The Ink gene cluster consisting of mlrA, mlrB, mlrC, and mlrD is involved in the degradation of the cyanobacterial toxin microcystin. However, it is unclear which degradation intermediates are metabolized by MlrB and MlrC. To address these questions, we constructed recombinant Escherichia coil to overproduce MlrB and MlrC from Sphingopyxis sp. C-1, and determined which intermediates were degraded in cell-free extracts. The cell-free extract containing MlrB degraded linearized microcystin-LR, giving rise to a tetrapeptide. The cell-free extract of MlrC degraded linearized microcystin-LR and also degraded the tetrapeptide to the amino acid Adda. These results indicate that linearized microcystin-LR is degraded by both MlrB and MlrC, and tetrapeptide is degraded by specifically by MlrC in Sphingopyxis sp. C-1. (C) 2012, The Society for Biotechnology, Japan. All rights reserved.