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机构地区:[1]中国水产科学研究院黑龙江水产研究所,黑龙江哈尔滨150070 [2]哈尔滨工业大学理学院,黑龙江哈尔滨150001
出 处:《大连海洋大学学报》2012年第6期558-563,共6页Journal of Dalian Ocean University
基 金:科技部农业科技基金资助项目(02EFN216900727);哈尔滨市科技局优秀学科带头人项目(2010RFXYNO47)
摘 要:采用两种硫酸铵饱和梯度盐析方法对怀头鲇Silurus soldatovi胃黏膜蛋白酶激活液进行粗提,用6种组合层析条件对胃蛋白酶粗提液进行Sephadex G-75凝胶过滤层析,获得初步纯化的蛋白酶,并通过蛋白质浓度、蛋白酶活力及SDS-PAGE凝胶电泳对其分析鉴定。结果表明:在28%、56%、83%(NH4)2SO4饱和度条件下分级盐析效果比较理想;不同层析条件下层析峰分离液的蛋白酶活力测定均显示出酶活性,层析峰蛋白酶比活力较粗提液提高5~9倍,60 cm柱(层析柱高52 cm、直径1.6 cm)层析峰分离效果较好。从盐析粗提液电泳结果,以及在60 cm层析柱、pH为3.3的0.01 mol/Lβ-丙氨酸-甲酸缓冲液、0.8mL/min流速条件下的层析峰蛋白分管收集液酶活力峰值和层析峰峰顶形状判断,怀头鲇胃蛋白酶可能有两种蛋白组分存在,并测得相对分子质量为26 000~47 000。The crude protease activated fluid (pH 2.8, 2 h) was extracted from gastric mucosa of northern sheat- fish Silirus soldatovi by two methods of (NH4 )2SO4 saturated salt gradient. Pepsin crude extracts were dealt with six kinds of combination chromatography conditions, and analyzed by gel filtration chromatography on Sephadex G-75 to produce preliminary purification proteases, and to be identified by protein concentration, protease activity and SDS-polyacrylamide gel electrophoresis analysis. The results showed that good isolation and purification were ob- served at 28%, 56% , and 83% (NH,)2SO4 saturated salt gradient. The chromatographic-peak separation solu- tions under different chromatographic conditions revealed that the specific activity of chromatography-peak protease was increased by about five to nine times compared with that of crude extracts, and the effect of 60 cm( ~1.6 cmx 52 cm)column chromatography peak separation was quite good. From the crude extract electrophoresis and chroma- tography-peak shapes under the condition of 0.8 mL/min flowing chromatography peak and the 60 cm chromatogra- phy column, and 0.01 mol/L 13-alanine-formate buffer( pH 3.3 ), there were two protein components with molec- ular weight from 26 000 to 47 000 in stomach proteases of northern sheatfish.
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