A new aminopeptidase from the keratin-degrading strain Streptomyces fradiae var. k11.
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 degrees 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 Ca(2+) and Co(2+). This is the first study to report the gene cloning and expression of a leucine aminopeptidase from S. fradiae.
Wu
Bo
B
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing 100081, People's Republic of China.
Shi
Pengjun
P
Li
Jiang
J
Wang
Yaru
Y
Meng
Kun
K
Bai
Yingguo
Y
Luo
Huiying
H
Yang
Peilong
P
Zhou
Zhigang
Z
Yao
Bin
B
D000595:Amino Acid Sequence; D000626:Aminopeptidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005110:Extracellular Space / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D007633:Keratins / Q000378:metabolism*; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013302:Streptomyces / Q000145:classification / Q000166:cytology / Q000201:enzymology* / Q000235:genetics
D008670:Metals; D011994:Recombinant Proteins; D007633:Keratins; D000626:Aminopeptidases