| Glucuronic acid is one important biochemical substance with wide applications in pharmaceutical and health care products. However, the current production of glucuronic acid in industry is derived from the nitric acid oxidation of starch, however, this industrial process has many disadvantages, such as very poor oxidative selectivity, serious environmental pollution, high energy consumption, etc. In the present work, it is proposed to produce glucuronic acid by developing an environmentally friend and efficient method.In this thesis, the gene of myo-inositol oxygenase (MIOX) was synthesized and linked with vector pET28a(+), which resulted the construction of pET28a(+)-MIOX. By adopting recombinant E. coli BL21(DE3)/pET28a(+)-MIOX, the effects of different expression conditions were investigated systematically to improve the expression level. The results indicated that the highest activity of MIOX (45.46kU/L) was achieved when0.1mM isopropyl-thio-β-D-galactoside (IPTG) was added into the cell culture (OD6oo1.0) and induced at26℃for8hours.Due to its designed His tag, MIOX was purified by Ni-NTA affinity chromatography. After the affinity chromatography purification, high-purity enzyme with the specific activity of610U/mg was obtained with a good recovery rate of63%. The effects of pH and temperature on the bioactivity of purified MIOX were further investigated. The results showed that the optical catalytic pH of MIOX was6.5, and the optimum temperature was30℃The biosynthesis of myo-inositol toglucuronic acid was tried by using the whole cells as the enzyme. Firstly, the effects of whole cell and crude cell lysate on the bio conversion rate were investigated, which showed that the highest conversion rate was achieved when using the whole cells. Secondly, the effects of various reaction conditions were futher investigated. And the optimal reaction system was obtained in the following:2g/L myo-inositol,20g/L (DCW) whole cells and100mM MOPS (pH7.5), the shaking speed of100rpm at30℃. Under this condition,2.13g/L D-glucuronic acid with nearly99%conversion rate could be achieved. Finally, the effect of adding fresh whole cells during the reaction process on the conversion rate was studied. Using20g/L (DCW) cells as the bio-catalytic,50g/L myo-inositol as substrate, MOPS pH7.5, incubation at30℃, adding5g/L (DCW) fresh cells at2,4, and6h respectively. The results indicated that the yield of glucuronic acid remained unchanged after the third addition of fresh cells, and the highest yield of glucuronic acid was15.7g/L. |
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