The gene of enzyme（Ape1547） was cloned from hyperthermophilic archaeon Aeropyrum pernix K1 and expressed in Escherichia coil.The effect of calcium cation on the properties of Ape1547 was studied.Ape1547 exhibits both peptidase activity and esterase activity.The fluorescence spectrum shows that calcium cation quenches the fluorescence of the enzyme through static quenching mechanism,indicating that calcium cation was bound to the enzyme.Based on the study of calcium cation on CD ellipticity of Ape1547 by circular dichroism,we concluded that the change of enzyme structure induced by calcium cation may be responsible for the change of enzyme activity.Calcium cation has dual effects on Ape1547：it could activate the enzyme activity when its concentration was 0.1 mol/L,and the enzyme had the highest activity;however,when its concentration was higher than 0.2 mol/L,the enzyme activity was inhibited.The results indicate that the activity center of peptidase activity might involve more amino acid residues than that of esterase activity.
The aminopeptidase gene from thermophilic archaea Sulfolobustokodaii was cloned and expressed in Escherichia coli BL21 codon-plus（DE3）. To overexpress the aminopeptidase, the vector pET32a was constructed, in which the target gene was fused with the genes of histidine-tag and thioredoxin（Trx）. The expressed protein was purified using Ni^2＋-column affinity chromatography and ion exchange chromatography and cleft with enterokinase（EK） to obtain the purified aminopeptidase（ST1737）. The biochemical and enzymic properties of the expressed ST1737 were characterized. The results show that its optimal pH and temperature are 8 and 80 ℃, respectively. The half-life of ST1737（0.2 mg/mL） is about 85 h at 90 ℃, indicating that the enzyme exhibits an excellent thermostability. The activity of ST1737 could still maintain over 85% after its treatment at 25 ℃ in different buffers with a pH range of from 6.0 to 10.5 for 24 h, demonstrating that ST1737 is stable in neutral or slight alkali environment. The enzyme shows a high activity for the substrates such as unmodified peptide Asp-Ala, while the pNPC8 shows an optimal esterase substrate specificity. These results indicate that the enzyme is a bifunctional enzyme, and different from the aminopeptidase reported before.
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.
