Abstract
Glucuronoyl esterases (GEs) are recently discovered enzymes that are suggested to cleave the ester bond between lignin alcohols and xylan-bound 4-O-methyl-d-glucuronic acid. Although their potential use for enhanced enzymatic biomass degradation and synthesis of valuable chemicals renders them attractive research targets for biotechnological applications, the difficulty to purify natural fractions of lignin-carbohydrate complexes hampers the characterization of fungal GEs. In this work, we report the synthesis of three aryl alkyl or alkenyl d-glucuronate esters using lipase B from Candida antarctica (CALB) and their use to determine the kinetic parameters of two GEs, StGE2 from the thermophilic fungus Myceliophthora thermophila (syn. Sporotrichum thermophile) and PaGE1 from the coprophilous fungus Podospora anserina. PaGE1 was functionally expressed in the methylotrophic yeast Pichia pastoris under the transcriptional control of the alcohol oxidase (AOX1) promoter and purified to its homogeneity (63 kDa). The three d-glucuronate esters contain an aromatic UV-absorbing phenol group that facilitates the quantification of their enzymatic hydrolysis by HPLC. Both enzymes were able to hydrolyze the synthetic esters with a pronounced preference towards the cinnamyl-d-glucuronate ester. The experimental results were corroborated by computational docking of the synthesized substrate analogues. We show that the nature of the alcohol portion of the hydrolyzed ester influences the catalytic efficiency of the two GEs.
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Acknowledgments
The financial support of General Secretariat of Research and Technology (GSRT) of Greece-ESPA 2007–2013 is gratefully acknowledged. We are also grateful to Novo Nordisk A/S for the generous gift of C. antarctica lipase CALB and to Marina Roussaki for her expertise in obtaining the NMR spectra.
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Katsimpouras, C., Bénarouche, A., Navarro, D. et al. Enzymatic synthesis of model substrates recognized by glucuronoyl esterases from Podospora anserina and Myceliophthora thermophila . Appl Microbiol Biotechnol 98, 5507–5516 (2014). https://doi.org/10.1007/s00253-014-5542-9
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DOI: https://doi.org/10.1007/s00253-014-5542-9