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当前位置: 首页 > 文章 > 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 食品科学 2012,33 (23) 160-165
Position: Home > Articles > Optimization of Conditions for Disruption of Recombinant E. coli Cells and Determination of Lipoxygenase Activity FOOD SCIENCE 2012,33 (23) 160-165

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

作  者：
郭芳芳;应琦;张充;陆兆新;别小妹;赵海珍;吕凤霞
单  位：
南京农业大学食品科技学院
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
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。
译  名：
Optimization of Conditions for Disruption of Recombinant E. coli Cells and Determination of Lipoxygenase Activity
作  者：
GUO Fang-fang,YING Qi,ZHANG Chong,LU Zhao-xin,BIE Xiao-mei,ZHAO Hai-zhen,Lü Feng-xia(College of Food Science and Technology,Nanjing Agricultural University,Nanjing 210095,China)
关键词：
lipoxygenase;chemical permeation;freeze-thaw;enzymatic lysis;enzyme activity assay
摘  要：
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.

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