title: Purification, characterization, and genetic analysis of a leucine aminopeptidase from Aspergillus sojae.
abstract: Extracellular leucine aminopeptidase (LAP) from Aspergillus sojae was purified to protein homogeneity by sequential fast protein liquid chromatography steps. LAP had an apparent molecular mass of 37 kDa, of which approximately 3% was contributed by N-glycosylated carbohydrate. The purified enzyme was most active at pH 9 and 70 degrees C for 30 min. The enzyme preferentially hydrolyzed leucine p-nitroanilide followed by Phe, Lys, and Arg derivatives. The LAP activity was strongly inhibited by metal-chelating agents, and was largely restored by divalent cations like Zn(2+) and Co(2+). The lap gene and its corresponding cDNA fragment of the A. sojae were cloned using degenerated primers derived from internal amino acid sequences of the purified enzyme. lap is interrupted by three introns and is transcribed in a 1.3-kb mRNA that encodes a 377-amino-acid protein with a calculated molecular mass of 41.061 kDa. The mature LAP is preceded by a leader peptide of 77 amino acids, predicted to include an 18-amino-acid signal peptide and an extra sequence of 59 amino acids. Two putative N-glycosylation sites are identified in Asn-87 and Asn-288. Southern blot analysis suggested that lap is a single-copy gene in the A. sojae genome. The deduced amino acid sequence of A. sojae LAP shares only 11-33.1% identity with those of LAPs from 18 organisms.
authors.affiliation: Department of Microbiology, Chung Shan Medical University, Taichung 402, Taiwan.
authors.forename: Hung-Chien Roger
authors.initials: HC
authors.lastname: Chien
authors.forename: Long-Liu
authors.initials: LL
authors.lastname: Lin
authors.forename: Shiou-Huei
authors.initials: SH
authors.lastname: Chao
authors.forename: Chun-Chang
authors.initials: CC
authors.lastname: Chen
authors.forename: Wen-Ching
authors.initials: WC
authors.lastname: Wang
authors.forename: Chin-Ying
authors.initials: CY
authors.lastname: Shaw
authors.forename: Ying-Chie
authors.initials: YC
authors.lastname: Tsai
authors.forename: Hui-Yu
authors.initials: HY
authors.lastname: Hu
authors.forename: Wen-Hwei
authors.initials: WH
authors.lastname: Hsu
mesh_terms: D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology / Q000235:genetics*; D002712:Chlorides; D003001:Cloning, Molecular; D004492:Edetic Acid; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D007931:Leucyl Aminopeptidase / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010802:Phylogeny; D016415:Sequence Alignment; D013379:Substrate Specificity; D013696:Temperature; D017967:Zinc Compounds
chemical_list: D002712:Chlorides; D017967:Zinc Compounds; C016837:zinc chloride; D004492:Edetic Acid; D007931:Leucyl Aminopeptidase