreased dramatically than wild-type protein. These results strongly suggest that the three specific hydrogen bonds played an important role on maintaining the stability and activity of the esterase APE1547.展开更多
Hyperthermophilic enzyme APE1547 is an extremely thermostable recombinant protein from thermophilic archaeon Aeropyrumpernix K1. The Tyr444 located in the catalytic domain adjacent to the catalytic amino acid Ser445 a...Hyperthermophilic enzyme APE1547 is an extremely thermostable recombinant protein from thermophilic archaeon Aeropyrumpernix K1. The Tyr444 located in the catalytic domain adjacent to the catalytic amino acid Ser445 and formed hydrogen bond with Ile567. To study the effect of Tyr444 on the activity of APE1547, site-directed mutagenesis was applied. Two mutant enzymes T444S and T444G were created. Comparison of the mutant enzymes with wide enzyme, the thermostability of mutants T444S and T444G decreased by 10%-20%, but the catalytic efficiency of mutants toward pNPC8 and Ac-Leu-pNA increased 1.33 and 1.75 fold respectively. Molecular modeling shows that the elimination of hydrogen bond between Tyr444 and Ile567 is the cause of the decrease in thermostability and increase in catalytic efficiency. These observations suggest that Tyr444 plays an important role in the catalytic ability and thermostability of this enzyme.展开更多
The regioselective acylation of quercetin catalyzed by a novel thermophilic esterase（APE1547） from the archaeon Aeropyrum pernix K1 was successfully conducted in organic solvents.The effects of acyl donor,substrate ...The regioselective acylation of quercetin catalyzed by a novel thermophilic esterase（APE1547） from the archaeon Aeropyrum pernix K1 was successfully conducted in organic solvents.The effects of acyl donor,substrate ratio,organic solvent,temperature,and water activity were investigated.Under the optimum conditions,a yield of 74% for its mono ester could be achieved in the reaction for about 6 h.With the reaction time extending to about 30 h,the final conversion of quercetin was about 100% and three products were synthesized.展开更多
文摘Thermophilic esterase APE1547 from Aeropyrum pernix K1 contained a β-propeller with seven blades. There are three hydrogen bonding interactions(Thr127-Gly154,Leu182- Arg145-Glu122) between blades 3 and 4 in the β-propeller domain of the APE1547. To examine the role of these hydrogen bonds, we eliminated these three hydrogen bonds between blades 3 and 4, by site-directed mutagenesis. The analysis results of kinetics, thermostability and differential scanning calorimetry(DSC) of the mutants show that Kcat/Km value of each mutation increased, and stability decreased dramatically than wild-type protein. These results strongly suggest that the three specific hydrogen bonds played an important role on maintaining the stability and activity of the esterase APE1547.
基金Supported by the National Natural Science Foundation of China(Nos.30400081 and 20432010)
文摘Hyperthermophilic enzyme APE1547 is an extremely thermostable recombinant protein from thermophilic archaeon Aeropyrumpernix K1. The Tyr444 located in the catalytic domain adjacent to the catalytic amino acid Ser445 and formed hydrogen bond with Ile567. To study the effect of Tyr444 on the activity of APE1547, site-directed mutagenesis was applied. Two mutant enzymes T444S and T444G were created. Comparison of the mutant enzymes with wide enzyme, the thermostability of mutants T444S and T444G decreased by 10%-20%, but the catalytic efficiency of mutants toward pNPC8 and Ac-Leu-pNA increased 1.33 and 1.75 fold respectively. Molecular modeling shows that the elimination of hydrogen bond between Tyr444 and Ile567 is the cause of the decrease in thermostability and increase in catalytic efficiency. These observations suggest that Tyr444 plays an important role in the catalytic ability and thermostability of this enzyme.
基金Supported by the National Natural Science Foundation of China(Nos.31070708,21172093 and 20772046)the Natural Science Foundation of Jilin Province,China(No.201115038)
文摘The regioselective acylation of quercetin catalyzed by a novel thermophilic esterase（APE1547） from the archaeon Aeropyrum pernix K1 was successfully conducted in organic solvents.The effects of acyl donor,substrate ratio,organic solvent,temperature,and water activity were investigated.Under the optimum conditions,a yield of 74% for its mono ester could be achieved in the reaction for about 6 h.With the reaction time extending to about 30 h,the final conversion of quercetin was about 100% and three products were synthesized.
