Thesis (Ph.D.)--Chulalongkorn University, 2013

The regulation of pyruvate decarboxylase (PDC) and alcohol dehydrogenase (ADH) in the living culture of Rhizopus oryzae was studied. The in vitro enzyme inhibitors were selected from the literatures. They were added into the fermentation of immobilized R. oryzae and their concentration effects on growth, metabolite production, and changes in the specific enzyme activities were investigated during the shake flask culture. It was found that 1,2-diazole and 2,2,2-trifluoroethanol were found to be the effective ADH regulators. Nevertheless, regulating only ADH could not fully limit ethanol during fermentation by R. oryzae. PDC was further suppressed by various regulators. It was found that among the regulators studied, 0.1 mM 4-methylpyrazole and 1.0 μM β-hydroxypyruvate successfully provided the enhancement of lactic acid production as well as the decreasing ethanol formation. Later on, all of these potential 4 regulators were tested with dissolved oxygen for their combinatorial effects on growth, metabolite production, and the specific enzyme activities in the static bed bioreactor. The results revealed that the effect of enzyme regulators was more profound at the higher DO level. It was found that DO level of 80% in the fermentation medium containing 2,2,2-trifluoroethanol exhibited the highest lactic acid production as well as reducing ethanol production. This was about 24% increasing yield when compared with that of the control fermentation. However, the results shown in this study implied that the regulation of ethanol fermentative pathway by adding the regulators during fermentation did not only affect the targeted enzymes but it also caused the dynamic change in the conversion of all metabolites in the living R. oryzae in order to maintain the balanced flux for cellular growth and maintenance.