褐藻胶降解菌突变株的选育及降解特性研究

Study on Breeding and Degradation Characteristics of Algin Degrading Mutant Strains

摘要: 【目的】通过双重诱变获得高效降解褐藻胶的菌株，并确定该菌株发酵产酶的最优条件，同时研究其化学试剂抗性能力、降解效率，进而解析该菌株的降解特性。【方法】采用紫外和亚硝基胍(NTG)溶液对原始菌株Cobetia sp. cqz5-12进行双重诱变，利用平板初筛(氯化钙透明圈法)和摇瓶复筛(二硝基水杨酸法(DNS法))获得目的突变菌株。采用单因素试验优化其发酵条件，分别利用滤纸片法和氯化钙透明圈法分析化学试剂对菌株生长和降解褐藻胶能力的影响，利用间苯三酚分光光度计法测定菌株发酵液对不同底物的降解效率。【结果】筛选得到一株降解效率显著提高的诱变菌株，并将其命名为Cobeita sp. cqz5-12-M2。该诱变菌株最优发酵条件为pH 6.5、NaCl浓度为0、发酵温度为31℃、发酵时间为96 h，此时该突变株总酶活可以达到59.81 U/mL，比原始菌株的酶活提高50.84%。该突变菌株对褐藻胶的降解效率为42%，比原始菌株提高8.8%。该突变菌株和原始菌株均对多种待测试剂有敏感性。突变使得菌株对苯酚、氯霉素、阿莫西林或氨苄青霉素的敏感性降低，对过氧化氢、乙醇、碘伏等多种化学试剂的敏感性增强。当培养基中存在链霉素、氨苄青霉素或卡那霉素存在时，突变使得菌株对褐藻胶降解能力减弱，当培养基中存在阿莫西林、5%石炭酸、庆大霉素或头孢菌素时，突变使得菌株对褐藻胶降解能力增强。【结论】筛选得到的突变菌株对多种消毒剂和抗生素具有良好抗性，对环境耐受性强，是目前少见的高适应性褐藻胶降解菌。

Abstract: [Objective] The efficient algin degrading strain was obtained by double mutagenesis, and the optimal conditions for the fermentation enzyme production of the strain were determined. Meanwhile, the chemical reagent resistance and degradation efficiency were studied to analyze the degradation characteristics of the strain. [Methods] The original strain Cobetia sp. cqz5-12 was mutagenic by UV and nitrosoguin (NTG) solution, and the target mutant strain was obtained by plate screening (calcium chloride transparent ring method) and flask rescreening (dinitrosalicylic acid method (DNS method)). Single factor test was used to optimize the fermentation conditions. The effects of chemical reagents on the growth and degradation ability of algin were analyzed by filter paper method and calcium chloride transparent circle method, respectively. The degradation efficiency of different substrates was determined by phloroglucinol spectrophotometer. [Results] A mutagenic strain with significantly improved degradation efficiency was selected and named Cobeita sp. cqz5-12-M2. The optimal fermentation conditions of the mutagen strain were pH 6.5, NaCl concentration 0, fermentation temperature 31˚C and fermentation time 96 h. In this case, the total enzyme activity of the mutant strain could reach 59.81 U/mL, which was 50.84% higher than that of the original strain. The degradation efficiency of the mutant strain was 42%, which was 8.8% higher than that of the original strain. Both the mutant strain and the original strain were sensitive to a variety of agents to be tested. The mutation made the strain less sensitive to phenol, chloramphenicol, amoxicillin or ampicillin, and more sensitive to hydrogen peroxide, ethanol, iodophor and other chemical agents. When streptomycin, ampicillin or kanamycin were present in the medium, the degradation ability of algin was weakened by mutation; when amoxicillin, 5% carbolic acid, gentamicin or cephalosporin were present in the medium, the degradation ability of algin was enhanced by mutation. [Conclusion] The mutant strain was resistant to various disinfectants and antibiotics, and had strong environmental tolerance. It was a rare and highly adaptable algin degrading bacterium.