基于羧基载体LX-1000IDA的脂肪酶固定化研究

Immobilization Method of Lipase Based on Carboxyl Carrier LX-1000IDA

本文利用碳二亚胺EDC作为羧基载体LX-1000IDA的活化剂,通过脂肪酶的表面氨基,将脂肪酶固定于羧基载体上,制成固定化海洋脂肪酶IDA-LIPASE。通过单因素实验、Plackett Burman试验和正交试验,对载体-酶液和交联剂的添加顺序、缓冲液浓度、温度、pH、载体量、时间和碳二亚胺EDC浓度等进行优化和筛选,得到的最佳固定化条件为:pH4.5、载体量0.2 g、时间6 h、温度25℃、EDC质量分数0.6%,制备得到酶活力为114 U/g的固定化酶。对该条件进行了10倍扩大固定化,制备得到的固定化脂肪酶IDA-LIPASE酶活力为210 U/g。对制备的固定化酶酶学性质鉴定表明:固定化酶与游离酶相比,最适反应温度提高10℃,最适反应pH向碱性方向移动0.5,重复使用7次还保留40%左右的酶活,具有优异的热稳定性;固定化酶在4℃保存30 d后保留84.6%酶活。该固定化酶IDA-LIPASE相比于游离酶更能适应工业生产环境,具备实际生产应用的潜力和价值。

This paper used carbon diamine EDC as the activator of the carboxyl carrier LX-1000 IDA,and the marine lipase was fixed on the carboxyl carrier through the surface amino resin of lipase to generate the fixed marine lipase IDA-LIPASE.To achieve the best immobilization effect,conditions such as the order of addition of carriers,enzymes and crosslinking agents,buffer concentration,temperature,pH,carrier quantity,time and EDC concentration were optimized and screened.After using single-factor experiment,Plackett Burman experiment,and orthogonal experiment,the optimized conditions of immobilization were determined as follows:pH 4.5,carrier amount 0.2 g,time 6 h,temperature 25℃,0.6%EDC,The immobilized enzyme was prepared with enzyme activity of 114 U/g.After 10 times of expanded immobilization,the activity of the immobilized IDA-LIPASE reached 210 U/g.After comparing the prepared immobilized lipase with free enzyme,it was found that:the optimal reaction temperature increased by 10℃,the optimal reaction pH shifted 0.5 to the alkaline direction,and the reusability was better.About 40%of the enzyme activity was still left after the 7 th usage of immobilized catalysis,the immobilize lipase exhibited excellent thermal stability.In addition,the immobilized lipase remained 84.6%enzyme activity after preservation at 4℃for 30 d and exhibited good storage stability.Compared with free enzyme,the immobilized enzyme IDA-LIPASE was more suitable for industrial production environment and possessed the potential and value of practical production.