| Hydroxy acids are important dual functional building blocks and fine intermediates, which are conventionally used as materials of pharmaceuticals, agrochemicals, etc. Therefore, the studies on the preparation of hydroxy acids from diols by biocatalysis are very important both for the knowledge on biocatalytic mechanism and its application.Gluconobacter oxydans is known for its incomplete oxidation of a wide range of carbohydrates and alcohols in a process that is referred to as oxidative fermentation. The corresponding oxidative products are secreted almost completely into the medium. In this dissertation, two hydroxyl acids were selected as the target product of bioconversion. The culture conditions of G.oxydans DSM 2003, bioconversion process by the resting cells and purification of the product were studied in detail.In the first section, high cell density culture of Gluconobacter oxydans DSM 2003 was studied.(1) Single factor analysis and uniform design were used to optimize the medium components. The optimum medium compositions were as follows:sorbitol 73.0g/l, yeast extract 18.4g/l, (NH4)2SO4 1.5g/l, KH2PO4 1.52g/l, MgSO4·7H2O 0.47g/l. Under the optimal conditions, cell density was 9.6g/l in shaking flasks.(2) To enhance G.oxydans DSM 2003 cell growth to higher cell density, the process of fermentation was improved in 3.7L bioreactor based on the optimized parameters in shaking flasks. Cell density achieved was 44.3g/l, which was increased by 361% compared with that in shaking flasks. The final cell density was 54.2g/l (DCW,14.1g/l) from the improved fed-batch culture in 3.7L bioreactor. To the best of our knowledge, this was the highest cell density until now. Furthermore, the activity the resting cells was also improved by 40%. The cells of the high density and the high activity laid the foundation of biosynthesis of hydroxyl acids.In the second section, bioconversion of ethylene glycol to glycolic acid by Gluconobacter oxydans DSM 2003 was studied.(1) The mutant strain defective in alcohol dehydrogenase (ADH, GOX1067-1068) and the complementary strain were constructed and the results of bioconversion of ethylene glycol using the resting cells showed that the ADH was the key enzyme responsible for biooxidation of ethylene glycol to glycolic acid in G. oxydans DSM 2003. (2) G.oxydans DSM 2003 was used to synthesize glycolic acid through microbial oxidation of ethylene glycol. To enhance glycolic acid production, the process of bioconversion was improved in 3.7L bioreactor based on the optimized parameters in shaking flasks. The ethylene glycol was controlled accurately by maintaining corresponding feeding rate. The pH was well controlled automatically by computer. Dissolve oxygen (DO) was controlled by increasing agitation speed, airflow and bioreactor pressure to keep it over 30% air saturation. Under the optimized reaction conditions,74.5g/l glycolic acid was obtained with a molar conversion yield of 87.1% after a 50-h reaction. The inhibition of glycolic acid was a key limitation for the bioconversion.(3) To resolve the problem of product inhibition and improve glycolic acid yield, a bioconversion strategy using ion-exchange resin D315 was investigated.①In order to entrap the cells, PVA-sodium alginate was used as carrier for the immobilization of the cells of Gluconobacter oxydans. D315 resin was chosen to adsorb glycolic acid in situ during the bioconversion. Under the optimized reaction conditions, 90.2g/l glycolic acid was obtained with a molar conversion yield of 80.3% after a 72-h reaction.②A adsorptive bioconversion for glycolic acid production from ethylene glycol using resting cells of Gluconobacter oxydans in a hollow fiber membrane bioreaction system was developed by using D315 resin as the adsorbent for selective removal of glycolic acid from the reaction mixture. This approach allowed the yield of glycolic acid to be increased to 93.2g/l, compared to 74.5g/l obtained from a conventional fed-batch mode after a 50-h reaction. Microbial bioconversion of ethylene glycol by G.oxydans DSM 2003 was very important for the application development of G.oxydans DSM 2003 and biosynthesis of hydroxyl acids.(4) According to the reaction mixture of glycolic acid, the process of separation and purification was present in detail. After decolorization by active carbon, purification by D315 and D113 resins, the crystallization was carried out at 4℃, the crystalloid purity of glycolic acid was 97.3%, and the total yield was 85%.In the third section, study on microbial asymmetric oxidation of 2-methyl-1, 3-propanediol by Gluconobacter oxydans was investigated.(1) Firstly, purification and identification of the reaction products was carried out. Two byproducts were identified to be 2-methyl propenal and methacrylic acid, respectively. Secondly, the alcohol dehydrogenase was demonstrated to serve as the key enzyme for the oxidation reaction of 2-methyl-1,3-propanediol toβ-hydroxyisobutyric acid by gene disruption and complementation.(2) We optimized the reaction conditions for (R)-β-hydroxyisobutyric acid production from 2-methyl-1,3-propandiol using G.oxydans DSM 2003. A yield 50.2g/l of (R)-β-hydroxyisobutyric acid was obtained with a molar conversion rate of 90.5% and 93.2% enantiomeric excess within 24 h in a 2-1 batch reaction in a 3.7L fermentor. Microbial asymmetric oxidation of 2-methyl-1,3-propanediol by G.oxydans DSM 2003 provides a new biological methods to synthesis to (R)-β-hydroxyisobutyric acid, an important building block.
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