Production of a Thermostable Pullulanase in Bacillus subtilis by Optimization of the Expression Elements

Abstract Pullulanase is a debranching enzyme commonly used in the starch processing industry. Production of a thermostable pullulanase with high activity is desired due to the application of the enzyme to starch processing at high temperature. In this study, a thermostable pullulanase with high activity is overproduced in Bacillus subtilis . Via improvement of the plasmid backbones, promoters, and ribosome‐binding‐site (RBS) sequences, an optimal recombinant strain B. subtilis WB800/RBS7 is obtained with an extracellular pullulanase activity of 154.9 U mL −1 in a shake flask, which is 136.8 times higher than that of the wild type. When the strain is cultured in a 5‐L bioreactor, the pullulanase activity is significantly improved to 269.1 U mL −1 (2.74 mg mL −1 ). These results are expected to aid pullulanase production in industrial starch debranching applications.