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

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.

Classification of fungal glucuronoyl esterases (FGEs) and characterization of two new FGEs from Ceriporiopsis subvermispora and Pleurotus eryngii.

Fungal glucuronoyl esterases (FGEs) catalyze cleavage of the ester bond connecting a lignin alcohol to the xylan-bound 4-O-methyl-D-glucuronic acid of glucuronoxylans. Thus, FGEs are capable of degrading lignin-carbohydrate complexes and have potential for biotechnological applications toward woody biomass utilization. Therefore, identification and characterization of new FGEs are of critical importance. Firstly, in this study, we built a phylogenetic tree from almost 400 putative FGEs obtained on BLAST analysis and defined six main clades. In the phylogenetic tree, all the putative FGEs of ascomycetes cluster in clades I to IV, and most of the putative FGEs of basidiomycetes (B-FGEs) cluster in clades V to VI. Interestingly, several B-FGEs were found to cluster in clade II; most FGEs of clade II were found to have higher theoretical isoelectric points than those in the other five clades. To gain an insight into the putative FGEs in the clades that have not been characterized yet, we chose the FGEs of Ceriporiopsis subvermispora (CsGE) and Pleurotus eryngii (PeGE), which belong to clades V and II, respectively. The catalytic domains of both CsGE and PeGE were successfully expressed using Pichia pastoris, and then purified. Benzyl glucuronic acid was used as a substrate to confirm the activities of the CsGE and PeGE, and the hydrolyzed product, glucuronic acid, was quantified spectrophotometrically. Both CsGE and PeGE clearly exhibited the esterase activity. Additionally, we demonstrated that PeGE exhibits high tolerance toward several denaturing agents, which may make it a potentially more applicable enzyme.

Identification of key residues for activities of atypical glutathione S-transferase of Ceriporiopsis subvermispora, a selective degrader of lignin in woody biomass, by crystallography and functional mutagenesis.

Ceriporiopsis subvermispora (C. subvermispora) is a selective degrader of lignin in the woody biomass. Glutathione S-transferases (GSTs) are multifunctional enzymes that play important roles in cellular detoxification and metabolism. The crystal structures of a GST of C. subvermispora, CsGST83044, in GSH-free and -bound forms were solved at 1.95 and 2.19 Šresolution, respectively. The structure of the GSH-bound form revealed that CsGST83044 can be categorized as an atypical-type of GST. In the GSH-bound form of CsGST83044, Asn22, Asn24, and Tyr46 are located closest to the sulfur atom and form hydrogen bonds with the thiol group. The functional mutagenesis indicated that they are critical for the enzymatic activities of CsGST83044. The critical residues of an atypical-type GST belonging to the GSTFuA class were revealed for the first time. A previous study indicated that CsGST83044 and another GST, CsGST63524, differ in substrate preference; CsGST83044 prefers smaller substrates than CsGST63524 for its esterase activity. The GSH-bound pocket of CsGST83044 turns out to be small, which may explain the preference for smaller substrates. Protein engineering of GSTs of C. subvermispora in the light of the obtained insight may pave a path in the future for utilization of the woody biomass.