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目的研究阿卡波糖产生菌原生质体制备与再生的条件。方法通过溶菌酶破壁的方法制备原生质体,考察影响原生质体制备与再生的因素。结果和结论确认原生质体的制备条件为:一级培养采用SM2液体培养基,培养时间为33 h,转二级培养的转种体积分数为15%;二级培养采用R2YE培养基,培养时间为20 h,甘氨酸质量分数为0.7%;溶菌酶作用质量浓度为3 g.L-1,作用时间100 min,最大原生质体制备量达到8×1010个.L-1。考察了原生质体再生培养基的组成,在优化的再生培养基上,再生率达到9.2%。
Objective To study the preparation and regeneration conditions of acarbose-producing protoplasts. Methods Protoplasts were prepared by lysozyme broken wall, and the factors affecting the preparation and regeneration of protoplasts were investigated. RESULTS AND CONCLUSION The preparation of protoplasts was confirmed as follows: SM2 liquid culture medium was used for primary culture, the culture time was 33 h, and the transformant volume was 15% in secondary culture. R2YE medium was used for secondary culture, the culture time was 20 h, the mass fraction of glycine was 0.7%, the concentration of lysozyme was 3 gL-1, the action time was 100 min, and the maximum protoplast preparation was 8 × 1010 L-1. The composition of protoplast regeneration medium was investigated. The regeneration rate reached 9.2% on optimized regeneration medium.