Human cathepsin S production by recombinant Pichia pastoris using cod skin as the co-nitrogen source was investigated in this study. The addition of carbon sources of glycerol in the fed-batch phase and of methanol in the induction stage was also investigated. A new approach to the highly expression of human cathepsin S was developed using 90 g/L of cod skin (wet weight). After 24 h of the initial fermentation, 4% glycerol (v/v, glycerol/culture) was added once to enhance the cell density (OD600) in the cultivation. Then, adding and maintaining methanol at 0.5% (v/v, methanol/cultivation) after about 48 h of fermentation achieved a high expression of human cathepsin S in a 5-L bioreactor. The results demonstrate that the maximum activity of human cathepsin S in the fermentation supernatant reached 7,152 U/L after 96 h of methanol induction. The methylotrophic yeast P. pastoris grown in the medium containing cod skin (90 g/L) as the co-nitrogen source provided a 21% higher cell density (OD600) and 18.3% higher human cathepsin S yield than P. pastoris grown in BMGY medium. For the first time, human cathepsin S was successfully expressed by P. pastoris with cod skin as the co-nitrogen source. The glycerol fed-batch controlling strategy and method of maintaining methanol at a constant concentration of 0.5% (v/v, methanol/cultivation) in the induction stage was efficient for P. pastoris growth and the expression of human cathepsin S.

中文翻译：

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重组巴斯德毕赤酵母与鳕鱼皮作为有机辅氮源对人组织蛋白酶S的高表达。

本研究研究了利用鳕鱼皮作为共氮源的重组毕赤酵母生产人组织蛋白酶S。还研究了在补料分批阶段添加甘油的碳源和在诱导阶段添加甲醇的碳源。使用90 g / L的鳕鱼皮（湿重）开发了一种新的高表达人组织蛋白酶S的方法。初始发酵24小时后，一次添加4％甘油（v / v，甘油/培养物）以增强培养中的细胞密度（OD600）。然后，在发酵约48小时后添加并保持甲醇含量为0.5％（v / v，甲醇/培养量），从而在5-L生物反应器中实现了人组织蛋白酶S的高表达。结果表明，在诱导甲醇96 h后，人组织蛋白酶S在发酵上清液中的最大活性达到7,152 U / L。在含有鳕鱼皮（90 g / L）作为共氮源的培养基中生长的甲基营养酵母毕赤酵母比在BMGY中生长的毕赤酵母提供更高的细胞密度（OD600）和21％的人组织蛋白酶S产量。中。第一次，人类组织蛋白酶S由鳕鱼皮成功地以鳕鱼皮作为共氮源表达。在诱导阶段，甘油补料分批控制策略和将甲醇维持在0.5％恒定浓度（v / v，甲醇/培养）的方法和方法对于巴斯德毕赤酵母的生长和人组织蛋白酶S的表达都是有效的。人组织蛋白酶S由鳕鱼皮成功地表达，鳕鱼皮为辅助氮源。在诱导阶段，甘油补料分批控制策略和将甲醇保持在0.5％恒定浓度（v / v，甲醇/培养）的方法和方法对于巴斯德毕赤酵母的生长和人组织蛋白酶S的表达都是有效的。人组织蛋白酶S由鳕鱼皮成功地表达，鳕鱼皮为辅助氮源。在诱导阶段，甘油补料分批控制策略和将甲醇保持在0.5％恒定浓度（v / v，甲醇/培养）的方法和方法对于巴斯德毕赤酵母的生长和人组织蛋白酶S的表达都是有效的。