CwIQ (previous YjbJ) is one of the putative cell wall hydrolases in Bacillus subtilis. Its domain has an amino acid sequence similar to the soluble-lytic transglycosylase (SLT) of Escherichia coli Slt70 and also goose lysozyme (muramidase). To characterize the enzyme, the domain of CwIQ was cloned and expressed in E. coil. The purified CwIQ protein exhibited cell wall hydrolytic activity. Surprisingly, RP-HPLC, mass spectrometry (MS), and MS/MS analyses showed that CwIQ produces two products, 1,6-anhydro-N-acetylmuramic acid and N-acetylmuramic acid, thus indicating that CwIQ is a bifunctional enzyme. The site-directed mutagenesis revealed that glutamic acid 85 (Glu-85) is an amino acid residue essential to both activities. (C) 2010 Elsevier Inc. All rights reserved.