转氨酶ATA117基因在大肠杆菌BL21(DE3)中的重组表达及产酶条件优化  

The Expression of Recombinant Transaminase in Escherichia Coli BL21(DE3) and Optimization of the Fermentation Conditions

作　　者：王璐[1] 徐刚[1] 吴坚平[1] 杨立荣[1] 

机构地区：[1]浙江大学化学工程与生物工程学系,浙江杭州310027

出　　处：《化学反应工程与工艺》2014年第6期513-521,共9页Chemical Reaction Engineering and Technology

基　　金：国家重点基础研究发展计划(2011CB710800)

摘　　要：采用基因工程的方法构建重组大肠杆菌得到重组菌Eco(p ETDuet-ATA117),实现了转氨酶ATA117的重组表达。使用响应面法对重组大肠杆菌摇瓶发酵产酶培养基进行了优化并对转氨酶诱导条件进行研究。首先采用二水平部分因子设计筛选确定培养基中对转氨酶ATA117酶活有显著影响的3种培养基组分,即甘油、酵母粉和胰蛋白胨。然后进行最陡爬坡实验确定最佳响应面区域,最后通过Box-Behnken设计确定各成分的最佳浓度。采用该优化培养基,转氨酶酶活为391.7 U/L,分别是基础培养基和单因素优化培养基酶活的3.13倍和1.22倍。对转氨酶的诱导条件进行了研究,得出最适诱导温度24℃,光密度值(OD)为0.8,此条件下进行诱导,异丙基硫代半乳糖苷(IPTG)浓度1 mmol/L,诱导时间15 h,最终转氨酶活力达到713.7 U/L。The recombinant Eco（pETDuet-ATA117） was constructed for the functional expression of transaminase ATA117. After that, the response surface methodology was employed to optimize the fermentation conditions. A two-level fractional factor design was used to evaluate the effects of different components of medium. The results showed that glycerin, yeast extract and tryptone had important effects on transaminase activity, then the optimal response surface was determined by the Steepest ascent design, finally, the optimal medium composition was observed by Box-Behnken design： glycerin14.45 g/L, yeast extract 7.27 g/L and tryptone 5.72 g/L. Under the optimal conditions, the Enzyme activity（391.7 U/L） was much higher than that using either basal fermentation medium（125.4 U/L） or single variable optimization of fermentation medium（320.7 U/L）. Meanwhile, the inducing conditions was also studied, and under the optimal induction conditions of 24℃, optical densityof 0.8 under 600 nm, the final Enzyme activity was 713.7 U/Lafter 15 h induction time when isopropyl thiogalactoside was 1 mmol/L.

关 键 词：重组转氨酶 响应面法 优化培养基 

分 类 号：Q555.6[生物学—生物化学]