The Selective Oxidations Of1-phenyl-1,2-ethanodiol And Erythritol Catalyzed By Gluconobacter Oxydans

Gluconobacter oxydans (G. oxydans) is known for its regio-and stereoselective oxidation of a wide range of carbohydrates and alcohols to corresponding aldehydes, ketones or acids and the products can accumulate in the medium. And this microbiology has been used in a wide variety of biotechnological processes as the biocatalyst, such as the production of vitamin C,(keto-) gluconic acid, dihydroxyacetone (DHA) and so on. As the genome of G. oxydans621H was published in2005, the further investigations on the unknown or known enzymes in G. oxydans were developed, which was the base of the application development of G. oxydans.In this dissertation, the oxidations of (R, S)-1-phenyl-1,2-ethanodiol and erythritol which were responsible by the membrane-bound alcohol dehydrogenase and membrane-bound glycerol dehydrogenase respectively were investigated. And the processes and their key factors were studied in detail.In the first part, the resolution of racemic1-phenyl-1,2-ethanediol (PED) for the production of (S)-isomeride catalyzed by G. oxydans was performed. The resting cells of G. oxydans were able to catalyze the regio-and stereoselective concurrent oxidation of PED to the corresponding hydroxyl acid, mandelic acid. And due to the different reactive activities of cells to the two isomerides, optically pure (S)-PED was cumulated in the reaction resolution as the unreacted substrate.(1) The membrane-bound alcohol dehydrogenase (ADH) and membrane-bound aldehyde dehydrogenase (ALDH) in G. oxydans were testified to be the key enzymes in the conversion of PED to mandelic acid. The oxidation of PED was only in the charge of the ADH, and the ALDH was beneficial for the production of mandelic acid but had no effect on the oxidation of PED.(2) The kinetic parameters for the oxidation of (R)-and (S)-PED were obtained using Lineweaver-BurK method, respectively, which reflected the different activities of G. oxydans cells (ADH) on the two isomerides. And due to the differences, it was feasible to product (S)-PED from the raceme using G. oxydans cells.(3) Through the optimization of reaction conditions,12g/L of (R, S)-PED was able to be separated completely by50g/L cells in12h, with40.8%of the productivity and over96%e.e. of purity. The substrate, PED, and oxidative product, mandelic acid, were testified to have inhibition to the reaction. The inhibited concentration of PED was over30g/L; and mandelic acid of only5g/L was able to create serious and irreversible inhibition (toxicity), which was the key inhibitor.(3) In order to overcome the inhibition of mandelic acid, the anion exchange resin D301was selected and introduced as the adsorbent for in situ removal of inhibitor from the reaction system. This method allowed the concentration of substrate to be increased to60g/L, with39.5%of the productivity and over96%e.e. of purity for (S)-PED. Comparing with the conversion without resins, the final space-time yield increased by2-fold to1.18g L-1h-1from0.41g L-1h-1. This method was more effective for the production of (S)-PED than those from the reports.In the second part, a high activity strain of G. oxydans DSM2003for the oxidation of erythritol was screened based on the membrane-bound glycerol dehydrogenase (GDH) and was used in the production of erythrulose. The process of the conversion was investigated and optimized.(1) The membrane-bound glycerol dehydrogenase (GDH) of G. oxydans was testified to be in charge of the conversion of erythritol. And according to the relationship of GDH and ADH, a strain of G. oxydans DSM2003with high GDH activity was obtained through the regulation of the growth condition.(2) The conversion conditions were optimized and the effects of erythritol and erythrulose were investigated in flasks. The erythritol didn’t have effect on the reaction rate, but it would produce toxicity to the cells when the concentration was over50g/l. The erythrulose strongly inhibited on both of the reaction rate and the activities of the cells, and the higher the concentration of erythrulose was, the stronger the inhibition was.(3) The process from erythritol to erythrulose was operated in a7L bioreactor and the reaction conditions were determined (pH4.0,30℃,8L/min of aeration and600rpm of agitation).150g/L of erythritol was oxidized completely in20h by20g/L of G. oxydans cells, and the space-time yield was6.65g L-1h-1and the yield per unit of cells was7.31g/g cells.40g/L of G. oxydans cells were required when the concentration of substrate was up to200g/L. The reaction time was18h and the space-time yield and the yield per unit of cells were10.96g L-1h-1and4.93g/g cells respectively.(4) In order to reduce the effects of erythritol to G. oxydans cells, the substrate was added into the reaction solution in several times. The initial concentration of substrate was50g/L catalyzed by20g/L G. oxydans cells, and erythritol of20g/L was added per hour after2h. The maximal total concentration of erythritol was250g/L, which was able to be completely oxidized to erythrulose in20h. The space-time yield and the yield per unit of cells for the fed-batch conversion were11.88g L-1h-1and11.88g/g cells respectively, which were increased by78.6%and62.4%comparing with the conventional conversion catalyzed by the same concentration of G. oxydans cells。The method showed the good industrial application potential for the production of erythrulose.