Lytic Polysaccharide Monooxygenase from Talaromyces amestolkiae with an Enigmatic Linker-like Region: The Role of This Enzyme on Cellulose Saccharification.

Francisco J Plou,Florian Csarman,Roland Ludwig,María Jesús Martínez,Iván Ayuso-Fernández,Noa Míguez,Laura Isabel De Eugenio,Alicia Prieto,Juan Antonio Méndez-Líter

doi:10.3390/ijms222413611

Abstract

The first lytic polysaccharide monooxygenase (LPMO) detected in the genome of the widespread ascomycete Talaromyces amestolkiae (TamAA9A) has been successfully expressed in Pichia pastoris and characterized. Molecular modeling of TamAA9A showed a structure similar to those from other AA9 LPMOs. Although fungal LPMOs belonging to the genera Penicillium or Talaromyces have not been analyzed in terms of regioselectivity, phylogenetic analyses suggested C1/C4 oxidation which was confirmed by HPAEC. To ascertain the function of a C-terminal linker-like region present in the wild-type sequence of the LPMO, two variants of the wild-type enzyme, one without this sequence and one with an additional C-terminal carbohydrate binding domain (CBM), were designed. The three enzymes (native, without linker and chimeric variant with a CBM) were purified in two chromatographic steps and were thermostable and active in the presence of H2O2. The transition midpoint temperature of the wild-type LPMO (Tm = 67.7 °C) and its variant with only the catalytic domain (Tm = 67.6 °C) showed the highest thermostability, whereas the presence of a CBM reduced it (Tm = 57.8 °C) and indicates an adverse effect on the enzyme structure. Besides, the potential of the different T. amestolkiae LPMO variants for their application in the saccharification of cellulosic and lignocellulosic materials was corroborated.

Highlights

One of the most important applications for plant biomass valorization is related to the production of second-generation ethanol, based on the degradation of lignocellulosic biomass, where the sources of fermentable sugars are cellulose and hemicellulose [1]
A single gene coding for a putative lytic polysaccharide monooxygenase (LPMO) (TamAA9A protein) was detected in the annotated genome of T. amestolkiae
The exact role of this domain remains uncertain, a linker region that connects the catalytic domain and a CBM1 has been described in an LPMO produced by Hypocrea jecorina (HjLPMO9A) [35]

Summary

Introduction

One of the most important applications for plant biomass valorization is related to the production of second-generation ethanol, based on the degradation of lignocellulosic biomass, where the sources of fermentable sugars are cellulose and hemicellulose [1]. After deconstruction of the plant cell wall and removal of lignin [2], plant polysaccharides must be hydrolyzed into their corresponding monosaccharides by the concerted action of different types of enzymes, mostly glycosyl hydrolases (GHs). These enzymes catalyze the hydrolysis of the glycosidic bond between two monosaccharides, or between a carbohydrate and an aglycone [3]. The first step was found to be due to the action of fungal and bacterial copper-containing lytic polysaccharide monooxygenases (LPMOs) [8] These novel catalysts were included in the CAZy database [9], in the class of auxiliary activities, and currently belong to families AA9, 10, 11, 13, 14, 15 16, and 17. Despite the broad diversity of LPMOs, the number of enzymes isolated and characterized biochemically and catalytically up to date is still quite low

Methods

Results

Conclusion