Optimization Of The Synthetic Process Of Cyclic ?-1,2-glucans By Rhizobium Radiobacter ATCC 1333

Cyclic?-1,2-glucans?C?Gs?are cyclic glucans,consisting of glucose by?-1,2 linkage with a degree of polymerization ranging from 17⁴0 and of high water-solubility.C?Gs play significant roles in nodule formig,helping Brucella escape from the immune system.However,there was no efficient access to obtain C?Gs.To solve this problem,Rhizobium radiobacter ATCC 1333 was used to optimize the fermentation process for enhancing the yield of C?Gs,identify the structure of polysaccharides and explore the synthesis and secretion of the extracellular C?Gs under NaNO₃ condition.The main contents and results are as follows:?1?In order to enhance the yield of extracellular C?Gs,the medium composition and fermentation conditions were optimized.8 nitrogen sources and 6 carbon sources were screened and one-factor-at-a-time method was conducted.Then significant factors were selected by applying Plackett-Burman experiment.The central composite design experiment was performed to maximize the yield of extracellular C?Gs.The optimization fermentation conditions were as follows:mannitol 23 g·L^-1,glutamate acid 3.5 g·L^-1,NaCl 0.2 g·L^-1,32°C.Under these conditions,the yield of extracellular C?Gs was up to 1.78 g·L^-1,increased by206.9%compared to the pre-optimization.?2?In a 7 L bioreactor,pH control strategy was proposed to limit the browning reaction happened at high concentrated glutamate acid which inhibiting the increasing of cell concentration and affecting the yield of C?Gs.A two stage pH strategy which pH was controlled at 7.0 for cell growth and changed to 5.5 for glucans production shows that biomass was up to 6.36 g·L^-1,and the the yield of C?Gs was up to 2.79 g·L^-1.Under a two stage pH control with dissolved oxygen control strategy,the biomass was up to 8.94 g·L^-1,yield of C?Gs was up to 2.94 g·L^-1,the production intensity of bacterial was 0.34 g·g^-1,which was 138.3%higher than that before optimization,and the yield of C?Gs was increased by61.5%.?3?The oligosaccharides were separated and purified by ethanol fractional precipitation,solid-phase extraction and high-performance liquid-phase from fermentation broth under pH control.Then they were analyzed by matrix-assisted laser desorption/ionization time of flight mass spectrometry,monosaccharide composition analysis,electrospray ionization mass spectrometry,fourier transform infrared spectrometer and nuclear magnetic resonance spectroscopy for structural identification.The results showed that the oligosaccharides mainly have two groups.Group?are acidic polysaccharides consisting of seven glucose and one galactose with pyruvic acid and succinic acid substituents.Group?are neutral polysaccharides consisting of glucose by the?-1,2-glycosidic linkage with the degree of polymerization ranging from 17 to 22,mainly 19.?4?According to the comparison of NaNO₃ and glutamate acid,the nitrogen source NaNO₃ improved the extracellular secretion of C?Gs.The strategy that glutamate acid was used in the early stage of cell growth for increasing cell concentration,and fedding NaNO₃ at logarithmic phase was conducted,and the extracellular C?Gs was greatly increased.Two-dimensional electrophoresis was used to analyze the intracellular proteins difference between NaNO₃ and glutamate acid.The main different proteins were involved in the transport and metabolism of inorganic ions,synthesis of extracellular polysaccharides,amino acid metabolism and energy metabolism.ABC transport system plays a crucial role in the synthesis of C?Gs and extracellular secretion.