【作者】 赵玲；

【导师】 包永明；

【作者基本信息】 大连理工大学 ， 生物化工， 2008， 硕士

【摘要】 本文的工作是在丁二酮发酵代谢途径及机制分析的基础上,以代谢控制发酵理论为指导,进行丁二酮高产菌株的选育、发酵条件的优化、代谢酶学研究及代谢机制分析。以产气肠杆菌（Enterobacter aerogenes,CICC 10293）为出发菌株,采用紫外线诱变,并结合亮氨酸平皿和丁二酮平皿筛选方法,获得耐高浓度底物-葡萄糖的高产丁二酮突变菌株,命名为UV-3。利用气相色谱测定代谢产物量,分析诱变前后菌株代谢途径中碳源的流向,结果表明突变株UV-3的丁二酮产量提高18.7倍,达到1.045g/L,乙偶姻产量降低48.4%,乙醇产量降低71.4%,乙酸产量提高34.6%,且遗传性质稳定。基于Logistic和Luedeking-Piret方程,建立UV-3细胞生长动力学、底物消耗动力学和丁二酮生成动力学模型,得到动力学方程分别为:X（t）=0.0011e^0.3953t╱[1-（0.3953╱5.81）(1-e^0.3953t)]、S（t）=100-0.0051×[X（t）-0.0011]-0.6594×5.81╱0.3956 ln[1-（0.0011╱5.81）(1-e^0.3946t)]和P（t）=0.0034×5.81╱0.3956 ln[1-（0.0011╱5.81）(1-e^0.3946t)]+0.0557（Xt-0.0011）,实验数据的拟合度依次为99.4%、99.26%和98.75%。优化后丁二酮发酵条件如下:培养基组成为葡萄糖10%,蛋白胨0.5%,酵母粉0.5%,（NH₄）₂SO₄ 0.01%,柠檬酸0.1%,MnSO₄ 0.2%,MgSO₄ 0.2%;500mL摇瓶培养条件为接种量10%,装液量150mL,培养温度37℃,pH 7.0,摇瓶转速180r/min;UV-3摇瓶培养60小时,丁二酮产量达到1.15±0.03 g/L;5L发酵罐发酵条件为接种量10%,装罐量3L,培养温度37℃,pH 7.0,培养30小时,丁二酮产量达到1.35 g/L。丁二酮代谢途径中主要代谢酶的酶活分析表明:UV-3的乳酸脱氢酶、双乙酰还原酶和α-乙酰乳酸脱羧酶三种酶比活依次为0.024±0.002U/mg、0.036±0.002U/mg和0.087±0.007U/mg,均低于出发菌株10293;而出发菌株和突变株中的乙偶姻脱氢酶均低于0.001U/mg。乳酸脱氢酶、双乙酰还原酶和α-乙酰乳酸脱羧酶三种酶活最低的pH分别为7.0、7.0和7.5左右。温度为35℃左右时,乳酸脱氢酶酶活最低;温度范围35～40℃内,双乙酰还原酶酶活较低;α-乙酰乳酸脱羧酶酶活随温度升高逐步降低。Mn²⁺对三种酶有抑制作用;Fe²⁺、Zn²⁺和Mg²⁺对三种酶均有不同程度的激活作用;Co²⁺对乳酸脱氢酶和α-乙酰乳酸脱羧酶有抑制作用,而对双乙酰还原酶有激活作用;Ba²⁺、Ca²⁺和Cu²⁺强烈的激活双乙酰还原酶和乳酸脱氢酶活性,Cu²⁺是α-乙酰乳酸脱羧酶的强抑制剂,Ba²⁺和Ca²⁺对α-乙酰乳酸脱羧酶无明显影响。通过主要代谢酶的酶学性质分析,确定产气肠杆菌突变株UV-3积累丁二酮的代谢途径是由α-乙酰乳酸的非酶氧化直接生成。更多还原

【Abstract】 According to the theory of metabolic control fermentation,the dissertation mainly focuses on the breeding of high diacetyl-producing mutant,fenmentation optimization, enzymatic activities and mechanism of diacetyl accumulation.A mutant,designated as UV-3,was selected from wild-type Enterobacter aerogenes 10293 by application of UV irradiation in combination with cultivation in high glucose medium,leucine medium and diacetyl medium.Gas chromatography was employed to quantify the output of metabolization,and it was found that the yield of diacetyl for the mutant UV-3 was 18.7 fold to the wild-type 10293,up to 1.045g·L^-1.Acetoin and ethanol yields were decreased by 48.4%and 71.4%,respectively.The yield of acetate was increased by 34.6%.Based on the Logistic equation and Luedeking-Piret equation,three kinetic models were proposed for cell growth,product formation and substrate consumption.With the data from experiment to evaluate model parameters,the three kinetic models appeared to provide a reasonable description for the fermentation process.Optimum medium contained glucose10%,peptone 0.5%,yeast extract powder 0.5%, （NH₄）₂SO₄ 0.01%,citric 0.1%,MnSO₄ 0.2%,MgSO₄ 0.2%;Optimum fermentation conditions contained seed volume 10%,medium volume 150mL（500mL shake flask）,temperature 37℃, pH 7.0,rotational speed 180r/min.After adopts this process,the production yield of diacetyl for UV-3 was 1.15±0.03 g·L^-1at the end of 60 h.Based on the results in shake flasks,diacetyl fermentation was scaled up in a 5L fermentor,the production yield of diacetyl reached 1.35 g·L^-1at the end of 60 h,which was increased by 17.4%compared to that of the shake flask fermentation.According to studies on these enzymes,lactate dehydrogenase（Ldh）activity,diacetyl reductase（Dr）activity andα-acetolactate decarboxylase（Ald）activity in mutant strain UV-3 was 0.024±0.002U/mg,0.036±0.002U/mg and 0.087±0.007U/mg,respectively,lower than that of the parental strain 10293.Acetoin dehydrogenase（Ad）activity both in mutant strain UV-3 and the parental strain 10293 were lower than 0.001U/mg.The activities of Ald,Ldh and Dr in the mutant UV-3 show the lowest at pH from 7.0 to 7.5,between 36℃and 39℃. Only Mn²⁺was inhibitor of the three enzyme activities at the same time.Fe²⁺,Zn²⁺and Mg²⁺ were stimulators for the three enzyme activities.Co²⁺inhibited both Ldh and Ald activities but stimulated Dr activity.Ba²⁺,Ca²⁺and Cu²⁺severely stimulated Dr and Ldh activities while Ba（2+）and Ca²⁺had almost no effect on Aid activity.Cu²⁺showed a significant inhibitor effect on Aid activity.These results show the level of diacetyl produced in Enterobacter aerogenes UV-3 would be dependent on the nonenzymatic oxidative decarboxylation of ALA.更多还原