重组大肠杆菌高效催化肌醇合成葡萄糖醛酸的研究

High Level Conversion of Myo-inositol into Glucuronic Acid by Recombinant Escherichia coli

葡萄糖醛酸在医药和保健品等领域都有广泛的应用。该研究利用重组大肠杆菌(E.coli BL21(DE3)/pET28a(+)-MIOX)催化肌醇生成葡萄糖醛酸。首先对重组大肠杆菌培养条件进行优化以达到肌醇氧化酶高效表达的目标,以MBL作为发酵培养基,在对数生长期中期(OD600=5.5)加入0.1 mmol/L IPTG,27℃下诱导6 h,肌醇氧化酶的酶活达到100.5 kU/L。然后利用重组菌菌体作为催化剂转化肌醇生产葡萄糖醛酸,以2 g/L肌醇为底物,30℃下反应2 h,葡萄糖醛酸的产量达到1.7g/L,肌醇的转化率为84%。在此基础上,进一步考察了多次添加新鲜菌体对葡萄糖醛酸合成的影响,当肌醇浓度为50 g/L时,经过3次添加菌体葡萄糖醛酸的最终产量达到15.7 g/L。该研究为生物法生产葡萄糖醛酸打下了坚实的基础。

Glucuronic acid is one important biochemical substance with wide applications in pharmaceutical and health care fields. However,the current production of glucuronic acid in industry is mainly derived from the nitric acid oxidation of starch and this p~＇oeess has very poor oxidative seleqtivity and serious environmental pollution, as well as high energy consumption. Now a study aiming at developing an environmentally friendly and efficient method to produce glucuronic acid was presented,iusing the recombi- nant E. coli BL21 （DE3）/pET28a（ ＋ ）-M!OX as bio-eatalytic. Firstly,to improve the expression level of myo-inositol oxygenase in recombinant cells, the effect of different expression conditions was systematically studied. The optimal conditions were determined as follows：cultivation in MBL medium,induction with 0.1 mmolfL IPTG at the middle stage of exponential phase （OD6oo： = 5.5 ）, and then cultivation at 27℃ for 6 h. Under the optimal conditions, the highest bioactivity of MIOX reached 100.5 kU/L. Secondly, the conversion of myo-inositol into glucuronic acid was achieved by using the recombinant cells as the bio-catalytic~.In order to improve the production of glucuronic acid, various reaction conditions were investigated systematically. With the optimized system containing 2 g/L myo-inositol,20 g/L （DCW） whole cells and 100 mmolfL MOPS （pH 8.5 ） ,the reaction was executed 2 h at 30 ℃ ,and the final glucuronic acid concentration of 1.7 g/L was achieved with conversion rate of 84%. Furthermore, the effect of adding fresh whole cells during the reaction process on the conversion rate was studied. Using 20 g/L （DCW） ceils as initial bio-catalytic ,50 g/L myo-inositol as substrate, MOPS pH 7.5, incubation at 30℃, finally, 15.7 g/L of glucuronic acid was achieved by adding fresh cells three times during the reaction process. The present work provides a promising biosynthetic process for glucurgnic acid production.