Optimization of pullulanase production in Escherichia coli by regulation of process conditions and supplement with natural osmolytes

In this study, the effects of temperature, IPTG (Isopropyl β-d-1-thiogalactopyranoside) concentration, and osmolytes (proline, K-glutamate, and betaine) on cell growth and soluble pullulanase productivity of recombinant Escherichia coli were investigated. The yield of soluble pullulanase was found to be enhanced with decrease in cultivation temperature, lower IPTG concentration, and betaine supplementation in a shake flask. In addition, a modified two-stage feeding strategy was proposed and applied in a 3-L fermentor supplied with 20 mM betaine, which achieved a dry cell weight of 59.3 g L−1. Through this cultivation approach at 25 °C, the total soluble activity of pullulanase reached 963.9 U mL−1, which was 8.3-fold higher than that observed without addition of betaine at 30 °C (115.8 U mL−1). The higher expression of soluble pullulanase in a scalable semisynthetic medium showed the potential of the proposed process for the industrial production of soluble enzyme.