Metagenomics facilitates the isolation of novel enzymes from the unculturable microorganisms inhabiting a particular ecosystem. In the present study, solid tannery waste (STW) metagenome was explored as a potential source of microbial serine proteases. KEGG annotated functional profile of the UNK metagenome was exploited for in silico mining of candidate serine proteases. A novel serine protease was identified, namely Prt1SU, corresponding to an 1149 bp full-length gene encoding a 383 amino acid protein. The identity of the Prt1SU was evaluated by PSI-BLAST against the databases at NCBI and BLAST against the MEROPS database. Identities ranged from 30–45.7% to their respective nearest homologs of the serine protease family. Utilizing the DNA template isolated from the UNK metagenome, the candidate serine protease gene was cloned in the pET28a(+) vector and expressed in E. coli BL21 (DE3) strain. Although the Prt1SU was overexpressed at 18ºC using 0.1 mM IPTG, it remained inactive because of the formation of inclusion body. After solubilization with 8 M urea, the Prt1SU enzyme was purified to homogeneity using Ni-NTA chromatography under denaturing conditions and then renatured to retain protease activity. The purified Prt1SU with a molecular weight of ~ 40 kDa exhibited the maximum enzyme activity at pH 9.0 and temperature 37ºC. The enzyme activity of the Prt1SU was enhanced by the addition of organic solvents, and the enzyme was found stable in the presence of anionic and non-ionic surfactants and oxidizing agents, suggesting that Prt1SU is suitable for various industrial applications as well as wastewater and STW treatment.

宏基因组学有助于从居住在特定生态系统中的不可培养微生物中分离出新的酶。在本研究中，固体制革废料 (STW) 宏基因组被探索为微生物丝氨酸蛋白酶的潜在来源。UNK 宏基因组的 KEGG 注释功能谱被用于计算机挖掘候选丝氨酸蛋白酶。鉴定出一种新型丝氨酸蛋白酶，即 Prt1SU，对应于编码 383 个氨基酸蛋白质的 1149 bp 全长基因。Prt1SU 的身份通过 PSI-BLAST 对 NCBI 的数据库和 BLAST 对 MEROPS 数据库的数据库进行评估。身份范围从 30-45.7% 到它们各自最接近的丝氨酸蛋白酶家族同系物。利用从 UNK 宏基因组中分离的 DNA 模板，将候选丝氨酸蛋白酶基因克隆到 pET28a(+) 载体中并在大肠杆菌中表达BL21 (DE3) 菌株。尽管 Prt1SU 在 18ºC 下使用 0.1 mM IPTG 过表达，但由于包涵体的形成，它仍然没有活性。用 8 M 尿素溶解后，在变性条件下使用 Ni-NTA 色谱法将 Prt1SU 酶纯化至均一，然后复性以保留蛋白酶活性。分子量约为 40 kDa 的纯化 Prt1SU 在 pH 9.0 和温度 37ºC 下表现出最大的酶活性。Prt1SU 的酶活性通过添加有机溶剂而增强，并且发现该酶在阴离子和非离子表面活性剂和氧化剂存在下稳定，表明 Prt1SU 适用于各种工业应用以及废水和 STW治疗。