Dataset Information

Enzyme mediated nanofibrillation of cellulose by the synergistic actions of an endoglucanase, lytic polysaccharide monooxygenase (LPMO) and xylanase.

ABSTRACT: Physiochemical methods have generally been used to "open-up" biomass substrates/pulps and have been the main method used to fibrillate cellulose. However, recent work has shown that canonical cellulase enzymes such as endoglucanases, in combination with "amorphogenesis inducing" proteins such as lytic polysaccharide monooxygenases (LPMO), swollenin and hemicellulases, are able to increase cellulose accessibility. In the work reported here different combinations of endoglucanase, LPMO and xylanase were applied to Kraft pulps to assess their potential to induce fibrillation at low enzyme loading over a short time period. Although gross fiber properties (fiber length, width and morphology) were relatively unchanged, over a short period of time, the intrinsic physicochemical characteristics of the pulp fibers (e.g. cellulose accessibility/DP/crystallinity/charge) were positively enhanced by the synergistic cooperation of the enzymes. LPMO addition resulted in the oxidative cleavage of the pulps, increasing the negative charge (~100?mmol kg-1) on the cellulose fibers. This improved cellulose nanofibrilliation while stabilizing the nanofibril suspension (zeta potential ??=?~60?mV), without sacrificing nanocellulose thermostability. The combination of endoglucanase, LPMO and xylanases was shown to facilitate nanofibrillation, potentially reducing the need for mechanical refining while resulting in a pulp with a more uniform nanofibril composition.

SUBMITTER: Hu J

PROVIDER: S-EPMC5816652 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Enzyme mediated nanofibrillation of cellulose by the synergistic actions of an endoglucanase, lytic polysaccharide monooxygenase (LPMO) and xylanase.

Hu Jinguang J Tian Dong D Renneckar Scott S Saddler Jack N JN

Scientific reports 20180216 1

Physiochemical methods have generally been used to "open-up" biomass substrates/pulps and have been the main method used to fibrillate cellulose. However, recent work has shown that canonical cellulase enzymes such as endoglucanases, in combination with "amorphogenesis inducing" proteins such as lytic polysaccharide monooxygenases (LPMO), swollenin and hemicellulases, are able to increase cellulose accessibility. In the work reported here different combinations of endoglucanase, LPMO and xylanas ...[more]

PMID: 29453372

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Project description:Lytic polysaccharide monooxygenases (LPMOs) are crucial industrial enzymes required in the biorefinery industry as well as in the natural carbon cycle. These enzymes, known to catalyze the oxidative cleavage of glycosidic bonds, are produced by numerous bacterial and fungal species to assist in the degradation of cellulosic biomass. In this study, we annotated and performed structural analysis of an uncharacterized LPMO from Penicillium funiculosum (PfLPMO9) based on computational methods in an attempt to understand the behavior of this enzyme in biomass degradation. PfLPMO9 exhibited 75% and 36% sequence identities with LPMOs from Thermoascus aurantiacus (TaLPMO9A) and Lentinus similis (LsLPMO9A), respectively. Furthermore, multiple fungal genetic manipulation tools were employed to simultaneously overexpress LPMO and cellobiohydrolase I (CBH1) in a catabolite-derepressed strain of Penicillium funiculosum, PfMig188 (an engineered variant of P. funiculosum), to improve its saccharification performance toward acid-pretreated wheat straw (PWS) at 20% substrate loading. The resulting transformants showed improved LPMO and CBH1 expression at both the transcriptional and translational levels, with ∼200% and ∼66% increases in ascorbate-induced LPMO and Avicelase activities, respectively. While the secretome of PfMig88 overexpressing LPMO or CBH1 increased the saccharification of PWS by 6% or 13%, respectively, over the secretome of PfMig188 at the same protein concentration, the simultaneous overexpression of these two genes led to a 20% increase in saccharification efficiency over that observed with PfMig188, which accounted for 82% saccharification of PWS under 20% substrate loading.IMPORTANCE The enzymatic hydrolysis of cellulosic biomass by cellulases continues to be a significant bottleneck in the development of second-generation biobased industries. While increasing efforts are being made to obtain indigenous cellulases for biomass hydrolysis, the high production cost of this enzyme remains a crucial challenge affecting its wide availability for the efficient utilization of cellulosic materials. This is because it is challenging to obtain an enzymatic cocktail with balanced activity from a single host. This report describes the annotation and structural analysis of an uncharacterized lytic polysaccharide monooxygenase (LPMO) gene in Penicillium funiculosum and its impact on biomass deconstruction upon overexpression in a catabolite-derepressed strain of P. funiculosum Cellobiohydrolase I (CBH1), which is the most important enzyme produced by many cellulolytic fungi for the saccharification of crystalline cellulose, was further overexpressed simultaneously with LPMO. The resulting secretome was analyzed for enhanced LPMO and exocellulase activities and the corresponding improvement in saccharification performance (by ∼20%) under high-level substrate loading using a minimal amount of protein.

Project description:Background:The 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. Results:In 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 ?-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. Conclusions:TrAA9A 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.

Dataset Information

Enzyme mediated nanofibrillation of cellulose by the synergistic actions of an endoglucanase, lytic polysaccharide monooxygenase (LPMO) and xylanase.

Publications

Enzyme mediated nanofibrillation of cellulose by the synergistic actions of an endoglucanase, lytic polysaccharide monooxygenase (LPMO) and xylanase.

Similar Datasets

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