限氧与通氧条件下米根霉ADH突变株的碳代谢特性

【目的】乙醇脱氢酶（ADH）是米根霉发酵生产L-乳酸代谢途径中的支路关键酶，选育米根霉ADH突变株，降低ADH活力，可以提高乳酸转化率。【方法】利用亚硝基胍（NTG）诱变米根霉As3.3462，在含丙烯醇的YPD选择培养基上筛选获得ADH突变株，研究了突变菌株在限氧与通氧条件下的乙醇、乳酸、ADH与L-乳酸脱氢酶（LDH）的特性。【结果】 获得11株ADH突变株，其中mut-2突变株在通氧和限氧条件下对乳酸的转化率比出发菌株分别提高了84.6%和175%，而对乙醇的转化分别下降了39.5%和59.8%。mut-2突变株的最大ADH活力在通氧和限氧条件下均比出发菌株降低，而LDH活力在限氧条件下显著增加，通氧条件下变化不明显。【结论】氧气的供给对米根霉发酵产L-乳酸的产物组成比例影响很大，限氧发酵条件下ADH活力提高，乙醇积累。结果还表明，通过筛选米根霉ADH突变株，是获得L-乳酸高产菌株的一种简单快速的有效手段。

Carbon Metabolism Characteristics of Rhizopus oryzae ADH Mutant under Aeration and Oxygen-limiting Fermentations

【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.