Abstract 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.