A novel lipase gene, lipS221, was cloned from Streptomyces fradiae var. k11, which secretes multiple proteases. The 930-bp nucleotide sequence encodes 309 amino acid residues with a calculated molecular weight of 28.5 kDa. The protein sequence shows highest identity (82%) with a putative lipase of Streptomyces coelicolor A3(2). LipS221 was functionally expressed in Pichia pastoris and purified to homogeneity with a final specific activity of 569 U mg−1. The purified enzyme showed the maximum activity at 55 °C and pH 9.8 for hydrolysis of p-nitrophenyl palmitate. Importantly, lipS221 was stable towards degradation by alkaline and neutral proteases, but activity decreased when hydrolyzed by savinase or proteinase K together with surfactant (SDS or CTAB). These results indicate that the proteolytic resistance of lipS221 may be related to its surface charge distribution.

We report the initial characterization and expression of sfp2, a gene encoding a keratinolytic serine protease from Streptomyces fradiae var. k11. Recombinant SFP2 was expressed in and secreted from the yeast Pichia pastoris with a final yield of 78mg/L (136.2U/mL caseinolytic activity) after 25h of induction. The recombinant enzyme was purified using by ammonium sulfate precipitation and gel filtration chromatography to electrophoretic homogeneity, which was appropriately glycosylated and had a molecular mass of 26.0kDa. The purified recombinant SFP2 was characterized. The optimal pHs and temperatures of SFP2 for proteolysis of casein and keratin azure were pH 10.0, 60°C, and pH 9.0, 55°C, respectively. SFP2 activity was stable from pH 3.0 to pH 11.0. The enzyme activity was inhibited by Co2+ and Cr3+ and enhanced by Ni2+ and Cu2+. The Km of 0.45mmol/L and Vmax of 19.84mmol/minmg were calculated using N-succinyl-Ala-Ala-Pro-Phe-pNA as a substrate. We tested the activity of SFP2 with soluble and insoluble substrates; SFP2 was more specific for keratinous substrates compared with proteinase K and other commercial proteases.

The gene sfp1, which encodes a predicted serine proteinase designated SFP1, was isolated by the screening of a gene library of the feather-degrading strain Streptomyces fradiae var.k11. The open reading frame of sfp1 encodes a protein of 454 amino acids with a calculated molecular mass of 46.19 kDa. Sequence analysis reveals that SFP1 possesses a typical pre-pro-mature organization that consists of a signal sequence, an N-terminal propeptide region, and a mature proteinase domain. The pre-enzyme of SFP1 was expressed in Escherichia coli and consequently purified. The 25.6 kDa fraction with protease activity separated by gel filtration chromatography indicated that the mature enzyme of SFP1 was formed by autolysis of the propeptide after its expression. The purified SFP1 is active under a broad range of pH and temperature. SFP1 has pH and temperature optima of pH 8.5 and 65 degrees C for its caseinolytic activity and pH 9 and 62 degrees C for its keratinolytic activity. SFP1 was sharply inhibited by the serine proteinase inhibitor phenylmethyl sulfonyl fluoride and exhibited a good stability to solvents, detergents, and salts. Comparison of the protease activity of SFP1 with other commercial proteases indicates that SFP1 has a considerable caseinolytic and keratinolytic activity as does proteinase K.