Characterization of an AA9 LPMO from Thielavia australiensis, TausLPMO9B, under industrially relevant lignocellulose saccharification conditions

M. Akeroyd,M. A. van den Berg,V. G. H. Eijsink,A. Várnai,L. E. Bevers,F. Calderaro,M. Keser

doi:10.1186/s13068-020-01836-3

Abstract

BackgroundThe discovery of lytic polysaccharide monooxygenases (LPMO) has changed our perspective on enzymatic degradation of plant biomass. Through an oxidative mechanism, these enzymes are able to cleave and depolymerize various polysaccharides, acting not only on crystalline substrates such as chitin and cellulose, but also on other polysaccharides, such as xyloglucan, glucomannan and starch. Despite their widespread use, uncertainties related to substrate specificity and stereospecificity, the nature of the co-substrate, in-process stability, and the nature of the optimal reductant challenge their exploitation in biomass processing applications.ResultsIn this work, we studied the properties of a novel fungal LPMO from the thermophilic fungus Thielavia australiensis, TausLPMO9B. Heterologous expression of TausLPMO9B in Aspergillus niger yielded a glycosylated protein with a methylated N-terminal histidine showing LPMO activity. High sequence identity of the AA9 domain to that of MtLPMO9B (MYCTH_80312) from Myceliophthora thermophila (84%) indicated strictly C1-oxidizing activity on cellulose, which was confirmed experimentally by the analysis of products released from cellulose using HPAEC. The enzyme was stable and active at a pH ranging from 4 to 6, thus matching the conditions commonly used in industrial biomass processing, where a low pH (between 4 and 5) is used due to the pH-optima of commercial cellulases and a desire to limit microbial contamination.ConclusionWhile the oxidative cleavage of phosphoric acid swollen cellulose (PASC) by TausLPMO9B was boosted by the addition of H2O2 as a co-substrate, this effect was not observed during the saccharification of acid pretreated corn stover. This illustrates key differences between the lab-scale tests with artificial, lignin-free substrates and industrial settings with lignocellulosic biomass as substrate.

Highlights

The discovery of lytic polysaccharide monooxygenases (LPMO) has changed our perspective on enzymatic degradation of plant biomass
This study focused on the characterization of such an LPMO, TausLPMO9B, from T. australiensis, which is, together with T. terrestris, the only described thermophilic species of the genus Thielavia [41]
The product encoded by the 984-basepair-long gene was named TausLPMO9B and consists of a typical N-terminal AA9 domain, a linker region and a C-terminal CBM1 domain (Additional file 1: Table S1)

Summary

Introduction

The discovery of lytic polysaccharide monooxygenases (LPMO) has changed our perspective on enzymatic degradation of plant biomass. These enzymes are able to cleave and depolymerize various polysaccharides, acting on crystalline substrates such as chitin and cellulose, and on other polysaccharides, such as xyloglucan, glucomannan and starch. Despite their widespread use, uncertainties related to substrate specificity and stereospecificity, the nature of the co-substrate, in-process stability, and the nature of the optimal reductant challenge their exploitation in biomass processing applications. Cellobiohydrolases and endoglucanases cleave, respectively, at the end or in the internal part of the cellulose chains and release cellobiose and longer cello-oligosaccharides, which are hydrolyzed to glucose by β-glucosidases [4]. These enzymes are classified as glycoside hydrolases (GH) in the CAZy (Carbohydrate Active Enzyme) database [8], based on sequence similarity

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