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Doctoral thesis, 2023

Structure and function studies of GH45 glycoside hydrolases

Okmane, Laura

Abstract

The enzymatic hydrolysis of lignocellulosic material in nature is carried out by a plethora of cellulases. Glycoside hydrolase family 45 (GH45) enzymes are small cellulases most commonly found in fungi that catalyse the hydrolysis of β(1→4) linked glucans. Additionally, GH45 enzymes display a structural resemblance to non-hydrolytic protein groups such as expansins and loosenins. In this thesis, the distinctness of GH45 enzymes from different subfamilies was explored. The enzymatic activity of GH45 enzymes from Humicola insolens (HiCel45A), Mytilus edulis (MeCel45A), Trichoderma reesei (TrCel45A), Phanerochaete chrysosporium (PcCel45A), Gloeophyllum trabeum (GtCel45A) was demonstrated. Among the tested substrates were barley betaglucan, konjac glucomannan, carboxymethyl cellulose, and cellohexaose. Initial hydrolysis rates and hydrolysis yields were determined by reducing sugar assays, product formation was analysed using NMR spectroscopy and HPLC. The subfamily B and C enzymes exhibited mannanase activity, and the subfamily B enzyme MeCel45A appeared cold adapted in comparison to TrCel45A. GH45 enzymes are known to act using an inverting action mechanism. This action mechanism had not been experimentally demonstrated in subfamilies B and C, however. Here experimental evidence is provided for the inverting nature of GH45 enzymes from all subfamilies.

Comparisons of GH45 enzyme structures were carried out and the first crystal structure of a GH45 subfamily C enzyme from the brown-rot fungus G. trabeum was reported at 1.3 Å resolution. Furthermore, structure and function investigations were done on an isotopically labelled Cel45A from the white-rot fungus P. chrysosporium using NMR spectroscopy. PcCel45A was expressed in Pichia pastoris with 13C and 15N labelling. A nearly complete assignment of 1 H, 13C and 15N backbone resonances was obtained and the interaction of 15Nlabelled PcCel45A with cellobiose was studied.

Keywords

Cel45A; cellulase, DPBB; endoglucanase; GH45; glycoside hydrolase

Published in

Acta Universitatis Agriculturae Sueciae
2023, number: 2023:41
ISBN: 978-91-8046-134-4, eISBN: 978-91-8046-135-1
Publisher: Swedish University of Agricultural Sciences

    UKÄ Subject classification

    Biocatalysis and Enzyme Technology
    Biochemistry and Molecular Biology

    Publication identifier

    DOI: https://doi.org/10.54612/a.3f14k593rr

    Permanent link to this page (URI)

    https://res.slu.se/id/publ/121946