Fadnavis N W, Babu R L, Deshpande A
Biotransformations Laboratory, Discovery Block, Indian Institute of Chemical Technology, Tarnaka, Hyderabad, India.
Biochimie. 1998 Dec;80(12):1025-30. doi: 10.1016/s0300-9084(99)80007-8.
pH-Dependence of hydrolytic activity of trypsin has been studied in cationic reverse micellar system of cetyltrimethylammonium bromide (CTAB) in (50% v/v) chloroform/isooctane using a positively charged substrate N(alpha)-benzoyl-L-arginine ethyl ester (BAEE). The pH of the medium was varied from 4.0 to 8.5 with addition of 0.025 M citrate-phosphate buffer containing 1 mM CaCl2. Optimum pH for maximum enzyme activity, pH(opt) in reverse micelles is found to be similar to that observed in bulk aqueous solution (8.0-8.5). However, changes in activity of trypsin (k(cat)) as a function of water content W0 (W0 = [H2O]/[CTAB]) in reverse micelles are found to be pH dependent. At low pH (4.0) and low water content (W0 = 5) the enzyme is more active in reverse micelles than in bulk aqueous solution by a factor of 2. This 'superactivity' is lost at higher W0 values and the k(cat) in reverse micelles is found to be similar to that observed in aqueous bulk. At pH 5, the enzyme activity is found to be independent of W0 while at pH 6.0-6.5 the enzyme activity is low at W0 5 and increases with water content to a constant value which is still 50% lower than that in aqueous buffer. Above pH 7, the W0-activity profile becomes distinctly bell shaped with W0 optimum around 10-15. The enzyme activity at optimum W0 is close to that observed in aqueous bulk.
在十六烷基三甲基溴化铵（CTAB）的阳离子反胶束体系中，于（50% v/v）氯仿/异辛烷中，使用带正电荷的底物N-α-苯甲酰-L-精氨酸乙酯（BAEE），研究了胰蛋白酶水解活性的pH依赖性。通过添加含1 mM CaCl₂的0.025 M柠檬酸-磷酸盐缓冲液，将介质的pH从4.0变化至8.5。发现反胶束中胰蛋白酶最大酶活性的最佳pH（pH(opt)）与在本体水溶液中观察到的相似（8.0 - 8.5）。然而，发现反胶束中胰蛋白酶的活性（k(cat)）随水含量W₀（W₀ = [H₂O]/[CTAB]）的变化是pH依赖性的。在低pH（4.0）和低水含量（W₀ = 5）时，该酶在反胶束中的活性比在本体水溶液中高2倍。这种“超活性”在较高的W₀值时消失，并且发现反胶束中的k(cat)与在本体水中观察到的相似。在pH 5时，发现酶活性与W₀无关，而在pH 6.0 - 6.5时，在W₀ 5时酶活性较低，并随着水含量增加至一个恒定值，该值仍比在水性缓冲液中低50%。在pH 7以上，W₀-活性曲线明显呈钟形，最佳W₀约为10 - 15。最佳W₀时的酶活性接近在本体水中观察到的活性。
