Expression Of Protease Genes From Keratin-Degrading Strain Streptomyces Fradiae Var.k11

Keratin is a structural protein that is extensively present in nature in the forms of hair, scale, feather, hoof as well as horn of animals. It is chemically stable, insoluble in water and can't be degraded by ordinary proteases, such as trypsin, pepsin and papain. Microbial keratinases have robust activities in hydrolysis of many obstinate fibrins, such as keratin, elastin and collagen, thus have extensive application prospects. In this study, a serine proteinase gene sfp1 and an aminopeptidase gene sfap from Streptomyces fradiae var. k11, a strain showing high hydrolysis ability to keratin, were expressed in Escherichia coli BL21 (DE3), purified, and characterized, respectively.The serine proteinase gene sfp1 encoded a 454-amino acid protein with a calculated molecular mass of 46 kDa. Sequence analysis revealed that SFP1 had a typical pre-pro-mature structure consisting of a signal peptide, an N-terminal propeptide and a mature protein. The pre-enzyme of SFP1 was expressed in E. coli and consequently purified. The 25.6 kDa fraction with protease activity separated by gel filtration chromatography indicated the mature enzyme of SFP1 was formed by autolysis the propeptide after its expression. The purified recombinant SFP1 was active under a broad range of pH and temperature. When using casein as substrate, the enzyme exhibited optimal activity at pH 8.5 and 65°C, and showed maximum activity at pH 9.0 and 62°C with azurin. SFP1 was significantly inhibited by the serine proteinase inhibitor PMSF, and exhibited good stability in solvents, detergents and salts. Comparison of the protease activity of SFP1 with other commercial proteases indicated that SFP1 had a considerable caseinolytic and keratinolytic activity as proteinase K did, thus had potentials in some applications.The aminopeptidase gene sfap encoded a 461-amino acid polypeptide with a calculated molecular mass of 47 kDa, and comprised of three domains: a signal peptide, a mature region, and a C-terminal propeptide. The gene with/without the C-terminal propeptide coding sequence was successfully overexpressed in E. coli. Only the gene without C-terminal propeptide coding sequence encoded a functional enzyme SFAPM. The purified recombinant SFAPM exhibited optimal activity at pH 8.0 and 60°C, and had good thermostability and pH stability. The enzyme showed some metalloprotease characteristics, which was inhibited by EDTA but activated by Ca²⁺ and Co²⁺. This is the first report on expression and characterization of an aminopeptidase from S. fradiae.Through expression and characterization of two proteases from S. fradiae, the present study not only enriched the kinds of proteases potential for commercial applications, but also contributed to the understanding of the relationship between bacterial keratinolytic enzyme system and mechanism of keratin hydrolysis.