Jia Baolei, Li Zhengqun, Liu Jinliang, Sun Ying, Jia Xiaomeng, Xuan Yuan Hu, Zhang Jiayan, Jeon Che Ok
Department of Life Science, Chung-Ang University Seoul, South Korea.
College of Plant Sciences, Jilin University Changchun, China.
Front Microbiol. 2015 Dec 17;6:1380. doi: 10.3389/fmicb.2015.01380. eCollection 2015.
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.
嗜热栖热菌KOD1中的一种假定的锌依赖性蛋白酶（TK0512）与古锌金属蛋白酶具有保守基序，古锌金属蛋白酶是在古菌、细菌和真核生物中发现的金属蛋白酶。系统发育和序列分析表明，嗜热栖热菌科中的TK0512及其同源物代表古锌金属蛋白酶家族的新成员，我们将其命名为AMZ-tk。我们通过在大肠杆菌中过表达重组AMZ-tk并对纯化的酶进行表征，进一步从生化角度证实了其蛋白水解活性。在锌存在的情况下，纯化的酶可降解酪蛋白，而添加乙二胺四乙酸（EDTA）则强烈抑制酶活性。AMZ-tk还表现出需要Zn(2+)的自我切割活性。这些结果表明，AMZ-tk是古锌金属蛋白酶家族中的一种锌依赖性蛋白酶。该酶在pH值为7.0至10.0的碱性条件下具有活性，最适pH值为8.0。AMZ-tk催化活性的最适温度为55°C。定量逆转录PCR显示，将细胞暴露于55°C和65°C后，AMZ-tk的转录也会上调。突变分析表明，与锌结合的组氨酸和催化性谷氨酸在蛋白水解中起关键作用。在EDTA存在的情况下，AMZ-tk在70°C孵育4小时仍具有热稳定性。在实验室表面活性剂和商业洗衣粉存在的情况下，AMZ-tk也保留了其原始活性的50%以上。AMZ-tk还对几种细菌表现出抗菌活性。因此，鉴于其在碱性pH值、洗涤剂和热稳定性方面的活性，AMZ-tk在许多方面都具有相当大的研究价值。
