Study On The Conversion Of L-glutamic Acid To α-ketoglutarate Using Immobilized Cells

As an important organic acid,α-ketoglutaric acid（α-KG）is widely used in the fields of medicine,food,and fine chemicals.Currently,the production ofα-KG via L-glutamate oxidase（LGOX）conversion has received increasing attention.LGOX is also an important tool enzyme.Our laboratory successfully constructed an LGOX containing recombinant Escherichia coli（E.coli）to catalyze the production ofα-KG from L-Glu.And the activity of LGOX was improved by fermentation optimization.But there were still many problems,such as poor stability,difficult reusing.To improve the catalytic stability of recombinant E.coli,the immobilization method was used.The process conditions and catalytic performance of the immobilized recombinant E.coli to catalyze productα-KG was studied.The main research contents are as follows:（1）Different immobilized carriers were screened according to the standard of catalytic efficiency and activity recovery rate.And the process parameters of immobilized cells were determined and studied.Two composite immobilization materials,polyvinyl alcohol-sodium alginate（PVA-SA）and ZIF-8-glutaraldehyde（ZIF-8-GA）,were selected and optimized.The catalytic efficiency of the immobilized cells prepared by two composite materials was compared.It was found that ZIF-8-GA had higher catalytic efficiency,which could produce95.81 g·L^-1α-KG from 100 g·L^-1 L-Glu within 16 h.（2）Immobilized cells prepared by ZIF-8-GA were analyzed.Firstly,the effect of the material on cell morphology was analyzed by flow cytometry.It was found that the integrity of the cells remained 97.71%after adding ZIF-8 and GA.The structure of the immobilized cells was analyzed by X-ray diffractometer,fourier transform infrared spectrometer and scanning electron microscope,respectively.It was confirmed that ZIF-8 could be coated on the cell surface,and the structure did not change.（3）The catalytic properties of immobilized cells were investigated.ZIF-8-GA significantly improved the temperature and p H tolerance of free cells.Under the condition of50°C and p H=9.0,the activity of immobilized cells could still maintain 37.48%and 40.27%after incubation for 16 h.It was found that the immobilized cells were recycled to 10 batches,the yield ofα-KG could still reached 70.03 g·L^-1,which improved the problem of poor stability of free cells in the reaction.In addition,when E.coli@ZIF-8-GA was stored in a refrigerator at 4°C for 14 days,the activity of immobilized cells could still maintain 60.23%,while the activity of free cells was only 8.37%.（4）The conditions of immobilized enzymes were optimized.Using ZIF-8 to immobilize enzyme,the adsorption capacity was only 45%.By introducing polyaspartic acid（PASP）,the adsorption rate of the immobilized enzyme was found to be increased to 90%.The addition of ZIF-8 was determined.When the concentrations of 2-methylimidazole and zinc acetate were160 m M and 40 m M,the activity recovery rate of the immobilized enzyme was 89.66%.The protective effect of ZIF-8 on the enzyme was determined,under the conditions of trypsin(5g·L^-1),urea（5 M）,and high temperature（60°C）for 60 min,the relative activities of the immobilized enzyme were 87.16%,74.53%,and 81.07%,respectively.While the relative activities of free enzymes were only 27.72%,19.11%,and 16.89%,respectively.Finally,the catalytic durability of the immobilized enzyme was studied.The relative activity of the immobilized enzyme remained above 90%after repeated use of 10 batches.