[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.
Pan
Li-jun
LJ
School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009, China. panlijun@tom.com
Fu
Ping
P
Zheng
Zhi
Z
Luo
Shui-zhong
SZ
Jiang
Shao-tong
ST
D000426:Alcohol Dehydrogenase / Q000235:genetics* / Q000378:metabolism*; D018533:Biomass; D004789:Enzyme Activation / Q000235:genetics; D000431:Ethanol / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D019344:Lactic Acid / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism*; D009154:Mutation; D012233:Rhizopus / Q000201:enzymology* / Q000235:genetics*; D015032:Zinc / Q000378:metabolism*
D019344:Lactic Acid; D000431:Ethanol; D000426:Alcohol Dehydrogenase; D007770:L-Lactate Dehydrogenase; D008274:Magnesium; D015032:Zinc