作　　者： ; ; ; ;

机构地区： 华南理工大学生物科学与工程学院

出　　处： 《食品科学》 2009年第19期244-247,共4页

摘　　要： 目的:以蒜氨酸酶竞争性抑制剂S-烷基-L-半胱氨酸为配基,制备亲和层析胶分离纯化蒜氨酸酶。方法:以1,4-丁二醇二缩水甘油醚为活化剂,在碱性条件下,活化载体SepharoseCL-4B,偶联半胱氨酸,作为蒜氨酸酶的亲和层析胶。低温粉碎大蒜所得的大蒜液,经25%PEG-5000沉淀,得到粗酶。用制得的亲和胶分离纯化,并利用SDS-PAGE对酶纯度进行检验。结果:大蒜液用25%PEG-5000沉淀可以较简便地分离出粗酶,经制得的亲和胶层析,活力回收达到68.42%,比活力为9.493U/mg,纯化倍数达到22.17倍。提纯的蒜氨酸酶为电泳纯,亚基约为52kD。纯酶的最适温度为35℃,最适pH值为6.23,Km=9.845mmol/L,Vmax=24.93U/mg。结论:以S-烷基-L-半胱氨酸为配基制得了适合于蒜氨酸酶分离纯化的亲和载体。 Objective： To design bio-specific affinity chromatography for alliinase purification. Methods： Sepharose CL-4B coupled with competitive alliinase inhibitor, S-alkyl-L-cysteine, was developed as the affinity chromatography gel, which was activated by 1,4-butanediol diglycidyl ether in an alkaline condition. Crude enzyme was obtained through cryogenic comminution and precipitation with PEG-5000. Further purification of alliinase was conducted on prepared affinity chromatography column and its purity was examined by SDS-PAGE. Results： Alliinase was successfully purified through activated Sepharose CL-4B column coupled with S-alkyl-L-cysteine. A single band with 52 kD was shown in SDS-PAGE. A total amount of 0.1296 mg of alliinase with high purity （the specific activity was as high as 9.493 U/mg protein） was obtained from 1 g of garlic. Compared with the supernatant of comminuted garlic, its activity recovery rate was 68.42%, which exhibited 22.17-fold enhancement in purity. The optimal reaction temperature and pH for alliinase were 35℃ and 6.32, respectively. Km and Vmax were 9.845 mmol/L and 24.93 la mol/mg · min. Conclusion： S-alkyl-L-cysteine as the binding ligand can be applied to affinity chromatography for alliinase purification.

关 键 词： 蒜氨酸酶 分离纯化 亲和层析 烷基 半胱氨基

领　　域： [轻工技术与工程] [轻工技术与工程]