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

Abstract

Background The 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. Results In 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. Conclusion While 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.

In situ measurements of oxidation–reduction potential and hydrogen peroxide concentration as tools for revealing LPMO inactivation during enzymatic saccharification of cellulose

“…The abundance of redox-active compounds in such reactions will lead to a multitude of (possible) side reactions that likely cannot be monitored or controlled using the approaches described above [ 17 , 22 , 31 ]. In fact, so far, saccharification of lignin-rich substrates with controlled supply of external H 2 O 2 has not been particularly successful [ 17 , 32 ]. The present results suggest that improved harnessing of the potential of LPMOs should be possible also for this type of substrates.…”

Oxidative Power: Tools for Assessing LPMO Activity on Cellulose

“…Double-oxidized products normally elute later in the gradient. Here, C1-oxidizing NcLPMO9F [46] and TausLPMO9B [47] and C4-oxidizing NcLPMO9C [46] were incubated with phosphoric acid swollen cellulose (PASC) in the presence of ascorbic acid as an electron donor. In order to separate the oligosaccharides present in the reaction mixtures, alkaline pH is applied during the elution.…”

“…HPAEC-PAD is powerful tool that is also routinely applied for the identification of products released from other substrates, such as xyloglucan [11,48,52,57,58]. [47] before (blue line) and after (green line) treatment with BG; 1 µ M TausLPMO9B was incubated with 0.1% (w/v) PASC and 1 mM ascorbic acid in a 50 mM sodium acetate buffer pH 5 at 45 °C for 16 h. Reaction mixtures were centrifuged and filtered before the treatment of soluble products with 1 unit of BG from almonds (Sigma, St Louis, MO, USA) at 37 °C for 16 h in a 50 mM sodium acetate buffer pH 5. Glc1A, gluconic acid; GlcGlc1A, cellobionic acid; Glc2 Glc1A, cellotrionic acid; Glc3Glc1A, cellotetraonic acid; Glc5Glc1A, cellohexaoinic acid; Glc6Glc1A, celloeptaonic acid.…”

“…On top of that, the presence of metal ions in fermentation media can interfere with the assay, as shown for TausLPMO9B (Figure 7b). In [47], this enzyme was produced in A. niger grown in corn steep liquor (CSL). When fermentation broth was directly tested, the background absorbance values were too high to allow for the clear detection of the LPMO activity (Figure 7b, blue line).…”

“…The TausLPMO9B activity tested with the Amplex TM Red assay before (green and blue lines) and after (brown and red lines) enzyme purification. Reactions were run in 50 mM sodium phosphate buffer pH 7 and contained 5 U/mL of HRP, 100 µM Amplex TM Red, and 20 µL of purified TausLPMO [47] or diluted fermentation broth (1:4) in a final volume of 100 µL. Reactions were started by the addition of ascorbic acid (brown and green lines) to a final concentration of 50 µM.…”