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南极磷虾羧肽酶A的分离纯化及其酶学性质分析
作者:  晶1 2 周德庆2 王珊珊2  婷2 3 朱兰兰2 4* 
单位: (1上海海洋大学 食品学院 上海 201306 2中国水产科学研究院 黄海水产研究所 山东 青岛 266071  
关键词: 南极磷虾 羧肽酶A 分离纯化 酶学性质 
分类号:S985.21
出版年,卷(期):页码:2017,48(8):1470-1476
摘要:

【目的】建立南极磷虾羧肽酶A的分离纯化方法,并研究分析其酶学性质,为南极磷虾羧肽酶A的开发及应用打下基础。【方法】南极磷虾粗酶液经硫酸铵分级沉淀、HiTrap DEAE FF阴离子交换柱层析和SEC 70凝胶过滤柱层析,纯化得到的羧肽酶A,以SDS-PAGE分析其分子量;通过分析酶系反应温度、pH、金属离子、抑制剂对南极磷虾羧肽酶A活力的影响,明确其酶学性质和酶促动力学。【结果】南极磷虾羧肽酶A分子量为75.0 kD,其最适温度30 ℃,最适pH 8.0。Mg2+、Zn2+、Mn2+和Ni2+对南极磷虾羧肽酶A有显著的激活作用(P<0.05,下同),且金属离子浓度越高,激活效果越明显;Ca2+、Fe3+、Cu2+和Hg2+对南极磷虾羧肽酶A存在不同程度的抑制作用,以Hg2+的抑制作用最强,Fe3+的抑制作用最弱。金属蛋白酶抑制剂乙二胺四乙酸(EDTA)、3-苯基丙酸(3-phenylpropionic acid)和1,10-菲罗啉(1,10-phenathroline)对南极磷虾羧肽酶A活力有显著的抑制作用,且抑制剂浓度越高,抑制效果越明显。【结论】南极磷虾羧肽酶A具有金属蛋白酶特性,可开发成降解酶制剂在食品工业及医药行业中推广应用。

【Objective】The separation and purification method for carboxypeptidase A from Euphausia superba was established and its enzymatic properties were studied in order to provide technical support for the exploitation and utilization of carboxypeptidase A from E. superba. 【Method】Crude enzyme liquid of E. superba was purified by ammonium sulfate precipitation,column chromatographieson HiTrap DEAE FF ion exchange chromatography and SEC 70 gel chromatography. After purification, carboxypeptidase A was obtained. Its molecular weight was determined by SDS-PAGE electrophoresis analysis. The enzyme properties and enzymatic kinetics of carboxypeptidase A were studied through investigating the effects of temperature, pH, metal ions and inhibitors on activity of carboxypeptidase A. 【Result】Molecular weight of this protease was 75 kD, the optimal temperature for it was 30 ℃, and suitable pH was 8.0. Mg2+, Zn2+, Mn2+ and Ni2+ significantly activated carboxypeptidase A from E. superba(P<0.05, the same below). As the concentration of metal ions increased, the effects were more obvious. While Ca2+,Fe3+,Cu2+,Hg2+ inhibited the activity of carboxypeptidase A to various extents. The inhibition effects of Hg2+ was the strongest, but that of Fe3+was the weakest. Inhibitors metalloproteinases ethylenediaminetetraacetic acid(EDTA), 3-phenylpropionic acid and 1,10-phenathroline had significant inhibition effects on carboxypeptidase A from E. superba. As the concentrations were higher, the inhibition effects were enhanced. 【Conclusion】Carboxypeptidase A from E. superba contains features of metalloproteinase, therefore it can be developed into enzyme degradation product for utilization in food industry and pharmaceuticals industry.

基金项目:
国家自然科学基金项目(31571915,31201311);农业部南极海洋生物资源开发利用项目(2016)
作者简介:
*为通讯作者,朱兰兰(1981-),副研究员,主要从事水产品加工与质量安全研究工作,E-mail:chinaxiaolan@163.com。吴晶(1990-),研究方向为水产品质量安全,E-mail:47426387@qq.com
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