摘要
[目的]建立一种快速、高通量筛选3-甾酮-Δ^(1)-脱氢酶(KstD)突变体库的方法。[方法]基于已发表KstD蛋白晶体结构,设计饱和突变引物,构建突变体文库,优化蛋白表达条件,利用冻融法结合溶菌酶法获得粗酶液;基于分光光度法原理,优化检测反应体系,利用酶标仪检测吸光度的变化来反映酶活力。[结果]利用饱和突变引物成功构建突变体文库,表达KstD最佳条件为:IPTG终浓度0.3 mmol/L,20℃诱导20 h;缓冲液中溶菌酶浓度为0.5 mg/mL时上清目的蛋白含量最高。优化后的酶活检测条件为:Tris-HCl缓冲液50 mmol/L、pH 8.0、0.4 mmol/L DCPIP、1.5 mmol/L PMS、0.7 mmol/L雄烯二酮(4AD)和适量的酶液组成,温度30℃、酶反应时间5 min,于600 nm波长下测定。[结论]成功建立了一种5 min检测96个3-甾酮-Δ^(1)-脱氢酶(KstD)突变体的高通量筛选方法,重复性变异系数在3%以下。
[Objective]To establish a fast and high-throughput method for screening the mutant library of 3-sterone-Δ^(1)-dehydrogenase(KstD).[Method]Based on the crystal structure of published KstD protein,saturated mutation primers were designed,mutant library was constructed,protein expression conditions were optimized,and crude enzyme solution was obtained by freeze-thaw method combined with lysozyme method.Based on the principle of spectrophotometry,the detection reaction system was optimized,and the change of absorbance was detected by enzyme marker to reflect the enzyme activity.[Result]The mutant library was successfully constructed using saturated mutant primers.The optimal conditions for expression of KstD were 0.3 mmol/L final concentration of IPTG and induction at 20℃ for 20 h.When the concentration of lysozyme in buffer was O.5 mg/mL,the target protein content of supernatant was the highest.The optimized enzyme activity detection conditions are as follows:Tris-HCI buffer solution consisted of 50 mmol/L,pH 8.0,0.4 mmol/L DCPIP,1.5 mmol/L phenazine methyl sulfate(PMS),0.7 mmol/L stenodione and appropriate enzyme solution at 30℃ and reaction time of 5 min,and was determined at 600 nm wavelength.[Conclusion]A fast and high-throughput method for screening the mutant library of 3-sterone-Δ^(1)-dehydrogenase(KstD)was successfully established.
作者
董新林
胡青
何建新
吕德强
宋士奎
苏正定
DONG Xinlin;HU Qing;HE Jianxin;LYU Deqiang;SONG Shikui;SU Zhengding(College of Bioengineering and Food Science,Hubei University of Technology,Wuhan 430068;Key Laboratory of Fermentation Engineering,Ministry of Education,Wuhan 430068,China)
出处
《生物技术》
CAS
2024年第2期158-164,共7页
Biotechnology
关键词
3-甾酮-Δ^(1)-脱氢酶
定点饱和突变
高通量筛选
半理性设计
雄烯二酮
酶标仪
酶法脱氢
突变体文库
3-sterone-△'-dehydrogenase
site-saturation mutation
high throughput screening
semi-rational design
androstenedione
microplate reader
enzymatic dehydrogenation
mutant library
