Roig M G, Rashid D H, Kennedy J F
Departamento de Quimica Fisica, Facultad de Farmacia, Universidad de Salamanca, Spain.
Appl Biochem Biotechnol. 1995 Nov;55(2):95-121. doi: 10.1007/BF02783552.
High-alkaline protease (HAP) has been entrapped in Manugel DMB (an alginate gel) and assayed with two sizes and types of substrates: neutral protein casein and synthetic chromogenic tripeptide substrate, Z-Gly-Pro-Cit-PNA. Increasing the concentration of calcium chloride used for capsule formation decreased the measured enzyme activity with both substrates. Capsules were found to be stable in water for long periods of time, but they dissolved in both phosphate and carbonate-bicarbonate buffers. The pH vs activity profiles of encapsulated enzyme showed pH optima between 10 and 11 with both substrates. The calcium alginate matrix surrounding the enzyme was quite effective in stabilizing the enzyme at 20-25 degrees C and even more so at 4 degrees C. Enzyme stability at 50 degrees C was quite impressive, some enzyme activity being evident even after remaining for 1 wk at this temperature in water. Increasing concentrations of sodium dodecyl sulfate (SDS) were also found to inhibit the protease progressively, whereas a polyhexamethylene biguanidium chloride (PHMBH+Cl-) and SDS:PHMBH+Cl- combination showed the opposite effect. Optical microscopy, especially polarized light microscopy, provided a sensitive physical means of ascertaining some of the structural properties (sphericity, disorganization or organization, distinct layer enveloping the capsules, intensity of the maltese cross) of the capsules with and without enzyme before and after different chemical treatments and the presence or absence of the substrate.
高碱性蛋白酶（HAP）被包埋在Manugel DMB（一种藻酸盐凝胶）中，并使用两种大小和类型的底物进行测定：中性蛋白质酪蛋白和合成发色三肽底物Z-Gly-Pro-Cit-PNA。用于胶囊形成的氯化钙浓度增加会降低两种底物测得的酶活性。发现胶囊在水中长时间稳定，但它们会溶解在磷酸盐和碳酸盐-碳酸氢盐缓冲液中。包封酶的pH与活性曲线显示，两种底物的最适pH在10至11之间。酶周围的藻酸钙基质在20-25摄氏度下能非常有效地稳定酶，在4摄氏度时效果更佳。在50摄氏度下酶的稳定性相当可观，即使在该温度下于水中放置1周后仍有一些酶活性。还发现增加十二烷基硫酸钠（SDS）的浓度会逐渐抑制蛋白酶，而聚六亚甲基双胍氯化物（PHMBH⁺Cl⁻）与SDS：PHMBH⁺Cl⁻的组合则显示出相反的效果。光学显微镜，尤其是偏光显微镜，提供了一种灵敏的物理方法，可确定在不同化学处理前后有酶和无酶的胶囊的一些结构特性（球形度、无序或有序、包裹胶囊的明显层、马耳他十字的强度）以及底物的存在与否。
