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首页> 外文期刊>Fruit, Vegetable and Cereal Science and Biotechnology >Purification and Properties of the Major Leueyl Aminopeptidase from Solanum tuberosum Tubers
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Purification and Properties of the Major Leueyl Aminopeptidase from Solanum tuberosum Tubers

机译：马铃薯块茎中主要的脂酰氨基肽酶的纯化及性质

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摘要

Zymogram analysis revealed three isoforms of leucyl aminopeptidase (LAP) using leucine-p-nitroanilide as a substrate in the crude extracts of different potato tuber cultivars. The major LAP isoform, detected in all analyzed potato cultivars, was alsoactive using methionine-p-nitroanilide, alanine-p-nitroanilide and lysine-p-nitroanilide as a substrates. The major LAP from potato tuber was subsequently purified and characterized. Specific LAP activity increased 160-fold by purification of the crude extract. The purified enzyme had a pH optimum of 9.0 and temperature optimum of 40 deg C. LAP hydrolyzed leucine-p-nitroanilide with a K_M of 0.048 m M and a V_(max) of 31.56 mM/min. Among a number of inhibitors tested, the most efficient was 1,10-phenanthroline, while ethylene glycol tetraacetic acid (EGTA) stimulated LAP activity. Almost all divalent cations tested, significantly inhibited the enzyme activity. LAP, purified by gel-filtration on Superose 12-FPLC column, had molecular mass of 90 kDa andisoelectric point of 5.45. Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed one band of 48 kDa. LAP from potato tubers displayed some novel properties among plant LAPs.
机译：形态学分析显示使用亮氨酸对硝基苯胺作为底物，在不同马铃薯块茎品种的粗提物中使用了亮氨酰氨肽酶（LAP）的三种同工型。使用所有蛋氨酸对硝基苯胺，丙氨酸对硝基苯胺和赖氨酸对硝基苯胺作为底物，在所有分析过的马铃薯品种中都发现了主要的LAP亚型。随后纯化并鉴定了马铃薯块茎的主要LAP。通过提纯粗提取物，比LAP活性增加了160倍。纯化的酶的最适pH值为9.0，最适温度为40℃。LAP水解的亮氨酸-对硝基苯胺的K_M为0.048 mM，V_（max）为31.56 mM / min。在测试的许多抑制剂中，最有效的是1,10-菲咯啉，而乙二醇四乙酸（EGTA）刺激了LAP活性。几乎所有测试的二价阳离子都显着抑制了酶的活性。通过在Superose 12-FPLC柱上的凝胶过滤纯化的LAP具有90kDa的分子量和5.45的等电点。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）显示48 kDa的一条带。马铃薯块茎的LAP在植物LAP中显示出一些新特性。

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