High yield production of fungal manganese peroxidases by E. coli through soluble expression, and examination of the activities

Abstract

Oxidative enzymes of white-rot fungi play a key role in lignin biodegradation. Among those fungus, Ceriporiopsis subvermispora degrades lignin before cellulose in wood; C. subvermispora is the only fungus that secretes all known types of manganese peroxidases (CsMnPs). Utilization of lignin-degrading peroxidases has been limited so far due to the lack of efficient preparation methods and intensive characterization. In this study, we developed a highly efficient method to prepare active CsMnPs through soluble expression by E. coli, which had long been impossible. The genes of MnPs selected from each subfamily were codon-optimized and expressed under the control of a cold shock promoter. A proper level of heme incorporation was achieved by continuous addition of hemin during cultivation. As much as 3 mg of purified MnPs was obtained from 100 mL culture, which is an about 20-fold higher yield than that from inclusion bodies through refolding. Further improvement of the solubility on the expression was achieved by combinatorial coexpression of chaperones. All obtained MnPs had heme-to-protein ratios as high as those of native MnPs. They were all active below pH 5. Our method is applicable to other fungal-secreted enzymes should help the progress of their basic characterization and application for better utilization of woody biomass.