【作者】 郎亚军；

【导师】 张苓花；

【作者基本信息】 大连海事大学 ， 环境科学， 2010， 博士

【摘要】 Ectoines (1,4,5,6-四氢-2-甲基-4-嘧啶羧酸,Ectoine;1,4,5,6-四氢-2-甲基-5-羟基-4-嘧啶羧酸,Hydroxyectoine)是中度嗜盐菌在渗透压胁迫下合成的一类渗透压补偿溶质。Ectoines在高温、冷冻和干燥等逆环境下,对酶、蛋白质、核酸及整个细胞具有保护作用,被广泛应用于化妆品、生物制剂稳定剂、药物制剂等领域的研究与应用,Ectoines的制备技术日益受到关注。本文分离鉴定Ectoines合成菌株,获得了Ectoine分泌型菌株和耐受渗透压冲击的Hydroxyectoine合成菌株。研究高效制备Ectoines的新工艺。同时研究了Ectoine在高浓度乙醇发酵中的应用。从盐场盐池中分离筛选获得了22株Ectoines合成菌株。HPLC方法检测到了菌株DLB06和DLS12的发酵培养基中存在Ectoine特征峰物质。菌株DLC31经HPLC和1H-NMR鉴定合成Hydroxyectoine,生理生化鉴定该菌株为Cobetia marina。克隆了C. marina的Ectoine合成酶基因,并进行了基因结构分析。HPLC和LC-MS方法发现并确认了分离菌株DLB06和DLS12的Ectoine分泌现象。考察了NaCl浓度对菌株DLS12 Ectoine分泌的影响。Ectoine分泌量随NaCl浓度的降低而升高。菌株DLS12对NaCl胁迫的应答、Ectoine合成浓度阈值等与已有文献报道的菌株显著不同,是Ectoine合成菌株的一个新类型。10 L发酵罐Ectoine分批发酵,Ectoine分泌型菌株DLS12的Ectoine总合成浓度是H. elongataDSM 2581总合成浓度的2.7倍。本文分离鉴定的C. marina,耐受渗透压冲击,适合细菌挤奶工艺条件,利用该菌株进行的Hydroxyectoine细菌挤奶,制备效率较分批发酵提高了71.9%,Hydroxyectoine对底物的转化率是分批发酵的2.5倍。高葡萄糖浓度对Zymomonas mobilis CICC 10232的乙醇发酵具有抑制作用。在Z. mobilis CICC 10232的250 g/L葡萄糖浓度乙醇发酵中添加1 mmol/L Ectoine,与未添加Ectoine相比,发酵终了时的糖醇转化率、乙醇发酵度和发酵效率分别提高了16.7%、18.2%和57.1%。Ectoines显著促进了细胞生长和葡萄糖的利用,提高了葡萄糖激酶、6-磷酸葡萄糖脱氢酶和乙醇脱氢酶在高渗透压下的酶活性。更多还原

【Abstract】 Ectoines (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid, ectoine; 1,4,5,6-tetrahydro-2-methyl-5-hydroxy-4-pyrimidinecarboxylic acid, hydroxyectoine) were compatible solutes synthesized by moderate halophiles under osmotic stress. Ectoines protected enzyme, protein, nucleic acid and the whole cell against high temperature, freeze and dryness, which had been applied in cosmetics, biological preparations and pharmaceutical industry, and other fields. The technology of ectoines preparation had increasingly attracted attention. In this paper, we screened and identified the strains to synthesize ectoines, and obtained ectoine-excreting wild-type strain. Based on this, efficient preparation of ectoines and its application during high-density fermentation of ethanol.The 22 strains of ectoines synthesis are screened from salt lakes. The characteristic peak of ectoine by DLB06 and DLS12 was measured by HPLC in medium. The DLC31 synthesized hydroxyectoine by HPLC and 1H-NMR analyses, and the strain was identified as Cobetia marina based on physiological and biochemical characteristics. The genes of ectoine synthase by C. marina were cloned and the structure of the genes was analyzed.The strain DLS12 excreted ectoine into outside cells under constant osmotic pressure at the exponential phase of growth by HPLC and LC-MS analyses. The excreted concentration of ectoine increased with decreasing of NaCl concentration. The response on NaCl stress and the threshold concentration of ectoine synthesis. for ectoine-excreting wild-type strain DLS12 were significantly different from those of other strains reported, which was a new type of strains of ectoine synthesis. The batch fermentation in 10 L fermentor was carried out, the total ectoine concentration was 2.7 times higher than that of ectoine non-excreting wild-type strain(Halomonas elongata DSM2581).C. marina screened and identified in this paper was enough for bacteria milking. Using this strain, bacteria milking system of hydroxyectoine synthesis was established. The productivity and convertion rate of substrate were significantly increased. The productivity increased by 71.9%, and the conversion rate of subtrate was 2.5 times compared with batch fermentation.The high concentration glucose inhibited ethanol fermentation by Zymomonas mobilis CICC10232. During ethanol fermentation in the presence of 250 g/L of glucose, the final sugar-ethanol conversion, ethanol concentration and productivity was elevated by 16.7%,18.2% and 57.1%, respectively, compared to those in the absence of ectoine, respectively. Ectoine significantly promoted cell growth and glucose consumption and increased enzyme activities of glucokinase (GK), glucose-6-phosphate dehydrogenase (G-6-PDH) and alcohol dehydrogenase (ADH).更多还原