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

作　　者：王慧敏 姚倩倩 李月月 蔡溯林 孙明珠 顾向阳[1] WANG Hui-Min;YAO Qian-Qian;LI Yue-Yue;CAI Su-Lin;SUN Ming-Zhu;GU Xiang-Yang(College of Life Seiences, Key Laboratory of Microbiological Engineering of Agricultural Environments, Ministry of Agriculture, Nanjing Agricultural University, Nanjing , Jiangsu 210095, China)

机构地区：[1]南京农业大学生命科学学院农业部农业环境微生物工程重点实验室,江苏南京210095

出　　处：《微生物学通报》2018年第4期744-752,共9页Microbiology China

基　　金：国际科学基金项目(W4260-1)~~

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。[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

关 键 词：中度嗜盐菌 四氢嘧啶 羟基四氢嘧啶 细菌泌乳 高渗-低渗冲击 

分 类 号：Q936[生物学—微生物学]