海藻糖合酶的自诱导表达及其催化生产海藻糖的新工艺

Auto-induced expression of trehalose synthetase and novel process for catalytic production of trehalose

海藻糖合成酶一步合成法是制备海藻糖最具工业化前景的方法之一。在前期研究基础上,本研究从发酵产酶和酶催化两方面进一步降低海藻糖生产成本,提高生产效率。首先,采用自诱导表达系统,在摇瓶体系中对含有海藻糖合成酶的大肠杆菌BL21(DE3)进行三阶段温度控制发酵,即以自诱导培养基发酵培养重组大肠杆菌,在37℃培养2.5h,25℃培养10.5h,30℃继续培养至发酵终点;此时,酶活达到1.42×10~4U/mL,比自诱导恒温提高了57.4%。在此基础上,在5L反应器上进行三阶段温控发酵验证实验。通过连续发酵40h后OD达到31,细胞干重浓度达到15.32g/L,酶活达到最高值1.81×10~4U/mL,比摇瓶发酵提高了28.6%。其次,开展全细胞催化生产海藻糖工艺优化,以5%甲苯处理45min的大肠杆菌细胞为研究对象,考察了pH、反应温度、底物浓度对全细胞催化合成海藻糖的影响以及全细胞催化的重复使用性能。结果发现:催化反应在pH为7.5、温度为30℃、麦芽糖初始浓度为350g/L时,海藻糖的得率达到74%以上;全细胞催化反应10个批次后,细胞仍保持对麦芽糖64.1%的转化率。本研究为海藻糖的工业化生产提供了一种经济、高效的工艺路线。

One-step catalysis by the trehalose synthase is one of the most promising methods for the trehalose production.Based on the previous researches,this study started with two aspects of enzyme expression and catalysis to reduce the cost and improve the productivity of trehalose production.Firstly,Escherichia coli BL21(DE3)containing trehalose synthase was cultured using auto-induced expression system and three-stage temperature controlling strategy in shake flasks.The recombinant E.coli cells were sustainedly cultivated in 37℃for 2.5h,25℃for 10.5 h,and 30℃till to the end of cultivation,respectively.The enzyme activity reached 1.42×104U/mL,which was 57.4%higher than that obtained from the process of the constant temperature incubation.Furthermore,this cultivating strategy was conducted on a 5L bioreactor,where the fermentation lasted for 40h.At the end of fermentation,the OD value reached 31,which was equivalent to the cell dry weight concentration of 15.32g/L.Meanwhile,the enzyme activity reached 1.81×104 U/mL,which was 28.6%higher than the value obtained in shake flasks.Second,the recombinant E.coli cells treated with 5%toluene for 45min were used to optimize the process of whole cell production of trehalose,where the effects of pH,reaction temperature and substrate concentration on trehalose synthesis as well as the reuse efficiency of the whole-cell catalysis were investigated.It was found that the yield of trehalose reached more than 74%under the catalytic conditions of pH 7.5,30℃and 350g/L of maltose,and remained as high as 64.1%at the 10th batch cycle.The results indicated that the new process could be a more economical and efficient process for the mass production of trehalose in the near future.