Optimization of protoplast preparation for an industrial glucoamylase-producing strain Aspergillus niger CBS 513.88
Journal Title: Journal of Henan Agricultural University - Year 2024, Vol 58, Issue 4
Abstract
[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 CaCl2. 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-1 KCl, whose maximum regeneration rate was 52%. The transformation efficiency was up to 206 transformants 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.
Authors and Affiliations
Jun ZHAO, Zhen ZHANG, Feiyu DONG, Yifan XU, Hongge CHEN
Keywords
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- EP ID EP769435
- DOI 10.16445/j.cnki.1000-2340.20240227.001
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