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A C4-oxidizing lytic polysaccharide monooxygenase cleaving both cellulose and cello-oligosaccharides.

ABSTRACT: Lignocellulosic biomass is a renewable resource that significantly can substitute fossil resources for the production of fuels, chemicals, and materials. Efficient saccharification of this biomass to fermentable sugars will be a key technology in future biorefineries. Traditionally, saccharification was thought to be accomplished by mixtures of hydrolytic enzymes. However, recently it has been shown that lytic polysaccharide monooxygenases (LPMOs) contribute to this process by catalyzing oxidative cleavage of insoluble polysaccharides utilizing a mechanism involving molecular oxygen and an electron donor. These enzymes thus represent novel tools for the saccharification of plant biomass. Most characterized LPMOs, including all reported bacterial LPMOs, form aldonic acids, i.e., products oxidized in the C1 position of the terminal sugar. Oxidation at other positions has been observed, and there has been some debate concerning the nature of this position (C4 or C6). In this study, we have characterized an LPMO from Neurospora crassa (NcLPMO9C; also known as NCU02916 and NcGH61-3). Remarkably, and in contrast to all previously characterized LPMOs, which are active only on polysaccharides, NcLPMO9C is able to cleave soluble cello-oligosaccharides as short as a tetramer, a property that allowed detailed product analysis. Using mass spectrometry and NMR, we show that the cello-oligosaccharide products released by this enzyme contain a C4 gemdiol/keto group at the nonreducing end.

SUBMITTER: Isaksen T

PROVIDER: S-EPMC3908397 | biostudies-literature | 2014 Jan

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe high cost of enzymes is one of the key technical barriers that must be overcome to realize the economical production of biofuels and biomaterials from biomass. Supplementation of enzyme cocktails with lytic polysaccharide monooxygenase (LPMO) can increase the efficiency of these cellulase mixtures for biomass conversion. The previous studies have revealed that LPMOs cleave polysaccharide chains by oxidization of the C1 and/or C4 carbons of the monomeric units. However, how LPMOs enhance enzymatic degradation of lignocellulose is still poorly understood.ResultsIn this study, we combined enzymatic assays and real-time imaging using atomic force microscopy (AFM) to study the molecular interactions of an LPMO [TrAA9A, formerly known as TrCel61A) from Trichoderma reesei] and a cellobiohydrolase I (TlCel7A from T. longibrachiatum) with bacterial microcrystalline cellulose (BMCC) as a substrate. Cellulose conversion by TlCel7A alone was enhanced from 46 to 54% by the addition of TrAA9A. Conversion by a mixture of TlCel7A, endoglucanase, and β-glucosidase was increased from 79 to 87% using pretreated BMCC with TrAA9A for 72 h. AFM imaging demonstrated that individual TrAA9A molecules exhibited intermittent random movement along, across, and penetrating into the ribbon-like microfibril structure of BMCC, which was concomitant with the release of a small amount of oxidized sugars and the splitting of large cellulose ribbons into fibrils with smaller diameters. The dividing effect of the cellulose microfibril occurred more rapidly when TrAA9A and TlCel7A were added together compared to TrAA9A alone; TlCel7A alone caused no separation.ConclusionsTrAA9A increases the accessible surface area of BMCC by separating large cellulose ribbons, and thereby enhances cellulose hydrolysis yield. By providing the first direct observation of LPMO action on a cellulosic substrate, this study sheds new light on the mechanisms by which LPMO enhances biomass conversion.

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A C4-oxidizing lytic polysaccharide monooxygenase cleaving both cellulose and cello-oligosaccharides.

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