Devi Selvaraju Gayathri, Fathima Anwar Aliya, Sanitha Mary, Iyappan Sellamuthu, Curtis Wayne R, Ramya Mohandass
Department of Genetic Engineering, SRM University, Tamilnadu 603203, India.
Department of Chemical Engineering, The Pennsylvania State University, University Park, PA 16802, USA.
J Biosci Bioeng. 2016 Dec;122(6):694-700. doi: 10.1016/j.jbiosc.2016.05.012. Epub 2016 Jun 17.
Metagenomics has the potential to facilitate the discovery of novel enzymes; however, to date, only a few alkaline proteases have been characterized from environmentally-sourced DNA. We report the identification and characterization of an alkaline serine protease designated as Prt1A from the metagenomic library of tannery activated sludge. Sequence analysis revealed that Prt1A is closely related to S8A family subtilisins with a catalytic triad of Asp, His and Ser. The putative protease gene (prt-1A) was subcloned in pET 28a (+) vector and overexpressed in Escherichia coli BL21(DE3)pLysS cells. This 38.8 KDa recombinant protease was purified to homogeneity by nickel affinity chromatography and exhibited optimal enzyme activity at elevated pH (11.0) and temperature (55°C). The enzyme activity was enhanced by the addition of 5 mM Ca ions, and was stable in the presence of anionic detergent, oxidizing agent and various organic solvents. The enzyme displayed high affinity and catalytic efficiency for casein under standard assay conditions (V = 279 U/mg/min, K = 1.70 mg/mL) and was also compatible with commercial detergents. These results suggest that Prt1A protease could act as an efficient enzyme in various industrial applications.
宏基因组学有潜力促进新型酶的发现；然而，迄今为止，仅从环境来源的DNA中鉴定出了少数几种碱性蛋白酶。我们报告了从制革厂活性污泥宏基因组文库中鉴定并表征了一种名为Prt1A的碱性丝氨酸蛋白酶。序列分析表明，Prt1A与S8A家族枯草杆菌蛋白酶密切相关，具有由天冬氨酸、组氨酸和丝氨酸组成的催化三联体。将假定的蛋白酶基因（prt-1A）亚克隆到pET 28a（+）载体中，并在大肠杆菌BL21(DE3)pLysS细胞中过表达。这种38.8 kDa的重组蛋白酶通过镍亲和层析纯化至同质，在升高的pH值（11.0）和温度（55°C）下表现出最佳酶活性。添加5 mM钙离子可增强酶活性，并且在阴离子洗涤剂、氧化剂和各种有机溶剂存在下稳定。在标准测定条件下，该酶对酪蛋白表现出高亲和力和催化效率（V = 279 U/mg/min，K = 1.70 mg/mL），并且也与市售洗涤剂兼容。这些结果表明，Prt1A蛋白酶可在各种工业应用中作为一种高效酶发挥作用。
