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南极磷虾粗酶液中蛋白酶的基础酶学性质研究及抑制剂的开发
陈怡炫1,2, 陈 昕2,3, 王 芳2, 惠彦行1,2, 盛 军2, 徐海波1, 徐甲坤2,4
1.中国海洋大学化学化工学院 青岛 266100;2.农业农村部极地渔业开发重点实验室 中国水产科学研究院黄海水产研究所 青岛 266071;3.上海海洋大学食品学院 上海 201306;4.青岛海洋科学与技术试点国家实验室海洋药物与生物制品功能实验室 青岛 266071
摘要:
本研究探讨了反应温度、pH、金属离子(Zn2+、Mg2+、Cu2+、Fe3+、Al3+和Ca2+)对南极磷虾粗酶液中蛋白酶活性的影响,研究了苯甲基磺酰氟(PMSF)、EDTA∙2Na、碘乙酰胺(IAM)和甲苯磺酰–苯丙氨酸氯甲基酮(TPCK)对蛋白酶活性的抑制效果。结果显示,南极磷虾粗酶液中蛋白酶的最适反应温度为40℃;最适反应pH值为8.0;当金属离子浓度为0.5 mmol/L时,Zn2+、Mg2+、Cu2+、Fe3+和Al3+金属离子对蛋白酶酶活的抑制率分别为55.02%、55.02%、35.39%、20.67%和41.12%,而Ca2+离子对蛋白酶活有明显的促进作用;PMSF和EDTA∙2Na对蛋白酶酶活具有较为显著的抑制作用,PMSF的浓度为8.0 mmol/L时,抑制率为60%;EDTA∙2Na浓度为0.6 mmol/L时,抑制率为86.67%;IAM在低浓度时有一定的抑制作用;TPCK低浓度时抑制效果不明显,浓度升高时有一定的抑制作用。在5~30℃的温度范围内,随着温度升高,EDTA∙2Na对蛋白酶酶活的抑制效果逐渐增加。本研究阐明了影响南极磷虾粗酶液蛋白酶酶活的因素,开发了针对南极磷虾粗酶液中蛋白酶活性的抑制剂,为南极磷虾在食品领域的开发利用提供了基础理论数据。
关键词:  南极磷虾  粗酶液  基础酶学性质  抑制剂
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Study on the Basic Enzymatic Properties of Protease in Crude Enzyme of Antarctic Krill and the Development of Inhibitors
CHEN Yixuan1,2, CHEN Xin2,3, WANG Fang2, HUI Yanxing1,2, SHENG Jun2, XU Haibo1,2, XU Jiakun2,4
1.College of Chemistry and Chemical Engineering, Ocean University of China, Qingdao 266100;2.Key Laboratory of Sustainable Development of Polar Fisheries, Ministry of Agriculture and Rural Affairs, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071;3.College of Food Sciences and Technology, Shanghai Ocean University, Shanghai 201306;4.Laboratory for Marine Drugs and Byproducts of Pilot National Laboratory for Marine Science and Technology (Qingdao), Qingdao 266071
Abstract:
The effects of reaction temperature, pH, and metal ions (Zn2+, Mg2+, Cu2+, Fe3+, Al3+, and Ca2+) on the protease activity of crude enzymes of Antarctic krill were studied, and the inhibitory effects of phenylmethanesulfonyl fluoride (PMSF), EDTA∙2Na, iodoacetamide (IAM), and toluenesulfonyl- phenylalanine chloromethyl ketone (TPCK) on protease activity also were investigated. The experimental results indicated the optimum reaction temperature of the protease in the crude enzyme of Antarctic krill was 40℃, and the optimum pH was 8.0; when the metal ion concentration was 0.5 mmol/L, the inhibitory rates of Zn2+, Mg2+, Cu2+, Fe3+, and Al3+ on the protease activity of Antarctic krill were 55.02%, 55.02%, 35.39%, 20.67%, and 41.12% respectively, while protease activity increased in the presence of Ca2+. The protease activity was significantly inhibited by PMSF and EDTA∙2Na. When the concentration of PMSF was 8.0 mmol/L, the inhibition rate was 60%; when the concentration of EDTA∙2Na was 0.6 mmol/L, the inhibition rate was 86.67%. IAM had a certain inhibitory effect at low concentrations, while TPCK showed no inhibitory effect. With the increase in temperature, the inhibitory effect of EDTA∙2Na on enzyme activity was gradually increased in the temperature range of 5–30℃. This study clarified the factors affecting the enzyme activity of Antarctic krill crude enzyme and developed an inhibitor of protease activity thereof, which provides the basic theoretical data for the development and utilization of Antarctic krill in the food industry.
Key words:  Antarctic Krill  Crude enzyme  Basic enzymatic properties  Inhibitors