title: Characterization of a metagenome-derived protease from contaminated agricultural soil microorganisms and its random mutagenesis.
abstract: Proteases are typical key enzymes that hydrolyze proteins into amino acids and peptides. Numerous proteases have been studied, but the discovery of metagenome-derived proteases is still significant for both commercial applications and basic research. An unexplored protease gene sep1A was identified by function-based screening from a plasmid metagenomic library derived from uncultured contaminated agricultural soil microorganisms. The putative protease gene was subcloned into pET-32a (+) vector and overexpressed in E. coli BL21(DE3) pLysS, then the recombinant protein was purified to homogeneity. The detailed biochemical characterization of the Sep1A protein was performed, including its molecular characterization, specific activity, pH-activity profile, metal ion-activity profile, and enzyme kinetic assays. Furthermore, the protein engineering approach of random mutagenesis via error-prone PCR was applied on the original Sep1A protein. Biochemical characterization demonstrated that the purified recombinant Ep48 protein could hydrolyze casein. Compared with the original Sep1A protein, the best variant of Ep48 in the random mutagenesis library, with the Gln307Leu and Asp391Gly changes, exhibited 2.62-fold activity at the optimal reaction conditions of 50 °C and pH 9.0. These results are the first step toward a better understanding of the properties of Sep1A protein. Protein engineering with error-prone PCR paves the way toward the metagenome-derived genes for biotechnological applications.
authors.affiliation: State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and Technology, Guangxi University, 100 Daxue East Road, Nanning, Guangxi, 530004, China. jiangcj0520@gmail.com.|Key Laboratory of Marine Bioactive Substance and Modern Analytical Techniques, SOA, 6 Xianxialing Road, Qingdao, Shandong, 266061, China. jiangcj0520@gmail.com.
authors.forename: Chengjian
authors.identifier: http://orcid.org/0000-0001-6887-7560
authors.initials: C
authors.lastname: Jiang
authors.affiliation: State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and Technology, Guangxi University, 100 Daxue East Road, Nanning, Guangxi, 530004, China.
authors.forename: Liang
authors.initials: L
authors.lastname: Zhang
authors.affiliation: State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and Technology, Guangxi University, 100 Daxue East Road, Nanning, Guangxi, 530004, China.
authors.forename: Fajia
authors.initials: F
authors.lastname: Li
authors.affiliation: State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and Technology, Guangxi University, 100 Daxue East Road, Nanning, Guangxi, 530004, China.
authors.forename: Can
authors.initials: C
authors.lastname: Meng
authors.affiliation: State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and Technology, Guangxi University, 100 Daxue East Road, Nanning, Guangxi, 530004, China.
authors.forename: Rong
authors.initials: R
authors.lastname: Zeng
authors.affiliation: State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and Technology, Guangxi University, 100 Daxue East Road, Nanning, Guangxi, 530004, China.
authors.forename: Jie
authors.initials: J
authors.lastname: Deng
authors.affiliation: State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and Technology, Guangxi University, 100 Daxue East Road, Nanning, Guangxi, 530004, China.
authors.forename: Peihong
authors.initials: P
authors.lastname: Shen
authors.affiliation: State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and Technology, Guangxi University, 100 Daxue East Road, Nanning, Guangxi, 530004, China.
authors.forename: Qian
authors.initials: Q
authors.lastname: Ou
authors.affiliation: State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and Technology, Guangxi University, 100 Daxue East Road, Nanning, Guangxi, 530004, China.
authors.forename: Bo
authors.initials: B
authors.lastname: Wu
mesh_terms: D000595:Amino Acid Sequence; D001419:Bacteria / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D015723:Gene Library; D007700:Kinetics; D054892:Metagenome; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D012988:Soil Microbiology; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D010447:Peptide Hydrolases
keywords: Biochemical characterization; Error-prone PCR; Metagenome-derived protease; Soil microorganisms