Purification and identification of a novel leucine aminopeptidase from Bacillus thuringiensis israelensis.
A novel leucine aminopeptidase was purified from a Bacillus thuringiensis israelensis (Bti) culture. The purification stages included heating the concentrated supernatant to 65 degrees C for 90 min, anion-exchange chromatography by DEAE cellulose, and hydrophobic chromatography by phenyl Sepharose. The specific activity of leucine aminopeptidase after the hydrophobic chromatography increased by 215.5-fold and the yield was 16%. The molecular weight of the active enzyme was 59 kDa. Mass spectrometry analysis of the 59-kDa leucine aminopeptidase revealed that this protein has at least 41% homology with the cytosol leucine aminopeptidase produced by Bacillus cereus. Maximal leucine aminopeptidase activity occurred at 65 degrees C, pH 10 toward leucine as the amino acid terminus. The enzyme was strongly inhibited by bestatin, dithiothreitol, and 1,10-phenanthroline, indicating that the enzyme might be considered as a metallo-aminopeptidase that has disulfide bonds at the catalytic site or at a region that influences its configuration. Examination of the purified leucine aminopeptidase's effect on the activation of the protoxin Cyt1Aa from Bti revealed that when it acts synergistically with Bti endogenous proteases, it has only a minor role in the processing of Cyt1Aa into an active toxin.
Cahan
Rivka
R
Department of Chemical Engineering and Biotechnology, College of Judea and Samaria, Ariel 44837, Israel. rivkac@yosh.ac.il
Hetzroni
Efrat
E
Nisnevitch
Marina
M
Nitzan
Yeshayahu
Y
D000626:Aminopeptidases / Q000737:chemistry / Q000378:metabolism; D000818:Animals; D001413:Bacillus thuringiensis / Q000201:enzymology* / Q000235:genetics; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism; D002384:Catalysis / Q000187:drug effects; D002852:Chromatography, Ion Exchange; D004229:Dithiothreitol / Q000494:pharmacology; D004731:Endotoxins / Q000737:chemistry / Q000378:metabolism; D006460:Hemolysin Proteins / Q000737:chemistry / Q000378:metabolism; D006461:Hemolysis; D006863:Hydrogen-Ion Concentration; D007930:Leucine / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D007931:Leucyl Aminopeptidase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010618:Phenanthrolines / Q000494:pharmacology; D011817:Rabbits; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D001427:Bacterial Toxins; D004731:Endotoxins; D006460:Hemolysin Proteins; D010618:Phenanthrolines; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D000626:Aminopeptidases; D007931:Leucyl Aminopeptidase; D007930:Leucine; C012211:ubenimex; D004229:Dithiothreitol; C025205:1,10-phenanthroline