title: A Zinc-Dependent Protease AMZ-tk from a Thermophilic Archaeon is a New Member of the Archaemetzincin Protein Family.
abstract: A putative zinc-dependent protease (TK0512) in Thermococcus kodakarensis KOD1 shares a conserved motif with archaemetzincins, which are metalloproteases found in archaea, bacteria, and eukarya. Phylogenetic and sequence analyses showed that TK0512 and its homologues in Thermococcaceae represent new members in the archaemetzincins family, which we named AMZ-tk. We further confirmed its proteolytic activity biochemically by overexpression of the recombinant AMZ-tk in Escherichia coli and characterization of the purified enzyme. In the presence of zinc, the purified enzyme degraded casein, while adding EDTA strongly inhibited the enzyme activity. AMZ-tk also exhibited self-cleavage activity that required Zn(2+). These results demonstrated that AMZ-tk is a zinc-dependent protease within the archaemetzincin family. The enzyme displayed activity at alkaline pHs ranging from 7.0 to 10.0, with the optimal pH being 8.0. The optimum temperature for the catalytic activity of AMZ-tk was 55°C. Quantitative reverse transcription-PCR revealed that transcription of AMZ-tk was also up-regulated after exposing the cells to 55 and 65°C. Mutant analysis suggested that Zn(2+) binding histidine and catalytic glutamate play key roles in proteolysis. AMZ-tk was thermostable on incubation for 4 h at 70°C in the presence of EDTA. AMZ-tk also retained >50% of its original activity in the presence of both laboratory surfactants and commercial laundry detergents. AMZ-tk further showed antibacterial activity against several bacteria. Therefore, AMZ-tk is of considerable interest for many purposes in view of its activity at alkaline pH, detergents, and thermostability.
authors.affiliation: Department of Life Science, Chung-Ang University Seoul, South Korea.
authors.forename: Baolei
authors.initials: B
authors.lastname: Jia
authors.affiliation: College of Plant Sciences, Jilin University Changchun, China.
authors.forename: Zhengqun
authors.initials: Z
authors.lastname: Li
authors.affiliation: College of Plant Sciences, Jilin University Changchun, China.
authors.forename: Jinliang
authors.initials: J
authors.lastname: Liu
authors.affiliation: Department of Life Science, Chung-Ang University Seoul, South Korea.
authors.forename: Ying
authors.initials: Y
authors.lastname: Sun
authors.affiliation: Department of Life Science, Chung-Ang University Seoul, South Korea.
authors.forename: Xiaomeng
authors.initials: X
authors.lastname: Jia
authors.affiliation: College of Plant Protection, Shenyang Agricultural University Shenyang, China.
authors.forename: Yuan Hu
authors.initials: YH
authors.lastname: Xuan
authors.affiliation: College of Plant Sciences, Jilin University Changchun, China.
authors.forename: Jiayan
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Life Science, Chung-Ang University Seoul, South Korea.
authors.forename: Che Ok
authors.initials: CO
authors.lastname: Jeon
keywords: archaemetzincin; biotechnological applications; enzymatic property; thermophilic archaeon; zinc-dependent protease