金乌贼肌肉中三甲胺脱甲基酶的分离纯化及酶学性质  

Purification and enzymatic properties of TMAOase from muscles of Sepia esculenta

作　　者：金洋[1] 薛张芝 张洪超[1] 宋正规 朱仁义[1] 步婷婷 李和生[1] 

机构地区：[1]宁波大学海洋学院,浙江宁波315211

出　　处：《水产学报》2017年第6期845-853,共9页Journal of Fisheries of China

基　　金：浙江省重大科技专项(2009C03017-3);"水产"浙江省重中之重学科开放基金(xkzsc1523);宁波市自然科学基金(013A610156)~~

摘　　要：为了提取纯化金乌贼肌肉中的三甲胺脱甲基酶(TMAOase),本研究采用含有0.1 mol/L NaCl、pH 7.0、浓度为20 mmol/L的三羟甲基氨基甲烷(Tris)-醋酸缓冲液提取粗酶液,经透析、浓缩处理后,通过DEAE-52阴离子交换柱层析和Sephacryl S-300柱层析得到了纯化的TMAOase,并对其酶学性质进行了研究。结果显示,经Sephacryl S-300柱层析的TMAOase相比粗酶纯化了209.54倍;粗酶和纯化酶的最适温度分别为55和50°C,当温度高于最适温度时,酶活性开始出现显著下降,粗酶在80°C仍残留21.9%的活性;而纯化酶在80°C时,几乎检测不到酶活;粗酶和纯化酶的最适p H均为7.0,中性条件下表现稳定,在酸性和碱性条件下稳定性下降,p H为9.0时,粗酶残留60.7%的活性,而纯化酶的活性仅为20.5%。以双倒数作图法(Lineweaver-Burk法)测得纯化的TMAOase的Km值为22.8 mmol/L;经SDS-PAGE电泳分析,测得其分子量为21.3 ku;在化学物质中,柠檬酸和CaCl_2对酶活性具有显著的促进作用,H_2O_2和Na_2S对TMAOase活性有显著抑制作用。In order to extract and purify TMAOase, acetate buffer made of Na Cl（0.1 mol/L） and Tris-CH3COOH（p H 7.0, 20 mmol/L） was used to extract from the muscles of cuttlefish.After the process of acid, alkali, dialysis and concentrated treatment, the extract was further purified by DEAE-52 Sephacel and Sephacryl S-300 chromatography. The enzymatic properties of TMAOase were also studied. The results revealed that the enzyme purified by Sephacryl S-300 was 209.54 times purer than the crude enzyme. The optimum temperature of the curde and purified enzyme was 55 and 50 ℃, respectively. Moreover, their thermal stability decreased significantly over this temperature. As the temperature reached 80 ℃, crude enzyme still retaimed 21.9% of its activity. Whereas purified enzyme lost its activity totally. The optimum p H of the purified enzyme was 7.0, which stayed well at this p H, but decreased in acidic and alkaline conditions. As the p H increased to 9.0,especially, the activity of crude and pure enzyme reduced to 60.7% and 20.5%, respectively. The Km of TMAOase measured by Lineweaver-Burk double reciprocal mapping method was 22.8 mmol/L. The purity of the obtained TMAOase was proved by SDSPAGE, with a molecular weight of 21.3 ku. Chemicals of citric acid and CaCl2 remarkably promoted the activity of the enzyme, while H2O2 and Na2S significantly inhibited TMAOase activity.

关 键 词：金乌贼 肌肉 三甲胺脱甲基酶 纯化 酶学性质 

分 类 号：S912[农业科学—水产科学] TS254.1[轻工技术与工程—水产品加工及贮藏工程]