产碱性果胶酶的枯草芽孢杆菌zgl14原生质体制备与再生条件研究.docVIP

产碱性果胶酶枯草芽孢杆菌ZGL14原生质体制备与再生条件研究* 于 平** 徐超超 浙江工商大学食品与生物工程学院 浙江 杭州 310035 摘 要 原生质体融合技术是一种重要的微生物育种方法。本文以枯草芽孢杆菌ZGL14为出发菌株，探讨影响其原生质体制备和再生的主要因素。通过研究菌龄、溶菌酶浓度、酶解时间、酶解温度、渗透压稳定剂的pH值、再生培养基类型和培养方式等对枯草芽孢杆菌ZGL14原生质体制备与再生的影响，确定其最优化条件。结果显示：枯草芽孢杆菌ZGL14原生质体制备的最优条件为：菌龄9 h，溶菌酶浓度0.2 mg/mL，酶解时间10 min，酶解温度32 ℃，渗透压稳定剂的pH值 7.0。在此条件下，枯草芽孢杆菌ZGL14原生质体的形成率为98%，再生率达30%。枯草芽孢杆菌ZGL14原生质体再生培养基以琥珀酸钠作为渗透压稳定剂并结合Ca2+再生效果较好，再生方式以单层培养较好。研究结果为实现枯草芽孢杆菌ZGL14的原生质体融合育种提供了技术支持，具有重要的理论意义和实践价值。图7表1参15 关键词 枯草芽孢杆菌；原生质体；制备；再生；条件优化 CLC Q813.2 收稿日期 Received: 接受日期 Accepted: *浙江省自然科学基金(M303087)和食品科学与工程浙江省重中之重一级学科项目(2017SIAR214)资助Supported by the Zhejiang Province National Nature Science Foundation (M303087) and the Food Science and Engineering-the most important discipline of Zhejiang province (2017SIAR214). **通讯作者Corresponding author (E-mail: yup9202@) Studies on the protoplast preparation and regeneration conditions of Bacillus subtilis ZGL14 producing alkaline pectinase* YU Ping** XU Chaochao College of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou 310035, China Abstract Objectives: The protoplast fusion technique is one of the important microbial breeding methods. The main factors which affect the protoplast preparation and regeneration using Bacillus subtilis ZGL14 as the starter strain were investigated in this study. Methods: The optimal conditions for the protoplast preparation and regeneration of Bacillus subtilis ZGL14 were determined in this study by optimizing the cell age, the lysozyme concentration, the enzymolysis time, the enzymolysis temperature, the pH values of the osmotic pressure stabilizer, the regeneration medium types and the culture type, etc. Results: The results indicated that the optimal conditions for the protoplast preparation of Bacillus subtilis ZGL14 were as follows: cell age 9 h, lysozyme concentration 0.2 mg/mL, enzymolysis time 10 min, enzymolysis temperature 32 ℃, pH value of osmotic pressure stabilizer 7.0. Under the above conditions, t