【作者】 刘西之； 邱晓燕； 蔡秋凤； 刘光明； 苏文金； 曹敏杰；

【Author】 Xizhi Liu~(a,b,c) Xiaoyan Qiu~(a,b) Qiu-feng Cai~(a,b,c) Guang ming Liu~(a,b) Wenjin Su~(a,b) Minjie Cao~(a,b) (a College of Biological Engineering,Jimei University,Xiamen 361021,China;b Key Laboratory of Science and Technology for Acquaculture and Food Safety,Jimei University,Xiamen 361021,China; c College of Food Science and Technology,Shanghai Ocean University,Shanghai 201306,China)

【机构】 集美大学生物工程学院； 福建省高校水产科学技术与食品安全重点实验室； 上海海洋大学食品学院；

【摘要】 采用硫酸铵盐析、DEAE-Sephacel,DEAE-Sepharose阴离子交换柱层析、SP-Sepharose阳离子交换柱层析和Sephacryl S-200凝胶过滤柱层析,从青石斑鱼（Epinephelus awoara）的胃中分离纯化到三种胃蛋白酶原（PG-1、PG-2和PG-3）,SDS-PAGE结果显示,它们的分子量分别为35、36和37 kDa。在pH 2.0下,三种胃蛋白酶原转化为有活性的胃蛋白酶P-1、P-2和P-3,分子量分别约为33、33和34kDa。三种酶的最适pH分别为3.0、2.5和2.5,最适温度均为40℃,且均能被典型的天冬氨酸蛋白酶抑制剂pepstatin A有效抑制。免疫交叉反应结果表明,三种胃蛋白酶原能与大鼠抗黄鳍鲷PG-Ⅰ,PG-Ⅱ,PG-3b,PG-3a,PG-4a及PG-4b多克隆抗体发生不同程度的免疫交叉反应。动力学参数测定结果表明,P-1、P-2和P-3对酸变性牛血红蛋白的K_m、k_cat及k_cat/K_m值分别为7.0×10^-5M,17.6 s^-1,2.5×10⁵M^-1·^-1;5.5×10^-5M,22.8 s^-1,4.1×10⁵M^-1·s^-1和5.2×10^-5 M,18.7 s^-1,3.6×10⁵M^-1·s^-1。更多还原

【Abstract】 Three pepsinogens（PG-1、PG-2 and PG-3 ）were highly purified from the stomach of Epinephelus awoara by ammonium sulfate fractionation,DEAE-Sephacel and DEAE-Sepharose anionic exchange column chromatography,SP-Sepharose cationic exchange column chromatography,and Sephacryl S-200 gel filtration.Their molecular masses were estimated to be 35,36 and 37 kDa,respectively,by SDS PAGE. Pepsinogens converted into their active form pepsins（P-KP-2 and P-3）with molecular masses of approximately 33,33 and 34 kDa,respectively,under pH 2.0.They showed maximal activity at pH 3.0,2.5 and 2.5.And their optimal temperatures were 40℃.All three pepsins were completely inhibited by pepstatin A,a typical aspartic proteinase inhibitor.Western blot analysis revealed that these pepsinogens had different cross reaction with anti-sea bream PG-I,PG-II,PG-3b,PG-3a,PG-4a and PG-4b polyclonal antibodies.The kinetic constants of K_m,k_cat,and k_cat/K_m of pepsins（P-1、P-2 and P-3）for acidic-denatured bovine hemoglobin were calculated as 7.0×10^-5 M,17.6 s^-1,2.5×10⁵ M^-1·s^-1;5.5×10^-5 M,22.8 s^-1,4.1×10⁵ M^-1·s^-1 and 5.2×10^-5 M,18.7 s^-1,3.6×10⁵ M^-1·s^-1,respectively.更多还原