原生质体制备技术在阿卡波糖菌种筛选中的应用

Application of the protoplast preparation technology in the strain separation of acarbose

通过原生质体诱变,筛选得到阿卡波糖高产菌株,并利用原生质体制备技术解决菌种难以纯化,容易衰退的问题。实验用溶菌酶破壁的方法制备原生质体,并对影响原生质体制备的关键因素进行了研究,最终确定原生质体制备条件:一级菌丝培养时间为44~48 h,二级培养添加甘氨酸质量分数为0.3%,培养时间为40~44 h;溶菌酶质量浓度为3 mg/mL,作用时间为2 h,该条件下原生质体制备量可达1.2×10^(5)个/mL;通过对制备的原生质体进行紫外诱变,筛选获得了3株阿卡波糖高产菌株,且中试放大后仍能保持高水平的发酵能力,10 m 3罐发酵水平较出发菌株分别提高了19.0%,22.2%,24.8%。

The purpose of this experiment was to screen the strains with high yield of acarbose by protoplast mutagenesis,and to solve the problem that the strains were difficult to purify and easy to decay by using protoplast preparation technology.The protoplast was prepared by lysozyme wall breaking method and the key factors affecting the protoplast preparation were investigated.Finally,the preparation conditions of protoplasm system were determined as follows:the time of primary mycelium culture was 4448 h;the time of secondary mycelium culture was 4044 h with 0.3%glycine added;the concentration of lysozyme was 3 mg/mL and the reaction time was 2 h.Under these conditions,the preparation amount of protoplasm reached 1.2×10^(5) pieces/mL.Three strains were obtained,after screening,with high-yield of acarbose from prepared protoplasts with UV mutagenesis.The strains kept high level of fermentation capacities after pilot-scale amplification cultivation.Compared with the original strain,the fermentation level of 10 m 3 tank increased by 19.0%,22.2%and 24.8%,respectively.