响应面法优化重组亚油酸异构酶产酶条件

Optimizition of Linoleic Acid Isomerase Producing Conditions from Recombinant E.coli by Response Surface Method

摘要:

共轭亚油酸（CLA）广泛存在于多种食物中，具有减肥、抗癌、抗动脉粥状硬化和抗糖尿病等诸多生理活性。为了获得活性CLA高产，本文将突变的亚油酸异构酶克隆到大肠杆菌中表达，单因素试验确定最优产酶条件为发酵时间18 h、IPTG诱导浓度0.2 mmol/L、20%装液、培养基初始pH7、诱导前菌体生物量OD600=0.4、LA浓度0.5 mg/mL、离子浓度0.02%；在此基础上采用响应曲面法优化重组大肠杆菌培养条件提高生产亚油酸异构酶的能力，根据响应面法实验结果分析显示，发酵时间、诱导前生物量和LA浓度对重组亚油酸异构酶的表达有显著影响且均为正效应。三个影响因素最佳组合为发酵时间19.5 h、诱导前生物量OD600=0.47和LA浓度0.57 mg/mL，此时预测亚油酸异构酶催化LA转化为CLA最大量为143.33 μg/mL。验证显示，发酵CLA的产量为141.75±0.14 μg/mL，与预测产量相符;通过优化设计，提高了107.5%。

Abstract:

Conjugated linoleic acid (CLA) is widely found in a variety of foods. In order to obtain high yield of active CLA, the mutant linoleic acid isomerase was cloned and expressed in escherichia coli in this work. The single factor test determined that the optimal conditions for enzyme production were as follows: fermentation time 18 h, IPTG induced concentration 0.2 mmol/L, 20% liquid loading, initial pH7 of the medium, pre-induction bacterial biomass OD600=0.4, LA concentration 0.5 mg/mL, and ion concentration 0.02%. The response surface method was used to optimize the culture conditions of recombinant escherichia coli to improve the capacity of producing linoleic acid isomerase. According to the response surface method analysis results, the fermentation time, pre-induction biomass and LA concentration had significant effects on the expression of recombinant linoleic acid isomerase, and all of them were positive effects. The optimal combination of the three influencing factors was as follows: 19.5 h fermentation time, pre-induction biomass OD600=0.47, and LA concentration 0.57 mg/mL. Unde this condition, the maximum amount of LA to CLA catalyzed by linoleic acid isomerase was predicted to be 143.33 μg/mL. The results showed that the yield of CLA was 141.75±0.14 μg/mL, which increased by 107.5%.