Improvement of expression level of keratinase Sfp2 from Streptomyces fradiae by site-directed mutagenesis of its N-terminal pro-sequence.

The keratinase Sfp2, produced by Streptomyces fradiae var. k11, is a serine alkaline protease first synthesized as pre-pro-mature precursor, of which the N-terminal propeptide must be autocatalytically cleaved on the C-terminal of P1 amino acid to produce mature enzyme. Single amino acid substitutions were introduced at positions -1 and -2 to improve the expression level of mature Sfp2. The specific activity of L(-1)F mutant (48935 U/mg) was nine times that of wild-type Sfp2, whereas the mutants L(-1)D, L(-1)G, L(-1)H, K(-2)E, and K(-2)L had 2-52 % of the specific activity of wild-type. The yield of mature Sfp2 of L(-1)F mutant was estimated to be 800 μg/mg total protein and 112 mg/l culture supernatant, nine and twice that of wild-type, respectively. The L(-1)F mutant exhibited similar enzymatic properties to wild-type.