| Owe to their unique gene types, special physiological mechanism and metabolic product, archaea provide a clue to reveal origin of life and phylogenic evolution, thereby attracting peoples' increasing attentions. Most thermostable enzymes are produced from thermophilic archaea and they often have high thermostabilities and are resistant to organic solvents or denaturants. Research on thermophilic archaea would not only lend further evidence for biological evolution but also offer attractive industrial applications. Hyperthermophilic archaeon Aeropyrum pernix Kl was the first strictly aerobe archaea ever found and research on its thermophilic enzymes would have vital meanings.Phospholipase A2 widely exists in eukaryotes, especially in mammalian pancreas, snake venoms and plant tissues. It plays essential roles in the signal transduction and phospholipid metabolism in vivo. Compared to those from eukaryotes, study on phospholipase A2 from microorganisms is relatively short. Till now, only Sugiyama's group reported a novel phospholipase A2 from prokaryote Strptomyces violaceoruber. In vitro, the phospholipase A2was used in the degumming process of oil refineries, which reduced cost and environment pollution caused by chemicals. However, the enzyme denatures easily when the temperature reaches over 65 Thus it is necessary to explore the novel thermostable phospholipase A2 at elevated reaction temperature of the degumming process.Aeropyrum pernix Kl was isolated in 1993 from coastal solfotaricthermal vent in Kodakara-jima Island in Kyusyu, Japan. In the genomesequences of Aeropyrum pernix Kl, we found two genes that were predictedto be able to express PLAa- APE2325 is one of them and is selected for furtherstudy. The genome of A. pernix Kl was isolated and used as template for PCRamplification. APE2325 DNA was then ligated to pET15b vector andexpressed in the E. coli BL21 (DE3) Codon Plus -RIL. This is the first timethat PLA2 gene was successfully cloned from thermophilic archaea. Wesimplified the purification steps by taking advantage of enzyme'sthermostability. Heat treatment at 85 for 30 min followed by centrifugationremoved most of proteins from E. coli. The recombinant protein was furtherpurified by Ni-chelation affinity chromatography. Thus, the enzyme waspurified to homogeneity through only two steps, leading to the overallpurification enrichment of more than 60 fold. The molecular weight of theprotein determined by 12% SDS-PAGE gel is approximate 18 kDa. Therecombinant protein shows both PLA2 and esterase activities. Maximumactivities of both PLA2 and esterase were observed at 90癈. The optimumsubstrate for esterase activity is p-nitrophenol propionate. APE2325 has itsbest esterase activity at pH 8.0 and it is stable at alkalinous environment. Therecombinant protein is thermostable. When it is incubated at 80, 90and 95for 1 h, the enzyme retains 80%, 70% and 55% of the maximum activities,respectively. And the half-life of APE2325 at 100癈 is about 1 hour. Theamount of hydrophobic (47.7%) and charged (32.5%) residues of APE2325 are higher than those of mesophilic enzymes. This result suggests that hydrophobic and ionic interactions may contribute to the stability of APE2325. Kinetic analysis revealed that Km, kcat, and Vm for the /?-nitrophenyl propionate substrate are 103 uM, 39 s"1, and 249 umol min"1 mg"1, respectively. The enzyme is partially inhibited by 1 mM Na+ and completely inhibited by 1 mM Zn2+. Ca2+ does not activate the APE2325's PLAa activity; on the contrary, 5 mM Ca2+ partially inhibits its activity. The results indicate APE 2325 is Ca2+- independent PLAa.Esterase is widely distributed in animals, plants and microorganisms. For it is active in both aqueous and nonaqueous solvent systems, esterase has been developed into the most widely used class of enzymes in various industrial processes, including stereospecific hydrolysis, transesterification, ester synthesis, improvement of physicochemical properties of tr...
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