Department of Animal Science, McGill University (Macdonald Campus), 21,111 Lakeshore Road, Ste. Anne de Bellevue, QC, H9X 3V9, Canada,
Fish Physiol Biochem. 2010 Dec;36(4):1041-60. doi: 10.1007/s10695-010-9382-y. Epub 2010 Feb 9.
Lipases were purified from delipidated pyloric ceca powder of two New Zealand-sourced fish, Chinook salmon (Oncorhynchus tshawytscha) and hoki (Macruronus novaezelandiae), by fractional precipitation with polyethylene glycol 1000, followed by affinity chromatography using cholate-Affi-Gel 102, and gel filtration on Sephacryl S-300 HR. For the first time, in-polyacrylamide gel activity of purified fish lipases against 4-methylumbelliferyl butyrate has been demonstrated. Calcium ions and sodium cholate were absolutely necessary both for lipase stability in the gel and for optimum activity against caprate and palmitate esters of p-nitrophenol. A single protein band was present in native polyacrylamide gels for both salmon and hoki final enzyme preparations. Under denaturing conditions, electrophoretic analysis revealed two bands of 79.6 and 54.9 kDa for salmon lipase. It is proposed that these bands correspond to an uncleaved and a final form of the enzyme. One band of 44.6 kDa was seen for hoki lipase. pI values of 5.8±0.1 and 5.7±0.1 were obtained for the two salmon lipase forms. The hoki lipase had a pI of 5.8±0.1. Both lipases had the highest activity at 35°C, were thermally labile, had a pH optimum of 8-8.5, and were more acid stable compared to other fish lipases studied to date. Both enzymes were inhibited by the organophosphate paraoxon. Chinook salmon and hoki lipases showed good stability in several water-immiscible solvents. The enzymes had very similar amino acid composition to mammalian carboxyl ester lipases and one other fish digestive lipase. The salmon enzyme was an overall better catalyst based on its higher turnover number (3.7±0.3 vs. 0.71±0.05 s(-1) for the hoki enzyme) and lower activation energy (2.0±0.4 vs. 7.6±0.8 kcal/mol for the hoki enzyme) for the hydrolysis of p-nitrophenyl caprate. The salmon and hoki enzymes are homologous with mammalian carboxyl ester lipases.
从两种新西兰来源的鱼类（奇努克三文鱼（Oncorhynchus tshawytscha）和竹荚鱼（Macruronus novaezelandiae））的脱脂幽门盲囊粉末中，通过聚乙二醇 1000 分级沉淀、胆酸钠亲和层析和 Sephacryl S-300 HR 凝胶过滤，纯化得到脂肪酶。首次在聚丙烯酰胺凝胶中证明了纯化的鱼脂肪酶对 4-甲基伞形酮丁酸的活性。钙离子和胆酸钠对脂肪酶在凝胶中的稳定性以及对癸酸和棕榈酸对硝基苯酚酯的最佳活性都是绝对必要的。在天然聚丙烯酰胺凝胶中，鲑鱼和竹荚鱼的最终酶制剂均存在单一的蛋白质带。在变性条件下，电泳分析显示鲑鱼脂肪酶有 79.6 和 54.9 kDa 的两条带。据推测，这些带对应于未切割的和酶的最终形式。竹荚鱼脂肪酶可见 44.6 kDa 的一条带。鲑鱼两种酶形式的等电点（pI）分别为 5.8±0.1 和 5.7±0.1。竹荚鱼脂肪酶的 pI 为 5.8±0.1。两种脂肪酶在 35°C 时活性最高，热不稳定，最适 pH 为 8-8.5，与迄今为止研究的其他鱼类脂肪酶相比，酸性稳定性更高。两种酶均被有机磷敌百虫抑制。奇努克三文鱼和竹荚鱼脂肪酶在几种水不混溶溶剂中表现出良好的稳定性。这些酶与哺乳动物羧酸酯脂肪酶和另一种鱼类消化脂肪酶具有非常相似的氨基酸组成。根据其更高的周转率（3.7±0.3 对 0.71±0.05 s(-1) 对竹荚鱼酶）和更低的活化能（2.0±0.4 对 7.6±0.8 kcal/mol 对竹荚鱼酶），鲑鱼酶是一种更好的水解对硝基苯癸酸酯的催化剂。鲑鱼和竹荚鱼酶与哺乳动物羧酸酯脂肪酶同源。
