Abstract: 【Objective】Ethanol dehydrogenase (ADH) is an important branch pathway enzyme that catalyzes the transformation of ethanol from pyruvate in Rhizopus oryzae, which decrease the conversion rate of glucose to L-lactic acid. So, screening the mutants with lower ADH activity may markedly increase lactate production.【Method】 Mutation of Rhizopus oryzae As3.3462 was induced with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) and mutants with lower ADH activity were screened out, then the characteristics of mutant under aeration and oxygen-limiting fermentations were investigated. 【Result】 There were 11 Rhizopus oryzae strains of ADH mutants were obtained. After preliminary fermentation, mut-2 was selected for further study on the fermentation characteristics because of its high lactate conversion rate of 84.6% and 175% as compared with parent strain under aeration and oxygen-limiting fermentation, respectively. The ethanol concentration of mut-2 decreased by 39.5% and 59.8% under aeration and oxygen-limiting fermentation. The ADH specific activities of mut-2 under aeration and oxygen-limiting fermentation were lower than parent strain. The LDH specific activities under oxygen-limiting condition were markedly higher than parent strain, but the difference was not distinct under aeration fermentation. 【Conclusion】 The product compositions of Rhizopus oryzae carbon metabolism were remarkably determined by the supply of oxygen. Oxygen-limiting fermentation would result in a higher ADH activity and the accumulation of ethanol. The results also indicated that screening Rhizopus oryzae ADH mutant was an effective method for increase the conversion rate of L-lactic acid.

Key words: Rhizopus oryzae, ADH, Ethanol, L-lactic acid