Enhanced extracellular production of recombinant Bacillus deramificans pullulanase in Escherichia coli through induction mode optimization and a glycine feeding strategy

• The pullulanase secretion was enhanced by optimized induction and glycine supplement.
• The pullulanase activity represented the highest reported so far.
• The approaches are valuable for extracellular secretion of other proteins in E. coli.

Process optimization strategies were developed to improve extracellular production of recombinant Bacillus deramificans pullulanase in Escherichia coli. Cell growth and pullulanase production in shake-flask cultures were investigated as a function of the concentration of added glycine, and the type and concentration of inducer. From the results of these experiments, a fed-batch fermentation strategy for high-cell-density cultivation was applied in a 3-L fermentor. The gradual addition of lactose was utilized for the induction of protein expression. The optimal lactose feeding rate and induction point were 0.4 g L−1 h−1 and a dry cell weight (DCW) of 15 g L−1, respectively. Furthermore, a glycine feeding strategy was formulated to promote the secretion of recombinant protein. The optimal total and extracellular pullulanase activity were 2523.5 and 1567.9 U mL−1, respectively, which represent 1.2 and 22.6-fold increases compared with those observed under unoptimized conditions.