Enzymatic synthesis of model substrates recognized by glucuronoyl esterases from [i]Podospora anserina[/i] and [i]Myceliophthora thermophila[/i]

• Published in: Applied Microbiology and Biotechnology
• Main Authors: Katsimpouras, Constantinos, Benarouche, Anaïs, Navarro, David, Karpusas, Michael, Dimarogona, Maria, Berrin, Jean-Guy, Christakopoulos, Paul, Topakas, Evangelos
• Other Authors: School of Chemical Engineering, Biotechnology Laboratory, National Technical University of Athens Athens (NTUA), Biodiversité et Biotechnologie Fongiques (BBF), Institut National de la Recherche Agronomique (INRA)-Aix Marseille Université (AMU)-École Centrale de Marseille (ECM), Department of Biotechnology, Physics Laboratory, Agricultural University of Athens, Department of Civil Environmental and Natural Resources Engineering, Division of Sustainable Process Engineering, Luleå University of Technology (LUT), General Secretariat of Research and Technology (GSRT) of Greece-ESPA
• Format: Article in Journal/Newspaper
• Language: English
• Published: HAL CCSD 2014
• Subjects: Glucuronoyl esterase; Myceliophthora thermophila; Podospora anserina; Pichia pastoris; Substrate specificity; Aryl alkyl or alkenyl D-glucuronates; [SDV]Life Sciences [q-bio]; Antarc*; Antarctica
• Online Access: https://hal.archives-ouvertes.fr/hal-01268764; https://doi.org/10.1007/s00253-014-5542-9

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.