High performance gel filtration chromatography with several on line detectors (refractometer. polarimeter, laser-light scattering, viscosimeter) revealed to be useful tools in the production study and characterization of exopolysaccharides from Rhizobium meliloti M5N1 strain.
The effect of the bleed ratio on acetic acid production in the cell-recycle culture of Acetobacter aceti was simulated, particularly with regard to the acetic acid production rate and the concentrations of viable cells, total cells, ethanol and acetic acid. The result of simulation agrees well with the experimental data obtained at the dilution rate of 0.73h-1. The theoretical maximum acetic acid production rate (123.1g/l/h) was obtained when the cell-recycle culture was carried out without bleeding at the dilution rate of 3.52h-1. A chart is presented to predict the dilution rate and bleed ratio for obtaining cultures of relatively high concentrations of viable cells and acetic acid (>40g/l). It is claimed that the bleed from such a culture can be used to supply active cells at high concentration to the second-stage bioreactor in a two-stage culture.
The effect of various metal ions on the anaerobic synthesis of superoxide dismutase (SOD) in Escherichia coli was examined. When cells were grown anaerobically by fermentation, adding CuSO4 in the culture medium resulted in the synthesis of manganese-containing SOD (Mn SOD), but adding CrCl3, CoCl2, NiSO4, ZnSO4 or Ga(NO3)3 did not. The presence of nitrate in culture medium stimulated the anaerobic synthesis of Mn SOD by CuSO4. During the incubation of resting cells under anaerobic conditions, the addition of CuSO4 and KNO3 increased Mn SOD activity, which was inhibited by chloramphenicol. Adding CrCl3 or Ga(NO3)3 with KNO3 increased Mn SOD activity during anaerobic incubation of resting cells, but adding MnCl2, (NH4)2Fe(SO4)2, K3Fe(CN)6, CoCl2, NiSO4 or ZnSO4 with KNO3 did not increase Mn SOD activity. Instead, they decreased it. The increase of Mn SOD activity in resting cells by Cr ions or Ga ions with nitrate was inhibited by ferric citrate but that by Cu ions was not. When cells were grown anaerobically with CuSO4 or when resting cells were incubated anaerobically with CuSO4, a new activity band of SOD appeared in the polyacrylamide gel electrophoreogram, regardless of the operation of nitrate respiration. The SOD of the new activity band was insensitive to cyanide, H2O2 or diethyldithiocarbamate. This new SOD and Mn SOD had the same molecular size but had different charges.
Exoglucanase (EC 18.104.22.168) was partially purified from Aspergillus niger. The enzyme was stable at room temperature in the pH range of 4.0-6.0 for 24h, with the optimum at 5.5. The enzyme had an optimum temperature of 50°C and a t1/2 at 65°C was 70min. Km and Vmax values were found to be 55.5mg/ml and 0.9μM/min, respectively. Glycerol protected the enzyme from inactivation on storage and from denaturation due to freezing and thawing. The effect of the sulfhydryl group reagents tested suggested the presence of -SH on the active site of the enzyme. Mn2+ and Co2+ were good activators of the enzyme, whereas Hg2+ and Pb2+ were potent inhibitors. The enzyme was a metalloprotein, or it requires certain metal ions for activation.
Cell-wall polysaccharides purified from four species of the genus Microbacterium were characterized in relation to their taxonomy, based on 1H-NMR and 13C-NMR spectrometry, methylation analysis and Smith degradation. The polysaccharide isolated from M. imperiale IFO 12610 contained a backbone consisting of heptasaccharide repeating-units, to which side chains of L-rhamnopyranosyl-(1→2)-D-mannopyranosyl-(1→2)-D-mannopyranoses as branches were attached to position 6 of the 3-linked D-galactopyranose residues. The polysaccharide of M. lacticum IFO 14135 contained hexasaccharide repeating-units, to which disaccharide groups of L-rhamnopyranosyl-(1→2)-L-rhamnopyranoses were attached to position 6 of the 3-linked D-galactopyranose residues to form branches. The polysaccharide of M. arborescens IFO 3750 consists of pentasaccharide repeating-units, and single D-mannopyranosyl groups were attached as branches to position 6 of the 3-linked D-galactofuranose residues. The polysaccharide of M. laevaniformans IFO 14471 had a more complicated structure consisting of decasaccharide repeating-units, and branches occur at three different positions. Thus, the cell-wall polysaccharides of the former three species were similar in their basic structures, but that of M. laevaniformans has a significantly different structure. These results indicated that the structures of cell-wall polysaccharides are heterogeneous in the genus Microbacterium and may characterize the species in the genus.