커들란 생산의 우라실 첨가 영향

이중헌1, 이인영2, 김미경2, 박영훈2

1조선대학교 화학공학과, 2생명공학연구소 생물공정연구부

The Effect of Uracil Addition on Curdlan Production

Jung-heon Lee1, In-Young Lee2, Mi-Kyung Kim2, Young-Hoon Park2

1 Department of Chemical Engineering, Chosun University, Kwangju, KOREA

2 Bioprocess Research Unit, KRIBB, Taejeon, KOREA

Curdlan was synthesized with UDP-glucose by glucanase. Since the UDP-glucose is a precursor of beta-1,3-glucan, it is necessary to activate UDP-glucose production. Since uracil is a component of UDP-glucose, it is added to increase the production of curdlan.

With the addition of sucrose and uracil, the curdlan production was increased to 93 g/l. The addition time was important to increase production and it was related with the activity of glucose and ATP.

Introduction

Curdlan is an insoluble linear polysaccharide composed of   □-1,3-linked glucose, and synthesized by Agrobacterium species under the nitrogen-limiting conditions. Curdlan's linear structure allows it to be resistant to heat and other external forces, including pH. Curdlan can be used for an admixture to concrete to enhance the fluidity of concrete. To have economic feasibility, it is important to increase curdlan productivity by manipulating the environmental conditions and its production cost should be minimized to compete with other polysaccharides.

The viscosities of some polysaccharides such as xanthan, gellan, and welan in the neutral condition are very high and it was an obstacle to increase polysaccharide production. Since curdlan is insoluble under neutral condition and the viscosity of culture broth is low unlike other polysaccharide, it is possible to increase curdlan concentration without problems due to high viscosity of culture broth.

For the synthesis of most polysaccharides, sugar nucleotides serve as glycosyl donors. UDP-glucose serves not only as an activated precursor for glycosyl moieties but also as a precursor in the formation of other monosaccharides. Cellular nucleotides, involved in cellular metabolism and have widespread regulatory potential, seem to play an important role in the synthesis of sugar nucleotides. Therefore, the level of nucleotide may be important for the production of curdlan.

In this article, the uracil is added to increase the nucleotide level in the cells and the effect of uracil addition on the production of curdlan is studied. The effect of sucrose addition on the curdlan production will also be discussed.

Material and Methods

The concentrations of cells and curdlan were determined by measuring the dry weight.  A suitably diluted sample was centrifuged at 5,000×g at 4℃ for 30 min.  The pellet consisting of cells and curdlan was washed with 0.01N HCl, and harvested by recentrifugation. Then, 0.5N NaOH was added to solubilized curdlan for 1 h. Cells were separated by centrifugation at 5,000×g for 30 min. Curdlan present in the supernatant phase was precipitated under acidic conditions by adding an appropriate volume of 2.0N HCl. Both cells and curdlan were washed and then dried to a constant weight. Sucrose concentration was determined by the dinitrosalicylic acid method (Miller, 1959) after hydrolyzing samples with 2N HCl at 100℃ for 15 min. Ammonium concentration was determined by the indophenol method (Srienc et al., 1984). Nucleotide concentrations were measured by an HPLC gradient system (Ryll and Wagner, 1991). The chromatographic system consisted of two HPLC pumps (Waters 501, Miliford, MA), a tunable absorbance detector (Waters 486), an automated gradient controller (Waters 680), and a data module (Waters 746). The separations were carried out on a reverse phase ODS column (150×4.6 mm i.d., 4 mm-particle size, YMC Inc.). Two buffer solutions were used. Buffer A consisted of 100 mM phosphate buffer, pH 6.0, supplemented with 8mM tetrabutylammonium hydrogensulfate as an ion pair reagent.

Results and Discussion

(1) No Uracil Addition

The productivity of curdlan decreased rapidly without uracil addition when the sucrose was added during fermentation. The curdlan production may be inhibited when the sucrose was dumped into fermentor. When the sucrose concentration increased, the cell was affected by high sucrose concentration and the curdlan productivity was decreased. The final curdlan concentration was lower than that of batch fermentation.

(2) Uracil Addition after Ammonium Depletion (at 26 hrs)

The uracil was added when the ammonium was depleted. For this case, uracil was used as one of nutrient source and the cell mass was further increased. However, curdlan production was not increase and affected by the sucrose addition. Uracil was consumed in 24 hrs after addition.

(3) Uracil Addition at 46 hrs

When the uracil was added one day after ammonium depletion, the productivity of the cell was maintained and the curdlan production was increased up to 93 g/l. The productivity of the cell was maintained with the addition of uracil and it may be used for cell growth and increasing productivity. Although we tried to control dissolved oxygen level, it was not possible for the high curdlan concentration due to the viscosity of culture broth. For the high curdlan production, special mixing apparatus is necessary to increase dissolved oxygen level.

Conclusion

The productivity of cells is important to increase curdlan production. Uracil was one of the important nutrients for Agrobacterium sp. to maintain the productivity. It was possible to increase curdlan production with uracil addition. The maximum curdlan concentration of 93 g/l was obtained when the uracil was added after 46 hrs.

Figure 1. Profiles of curdlan production

References

1. Lee IY, Seo WT, Kim GJ, Kim MK, Park CS, Park YH, Production of Curdlan using Sucrose or Sugar Cane Molasses by two-step Fed-batch Cultivation of Agrobacterium species, Journal of Industrial Microbiology & Biotechnology, 18 :5-8,1997.

2. Kim MK, Lee IY, Ko JH, Rhee YH, Park YH Higher intracellular levels of uridinemonophosphate under nitrogen-limited conditions enhance metabolic flux of curdlan synthesis in Agrobacterium species, Biotechnology & Bioengineering, 62: 317-323, 1999.

3. Lee JH, Lee IY, Kim MK, Park YH, Optimal pH control of batch processes for production of curdlan by Agrobacterium species, Journal of Industrial Microbiology & Biotechnology, 23:143-148, 1999.