Abstract
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.
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Funding
This work was supported by JSPS KAKENHI grants to M.K. (#18H04550 and #18K19397) and to T.N. (#17K07307 and #17H05878), JST/ALCA, the Joint Usage/Research Program on Zero-Emission Energy Research (ZE30A-36), and the Collaboration Program of the Laboratory for Complex Energy Processes.
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Lin, MI., Hiyama, A., Kondo, K. et al. Classification of fungal glucuronoyl esterases (FGEs) and characterization of two new FGEs from Ceriporiopsis subvermispora and Pleurotus eryngii. Appl Microbiol Biotechnol 102, 9635–9645 (2018). https://doi.org/10.1007/s00253-018-9318-5
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DOI: https://doi.org/10.1007/s00253-018-9318-5