[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 12th 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 Zn2+ and Mg2+, but showed opposite effects. Added with Zn2+ to the concentration of 0.01% improves the ADH activity dramatically, but inhibits the activity of LDH. By contraries, added with Mg2+ improves the LDH activity markedly, but inhibits the ADH activity slightly. In fermentation experiment, the addition of Zn2+ and Mg2+ show different effects on the accumulation of ethanol, L-lactic acid and the biomass in mutant HBF-12. When improve the concentration of Zn2+, 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 Mg2+, the production of lactic acid and biomass increase markedly, but no effect on the production of ethanol. When ferment under the concentrations of Zn2+ 0.01% and Mg2+ 0.04% in fermentation medium, the lactate production of HBF-12 reached the highest level, 96.21 g/L.