重组脂肪氧合酶基因工程菌破碎条件优化及其酶活力测定方法研究  

Optimization of Conditions for Disruption of Recombinant E. coli Cells and Determination of Lipoxygenase Activity

作　　者：郭芳芳[1] 应琦[1] 张充[1] 陆兆新[1] 别小妹[1] 赵海珍[1] 吕凤霞[1] 

机构地区：[1]南京农业大学食品科技学院,江苏南京210095

出　　处：《食品科学》2012年第23期160-165,共6页Food Science

基　　金：国家自然科学基金面上项目(31071605);江苏省科技支撑计划项目(BE2011390);苏州市科技支撑计划项目(SN201105)

摘　　要：建立适用于从重组E.coli中提取重组脂肪氧合酶非机械法的破碎条件。采用化学渗透法、冻融法和酶解法破碎重组E.coli细胞,并采用单因素试验和L25(56)正交试验对细胞破碎条件进行优化。结果表明:溶菌酶添加量为1.5mg/mL、溶菌酶处理时间40min、乙二胺四乙酸二钠(EDTA-2Na)添加量为2.0mmol/L、吐温-60添加量为2%、冻融温度(-70℃冷冻37℃融解)、冻融次数3次,效果最佳,获得粗酶液的脂肪氧合酶(LOX)酶活力为6840U/mL,比优化前(4750U/mL)提高了0.44倍;并在此条件下,比较分光光度法、碘化钾-淀粉法和二甲苯酚橙法测定LOX酶活力,其中碘化钾-淀粉法简单、灵敏、快速,且测定体系具备肉眼可辨的特征颜色,可用于LOX酶活性高通量筛选。Recombinant E.coli cells were disrupted by combined non-mechanical methods,such as chemical permeation,repeated freeze-thaw and enzymatic lysis for the extraction of recombinant lipoxygenase（LOX）.Based on one-factor-at-a-time experiments,an L25（56） orthogonal array design was employed to optimize five process parameters.The best results for bacterial cell disruption were achieved through enzymatic lysis with 1.5 mg/mL of lysozyme for 40 min in the presence of 2.0 mmoL/L EDTA2Na and 2% Tween-60 followed by 3 repeated freeze-thaw cycles,yielding an LOX activity of 6840 U/mL in crude enzyme solution,which was 1.44-fold higher than before the optimization.Compared with spectrophotometry and xylenol orange method,potassium iodide-starch method was more simple,sensitive and rapid and the reaction system showed characteristic color visible to the naked eye.Thus,this method is suitable for high throughput screening of LOX activity.

关 键 词：脂肪氧合酶 化学渗透法 冻融法 酶解法 酶活力测定法 

分 类 号：TS201.3[轻工技术与工程—食品科学]