Purification, characterization, and genetic analysis of a leucine aminopeptidase from Aspergillus sojae.
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.
Chien
Hung-Chien Roger
HC
Department of Microbiology, Chung Shan Medical University, Taichung 402, Taiwan.
Lin
Long-Liu
LL
Chao
Shiou-Huei
SH
Chen
Chun-Chang
CC
Wang
Wen-Ching
WC
Shaw
Chin-Ying
CY
Tsai
Ying-Chie
YC
Hu
Hui-Yu
HY
Hsu
Wen-Hwei
WH
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
D002712:Chlorides; D017967:Zinc Compounds; C016837:zinc chloride; D004492:Edetic Acid; D007931:Leucyl Aminopeptidase