scholarly journals Four cellulose-active lytic polysaccharide monooxygenases from Cellulomonas species

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
James Li ◽  
Laleh Solhi ◽  
Ethan D. Goddard-Borger ◽  
Yann Mathieu ◽  
Warren W. Wakarchuk ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Morphological Changes ◽  
Cellulose Degradation ◽  
High Surface ◽  
High Surface Area ◽  
Hydrolytic Enzymes ◽  
Lignocellulose Degradation ◽  
Cellulomonas Flavigena ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases

Abstract Background The discovery of lytic polysaccharide monooxygenases (LPMOs) has fundamentally changed our understanding of microbial lignocellulose degradation. Cellulomonas bacteria have a rich history of study due to their ability to degrade recalcitrant cellulose, yet little is known about the predicted LPMOs that they encode from Auxiliary Activity Family 10 (AA10). Results Here, we present the comprehensive biochemical characterization of three AA10 LPMOs from Cellulomonas flavigena (CflaLPMO10A, CflaLPMO10B, and CflaLPMO10C) and one LPMO from Cellulomonas fimi (CfiLPMO10). We demonstrate that these four enzymes oxidize insoluble cellulose with C1 regioselectivity and show a preference for substrates with high surface area. In addition, CflaLPMO10B, CflaLPMO10C, and CfiLPMO10 exhibit limited capacity to perform mixed C1/C4 regioselective oxidative cleavage. Thermostability analysis indicates that these LPMOs can refold spontaneously following denaturation dependent on the presence of copper coordination. Scanning and transmission electron microscopy revealed substrate-specific surface and structural morphological changes following LPMO action on Avicel and phosphoric acid-swollen cellulose (PASC). Further, we demonstrate that the LPMOs encoded by Cellulomonas flavigena exhibit synergy in cellulose degradation, which is due in part to decreased autoinactivation. Conclusions Together, these results advance understanding of the cellulose utilization machinery of historically important Cellulomonas species beyond hydrolytic enzymes to include lytic cleavage. This work also contributes to the broader mapping of enzyme activity in Auxiliary Activity Family 10 and provides new biocatalysts for potential applications in biomass modification.

Understanding Micro-Droplet Interface Bilayers for Developing Bioinspired Sensors

2013 ◽  
Author(s):  
Guru Venkatesan ◽  
Andy Sarles
Keyword(s):  
Water Droplet ◽  
Morphological Changes ◽  
High Surface ◽  
High Surface Area ◽  
Material System ◽  
Modes Of Operation ◽  
New Class ◽  
Wide Range ◽  
Droplet Sizes ◽  
The Stability

Droplet-based biomolecular arrays form the basis for a new class of bioinspired material system, whereby decreasing the sizes of the droplets and increasing the number of droplets can lead to higher functional density for the array. In this paper, we report on a non-microfluidic approach to form and connect nanoliter-to-femtoliter, lipid-coated aqueous droplets in oil to form micro-droplet interface bilayers (μDIBs). Two different modes of operation are reported for dispensing a wide range of droplet sizes (2–200μm radius). Due to the high surface-area-to-volume ratios of microdroplets at these length scales, droplet shrinking is prominent, which affects the stability and lifetime of the bilayer. To better quantify these effects, we measure the shrinkage rates for 8 different water droplet/oil compositions and study the effect of lipid placement and lipid type on morphological changes to μDIBs.

A Multiscale Simulation Approach for the Mechanical Response of Copper/Nickel Nanofoams With Experimental Validation

2021 ◽  
pp. 1-27
Author(s):  
Hang Ke ◽  
Alexandra Loaiza ◽  
Andres Jimenez ◽  
David Bahr ◽  
I. Mastorakos
Keyword(s):  
Mechanical Response ◽  
Morphological Changes ◽  
High Surface ◽  
High Surface Area ◽  
Multiscale Simulation ◽  
Woven Fabric ◽  
Multiscale Approach ◽  
Brittle Behavior ◽  
Copper Nickel ◽  
Dislocation Movement

Abstract Metallic nanofoams, cellular structures consisting of interlinked thin nanowires and empty pores, create low density, high surface area materials. These structures can suffer from macroscopically brittle behavior. In this work, we present a multiscale approach to study and explain the mechanical behavior of metallic nanofoams obtained by an electrospinning method. In this multiscale approach, atomistic simulations were first used to obtain the yield surfaces of different metallic nanofoam cell structures. Then, a continuum plasticity model using finite elements was used to predict the alloy nanofoam's overall strength in compression. The manufactured metallic nanofoams were produced by electrospinning a polymeric non-woven fabric containing metal precursors for alloys of copper-nickel and then thermally processing the fabric to create alloy metallic nanofoams. The nanofoams were tested with nanoindentation. The experimental results suggest that the addition of nickel increases the hardening of the nanofoams. The multiscale simulation modeling results agreed qualitatively with the experiments by suggesting that the addition of the alloying can be beneficial to the hardening behavior of the metallic nanofoams, and helps to isolate the effects of alloying from morphological changes in the foam. This behavior was related to the addition of solute atoms that prevent the free dislocation movement and increase the strength of the structure.

scholarly journals C-type cytochrome-initiated reduction of bacterial lytic polysaccharide monooxygenases.

2021 ◽  
Author(s):  
Jessie Branch ◽  
Badri S Rajagopal ◽  
Alessandro Paradisi ◽  
Nick Yates ◽  
Peter J Lindley ◽  
...  
Keyword(s):  
Cellulose Degradation ◽  
H2o2 Production ◽  
Electron Sources ◽  
Lytic Polysaccharide Monooxygenases ◽  
Energy Needs ◽  
Polysaccharide Monooxygenases ◽  
Cellvibrio Japonicus ◽  
New Research ◽  
The Impact ◽  
Insight Into

The release of glucose from lignocellulosic waste for subsequent fermentation into biofuels holds promise for securing humankind's future energy needs. The discovery of a set of copper dependent enzymes known as lytic polysaccharide monooxygenases (LPMOs) has galvanized new research in this area. LPMOs act by oxidatively introducing chain breaks into cellulose and other polysaccharides, boosting the ability of cellulases to act on the substrate. Although several proteins have been implicated as electron sources in fungal LPMO biochemistry, no equivalent bacterial LPMO electron donors have been previously identified, although the proteins Cbp2D and E from Cellvibrio japonicus have been implicated as potential candidates. Here we analyze a small c-type cytochrome (CjX183) present in Cellvibrio japonicus Cbp2D, and show that it can initiate bacterial CuII/I LPMO reduction and also activate LPMO-catalyzed cellulose-degradation. In the absence of cellulose, CjX183-driven reduction of the LPMO results in less H2O2 production from O2, and correspondingly less oxidative damage to the enzyme than when ascorbate is used as the reducing agent. Significantly, using CjX183 as the activator maintained similar cellulase boosting levels relative to the use of an equivalent amount of ascorbate. Our results therefore add further evidence to the impact that the choice of electron source can have on LPMO action. Furthermore, the study of Cbp2D and other similar proteins may yet reveal new insight into the redox processes governing polysaccharide degradation in bacteria.

scholarly journals Colorimetric LPMO assay with direct implication for cellulolytic activity

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Søren Brander ◽  
Stine Lausten ◽  
Johan Ø. Ipsen ◽  
Kristoffer B. Falkenberg ◽  
Andreas B. Bertelsen ◽  
...  
Keyword(s):  
High Throughput ◽  
High Throughput Screening ◽  
Biochemical Characterization ◽  
Cellulose Degradation ◽  
Microtiter Plate ◽  
Enzyme Characterization ◽  
Cellulolytic Activity ◽  
Polysaccharide Monooxygenases ◽  
High Throughput Assay

Abstract Background Lytic polysaccharide monooxygenases (LPMOs) are important industrial enzymes known for their catalytic degradation of recalcitrant polymers such as cellulose or chitin. Their activity can be measured by lengthy HPLC methods, while high-throughput methods are less specific. A fast and specific LPMO assay would simplify screening for new or engineered LPMOs and accelerate biochemical characterization. Results A novel LPMO activity assay was developed based on the production of the dye phenolphthalein (PHP) from its reduced counterpart (rPHP). The colour response of rPHP oxidisation catalysed by the cellulose-specific LPMO from Thermoascus aurantiacus (TaAA9A), was found to increase tenfold by adding dehydroascorbate (DHA) as a co-substrate. The assay using a combination of rPHP and DHA was tested on 12 different metallo-enzymes, but only the LPMOs catalysed this reaction. The assay was optimized for characterization of TaAA9A and showed a sensitivity of 15 nM after 30 min incubation. It followed apparent Michaelis–Menten kinetics with kcat = 0.09 s−1 and KM = 244 µM, and the assay was used to confirm stoichiometric copper–enzyme binding and enzyme unfolding at a temperature of approximately 60 °C. DHA, glutathione and fructose were found to enhance LPMO oxidation of rPHP and in the optimized assay conditions these co-substrates also enabled cellulose degradation. Conclusions This novel and specific LPMO assay can be carried out in a convenient microtiter plate format ready for high-throughput screening and enzyme characterization. DHA was the best co-substrate tested for oxidation of rPHP and this preference appears to be LPMO-specific. The identified co-substrates DHA and fructose are not normally considered as LPMO co-substrates but here they are shown to facilitate both oxidation of rPHP and degradation of cellulose. This is a rare example of a finding from a high-throughput assay that directly translate into enzyme activity on an insoluble substrate. The rPHP-based assay thus expands our understanding of LPMO catalysed reactions and has the potential to characterize LPMO activity in industrial settings, where usual co-substrates such as ascorbate and oxygen are depleted.

scholarly journals Quantifying oxidation of cellulose-associated glucuronoxylan by two lytic polysaccharide monooxygenases from Neurospora crassa

2021 ◽  
Author(s):  
Olav A. Hegnar ◽  
Heidi Østby ◽  
Dejan M. Petrović ◽  
Lisbeth Olsson ◽  
Anikó Várnai ◽  
...  
Keyword(s):  
Neurospora Crassa ◽  
Plant Cell Wall ◽  
Plant Biomass ◽  
Hydrolytic Enzymes ◽  
Structural Features ◽  
Cell Wall Polysaccharides ◽  
Functional Variation ◽  
Lytic Polysaccharide Monooxygenases ◽  
Polysaccharide Monooxygenases ◽  
Oxidized Products

Family AA9 lytic polysaccharide monooxygenases (LPMOs) are abundant in fungi where they catalyze oxidative depolymerization of recalcitrant plant biomass. These AA9 LPMOs cleave cellulose, and some also act on hemicelluloses, primarily other (substituted) β-(1→4)-glucans. Oxidative cleavage of xylan has been shown for only a handful AA9 LPMOs, and it remains unclear whether this activity is a minor side reaction or primary function. Here, we show that Nc LPMO9F and the phylogenetically related, hitherto uncharacterized Nc LPMO9L from Neurospora crassa are active on both cellulose and cellulose-associated glucuronoxylan, but not on glucuronoxylan alone. A newly developed method for simultaneous quantification of xylan-derived and cellulose-derived oxidized products showed that Nc LPMO9F preferentially cleaves xylan when acting on a cellulose–beechwood glucuronoxylan mixture, yielding about three times more xylan-derived than cellulose-derived oxidized products. Interestingly, under similar conditions, Nc LPMO9L and previously characterized Mc LPMO9H from Malbranchea cinnamomea showed different xylan-to-cellulose preferences, giving oxidized product ratios of about 0.5:1 and 1:1, respectively, indicative of functional variation among xylan-active LPMOs. Phylogenetic and structural analysis of xylan-active AA9 LPMOs led to the identification of characteristic structural features, including unique features that do not occur in phylogenetically remote AA9 LPMOs, such as four AA9 LPMOs whose lack of activity towards glucuronoxylan was demonstrated in the present study. Taken together, the results provide a path towards discovery of additional xylan-active LPMOs and show that the huge family of AA9 LPMOs has members that preferentially act on xylan. These findings shed new light on the biological role and industrial potential of these fascinating enzymes. Importance Plant cell wall polysaccharides are highly resilient to depolymerization by hydrolytic enzymes, partly due to cellulose chains being tightly packed in microfibrils that are covered by hemicelluloses. Lytic polysaccharide monooxygenases (LPMOs) seem well suited to attack these resilient co-polymeric structures, but the occurrence and importance of hemicellulolytic activity among LPMOs remains unclear. Here we show that certain AA9 LPMOs preferentially cleave xylan when acting on a cellulose–glucuronoxylan mixture, and that this ability is the result of protein evolution that has resulted in a clade of AA9 LPMOs with specific structural features. Our findings strengthen the notion that the vast arsenal of AA9 LPMOs in certain fungal species provides functional versatility, and that AA9 LPMOs may have evolved to promote oxidative depolymerization of a wide variety of recalcitrant, co-polymeric plant polysaccharide structures. These findings have implications for understanding the biological roles and industrial potential of LPMOs.

In Situ microscopy of the anodic etching of silicon

1995 ◽  
Vol 53 ◽  
pp. 232-233
Author(s):  
Frances M. Ross ◽  
Peter C. Searson
Keyword(s):  
Composite Structures ◽  
High Surface ◽  
High Surface Area ◽  
Chemical Properties ◽  
Selective Etching ◽  
Silicon On Insulator ◽  
Second Phase ◽  
Porous Layers ◽  
New Class ◽  
Porous Semiconductors

Porous semiconductors represent a relatively new class of materials formed by the selective etching of a single or polycrystalline substrate. Although porous silicon has received considerable attention due to its novel optical properties1, porous layers can be formed in other semiconductors such as GaAs and GaP. These materials are characterised by very high surface area and by electrical, optical and chemical properties that may differ considerably from bulk. The properties depend on the pore morphology, which can be controlled by adjusting the processing conditions and the dopant concentration. A number of novel structures can be fabricated using selective etching. For example, self-supporting membranes can be made by growing pores through a wafer, films with modulated pore structure can be fabricated by varying the applied potential during growth, composite structures can be prepared by depositing a second phase into the pores and silicon-on-insulator structures can be formed by oxidising a buried porous layer. In all these applications the ability to grow nanostructures controllably is critical.

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