Purification and characterization of a thermostable thiol protease from a newly isolated hyperthermophilic Pyrococcus sp

Abstract

A hyperthermophilic archaeon strain, KODI, was isolated from a solfatara at a wharf on Kodakara Island, Kagoshima, Japan. The growth temperature of the strain ranged from 65 to 100°C, and the optimal temperature was 950C. The anaerobic strain was an So-dependent heterotroph. Cells were irregular cocci and were highly motile with several polar flagella. The membrane lipid was of the ether type, and the GC content of the DNA was estimated to be 38 mol%. The 16S rRNA sequence was 95% homologous to that of Pyrococcus abyssi. The optimum growth pH and NaCl concentration of the strain KOD1 were 7.0 and 3%, respectively. Therefore, strain KOD1 was identified as a Pyrococcus sp. Strain KOD1 produced at least three extracellular proteases. One of the most thermostable proteases was purified 21-fold, and the molecular size was determined to be 44 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 45 kDa by gel filtration chromatography. The specific activity of the purified protease was 2,160 U/mg of protein. The enzyme exhibited its maximum activity at approximately pH 7.0 and at a temperature of 110°C, with azocasein as a substrate. The enzyme activity was completely retained after heat treatment at 90°C for 2 h, and the half-life of enzymatic activity at 100°C was 60 min. The proteolytic activity was significantly inhibited byp-chloromercuribenzoic acid or E-64 but not by EDTA or phenylmethylsulfonyl fluoride. Proteolytic activity was enhanced threefold in the presence of 8 mM cysteine. These experimental results indicated that the enzyme was a thermostable thiol protease.

Identification and characterization of an archaeal ketopantoate reductase and its involvement in regulation of coenzyme A biosynthesis

“…Cultivation of T. kodakarensis KOD1 (Morikawa et al, 1994;Atomi et al, 2004;Fukui et al, 2005) and its derivative strains was performed under anaerobic conditions at 85°C in a nutrient-rich medium (ASW-YT) or a synthetic medium (ASW-AA). ASW-YT medium consists of 0.8× artificial seawater (ASW), 5.0 g l −1 yeast extract, 5.0 g l −1 tryptone and Fig.…”

Structure of the archaellar motor and associated cytoplasmic cone inThermococcus kodakaraensis

“…strain KOD1 and later identified as belonging to the Thermococcus genus [14]; also known as T. kodakarensis ) is one of the best-studied archaeal species. It was isolated from a Japanese solfatara in 1994 [15], and has proven readily amenable to genetics (well-developed gene manipulation techniques exploit its natural competence [16]) and the isolation of thermostable enzymes (e.g. high-fidelity DNA polymerase for PCR [17]).…”

An alternative mature form of subtilisin homologue, Tk‐SP, from Thermococcus kodakaraensis identified in the presence of Ca2+

“…The enzymatic activity of ProC-Tk-S359C was higher than (but comparable to) that of Tk-S359C, suggesting that the C-propeptide is not important for activity. However, the T m value of ProC-Tk-S359C determined by far-UV CD spectroscopy was higher than that of Tk-S359C by 25.9°C in the absence of Ca 2+ and 7.5°C in the presence of Ca 2+ , indicating that the C-propeptide contributes to the stabilization of ProC-Tk-S359C.…”