College of Life Sciences, Anhui Normal University, Wuhu, 241001, Anhui, China.
Anhui Provincial Key Laboratory of Molecular Enzymology and Mechanism of Major Diseases, College of Life Sciences, Anhui Normal University, Wuhu, 241001, Anhui, China.
World J Microbiol Biotechnol. 2024 Oct 3;40(11):340. doi: 10.1007/s11274-024-04149-x.
Extracellular proteases from haloarchaea, also referred to as halolysins, are in increasing demand and are studied for their various applications in condiments and leather industries. In this study, an extracellular protease encoding gene from the haloarchaeon Halorubellus sp. PRR65, hly65, was cloned and heterologously expressed in E. coli. The novel halolysin Hly65 from the genus Halorubellus was characterized by complete inhibition of phenylmethanesulfonyl fluoride (PMSF) on its enzyme activity. Experimental determination revealed a triad catalytic active center consisting of Asp-His-Ser. Deletion of the C-terminal extension (CTE) resulted in loss of enzyme activity, while dithiothreitol (DTT) did not inhibit the enzyme activity, suggesting that Hly65 may function as a monomer. The K, V and K for the Hly65 were determined to be 2.91 mM, 1230.47 U·mg and 1538.09 S, respectively, under 60 °C, pH 8.0 and 4.0 M NaCl using azocasecin as a substrate. Furthermore, a three-dimensional structure prediction based on functional domains was obtained in this study which will facilitate modification and reorganization of halolysins to generate mutants with new physiological activities.
从嗜盐古菌中提取的细胞外蛋白酶，也称为嗜盐菌素，由于其在调味品和制革工业中的各种应用而受到越来越多的关注。在这项研究中，从嗜盐古菌 Halorubellus sp. PRR65 中克隆并异源表达了一种细胞外蛋白酶编码基因 hly65。该新型嗜盐菌素 Hly65 完全被苯甲基磺酰氟（PMSF）抑制，实验确定其具有由 Asp-His-Ser 组成的三联催化活性中心。C 端延伸（CTE）缺失导致酶活性丧失，而二硫苏糖醇（DTT）不会抑制酶活性，表明 Hly65 可能作为单体发挥作用。在 60°C、pH 8.0 和 4.0 M NaCl 下，以偶氮酪蛋白为底物，测定了 Hly65 的 K、V 和 K 值，分别为 2.91 mM、1230.47 U·mg 和 1538.09 S。此外，本研究还获得了基于功能域的三维结构预测，这将有助于对嗜盐菌素进行修饰和重组，从而产生具有新生理活性的突变体。
