Functional expression of the keratinolytic serine protease gene sfp2 from Streptomyces fradiae var. k11 in Pichia pastoris.
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 78 mg/L (136.2 U/mL caseinolytic activity) after 25 h 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 26.0 kDa. 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 degrees C, and pH 9.0, 55 degrees C, respectively. SFP2 activity was stable from pH 3.0 to pH 11.0. The enzyme activity was inhibited by Co(2+) and Cr(3+) and enhanced by Ni(2+) and Cu(2+). The K(m) of 0.45 mmol/L and V(max) of 19.84 mmol/min mg 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.
Li
Jiang
J
Microbial Engineering Department, Feed Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China.
Shi
Peng-Jun
PJ
Han
Xiao-Yu
XY
Meng
Kun
K
Yang
Pei-Long
PL
Wang
Ya-Ru
YR
Luo
Hui-Ying
HY
Wu
Ning-Feng
NF
Yao
Bin
B
Fan
Yun-Liu
YL
D018929:Cell Culture Techniques; D003001:Cloning, Molecular; D005285:Fermentation; D006863:Hydrogen-Ion Concentration; D007633:Keratins / Q000737:chemistry*; D019216:Metals, Heavy / Q000737:chemistry; D009842:Oligopeptides / Q000737:chemistry; D010843:Pichia / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000302:isolation & purification; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D012995:Solubility; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity; D013696:Temperature
D019216:Metals, Heavy; D009842:Oligopeptides; D011994:Recombinant Proteins; C069564:succinimidyl-alanyl-phenylalanyl-prolyl-phenylalanine 4-nitroanilide; D007633:Keratins; D012697:Serine Endopeptidases