| 金乌贼肌肉中三甲胺脱甲基酶的分离纯化及酶学性质 |
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| 引用本文： | 金洋,薛张芝,张洪超,宋正规,朱仁义,李和生,步婷婷.金乌贼肌肉中三甲胺脱甲基酶的分离纯化及酶学性质[J].水产学报,2017,41(6):845-853. |
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| 作者姓名： | 金洋 薛张芝 张洪超 宋正规 朱仁义 李和生 步婷婷 |
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| 作者单位： | 宁波大学海洋学院,浙江 宁波,315211 |
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| 基金项目： | 浙江省重大科技专项项目(2009C03017-3)；“水产”浙江省重中之重学科开放(xkzsc1426, xkzsc1523)；宁波市自然科学(013A610156) |
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| 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。
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| 关 键 词： | 金乌贼 肌肉 三甲胺脱甲基酶 纯化 酶学性质 |
| 收稿时间： | 2016/12/14 0:00:00 |
| 修稿时间： | 2017/3/7 0:00:00 |
Purification and enzymatic properties of TMAOase from muscles of Sepia esculenta |
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| JIN Yang,XUE Zhangzhi,ZHANG Hongchao,SONG Zhenggui,ZHU Renyi,LI Hesheng and BU Tingting.Purification and enzymatic properties of TMAOase from muscles of Sepia esculenta[J].Journal of Fisheries of China,2017,41(6):845-853. |
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| Authors: | JIN Yang XUE Zhangzhi ZHANG Hongchao SONG Zhenggui ZHU Renyi LI Hesheng and BU Tingting |
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| Institution: | Department of Food Science and Engineering,Ningbo University,Zhejiang Ningbo,315211,,,,Department of Food Science and Engineering,Ningbo University,Zhejiang Ningbo,315211 |
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| Abstract: | In order to extract and purify TMAOase, acetate buffer made of NaCl (0.1 mol/L) and Tris-CH3COOH (pH 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 pH of the purified enzyme was 7.0, which stayed well at this pH, but decreased in acidic and alkaline conditions. As the pH 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 SDS-PAGE, 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. |
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| Keywords: | cuttlefish TMAOase purification enzymatic properties |
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