Characterization of a metagenome-derived protease from contaminated agricultural soil microorganisms and its random mutagenesis

doi:10.1007/s12223-017-0522-y

	Characterization of a metagenome-derived protease from contaminated agricultural soil microorganisms and its random mutagenesis
	Jiang, Chengjian 1,2; Zhang, Liang 1; Li, Fajia 1; Meng, Can 1; Zeng, Rong 1; Deng, Jie 1; Shen, Peihong 1; Ou, Qian 1; Wu, Bo 1
刊名	FOLIA MICROBIOLOGICA
	2017-11
卷号	62 期号:6 页码:499-508
关键词	Metagenome-derived protease Soil microorganisms Biochemical characterization Error-prone PCR
ISSN号	0015-5632
DOI	10.1007/s12223-017-0522-y
英文摘要	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 A degrees 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.
资助项目	Key Lab of Marine Bioactive Substance and Modern Analytical Technique, SOA[MBSMAT-2015-02]
WOS研究方向	Biotechnology & Applied Microbiology ; Microbiology
语种	英语
出版者	SPRINGER
WOS记录号	WOS:000412456500007
内容类型	期刊论文
源URL	[http://ir.fio.com.cn/handle/2SI8HI0U/3060]
专题	自然资源部第一海洋研究所
作者单位	1.Guangxi Univ, State Key Lab Conservat & Utilizat Subtrop Agrobi, Coll Life Sci & Technol, 100 Daxue East Rd, Nanning 530004, Guangxi, Peoples R China; 2.SOA, Key Lab Marine Bioact Subst & Modern Analyt Techn, 6 Xianxialing Rd, Qingdao 266061, Shandong, Peoples R China
推荐引用方式 GB/T 7714	Jiang, Chengjian,Zhang, Liang,Li, Fajia,et al. Characterization of a metagenome-derived protease from contaminated agricultural soil microorganisms and its random mutagenesis[J]. FOLIA MICROBIOLOGICA,2017,62(6):499-508.
APA	Jiang, Chengjian.,Zhang, Liang.,Li, Fajia.,Meng, Can.,Zeng, Rong.,...&Wu, Bo.(2017).Characterization of a metagenome-derived protease from contaminated agricultural soil microorganisms and its random mutagenesis.FOLIA MICROBIOLOGICA,62(6),499-508.
MLA	Jiang, Chengjian,et al."Characterization of a metagenome-derived protease from contaminated agricultural soil microorganisms and its random mutagenesis".FOLIA MICROBIOLOGICA 62.6(2017):499-508.