黑曲霉糖化酶工业菌株CBS 513.88原生质体制备条件的优化OA北大核心CSTPCD

[Objective]The conditions of protoplast preparation were optimized for the industrial glu-coamylase-producing strain Aspergillus niger CBS 513.88,thus facilitating its basic research and mo-lecular 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 for-mation 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 prepa-ration of Aspergillus niger CBS 513.88,the optimal protoplast preparation material was the mycelium cul-tivated 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-1KCl+0.27 mol·L-1CaCl2.Under the above conditions,the concentration of protoplast suspension can reach up to 2 × 107 protoplasts·mL-1.Besides,the best protoplasts regeneration medium was ME medium supplemented with 0.7 mol·L-1KCl,whose maximum regeneration rate was 52％.The transformation efficiency was up to 206 trans-formants per μg 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.