Surface plasmon resonance imaging of the enzymatic degradation of cellulose microfibrils (original) (raw)
Related papers
Visualization of enzymatic hydrolysis of cellulose using AFM phase imaging
Enzyme and Microbial Technology, 2009
Complete cellulase, an endoglucanase (EGV) with cellulose-binding domain (CBD) and a mutant endoglucanase without CBD (EGI) were utilized for the hydrolysis of a fully bleached reed Kraft pulp sample. The changes of microfibrils on the fiber surface were examined with tapping mode atomic force microscopy (TM-AFM) phase imaging. The results indicated that complete cellulase could either peel the fibrillar bundles along the microfibrils (peeling) or cut microfibrils into short length across the length direction (cutting) during the process. After 24 h treatment, most orientated microfibrils on the cellulose fiber surface were degraded into fragments by the complete cellulase. Incubation with endoglucanase (EGV or EGI) also caused peeling action. But no significant size reduction of microfibrils length was observed, which was probably due to the absence of cellobiohydrolase. The AFM phase imaging clearly revealed that individual EGV particles were adsorbed onto the surface of a cellulose fiber and may be bound to several microfibrils.
FEBS Journal, 2014
Highly heterogeneous and usually weakly defined substrate morphologies complicate the study of enzymatic cellulose hydrolysis. The cellulose surface has a non-uniform shape in particular, with consequent impacts on cellulase adsorption and activity. We have therefore prepared a cellulosic model substrate which is shown by atomic force microscopy (AFM) to display a completely smooth surface, the residual squared mean roughness being 10 nm or lower, and applied it for kinetic analysis of cellulase action. The substrate consists of an amorphous cellulose matrix into which variably sized crystalline fibers are distributed in apparently irregular fashion. Its conversion into soluble sugars by Trichoderma sp. cellulase at 50°C proceeded without apparent limitation up to 70% completion and was paralleled by a steady increase in cellulase adsorption to the cellulose. Individual cellulase components (CBH I, CBH II, EG) also showed strongly enhanced adsorption with progressing cellulose conversion, irrespective of their preference for degrading the amorphous or crystalline substrate parts as revealed by AFM. The specific activity of the adsorbed cellulases, however, decreased concomitantly. Cellulose surface morphologies evolving as a consequence of cellulase action were visualized by AFM. Three-dimensional surface degradation by the cellulases resulted in a large increase in cellulose surface area for enzyme adsorption. However, the decline in enzyme specific activity during conversion was caused by factors other than surface ablation and disruption. Based on kinetic evidence for enzymatic hydrolyses of the smooth-surface model substrate and microcrystalline cellulose (Avicel), we hypothesize that, due to gradual loss of productive dynamics in their interactions with the cellulose surface, individual cellulases get progressively confined to substrate parts where they are no longer optimally active. This eventually leads to an overall slow-down of hydrolysis.
Cellulase Activity on Thin Films of Cellulose by Qcm and Spr
2005
We monitored the enzymatic hydrolysis on thin films of cellulose, in situ and real time by using a piezoelectric sensing device (Quartz Crystal Microbalance, QCM) and Surface Plasmon Resonance (SPR). Cellulose thin films were deposited on piezoelectric resonators using spin coating technique. Films of different crystallinity were also prepared by using self assembly of cellulose-thiol derivatives on gold substrates. By using QCM and SPR we elucidated the role of hydration water in view of the dynamics of enzyme binding and substrate degradation. We focused on the activity of isolated endoglucanases as compared to multi-component commercial enzyme mix. The activity of each kind of cellulase was determined under different conditions of temperature, pH and enzyme dose.
Surface Plasmon Resonance Studies of Polysaccharide Self-Assembly on Cellulose
Wood is a multiphase material consisting of cellulose crystals embedded within a non-crystalline hetereopolysaccharide (hemicellulose) and lignin rich phase. The hierarchial arrangement of these three chief components in wood produces excellent properties like resistance to fracture and toughness. Through the study of polysaccharide self-assembly onto a model cellulose surface, further insight into the interactions between hemicelluloses and cellulose can be gained. In our study, we synthesized pullulan cinnamates with different degrees of substitution of cinnamoyl groups as a model for a hemicellulose with lignin-like moieties. Surface plasmon resonance measurements probe the self-assembly behavior of pullulan and pullulan cinnamate onto a cellulose coated gold surface. Our results suggest that pullulan does not adsorb onto the model cellulose surface, whereas pullulan cinnamate does. These preliminary results signify the important role that lignin-like substituents play on hemicel...
Langmuir, 2008
Model films of native cellulose nanofibrils, which contain both crystalline cellulose I and amorphous domains, were used to investigate the dynamics and activities of cellulase enzymes. The enzyme binding and degradation of nanofibril films were compared with those for other films of cellulose, namely, Langmuir-Schaefer and spin-coated regenerated cellulose, as well as cellulose nanocrystal cast films. Quartz crystal microbalance with dissipation (QCM-D) was used to monitor the changes in frequency and energy dissipation during incubation at varying enzyme concentrations and experimental temperatures. Structural and morphological changes of the cellulose films upon incubation with enzymes were evaluated by using atomic force microscopy. The QCM-D results revealed that the rate of enzymatic degradation of the nanofibril films was much faster compared to the other types of cellulosic films. Higher enzyme loads did not dramatically increase the already fast degradation rate. Real-time measurements of the coupled contributions of enzyme binding and hydrolytic reactions were fitted to an empirical model that closely described the cellulase activities. The hydrolytic potential of the cellulase mixture was found to be considerably affected by the nature of the substrates, especially their crystallinity and morphology. The implications of these observations are discussed in this report.
The Journal of biological chemistry, 2014
Lytic polysaccharide monooxygenase (LPMO) represents a unique principle of oxidative degradation of recalcitrant insoluble polysaccharides. Used in combination with hydrolytic enzymes, LPMO appears to constitute a significant factor of the efficiency of enzymatic biomass depolymerization. LPMO activity on different cellulose substrates has been shown from the slow release of oxidized oligosaccharides into solution, but an immediate and direct demonstration of the enzyme action on the cellulose surface is lacking. Specificity of LPMO for degrading ordered crystalline and unordered amorphous cellulose material of the substrate surface is also unknown. We show by fluorescence dye adsorption analyzed with confocal laser scanning microscopy that a LPMO (from Neurospora crassa) introduces carboxyl groups primarily in surface-exposed crystalline areas of the cellulosic substrate. Using time-resolved in situ atomic force microscopy we further demonstrate that cellulose nano-fibrils exposed ...
Surface modification of cellulose fibers: towards wood composites by biomimetics
Comptes Rendus Biologies, 2004
A biomimetic approach was taken for studying the adsorption of a model copolymer (pullulan abietate, DS 0.027), representing the lignin-carbohydrate complex, to a model surface for cellulose fibers (Langmuir-Blodgett thin films of regenerated cellulose). Adsorption results were assayed using surface plasmon resonance spectroscopy (SPR) and atomic force microscopy (AFM). Rapid, spontaneous, and desorption-resistant surface modification resulted. This effort is viewed as a critical first step towards the permanent surface modification of cellulose fibers with a layer of molecules amenable to either enzymatic crosslinking for improved wood composites or thermoplastic consolidation. To cite this article: S.E. Gradwell et al., C. R. Biologies 327 (2004). Résumé Modification de la surface des fibres cellulosiques : vers la conception de composites du bois par la biomimétique. Une approche biomimétique a été utilisée pour étudier l'adsorption d'un copolymère modèle, représentant le complexe lignine-carbohydrate (pullulan abietate, DS 0.027) sur une surface cellulosique modèle (films de cellulose régénérée de type Langmuir-Blodgett). Le phénomène d'adsorption a été analysé par résonance de plasmon de surface (RPS) et microscopie de force atomique (AFM). Une modification rapide de la surface, spontanée et résistante à la désorption, a été obtenue. Ces ✩ This article is dedicated to Prof. Bernard Monties on the occasion of his retirement.
Enzymatic Treatment as a Pre-Step to Remove Cellulose Films from Sensors
Macromolecular Symposia, 2011
In this work an enzymatic treatment was proposed as a pre-step to remove cellulose film from surface sensors in a cleaning protocol. Quartz crystal gold sensors, coated with cellulose ultrathin films were used in polymer adsorption studies. They had to be cleaned with one of the two treatments with hot acid or ammoniac solutions available for their reuse. Both treatments showed advantages and some disadvantages for cellulose film removal. A mix of cellulase from Aspergillus species, supplied by Sigma, is proposed here as a pre-treatment to improve the cellulose film releasing from the quartz crystal surfaces. Two concentrations of salt solutions are considered in the enzymatic activity, 1 and 10 mM [NaCl], for each fixed conditions of enzyme solution, temperature and pH. It is found that after 80 min, the contact angle for both salt concentrations reaches the equilibrium. The ammoniac solution after the enzymatic treatment showed to be a very practical and safe way to remove the discharged cellulose model films on the gold sensor. This suggested protocol is very reliable and it is a low time demanding step for the researcher. The average contact angle after the integration of the enzymatic and ammoniac treatments was found to be enough to reuse the sensors, between 6.4° and 7.1°.
Layer-By-Layer Assembly of Enzymes and Nanoparticles onto Cellulose Support
Journal of Biosensors & Bioelectronics, 2018
Multilayered films of cellulose nanoparticles (NFC's) and modified multi-walled carbon nanotubes (MWCNT's) were assembled by means of alternate electrostatic adsorption with positively charged poly(ethyleneimine) (PEI) onto cellulose support. The free carboxylic groups of NFC's and MWCNT's were coupled with ethylenediamine. Glucose oxidase and laccase were immobilized by means of Schiff base reaction between aldehyde groups of glutaraldehyde and the free amino sites of the proteins. The immobilized enzymes on the surface of nanoparticles have higher value for the specific activity compared to the enzymes immobilized directly on the cellulose surface indicating the stabilization of the proteins by the nanoparticles. The kinetics of the enzymes, catalyzed reactions and reusability of the enzymes were investigated and were showing better properties for enzymes cross-linked with glutaraldehyde. After 5 days the initial enzyme activity of glucose oxidase was around 85%, but the initial enzyme activity of laccase was 60%. The kinetic investigations of the immobilized enzymes showed no significant difference in Michaelis constant but the maximum reaction rate is decreased.
Enzymatic hydrolysis of cellulose: visual characterization of the process
… Academy of Sciences of the United …, 1981
Cellulose from the Gram-neative bacterium Acetobacter xylinum has been used as a model substrate for visualizing the action of cellulase enzymes from the fungus Trichoderma reesei. High-resolution electron microscopy reveals that A. xylinum normally produces a ribbon of cellulose that is a composite of bundles of crystalline microfibrils. Visual patterns of the process of cellulose degradation have been established. Enzymes are initially observed bound to the cellulose ribbon. Within 10 min, the ribbon is split along its long axis into bundles of microfibrils which are subsequently thinned until they are completely dissolved within 30 min. Incubations with purified components of the cellulase enzyme system produced less dramatic changes in ribbon structure. Purified 1,4-(-D-glucan cellobiohydrolase I (D) (EC 3.2.1.91) produced no visible change in cellulose structure. Purified endo-1,4-l-D-glucanase IV (EC 3.2.1.4) produced some splaying of ribbons into microfibril bundles. In both cases, whole ribbons were present even after 60 min of incubation, visually confirming the synergistic mode of action of these enzymes.