一种利用甘氨酸氧化酶生产乙醛酸的方法

Production of glyoxylic acid by glycine oxidase

为实现生物法工业化生产乙醛酸,采用基因工程技术对甘氨酸氧化酶(ThiO)进行表达优化,使用SDS-PAGE电泳手段验证ThiO的可溶性表达,通过全细胞转化法检测重组菌体生产乙醛酸的能力,并优化了反应条件,在放大体系中考察其应用性。结果表明:构建的菌株Bacillus subtilis 168(pP43NMK-ThiO)实现了ThiO的可溶性表达,全细胞转化甘氨酸生产乙醛酸的产量超越了当前生物法制备的最高产量。全细胞催化剂的最佳反应条件为温度30℃和pH值8.3,在35℃内保持高热稳定性,保温8 h后仍保持90%以上反应活性。在最佳反应条件下添加终浓度1%的过氧化氢酶使得乙醛酸产量提升,相对产率提高了30%。3 L发酵罐上的结果与摇瓶上一致,显示出全细胞法制备乙醛酸的稳定的转化能力,为其工业化奠定了基础。

In order to realize the industrial production of glyoxylic acid,the expression of glycine oxidase(ThiO)was optimized using genetic engineering technology.The soluble expression of recombinant strains was verified by SDS-PAGE electrophoresis.The ability of recombinant bacteria to produce glyoxylic acid was tested by whole cell transformation.With the optimization of reaction conditions,its applicability in the enlarged transformation system was investigated.The strain Bacillus subtilis 168(pP43NMK-ThiO)achieved intracellular soluble expression and the yield of glyoxylic acid from glycine by the whole cell transformation surpassed the highest yield of glyoxylic acid produced by current biological methods.The best reaction conditions were 30℃and pH value 8.3.The whole cell catalyst maintained high thermal stability at 35℃,and more than still maintains more than reaction activity after 8 h incubation.Under the optimal reaction conditions,the yield was further increased to 30%by adding catalase at a final concentration of 1%.The results from the 3 L fermentation tank are consistent with those obtained from the shake flask,showing stable conversion capacity.The results lays the foundation for the industrialization of the preparation of glyoxylic acid by the biological method.