耐Mn~(2+)高产柠檬酸菌株的选育及其特性分析

诱变选育耐锰离子(Mn2+)的高产柠檬酸菌株,并研究了突变株的特性。采用紫外和亚硝基胍复合诱变,以含不同浓度Mn2+的培养基进行筛选,同时考察了不同浓度Mn2+对突变株柠檬酸发酵、关键酶酶活力及呼吸途径的影响。在以葡萄糖为唯一碳源进行摇瓶发酵时,突变株柠檬酸产量比野生菌株提高了92.4%,突变株UV-141对高浓度Mn2+有一定的耐受性,且Mn2+对其发酵生产柠檬酸有促进作用,与不添加Mn2+时相比,添加300 mg/L Mn2+时,柠檬酸产量及葡萄糖最大比消耗速率分别提高了107%和16.7%;乌头酸酶最高酶活下降了15.8%,NAD+-(NADP+-)异柠檬酸脱氢酶对Mn2+不敏感且在整个发酵过程中始终处于较低水平,有利于柠檬酸的积累;当侧系呼吸链(AOX)受到水杨基氧肟酸(SHAM)抑制时柠檬酸产量显著下降,而添加Mn2+则能够削弱其对柠檬酸产量的影响。突变株柠檬酸产量明显高于野生菌株,Mn2+能促进突变株葡萄糖代谢加快、乌头酸酶酶活下降,同时能削弱SHAM对AOX途径的影响,从而促进柠檬酸的大量积累。

Breeding and characterization of Mn2+-resistance and high citric acid-yielding strain

This paper was aimed to breed manganese ion(Mn2+)-resistance and high citric acid-yielding strain with mutation,and analyze its characteristics.A high yielding-citric acid and Mn2+-resistance strain was screened by ultraviolet(UV) radiation and nitrosoguanidine(NTG) treatment.The influences of Mn2+ concentration on the specific activities of key enzymes and respiratory pathway were investigated in the mutant strain during the citric acid fermentation.The citric acid production of the mutant UV-141 increased by 92.4% when it was cultured with glucose as the sole carbon source in shake flask fermentation.Compared with culture without Mn2+,the citric acid production and the maximum ratio of consumption rate of the mutant UV-141 increased by 107% and 16.7,respectively.The maximum specific activity of aconitase decreased by 15.8%,while the specific activity of NAD+-(NADP+-) isocitrate dehydrogenase maintained at a low level,which was in favor of the accumulation of citric acid.In addition,Mn2+ could weaken the decrease of citric acid,which was caused by the inhibition of alternative respiratory chain(AOX) in presence of salicylhydroxamic acid(SHAM).The citric acid production of the mutant strains was obviously higher than that of the wild strain.For the mutant strain,Mn2+ could promote glucose metabolism,decrease the specific activity aconitase,and weaken the influence of the SHAM on AOX pathway,thus lead to the abundant accumulation of citric acid.