Surinenaite Birute, Bendikiene Vida, Juodka Benediktas, Bachmatova Irena, Marcinkevichiene Liucija
Department of Biochemistry and Biophysics, Vilnius University, M. K. Ciurlionio 21, LT-2009, Vilnius, Lithuania.
Biotechnol Appl Biochem. 2002 Aug;36(1):47-55. doi: 10.1042/ba20020013.
The lipase from Pseudomonas mendocina 3121-1 was found to be homogeneous with a molecular mass of 30 kDa by SDS/PAGE. It is composed of two identical subunits. A molecular mass of 62 kDa was determined by gel chromatography on a Toyopearl HW-55F column. Some physicochemical properties of the lipase were investigated using p-nitrophenyl butyrate (p-NPB), Tween 80 solution and Sigma olive-oil emulsion as substrates. The optimum temperature was determined to be 52 degrees C with p-NPB, in the range 50-60 degrees C with Tween 80 and in the range 50-65 degrees C with olive-oil emulsion. The optimum pH was determined to be in the pH range 7.2-7.5, both with Tween and the emulsion, but was unusually alkaline (pH 9.5) with p-NPB. The enzyme was activated for p-NPB hydrolysis by thermal treatment up to 60 min at 60 degrees C, pH 7.0-8.2, but was rapidly inactivated at 70-80 degrees C and at pH 7.0. The lipase was shown to be more thermolabile at 60 degrees C with respect to other two substrates. Using the emulsified substrate, no activity was obtained after preincubating the enzyme for 30 min at 70 degrees C. The enzyme was found to be pH-tolerant when stored at 20 degrees C, pH 6.3-10.3 (100 mM Briton-Robson buffer) as the half-life (t(1/2)) was more than 240 h when p-NPB was used as the substrate. By contrast, the pH-stability range was more narrow (pH 8.0-10.5) with olive-oil emulsion. The effect of various metal ions and EDTA depended on the nature of the substrate.
通过SDS/PAGE分析发现，门多萨假单胞菌3121-1脂肪酶呈均一状态，分子量为30 kDa。它由两个相同的亚基组成。在Toyopearl HW-55F柱上进行凝胶色谱分析，测得其分子量为62 kDa。以对硝基苯丁酸酯（p-NPB）、吐温80溶液和Sigma橄榄油乳液为底物，对该脂肪酶的一些理化性质进行了研究。以p-NPB为底物时，确定最适温度为52℃；以吐温80为底物时，最适温度在50-60℃范围内；以橄榄油乳液为底物时，最适温度在50-65℃范围内。以吐温和乳液为底物时，确定最适pH在7.2-7.5范围内，但以p-NPB为底物时，最适pH异常呈碱性（pH 9.5）。在60℃、pH 7.0-8.2条件下热处理长达60分钟，该酶对p-NPB水解具有激活作用，但在70-80℃和pH 7.0条件下迅速失活。相对于其他两种底物，该脂肪酶在60℃时表现出更高的热不稳定性。使用乳化底物时，在70℃下将酶预孵育30分钟后未检测到活性。当在20℃、pH 6.3-10.3（100 mM Britton-Robson缓冲液）条件下储存时，以p-NPB为底物，该酶表现出pH耐受性，半衰期（t(1/2)）超过240小时。相比之下，以橄榄油乳液为底物时，pH稳定范围更窄（pH 8.0-10.5）。各种金属离子和EDTA的影响取决于底物的性质。
