摘要
褐藻胶裂解酶是制备褐藻胶寡糖的重要工具酶,利用工程菌进行褐藻胶裂解酶基因的克隆与高效表达是提高酶活的主要途径之一。工程酶位于胞内,需采取有效破碎手段获取。对重组褐藻胶裂解酶基因工程菌E.coli TOP10采用超声波破碎,分析了超声波破碎强度、总工作时间、NaCl浓度对菌体破碎程度及酶活力的影响。通过单因素试验和正交试验得出重组大肠杆菌的最佳破碎条件为:菌液浓度40 mg/mL,菌液体积50 mL,超声波工作时间4 s,间歇时间8 s,超声波破碎强度50%,总工作时间6min,NaCl浓度为0.15 mol/L。在该条件下获得褐藻胶裂解酶粗酶液的酶活为21 U/mL。
Alginate lyase is an important enzyme for production of alginate oligosaccharides.Using engineering bacteria to clone and express alginate lyase is a major way to improve enzyme activity.The engineering enzyme is endoenzyme,so it need use effective disruption to be obtained.The recombinant E.coli of alginate lyase was treated with ultrasonic wave disruption,and the influences of strength of ultrasonic wave disruption,total working time and concentration of NaCl on disruption degree of strain and enzyme activity were studied.The optimal disruption conditions for recombinant E.coli were determined by single-factor experiment and orthogonal experiment,which are as follows： concentration of cell suspension is 40 mg/mL;volume of cell suspension is 50 mL;radiation/intermission time is 4s/8s;strength of ultrasonic wave disruption is 50%;total working time is 6 min;concentration of NaCl is 0.15 mol/L.The enzyme activity of crude alginate lyase produced under the optimal conditions achieved 21 U/mL.
出处
《湖南农业科学》
2010年第11期98-101,104,共5页
Hunan Agricultural Sciences
基金
上海市教育委员会重点学科建设项目(J50704)
