title: A new aminopeptidase from the keratin-degrading strain Streptomyces fradiae var. k11.
abstract: 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.
authors.affiliation: 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.
authors.forename: Bo
authors.initials: B
authors.lastname: Wu
authors.forename: Pengjun
authors.initials: P
authors.lastname: Shi
authors.forename: Jiang
authors.initials: J
authors.lastname: Li
authors.forename: Yaru
authors.initials: Y
authors.lastname: Wang
authors.forename: Kun
authors.initials: K
authors.lastname: Meng
authors.forename: Yingguo
authors.initials: Y
authors.lastname: Bai
authors.forename: Huiying
authors.initials: H
authors.lastname: Luo
authors.forename: Peilong
authors.initials: P
authors.lastname: Yang
authors.forename: Zhigang
authors.initials: Z
authors.lastname: Zhou
authors.forename: Bin
authors.initials: B
authors.lastname: Yao
mesh_terms: 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
chemical_list: D008670:Metals; D011994:Recombinant Proteins; D007633:Keratins; D000626:Aminopeptidases