碱性果胶酶在重组毕赤酵母中高效表达的关键因素研究

Key Factors of High-level Production of Polygalacturonate Lyase in Recombinant Pichia pastoris

摘要:

为提高重组毕赤酵母生产碱性果胶酶的产量和生产强度, 在摇瓶条件下优化了重组毕赤酵母生产碱性果胶酶的关键因素。结果表明, 以下条件：初始甘油浓度40 g/L、初始甲醇浓度3.1 g甲醇/g DCW、每24 h添加0.51 g甲醇/g DCW、诱导表达周期72 h、250 mL三角瓶诱导培养基装液量30 mL、初始pH 6.0, 最适于菌体生长与产物表达。在此基础上, 7 L罐上通过恒速流加甘油进一步提高细胞密度, 诱导阶段甲醇采取前期恒速流加和后期DO-stat, 发酵结束菌体干重达80 g/L, 酶活为217 U/mL, 比摇瓶结果提高了66.2%。

Abstract:

The key factors on high-level polygalacturonate lyase (PGL) production in recombinant Pichia pastoris were investigated. In 250 mL shake flask, the optimal glycerol concentration, initial methanol concentration, methanol supplementation quantity, duration of induction, initial pH, medium volume were 40 g/L, 3.1 g Methanol/g DCW, 0.51 g Methanol/g DCW (in every 24 h), 72 h, pH6.0 and 30 mL, respectively. In 7 L fermentor, constant glycerol feeding, constant and DO-stat methanol feeding strategies were applied to enhance cell density and PGL production. Finally, the dry cell weight was 80 g/L, and the maximum yield of PGL was achieved to 217 U/mL, increased by 66.2% than that of flask level.