解淀粉芽孢杆菌BA-2原生质体的制备及纤维素酶高产突变菌株筛选

Protoplast mutagenesis was used to select and breed mutant strains with high yield of cellulase

中文关键词:  解淀粉芽孢杆菌  原生质体  诱变  纤维素酶  

英文关键词:Bacillus amyloliquefaciens  protoplasts  mutagenesis  cellulase

中文摘要:

      目的 确认解淀粉芽孢杆菌BA-2 (Bacillus amyloliquefaciens BA-2)原生质体制备的最佳条件, 并对其菌株进行复合诱变筛选获得耐高温性状产纤维素酶菌株。方法 通过探究菌龄、溶菌酶浓度、酶解时间、酶解温度、渗透压稳定剂的pH、再生培养基类型和培养方式等考察原生质体制备与再生条件的影响因素, 确定其最优条件。并用紫外线对原生质体进行诱变, 获得的再生突变菌株作为出发菌株进行后续诱变处理, 采用化学诱变剂硫酸二乙酯处理菌株, 设置培养温度梯度筛选菌株。结果 解淀粉芽孢杆菌BA-2原生质体制备的最优条件为菌龄8 h, 溶菌酶质量浓度1.5 mg/mL, 酶解时间65 min, 酶解温度37℃, 渗透压稳定剂pH 6.5。在此条件下, 解淀粉芽孢杆菌原生质体的形成率为98.85%±0.26%, 再生率为57.39%±0.76%。经过复合诱变获得突变菌株BA-DES4在培养温度55℃下酶活达到(76.58±1.19) U/mL, 滤纸酶活较出发菌株BA-2提高24.5%。结论 本研究获得了解淀粉芽孢杆菌BA-2原生质体制备的最佳条件, 并通过复合诱变筛选获得耐高温性状产纤维素酶菌株BA-DES4, 该菌株稳定性强、适应性好, 具有开发成为工业化生产纤维素酶的微生物潜力, 突变菌株产酶条件有待进一步研究, 以满足于工业化生产的需求。

英文摘要:

      Objective To identify the optimum conditions of plasmid preparation of Bacillus amyloliquefaciens BA-2, and to select its strains by complex mutagenesis to obtain cellulase resistant strains. Methods The influencing factors of the conditions in the preparation and regeneration of protoplasts were examined by exploring the age of the bacteria, lysozyme concentration, enzyme digestion time, enzyme digestion temperature, pH of the osmotic pressure stabilizer, regeneration medium type and cultivation method to determine the optimal conditions. And the protoplasts were mutagenized with ultraviolet light, and the regenerated mutant strains obtained were used as the starting strains for the subsequent mutagenesis treatment, and the strains were treated with the chemical mutagen diethyl sulfate, and the culture temperature gradient was set to screen the strains. Results The optimal conditions for the preparation of Bacillus amyloliquefaciens protoplasts were 8 h of bacterial age, lysozyme concentration of 1.5 mg/mL, enzyme digestion time of 65 min, enzyme digestion temperature of 37℃, and osmolality stabilizer pH 7.0. Under these conditions, the formation rate of Bacillus amyloliquefaciens protoplasts was 98.85%±0.26%, and the regeneration rate was 57.39%±0.76%. The mutant strain BA-DES4 was obtained after compound mutagenesis to reach an enzyme activity of (76.58±1.19) U/mL at an incubation temperature of 55°C, and the filter paper enzyme activity was increased by 24.5% compared with that of the departure strain BA-2. Conclusion In this study, the optimal conditions for the preparation of the protoplasmic system of Bacillus amyloliquefaciens BA-2, and the high-temperature-resistant trait cellulase-producing strain BA-DES4 through compound mutagenesis screening are obtained, which is stable and well-adapted, and has the potential to be developed into a microorganism for the industrial production of cellulase, and the conditions for the production of enzyme by mutant strains need to be further investigated to satisfy the needs of the industrialization of the production.