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Chaperonins suppress the denaturation of proteins and promote protein folding in vivo. Because hyperthermophilic chaperonins are expected to be used as a stabilizer for proteins, the effects of a group II chaperonin from a hyperthermophilic archaeum, Thermococcus strain KS-1 (T. KS-1 cpn), on the stabilization of mesophilic and thermophilic free enzymes and an enzyme co-immobilized with T. KS-1 cpn were studied. T. KS-1 cpn prevented the thermal inactivation of yeast alcohol dehydrogenase (ADH), jack bean urease, and Thermus flavus malate dehydrogenase (MDH) at high temperatures. T. KS-1 cpn also improved the long-term stability of ADH at lower temperatures. Moreover, the residual ADH activity of ADH co-entrapped with T. KS-1 cpn was improved and maintained at a higher level than that of the entrapped ADH without chaperonin. T. KS-1 cpn is useful for the stabilization of free and immobilized enzymes and applicable to various fields of biotechnology.

The biological treatment of wastewater discharged from a biodiesel fuel (BDF) production plant conducting alkali catalysis transesterification was investigated. BDF wastewater has a high pH and high hexane-extracted oil and low nitrogen concentrations, and inhibits the growth of microorganisms. The biological treatment of BDF wastewater is difficult because the composition of such wastewater is not suitable for microbial growth. To apply the microbiological treatment of BDF wastewater using an oil degradable yeast, Rhodotorula mucilaginosa, the pH was adjusted to 6.8 and several nutrients such as a nitrogen source (ammonium sulfate, ammonium chloride or urea), yeast extract, KH2PO4 and MgSO4.7H2O were added to the wastewater. The optimal initial concentration of yeast extract was 1 g/l and the optimal C/N ratio was between 17 and 68 when using urea as a nitrogen source. A growth inhibitor was also present in the BDF wastewater, and this growth inhibitor could be detected by measuring the solid content in an aqueous phase after the hexane extraction of the wastewater. Microorganisms could not grow at solid contents higher than 2.14 g/l in the wastewater. To avoid the growth inhibition, the BDF wastewater was diluted with the same volume of water. Oil degradation in the diluted BDF wastewater was observed and the best result was obtained under the determined optimal conditions. This treatment system is simple because no controllers, except for a temperature, are necessary. These results suggest that the biological treatment system developed for BDF wastewater is useful for small-scale BDF production plants.

Waste oil treatment was attempted using a compost fermentation process. To develop a simple method for waste oil treatment, cheap and simple materials were used as compost materials. The fermentation experiment was performed using a domestic composter to determine the optimum conditions of the fermentation. Adjustment of the pH value during the compost fermentation was also important for progression of the oil degradation. When the pH value was not controlled, the pH value decreased quickly and reached about 2 and the oil degradation was stopped. Adding caustic lime to the raw materials caused the pH value of the compost to stabilize at approximately 7. The addition of a nitrogen source had a large effect on oil degradation during the compost fermentation. The optimum value of the C N ratio of the raw materials with pH control was between 10 and 40. When the C N ratio of the materials was adjusted to 10, 20, and 40, the rate constants for oil degradation were very similar. The rate constants for NH4+ consumption were also similar. Oil degradation efficiency reached 83.5% relative to the initial oil content in the compost materials. Repeated batch operation of the compost fermentation was carried out and the compost system could maintain good efficiency for oil degradation over several repeated batch operations. Finally, the compost system was applied to the treatment of recalled mayonnaise, with favorable results being obtained.

DsbC, which catalyzes disulfide isomerization, was overproduced in the periplasm of Escherichia coli and purified from the periplasmic fraction by osmotic shock and anion-exchange chromatography. The active site of the purified DsbC was found to be an oxidized form (ox-DsbC) which could be converted to the reduced form (red-DsbC) by the addition of dithiothreitol. The effect of ox- and red-DsbC on the refolding of chemically denatured and reduced proteins with different numbers of disulfide bonds and free cysteine-thiol groups was investigated. Ox-DsbC facilitated the refolding of proteins with multiple disulfide bonds in both oxidative and reductive environments, while red-DsbC facilitated refolding only in the former. On the other hand, only red-DsbC facilitated the refolding of proteins with multiple free cysteine-thiol groups but either form of DsbC did not facilitate the refolding of proteins with only one cysteine-thiol group. It is therefore important to choose the form which suits the properties of the protein. Holo-chaperonin from Thermus thermophilus and DsbC demonstrated a synergistic effect on protein refolding.