Abstract
An aminopeptidase gene fragment was isolated from a keratin-degrading strain, Streptomyces fradiae var. k11, by PCR amplification using a degenerate primer set designed based on the partial amino acid sequence of the native enzyme. The gene, designated sfap, encoded a polypeptide of 461 amino acids comprised of three domains: a signal peptide, a mature region, and a C-terminal propeptide. The aminopeptidase, SFAP, had highest amino acid sequence identity (79%) with a putative aminopeptidase from Streptomyces griseus subsp. griseus NBRC 13350. The gene with and without C-terminal propeptide was successfully overexpressed in Escherichia coli BL21 (DE3), and the gene without C-terminal propeptide encoded a functional enzyme. Purified recombinant SFAP exhibited optimal activity at pH 8.0 and 60 °C, and retained >60% peak activity over a broad range of temperature. The enzyme was thermal and pH stable, and showed metalloprotease characteristics, which was inhibited by EDTA but activated by Ca2+ and Co2+. This is the first study to report the gene cloning and expression of a leucine aminopeptidase from S. fradiae.
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This work was supported by the Chinese National High Technology Research and Development Program (863 Program, Grant No. 2007AA100601) and National Key Technology R&D Program of China (No. 2006BAD12B05-03).
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Wu, B., Shi, P., Li, J. et al. A New Aminopeptidase from the Keratin-Degrading Strain Streptomyces fradiae var. k11. Appl Biochem Biotechnol 160, 730–739 (2010). https://doi.org/10.1007/s12010-009-8537-8
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DOI: https://doi.org/10.1007/s12010-009-8537-8