嗜热脂肪芽孢杆菌氨基酰化酶在大肠杆菌中的克隆、表达及细胞固定化研究  

Cloning and expression of aminoacylase from Bacillus stearothermophilus in E. coli and cell immobilization

作　　者：苏志鹏[1] 张培德[1] 

机构地区：[1]复旦大学生命科学学院,上海200433

出　　处：《工业微生物》2008年第5期27-33,共7页Industrial Microbiology

摘　　要：将嗜热脂肪芽孢杆菌的氨基酰化酶(amaA)基因克隆到E.coli中进行表达,同时利用渗透交联法固定化E.coli细胞,并对固定化细胞氨基酰化酶进行了温度和pH等理化性质的初步探讨。结果显示amaA基因在E.coli中获得了高效表达,酶活性达1043U/g湿菌体。最佳固定化条件为3%卡拉胶,30%细胞。当以1.25%多乙烯多胺渗透交联固定化细胞10min和0.1%戊二醛硬化处理20min,酶学性质研究表明,酶反应的最适温度为65℃,最适pH为7.0。细胞固定化后仍保留有83%活性,pH稳定范围更广,热稳定性更高,55℃酶活性不损失,4℃保存23d仍保留有固定化时73.6%的酶活性,连续10批次转化酶活性仅损失约20%,预示该固定化E.coli具有良好的操作和保存稳定性。The gene ama.A encoding aminoacylase from Bacillus Stearothermophilus was cloned in E. coll. The cloned cells were immobilized by permeating and crosslinking method. After cell immobilization, the temperature and pH characteristics of aminoacylase were preliminarily investigated. Results revealed that the gene encoding aminoacylase was effectively expressed in E. coli and the enzyme activity could reach 1043U/g cells. The optimized immobilization conditions were 30% cloned cells embed by 3% carrageenan, with 10 minutes＇ treatment of 1.25% polyethylene polyamines to permeate and crosslink and 20 minutes＇ treatment of 0.15 % glutaraldehyde to indurate. The enzyme properties indicated that the optimum temperature and pH were 65℃ and 7.0 respectively. After immobilization, the enzyme remained 83 % activity and had a higher thermal stability and wider range of pH stability, compared with that to free cells. The enzyme lost little activity at 5512. When placed at 412 for 23 days, the enzyme preserved 73.6 % activity of the original immobilized. Meanwhile, the enzyme activity lost only 20 % after 10 times continuously reaction. These indicated excellent preservation and operation stability of immobilized cells.

关 键 词：嗜热脂肪芽孢杆菌 克隆表达 氨基酰化酶 卡拉胶 渗透交联 固定化 

分 类 号：Q78[生物学—分子生物学]