Efficient Preparation Of Ectoines And Its Application

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).