Process optimization of high-level extracellular production of alkaline pectate lyase in recombinant Escherichia coli BL21 (DE3)

• A multi-step feeding strategy based on specific growth rate was developed.
• Strategies for high-level extracellular production of alkaline PL were established.
• The yield of alkaline PL represents the highest level reported to date.
• The bioprocess should be an appropriate method for the industrial production.

Eco-effective high-level production of alkaline pectate lyase (PL) is a necessary prerequisite for the application of this enzyme in large-scale industries, such as bioscouring. For this purpose, in this work, a multi-step glycerol feeding strategy based on specific growth rate for high cell density cultivation of recombinant Escherichia coli was first established, which could effectively control cell growth and acetate yield. Next, the effects of induction time, temperature, and inducer concentration on cell growth and extracellular secretory production of recombinant PL were investigated. When induced by IPTG (using either low IPTG concentration at 25 °C or high IPTG concentration at 30 °C), two different extracellular secretion strategies were found to be appropriate for extracellular PL production. On the other hand, when cultivations were induced by mild continuous lactose feeding strategy, the extracellular and total PL activities reached 4478 and 5337 U/mL, respectively, representing the highest PL yield reported to date.