Isolation, characterization and biological role of camelysin from Bacillus thuringiensis subsp. israelensis.
The present study reports a simple rapid method for isolating the zinc-containing metalloprotease camelysin from Bacillus thuringiensis subsp. israelensis (Bti) by extraction from intact bacterial cells with egg L-alpha-phosphatidylcholine containing monolamellar liposomes, followed by separation on a sucrose gradient. Characterization of the isolated camelysin revealed a molecular weight of 23 kDa and a pI of 6.2. The camelysin exhibited maximal activity against the substrate azocasein at a temperature of 37 degrees C and pH 7.5. However, the enzyme's activity remained high also at basic pH values (8-10). In a rich growth medium (LB), camelysin appeared at the late logarithmic phase of Bti growth and reached its maximum in the stationary phase. Camelysin was shown to activate the protoxins Cyt1Aa and Cyt2Ba produced by Bti. The hemolytic activity of Cyt1Aa increased from 40 to 70% and that of Cyt2Ba from 6 to 50% in the presence of 50% (w/w) camelysin. It is concluded that these protoxins can be activated not only by insect gut proteases, but also by the endogeneous metalloprotease camelysin of the Bti bacterium.
Nisnevitch
Marina
M
Department of Chemical Engineering and Biotechnology, Ariel University Center of Samaria, Ariel 44837, Israel. marinan@ariel.ac.il
Sigawi
Sasi
S
Cahan
Rivka
R
Nitzan
Yeshayahu
Y
D000595:Amino Acid Sequence; D001413:Bacillus thuringiensis / Q000201:enzymology*; D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins / Q000378:metabolism; D002364:Caseins / Q000378:metabolism; D002499:Centrifugation, Density Gradient / Q000379:methods; D003470:Culture Media / Q000737:chemistry; D004731:Endotoxins / Q000378:metabolism; D004795:Enzyme Stability; D020869:Gene Expression Profiling; D006460:Hemolysin Proteins / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D008081:Liposomes / Q000302:isolation & purification; D008666:Metalloendopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016415:Sequence Alignment; D013696:Temperature
D000083722:Bacillus thuringiensis Toxins; D001426:Bacterial Proteins; D002364:Caseins; D003470:Culture Media; D004731:Endotoxins; D006460:Hemolysin Proteins; D008081:Liposomes; C026018:azocasein; C030956:insecticidal crystal protein, Bacillus Thuringiensis; D008666:Metalloendopeptidases; C438729:camelysin