In this study, the effects of temperature, IPTG (Isopropyl beta-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 degrees 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 degrees 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. (c) 2013 Elsevier Ltd. All rights reserved.