亲和层析法分离纯化蒜氨酸酶
Food Science(2009)
Abstract
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。
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Key words
affinity chromatography,S-alkyl-L-cysteine,isolation and purification,alliinase
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