比色法中抑制残留脂肪酶活性的方法：一项比较研究。
Methods for inhibition of residual lipase activity in colorimetric assay: a comparative study.
作者信息
Kanwar Shamsher Singh, Kaushal Rajeev Kumar, Jawed Arshad, Gupta Reena, Chimni Swapandeep Singh
机构信息
Department of Biotechnology, Himachal Pradesh University, Summer-Hill, Shimla 171 005, India.
出版信息
Indian J Biochem Biophys. 2005 Aug;42(4):233-7.
A comparative study of various treatments for inhibition of the residual activity of a lipase (obtained from Bacillus coagulans MTCC-6375) in a colorimetric assay using p-nitrophenyl palmitate (pNPP) was made. Direct chilling of contents of reaction mixture or addition of chilled mixture of ethanol : acetone (1:1) decreased the residual lipase activity by 94.0 and 95.0% respectively, as compared to lipase incubated at 45 degrees C for 20 min (control). Amongst various ionic and non-ionic detergents, Triton X-100 (0.07%, v/v) and sodium lauryl sarcosine or SLS (0.25%, w/v) partially, and SDS (0.05%, w/v) completely blocked the residual lipase activity of B. coagulans lipase in colorimetric assay. Addition of a serine protease inhibitor, PMSF (15 mM) or EDTA (200 mM) inhibited residual lipase activity by 99.5 and 100%, respectively. However, addition of reducing agents viz., 2-mercaptoethanol and dithiothreitol caused decomposition of chromogenic substrate (pNPP) thus rendering the colorimetric method unfit for lipase assay. EDTA (200 mM) and SDS (0.05%, w/v) were also highly effective in inhibiting the residual activities of lipases of Pseudomonas aeruginosa MTCC-4713, P. cepacia and commercial grade lipolytic preparations such as lipozyme, lipolase and porcine pancreatic lipase. However, PMSF (15 mM) completely inhibited the residual activity of lipase of P. aeruginosa.
开展了一项比较研究，以探讨在使用对硝基苯基棕榈酸酯（pNPP）的比色测定中，抑制一种脂肪酶（从凝结芽孢杆菌MTCC - 6375获得）残留活性的各种处理方法。与在45℃孵育20分钟的脂肪酶（对照）相比，直接冷却反应混合物的内容物或添加乙醇:丙酮（1:1）的冷却混合物分别使残留脂肪酶活性降低了94.0%和95.0%。在各种离子和非离子洗涤剂中，Triton X - 100（0.07%，v/v）和月桂基肌氨酸钠或SLS（0.25%，w/v）部分地，而SDS（0.05%，w/v）完全阻断了凝结芽孢杆菌脂肪酶在比色测定中的残留脂肪酶活性。添加丝氨酸蛋白酶抑制剂苯甲基磺酰氟（PMSF，15 mM）或乙二胺四乙酸（EDTA，200 mM）分别抑制残留脂肪酶活性达99.5%和100%。然而，添加还原剂即2 - 巯基乙醇和二硫苏糖醇会导致显色底物（pNPP）分解，从而使比色法不适用于脂肪酶测定。EDTA（200 mM）和SDS（0.05%，w/v）在抑制铜绿假单胞菌MTCC - 4713、洋葱伯克霍尔德菌的脂肪酶残留活性以及商业级脂解制剂如脂肪酶、脂解酶和猪胰脂肪酶的残留活性方面也非常有效。然而，PMSF（15 mM）完全抑制了铜绿假单胞菌脂肪酶的残留活性。
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