黑曲霉糖化酶工业菌株CBS 513.88原生质体制备条件的优化

Optimization of protoplast preparation for an industrial glucoamylase-producing strain Aspergillus niger CBS 513.88

【目的】优化黑曲霉糖化酶工业菌株CBS 513.88原生质体制备条件,促进其基础研究和分子改造。【方法】通过考察酶解液成分与比例、酶解条件、渗透压稳定剂和菌体生长条件对黑曲霉CBS 513.88原生质体制备的影响,优化其制备条件,并通过聚乙二醇介导的原生质体转化方法测试原生质体的转化效率。【结果】适合黑曲霉CBS 513.88原生质体制备的材料为改良ME培养基培养10 h的菌丝,酶解条件为在10 g·L^(-1)溶壁酶Glucanex和10 g·L^(-1)蛋清溶菌酶的破壁酶溶液(pH值5.8)33℃、110 r·min^(-1)酶解1.5 h,渗透压稳定剂为0.7 mol·L^(-1)KCl+0.27 mol·L^(-1)CaCl_(2)。在此制备条件下,原生质体悬液含量最高可达2×10^(7)个·mL^(-1);原生质体再生培养基为添加0.7 mol·L^(-1)KCl的ME培养基,其最大再生率为52%;转化率最高为206个·μg-1 DNA(质粒),阳性率为94.4%。【结论】通过对黑曲霉CBS 513.88原生质体制备条件的优化,建立短时高效的原生质体介导遗传转化体系。

【Objective】The conditions of protoplast preparation were optimized for the industrial glucoamylase-producing strain Aspergillus niger CBS 513.88,thus facilitating its basic research and molecular modification.【Method】In this study,the protoplast preparation conditions of Aspergillus niger CBS 513.88 were optimized by examining the effects of enzymatic hydrolysis solution composition and proportion,enzymatic hydrolysis conditions,osmotic stabilizer,mycelium growth conditions on the formation of protoplasts.The protoplasts transformation efficiency of Aspergillus niger CBS 513.88 was also tested by polyethylene glycol-mediated protoplast transformation method.【Result】For protoplast preparation of Aspergillus niger CBS 513.88,the optimal protoplast preparation material was the mycelium cultivated in modified ME medium for 10 hours,and the best enzymolysis condition was carried out at 33℃and 110 r·min^(-1)for 1.5 hours in a solution(pH 5.8)of 10 g·L^(-1)Glucanex and 10 g·L^(-1)lysozyme,with the optimal osmotic stabilizer of 0.7 mol·L^(-1)KCl+0.27 mol·L^(-1)CaCl_(2).Under the above conditions,the concentration of protoplast suspension can reach up to 2×10^(7)protoplasts·mL^(-1).Besides,the best protoplasts regeneration medium was ME medium supplemented with 0.7 mol·L^(-1)KCl,whose maximum regeneration rate was 52%.The transformation efficiency was up to 206 transformants perμg DNA of plasmid,and the positive rate was 94.4%.【Conclusion】In this study,a short and efficient protoplast-mediated genetic transformation system was established by optimizing the protoplast preparation conditions of Aspergillus niger CBS 513.88.