Gene cloning and heterologous expression of a serine protease from Streptomyces fradiae var.k11.
The gene sfp1, which encodes a predicted serine proteinase designated SFP1, was isolated by the screening of a gene library of the feather-degrading strain Streptomyces fradiae var.k11. The open reading frame of sfp1 encodes a protein of 454 amino acids with a calculated molecular mass of 46.19 kDa. Sequence analysis reveals that SFP1 possesses a typical pre-pro-mature organization that consists of a signal sequence, an N-terminal propeptide region, and a mature proteinase domain. The pre-enzyme of SFP1 was expressed in Escherichia coli and consequently purified. The 25.6 kDa fraction with protease activity separated by gel filtration chromatography indicated that the mature enzyme of SFP1 was formed by autolysis of the propeptide after its expression. The purified SFP1 is active under a broad range of pH and temperature. SFP1 has pH and temperature optima of pH 8.5 and 65 degrees C for its caseinolytic activity and pH 9 and 62 degrees C for its keratinolytic activity. SFP1 was sharply inhibited by the serine proteinase inhibitor phenylmethyl sulfonyl fluoride and exhibited a good stability to solvents, detergents, and salts. Comparison of the protease activity of SFP1 with other commercial proteases indicates that SFP1 has a considerable caseinolytic and keratinolytic activity as does proteinase K.
Meng
Kun
K
Microbiological Engineering Laboratory, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancunnandajie Road, Beijing 100081, China.
Li
Jiang
J
Cao
Yanan
Y
Shi
Pengjun
P
Wu
Bo
B
Han
Xiaoyu
X
Bai
Yingguo
Y
Wu
Ningfeng
N
Yao
Bin
B
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D012697:Serine Endopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013302:Streptomyces / Q000201:enzymology*; D013696:Temperature
D015842:Serine Proteinase Inhibitors; D012697:Serine Endopeptidases