米根霉乙醇脱氢酶突变株的筛选及其锌镁离子的调控研究

利用亚硝基胍(NTG)对米根霉As3.3461进行诱变,在含丙烯醇0.6%的YPD平板上筛选获得21株乙醇含量降低的突变株,其中突变株HBF-12乳酸产量最高。与出发菌株相比,突变株HBF-12的乙醇产量和乙醇脱氢酶(ADH)活力分别降低了73.6%和76%,乳酸产量和乳酸脱氢酶(LDH)活力分别提高了41.2%和19.6%。研究Zn2 与Mg2 对HBF-12中ADH与LDH活性的调控,结果显示Zn2 对ADH有强烈的激活作用,但抑制LDH活性;Mg2 则轻微抑制ADH活性,促使LDH活性增强。考察两种离子影响末端产物乙醇与乳酸形成的实验说明:培养基中Zn2 浓度与乳酸积累基本上呈负相关性,与乙醇积累呈正相关性,浓度降低有利于生物量积累;Mg2 浓度增加可以促进乳酸积累和生物量增加,对于乙醇积累无明显作用。发酵培养基中添加0.01%Zn2 、0.04%Mg2 ,突变株产酸可达96.21g/L。

Screening of a low alcohol dehydrogenase activity mutant of Rhizopus oryzae and the regulation of Zr2+ 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.