Lee Y P, Chung G H, Rhee J S
Department of Biotechnology, Korea Advanced Institute of Science and Technology, Taejon.
Biochim Biophys Acta. 1993 Aug 11;1169(2):156-64. doi: 10.1016/0005-2760(93)90200-s.
Pseudomonas fluorescens SIK W1 lipase was expressed as a form of inclusion bodies in Escherichia coli, which was equivalent to 46% of total cell protein. The inclusion bodies isolated from other cell components were solubilized in the buffer containing 8 M urea and then refolded by diluting urea. The lipase with active conformation was purified by hydrophobic interaction chromatography, gel filtration, anion-exchange chromatography and hydroxyapatite chromatography from the refolded sample. By these purification steps, a single band for active lipase was detected on non-reducing SDS-PAGE and 10-fold purification was attained on the basis of specific activity. Specific activity of the purified lipase toward olive oil emulsion was found to be 7395 units per mg protein. The optimum pH and temperature of the lipase were pH 8.5 and 45-55 degrees C, respectively. The lipase showed higher lipolytic activity toward tricaproin (C6) and tricaprylin (C8) among the triacylglycerols examined and preferentially hydrolyzed ester bond of 1- and 3-position of triolein. Lipase activity was greatly increased by approx. 6-fold and stability for pH was shifted to alkaline pH by Ca2+ ion. The lipase was inhibited by Hg2+, Ag2+, p-chloromercuribenzoate, diethylpyrocarbonate and sodium dodecyl sulfate.
荧光假单胞菌SIK W1脂肪酶在大肠杆菌中以包涵体形式表达，其含量相当于总细胞蛋白的46%。从其他细胞成分中分离出的包涵体在含有8 M尿素的缓冲液中溶解，然后通过稀释尿素进行复性。具有活性构象的脂肪酶通过疏水相互作用色谱、凝胶过滤、阴离子交换色谱和羟基磷灰石柱色谱从复性样品中纯化。通过这些纯化步骤，在非还原SDS-PAGE上检测到单一条带的活性脂肪酶，基于比活性实现了10倍的纯化。纯化后的脂肪酶对橄榄油乳液的比活性为每毫克蛋白7395单位。该脂肪酶的最适pH和温度分别为pH 8.5和45 - 55℃。在所检测的三酰甘油中，该脂肪酶对三己酸甘油酯（C6）和三辛酸甘油酯（C8）表现出较高的脂解活性，并且优先水解三油精1位和3位的酯键。Ca2+离子可使脂肪酶活性大幅提高约6倍，且pH稳定性向碱性pH偏移。该脂肪酶受到Hg2+、Ag2+、对氯汞苯甲酸、焦碳酸二乙酯和十二烷基硫酸钠的抑制。
