生物素-亲和素系统定向固定糖化酶的研究  

Immobilization of Glucoamylase on Biotin-avidin System

作　　者：侯红萍[1] 张萍[1] 王如福[1] 

机构地区：[1]山西农业大学食品科学与工程学院,山西太谷030801

出　　处：《中国食品学报》2014年第3期106-114,共9页Journal of Chinese Institute Of Food Science and Technology

基　　金：山西省科技攻关项目(041030);国家"863"计划项目(2011AA10090401)

摘　　要：以生物素-亲和素系统定向固定糖化酶,首先,通过正交试验确定生物素标记糖化酶的最佳条件是:质量浓度为4 mg/mL的酶液2 mL、反应温度20℃、反应pH 4.6、搅拌时间2.5 h;其次,确定了亲和素-磁性琼脂糖微粒的最佳制备条件是:亲和素30μg/mg载体、反应时间20 min、pH 6.6;最后,确定了生物素-亲和素系统定向固定糖化酶的最佳条件是:生物素标记糖化酶液40μL/mg载体、反应时间20 min、反应温度20℃、pH 6.1。此条件下固定化酶活力为1 629.7 U/g,活力回收率为50.3%,固定化酶相对活力为64.7%。对固定化糖化酶的酶学性质的研究表明,固定化酶的最适作用温度65℃,最适作用pH 5.1,米氏常数Km为2.28 mg/mL,较游离糖化酶的米氏常数Km(5.56 mol/L)小,说明其与底物的亲和力提高。固定化酶连续使用5次,酶活仍保持71.8%,固定化酶的半衰期为84 d。The biotin-avidin system was used as carriers to immobilize glucoamylase.First,the optimum conditions of biotin marking glucoamylase were determined by the orthogonal experiments： 4mg/mL enzyme liquid 2mL,the reaction temperature was 20℃,the reactive pH was 4.6,stirring time was 2.5h.Second,the optimum preparation conditions of avidin-magnetic agarose microspheres were determined by the single factor-orthogonal experiments： avidin load was 3 μg/ mg carrier,reaction time was 20 min,pH was 6.6.Third,the conditions of oriented-immobilization enzyme with biotinavidin system were optimized by the single factor-orthogonal experiments： biotin marking glucoamylase load was 40 μL/mg carrier,the reaction time was 20 min,the reaction temperature was 20 ℃,pH was 6.1.Under these conditions,the activity of the immobilized enzyme reached 1 629.7 U/g.Activity recovery and relative activity of the immobilized enzyme were 50.3% and 64.7% respectively.Finally,the enzymatic properties of the immobilized glucoamylase were investigated.The results showed that the optimum temperature was 65 ℃,the optimum pH was 5.1.The Kmwas 2.28 mg/mL that less than free glucoamylase＇s.It mains that immobilized glucoamylase has a stronger affinity for substrate than free glucoamylase.And the activity of immobilized enzyme was still maintained 71.8% after used for five times.The half-life of the immobilized enzyme was 84 d.

关 键 词：糖化酶 定向固定化 生物素 亲和素 磁性琼脂糖微粒 

分 类 号：TS201.25[轻工技术与工程—食品科学]