Optimization Of Fermentation Conditions For Ectoine Synthesis From Moderately Halophilic Bacteria Halomonas Sp. TA-4 And Preliminary Study On Biological Functions Of Ectoine

Most microorganisms have evolved their own strategies to cope with the ever-present threat of losing osmotic equilibrium in either natural or artificial environment and among these strategies, accumulating intracellular compatible solutes to withstand the extracellular fluctuations in osmolarity and maintain normal cell metabolisms employed by halophilic bacterium is known as the most important one. As a sensitive, short term or instantaneous reaction which is totally controlled by the environmental osmotic changes, it became a preferable model and one of the heat spots for research on osmo-regulation mechanisms and being the principal fuctional compound of this reaction, compatible solutes also attracted great attention in recent years. Among the compatible solutes discovered so far, ectoine is recognized as the most common one in bacteria and is of great significance in scientific investigation and extensive prospects in applications because it serves not only as a key osmoprotectant but also as stress protectants for cells and macromolecules against adversities such as desiccation, freezing and radiation. In this study, an ectoine producing strain was isolated and studied upon its fermentation properties, and some of the biological functions of ectoine were also explored. These results supplied China with new materials in the research and development in this field.The bacteria TA-4, which produces ectoine as its main compatible solute, was isolated and studied on the optimal conditions for ectoine synthesis. The physiological and biological assays as well as 16S ribosome DNA (rDNA) analysis indicated that this strain was a moderately halophilic bacterium and had the highest similarity to the species salina which belongs to the Halomonas genus. With the ability to tolerate a broad salinity range, synthesize and release intracellular compatible solutes rapidly when subjected to osmotic changes and the low mortality after the osmotic upshock and dowshock alternation, TA-4 proved to be an advantageous strain for ectoine production by bacterial milking technics. In order to enhance the amount of ectoine in practical industrial production, the fermentation conditions for intracellular ectoine synthesis by strain TA-4 was studied and the optimal composition of the fermentation medium affording the highest ectoine productivity was determined to be the one that consisted of yeast extract (4.0g/l), monosodium glutamate (60g/l), NaCl (120g/l); the optimum cultivation conditions for ectoine accumulation was illustrated as follows: cells grown in the presence of 5%NaCl for 24 hours were transferred to the fermentation medium with initial pH value 8.0 and NaCl concentration 12% at the inoculation rate of 10% and were incubated under the temperature of 30℃and shaking speed of 220rpm for 48 hours. Then a second transfer of the cells to fresh medium containing 12%NaCl was carried out and the cells were cultivated for a further 24 hours. Under these optimized conditions, a maximum yield of ectoine, 633.1 mg/l, was obtained.As an important compatible solute in the domain Bacteria, ectoine has extensive biological functions that derserve to be further developed and applied to industrial uses. In this study, we investigated the protective effects of ectoine on cells and biological macromolecules under the conditions of thermal shock and hydroxyl radicals attack. The results showed that ectoine was an effective protectant against high temperature shock to both halophilic and non-halophilic bacteria. It could enhance the thermal tolerance of cells and eliminate inhibition of elevated temperature on their growth, moreover, the more ectoine was added, the greater this protection effect was observed. Ectoine was also an excellent antioxidant. The ectoine-salicylate competitive hydroxyl radicals trapping assay, the DNA nicking assay and the pyruvate kinase inactivation experiment proved that ectoine was not only a more efficient hydroxyl radicals scavenger than mannitol is, but also served as a protectant for pBluescriptⅡKS and PK that could greatly reduce the oxidative injuries on their structures and activities.