The extracellular nuclease, NucB, from Bacillus licheniformis, can digest extracellular DNA in biofilms, causing biofilm dispersal, and may therefore be used commercially to remove biofilms. However, producing quantities of this secreted peptide is difficult and our aim was therefore to improve its laboratory scale production. This study builds on our understanding of B. licheniformis physiology to enhance NucB production. The addition of manganese, which triggers sporulation and enhances NucB expression, lead to a 5-fold increase in NucB production. Optimisation via Placket-Burman design of experiments identified 3 significant medium components and a subsequent Central Composite Design, to determine the optimum levels of these components, resulted in a 10-fold increase to 471 U/ml. The optimal phosphate concentration was less than 0.3 mM as this is known to inhibit nuclease production. The use of physiologically relevant information combined with optimisation represents a promising approach to increased enzyme production, which may also be widely applicable. (C) 2012 Elsevier Ltd. All rights reserved.