重组大肠杆菌发酵条件优化提高D-阿洛酮糖3-差向异构酶活性的研究

Optimization of fermentation conditions of recombinant Escherichia coli to improve the production of D-allulose 3-epimerase

功能性稀有糖D-阿洛酮糖主要由基因工程菌株表达D-阿洛酮糖3-差向异构酶(D-allulose 3-epimerase,DAE)催化果糖生产。该研究以表达DAE的重组大肠杆菌为研究对象,利用单因素试验对发酵培养基的碳源、氮源、金属离子等成分进行优化,获得了最佳的培养基组合:蔗糖10 g/L、大豆蛋白胨15 g/L、(NH4)2SO_(4)3 g/L、KH2PO_(4)3 g/L、MgSO_(4)0.5 g/L、MnSO_(4)0.025 mmol/L。在此基础上,利用单因素试验对培养条件进行研究,确定了最佳发酵诱导时间(10 h)、接种量(3%,摇瓶)、装液量(30%)以及异丙基-β-D-硫代半乳糖苷(isopropyl-beta-D-thiogalactopyranoside,IPTG)添加浓度(1 mmol/L),使OD_(600)增加了1.46倍,酶活提高了2.57倍。在此基础上,利用5 L发酵罐进行放大发酵实验,确定了最佳诱导时间。在最佳条件下,经过18 h的诱导发酵,菌体OD_(600)最高值达51.8,干重达到21.5 g/L,酶活达到103.8 U/mL。当细胞添加量为0.014 g DCW/L时,以500 g/L D-果糖为底物,pH为7,50℃,1 h转化率达28.76%,D-阿洛酮糖最高生成量可达149.74 g/L。该研究对D-阿洛酮糖3-差向异构酶的发酵生产具有参考价值。

The functional rare sugar D-allulose is mainly produced from fructose by the catalyzation of D-allulose 3-epimerase(DAE)from genetically engineered microorganisms.In this study,a DAE expression recombinant Escherichia coli was chosen as the object,and single factor experiments were carried out to study the influence of carbon source,nitrogen source,and metal ions on the growth and DAE expression.The results indicated that the optimal medium formulation as:sucrose 10 g/L,soybean peptone 15 g/L,(NH 4)2SO_(4)3 g/L,KH 2PO_(4)3 g/L,MgSO_(4)0.5 g/L,MnSO_(4)0.025 mmol/L.On this basis,the cultivation conditions were further studied through single factor experiments.The optimal cultivation conditions were liquid loading amount of 30%in a flask,inoculation volume of 3%,IPTG concentration of 1 mmol/L,and induction time of 10 h.Under this condition,the OD_(600)of recombinant E.coli had a 1.46-fold increase,and the volume DAE activity had a 2.57-fold increase compared to the starting culture conditions.The culture medium and condition were further tested by batch fermentation in a 5 L bioreactor.After 18 h of induction,the OD_(600)of recombinant cells reached the highest value of 51.8,the dry cell weight reached 21.5 g/L,and the volume enzyme activity reached 103.8 U/mL.The reactions from 500 g/L D-fructose to D-allulose were carried out with different recombinant cell concentrations at pH 7,50℃.When the cell concentration was 0.014 g DCW/L,the product D-allulose reached the highest concentration at 149.74 g/L and conversion rate at 28.76%after 1 h reaction.This study has reference value for the fermentative production of DAE.