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.
State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China.
Junxia
J
Li
Dongdong
D
Chen
Zhanqiao
Z
Yu
Longmei
L
Zhao
Rijun
R
Zhang
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D009154:Mutation; D010447:Peptide Hydrolases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D013696:Temperature
D001426:Bacterial Proteins; D010447:Peptide Hydrolases; C035855:keratinase