渗透压冲击下中度嗜盐菌Halomonas sp. Y四氢嘧啶类相容性溶质的合成与释放

Synthesis and release of ectoines in a moderate halophile Halomonas sp. Y subjected to osmotic shocks

摘要:

【背景】四氢嘧啶类物质在高温、冷冻和干燥等逆境条件下，对酶、蛋白质、核酸及整个细胞具有良好的保护作用，已经应用于酶制剂、生物医药及护肤品等相关领域。目前此类物质只能依赖中度嗜盐菌采用细菌泌乳工艺进行商业化生产，因此四氢嘧啶类高产菌株及其发酵技术的研究日益受到国内外研究者关注。【目的】分离获得高产合成四氢嘧啶类相容性溶质的中度嗜盐细菌，研究渗透压冲击对其胞内四氢嘧啶合成与释放的影响，探索细菌泌乳法制备四氢嘧啶的可行性。【方法】采用涂布平板法分离中度嗜盐菌，对分离菌株进行形态、生理生化和16S rRNA基因序列分析，鉴定其种属；采用高效液相色谱法(HPLC)和质谱法(MS)分析四氢嘧啶类物质，细菌泌乳法制备四氢嘧啶类物质。【结果】从盐池土样中分离到一株以四氢嘧啶类物质为主要相容性溶质的中度嗜盐菌Y，鉴定为盐单胞菌(Halomonas sp.) Y。盐单胞菌Y能在NaCl质量浓度为10?250 g/L的培养基中生长，最适生长的NaCl浓度为100 g/L；HPLC-MS测试结果证明盐单胞菌Y可同时合成四氢嘧啶和羟基四氢嘧啶2种相容性溶质，在最适生长的盐浓度下其合成量分别达175.5 mg/g和47.9 mg/g；在NaCl质量浓度为0?30 g/L的低渗溶液中胞内四氢嘧啶类物质经5 min即可达到最大释放率，而细菌泌乳工艺中最适合诱导四氢嘧啶释放的低渗溶液为质量浓度为10 g/L的NaCl溶液；采用细菌泌乳工艺制备四氢嘧啶，经连续11轮的高渗/低渗冲击，四氢嘧啶总合成量为6.0 g/L，总释放量为5.7 g/L，平均释放率为64.5%，底物转化率为128.9 mg/g。【结论】盐单胞菌Y是一株较高产合成四氢嘧啶类的中度嗜盐菌，能够耐受反复的渗透压冲击，采用细菌泌乳工艺显著提高了四氢嘧啶的制备效率。

Abstract:

[Background] Ectoines have been applied in cosmetics, enzyme preparations and pharmaceutical industry due to their powerful stabilizing property of protecting enzyme, protein, nucleic acid and the whole cell against high temperature, freeze and dryness. Up to now, industrial-scale production of ectoines relies on bacterial milking process using moderately halophilic bacterium. Therefore, development of high-yield ectoine-producers and related fermentative technology has attracted considerable attention worldwide. [Objective] The objective of the present study was to isolate a moderately halophilic bacterium capable of synthesizing ectoines as main compatible solute, study the effects of osmotic shocks on synthesis and release of ectoines in this bacterium and explore the feasibility of producing ectoines by using bacterial milking process. [Methods] Moderately halophilic bacterium was isolated by spread-plate method and identified through analysis of its morphology, physicochemical properties and 16S rRNA gene sequence similarity. Ectoines were analyzed quantitatively and qualitatively by high performance liquid chromatography (HPLC) and mass spectrometry (MS), respectively. Ectoines were prepared by bacterial milking process. [Results] A moderately halophilic bacterium Y capable of synthesizing ectoines as main compatible solute was isolated from saltern sediment and identified as a member of the genus Halomonas according to its physiological properties and 16S rRNA gene sequence similarity. Halomonas sp. Y showed growth in lactate medium containing a wide range of NaCl concentrations of 10?250 g/L. Optimum growth was observed in the medium containing 100 g/L NaCl. HPLC-MS analysis revealed that this bacterium could synthesize both ectoine and hydroxyectoine as the main compatible solute. The yields of each compatible solute synthesized in the presence of 100 g/L NaCl were 175.5 mg/g and 47.9 mg/g for ectoine and hydroxyectoine, respectively. Maximum release rate of intracellular ectoines could be achieved within 5 min when Y cells experienced hypo-osmotic stress brought about by 0?30 g/L NaCl solutions. Hypo-osmotic saline solution with salinity of 10 g/L NaCl was the most appropriate for ectoine release during bacterial milking process. During the bacterial milking process eleven rounds of hyper/hypo osmotic shocks were exerted to the isolate and the total yields of synthesized and released ectoines were 6.0 g/L and 5.7 g/L, respectively. This corresponded to an average release rate of 64.5% and a high conversion rate of 128.9 mg/g. [Conclusion] Halomonas sp. Y is a moderately halophilic bacterium with quite high yield of ectoines. It could resist repeated osmotic shocks as characterized by ectoine release under hypo-osmotic stress and synthesis of ectoine under hyper-osmotic condition. Ectoine productivity was significantly improved by using bacterial milking process.