[Screening of a low alcohol dehydrogenase activity mutant of rhizopus oryzae and the regulation of Zn2+ and Mg2+].

Ethanol is the main by-product in the fermentation broth of Rhizopus oryzae As3.3461 for the production of high-optical purity L-lactic acid.Alcohol Dehydrogenase(ADH) is the branch pathway enzyme that catalyzes the transformation of ethanol from pyruvate in Rhizopus oryzae,which decreases the conversion rate of glucose to L-lactic acid.Thus,screening the mutants with lower ADH activity may increase lactate production dramatically.In present study,Rhizopus oryzae As3.3461 was mutated with N-methyl-N'-nitro-N-nitrosoguanidine(NTG), and 21 mutants which showed lower ADH activity were isolated with selective medium of Yeast-Peptone-Dextrose(YPD) containing 0.6% allyl alcohol(V/V).Compared with other mutants,the 12~(th) mutant strain(named as HBF-12) shows the highest conversion rate of L-lactic acid.By contrast with Rhizopus oryzae As3.3461,the parent strain,the ethanol production and the ADH activity of HBF-12 decrease 73.6% and 76%,respectively.Whereas,the L-lactic acid production and the LDH activity of HBF-12 increase 41.2% and 19.6% than those of the parent strain,respectively.The activities of ADH and LDH of HBF-12 were regulated by Zn~(2+)and Mg~(2+),but showed opposite effects.Added with Zn~(2+) to the concentration of 0.01% improves the ADH activity dramatically,but inhibits the activity of LDH.By contraries,added with Mg~(2+) improves the LDH activity markedly,but inhibits the ADH activity slightly.In fermentation experiment,the addition of Zn~(2+) and Mg~(2+) show different effects on the accumulation of ethanol,L-lactic acid and the biomass in mutant HBF-12.When improve the concentration of Zn~(2+),the accumulation of L-lactic acid and the biomass show the decreased trend,but the production of ethanol show positive effect.With the improvement of the concentration of Mg~(2+),the production of lactic acid and biomass increase markedly,but no effect on the production of ethanol.When ferment under the concentrations of Zn~(2+) 0.01% and Mg~(2+) 0.04% in fermentation medium,the lactate production of HBF-12 reached the highest level,96.21g/L.