Department of Chemical Engineering and Biotechnology, Ariel University Center of Samaria, Ariel 44837, Israel.
Curr Microbiol. 2010 Sep;61(3):176-83. doi: 10.1007/s00284-010-9593-6. Epub 2010 Feb 3.
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.
本研究报告了一种从苏云金芽孢杆菌亚种以色列（Bti）中提取含锌金属蛋白酶 Camelysin 的简单快速方法，方法是用含有单层脂小体的卵 L-α-磷脂酰胆碱从完整的细菌细胞中提取，然后在蔗糖梯度上分离。分离得到的 Camelysin 的特性表明其分子量为 23 kDa，等电点为 6.2。Camelysin 对底物偶氮酪蛋白的最适活性温度为 37°C，最适 pH 值为 7.5。然而，该酶在碱性 pH 值（8-10）下仍保持较高的活性。在丰富的生长培养基（LB）中，Camelysin 出现在 Bti 生长的对数后期，并在静止期达到最大值。结果表明，Camelysin 能激活 Bti 产生的原毒素 Cyt1Aa 和 Cyt2Ba。在存在 50%（w/w）Camelysin 的情况下，Cyt1Aa 的溶血活性从 40%增加到 70%，Cyt2Ba 的溶血活性从 6%增加到 50%。结论是这些原毒素不仅可以被昆虫肠道蛋白酶激活，还可以被 Bti 细菌内源性金属蛋白酶 Camelysin 激活。
