Kinetic characterization and fed-batch fermentation for maximal simultaneous production of esterase and protease from <i>Lysinibacillus fusiformis</i> AU01

The simultaneous production of intracellular esterase and extracellular protease from the strain <i>Lysinibacillus fusiformis</i> AU01 was studied in detail. The production was performed both under batch and fed-batch modes. The maximum yield of intracellular esterase and protease was obtained under full oxygen saturation at the beginning of the fermentation. The data were fitted to the Luedeking–Piret model and it was shown that the enzyme (both esterase and protease) production was growth associated. A decrease in intracellular esterase and increase in the extracellular esterase were observed during late stationary phase. The appearance of intracellular proteins in extracellular media and decrease in viable cell count and biomass during late stationary phase confirmed that the presence of extracellular esterase is due to cell lysis. Even though the fed-batch fermentation with different feeding strategies showed improved productivity, feeding yeast extract under DO-stat fermentation conditions showed highest intracellular esterase and protease production. Under DO-stat fed-batch cultivation, maximum intracellular esterase activity of 820 × 10³ U/L and extracellular protease activity of 172 × 10³ U/L were obtained at the 16th hr. Intracellular esterase and extracellular protease production were increased fivefold and fourfold, respectively, when compared to batch fermentation performed under shake flask conditions.本研究针对纺锤形溶杆菌（Lysinibacillus fusiformis）AU01菌株同步合成胞内酯酶与胞外蛋白酶的过程展开了详尽探究。该酶类的合成实验分别采用分批发酵与补料分批发酵两种模式开展。在发酵初始阶段通入完全饱和的氧气时，胞内酯酶与蛋白酶的产量达到峰值。将实验数据拟合至吕德金-皮雷模型（Luedeking–Piret model）后发现，酯酶与蛋白酶的合成都与菌体生长呈偶联关系。在稳定期后期，可观测到胞内酯酶含量下降，而胞外酯酶含量上升。稳定期后期胞外培养基中出现胞内蛋白，同时活菌数与生物量均出现下降，这一结果证实胞外酯酶的产生源于菌体裂解。尽管采用不同补料策略的补料分批发酵均提升了生产效率，但在溶氧反馈补料（DO-stat）条件下补加酵母提取物时，胞内酯酶与蛋白酶的产量达到最高。在溶氧反馈补料分批培养条件下，发酵至第16小时时，胞内酯酶活性峰值达820×10³ U/L，胞外蛋白酶活性峰值达172×10³ U/L。与摇瓶分批发酵相比，本实验中胞内酯酶与胞外蛋白酶的产量分别提升了5倍与4倍。