scholarly journals The use of fluorescent protein-tagged carbohydrate-binding modules to evaluate the influence of drying on cellulose accessibility and enzymatic hydrolysis

RSC Advances ◽  
2020 ◽  
Vol 10 (45) ◽  
pp. 27152-27160
Author(s):  
Drake Mboowa ◽  
Vinay Khatri ◽  
Jack N. Saddler
Keyword(s):  
Enzymatic Hydrolysis ◽  
Fluorescent Protein ◽  
Carbohydrate Binding ◽  
Fiber Structure ◽  
Micro Scale ◽  
Cellulose Accessibility ◽  
Carbohydrate Binding Modules

Drying restricts cellulose accessibility at macro- and micro-scale of fiber structure with restriction at macro-fiber significantly limiting accessibility to micro-fibrils.

scholarly journals Functionalization of Cellulose-Based Hydrogels with Bi-Functional Fusion Proteins Containing Carbohydrate-Binding Modules

Materials ◽  
2021 ◽  
Vol 14 (12) ◽  
pp. 3175
Author(s):  
Mariana Barbosa ◽  
Hélvio Simões ◽  
Duarte Miguel F. Prazeres
Keyword(s):  
Fusion Proteins ◽  
Fluorescent Protein ◽  
Protein A ◽  
Chemical Properties ◽  
Main Idea ◽  
Bioactive Molecules ◽  
Scaffolding Protein ◽  
Carbohydrate Binding ◽  
Biological Interactions ◽  
Carbohydrate Binding Modules

Materials with novel and enhanced functionalities can be obtained by modifying cellulose with a range of biomolecules. This functionalization can deliver tailored cellulose-based materials with enhanced physical and chemical properties and control of biological interactions that match specific applications. One of the foundations for the success of such biomaterials is to efficiently control the capacity to combine relevant biomolecules into cellulose materials in such a way that the desired functionality is attained. In this context, our main goal was to develop bi-functional biomolecular constructs for the precise modification of cellulose hydrogels with bioactive molecules of interest. The main idea was to use biomolecular engineering techniques to generate and purify different recombinant fusions of carbohydrate binding modules (CBMs) with significant biological entities. Specifically, CBM-based fusions were designed to enable the bridging of proteins or oligonucleotides with cellulose hydrogels. The work focused on constructs that combine a family 3 CBM derived from the cellulosomal-scaffolding protein A from Clostridium thermocellum (CBM3) with the following: (i) an N-terminal green fluorescent protein (GFP) domain (GFP-CBM3); (ii) a double Z domain that recognizes IgG antibodies; and (iii) a C-terminal cysteine (CBM3C). The ability of the CBM fusions to bind and/or anchor their counterparts onto the surface of cellulose hydrogels was evaluated with pull-down assays. Capture of GFP-CBM3 by cellulose was first demonstrated qualitatively by fluorescence microscopy. The binding of the fusion proteins, the capture of antibodies (by ZZ-CBM3), and the grafting of an oligonucleotide (to CBM3C) were successfully demonstrated. The bioactive cellulose platform described here enables the precise anchoring of different biomolecules onto cellulose hydrogels and could contribute significatively to the development of advanced medical diagnostic sensors or specialized biomaterials, among others.

scholarly journals Ubiquitous Carbohydrate Binding Modules Decorate 936 Lactococcal Siphophage Virions

Viruses ◽  
2019 ◽  
Vol 11 (7) ◽  
pp. 631 ◽  
Author(s):  
Stephen Hayes ◽  
Jennifer Mahony ◽  
Renaud Vincentelli ◽  
Laurie Ramond ◽  
Arjen Nauta ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Bioinformatic Analysis ◽  
Infection Process ◽  
Ecological Niches ◽  
Carbohydrate Binding ◽  
Binding Assays ◽  
Protein Fusion ◽  
Carbohydrate Binding Modules ◽  
Structural Assembly ◽  
Green Fluorescent

With the availability of an increasing number of 3D structures of bacteriophage components, combined with powerful in silico predictive tools, it has become possible to decipher the structural assembly and functionality of phage adhesion devices. In the current study, we examined 113 members of the 936 group of lactococcal siphophages, and identified a number of Carbohydrate Binding Modules (CBMs) in the neck passage structure and major tail protein, on top of evolved Dit proteins, as recently reported by us. The binding ability of such CBM-containing proteins was assessed through the construction of green fluorescent protein fusion proteins and subsequent binding assays. Two CBMs, one from the phage tail and another from the neck, demonstrated definite binding to their phage-specific host. Bioinformatic analysis of the structural proteins of 936 phages reveals that they incorporate binding modules which exhibit structural homology to those found in other lactococcal phage groups and beyond, indicating that phages utilize common structural “bricks” to enhance host binding capabilities. The omnipresence of CBMs in Siphophages supports their beneficial role in the infection process, as they can be combined in various ways to form appendages with different shapes and functionalities, ensuring their success in host detection in their respective ecological niches.

scholarly journals Quantifying cellulose accessibility during enzyme-mediated deconstruction using 2 fluorescence-tagged carbohydrate-binding modules

2019 ◽  
Vol 116 (45) ◽  
pp. 22545-22551 ◽  
Author(s):  
Vera Novy ◽  
Kevin Aïssa ◽  
Fredrik Nielsen ◽  
Suzana K. Straus ◽  
Peter Ciesielski ◽  
...  
Keyword(s):  
Laser Scanning ◽  
Degradation Mechanism ◽  
Length Distribution ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Crystalline Cellulose ◽  
Carbohydrate Binding ◽  
Distribution Analysis ◽  
Cellulose Accessibility ◽  
Carbohydrate Binding Modules

Two fluorescence-tagged carbohydrate-binding modules (CBMs), which specifically bind to crystalline (CBM2a-RRedX) and paracrystalline (CBM17-FITC) cellulose, were used to differentiate the supramolecular cellulose structures in bleached softwood Kraft fibers during enzyme-mediated hydrolysis. Differences in CBM adsorption were elucidated using confocal laser scanning microscopy (CLSM), and the structural changes occurring during enzyme-mediated deconstruction were quantified via the relative fluorescence intensities of the respective probes. It was apparent that a high degree of order (i.e., crystalline cellulose) occurred at the cellulose fiber surface, which was interspersed by zones of lower structural organization and increased cellulose accessibility. Quantitative image analysis, supported by 13C NMR, scanning electron microscopy (SEM) imaging, and fiber length distribution analysis, showed that enzymatic degradation predominates at these zones during the initial phase of the reaction, resulting in rapid fiber fragmentation and an increase in cellulose surface crystallinity. By applying this method to elucidate the differences in the enzyme-mediated deconstruction mechanisms, this work further demonstrated that drying decreased the accessibility of enzymes to these disorganized zones, resulting in a delayed onset of degradation and fragmentation. The use of fluorescence-tagged CBMs with specific recognition sites provided a quantitative way to elucidate supramolecular substructures of cellulose and their impact on enzyme accessibility. By designing a quantitative method to analyze the cellulose ultrastructure and accessibility, this study gives insights into the degradation mechanism of cellulosic substrates.

New insights into the enzymatic hydrolysis of lignocellulosic polymers by using fluorescent tagged carbohydrate-binding modules

2018 ◽  
Vol 2 (2) ◽  
pp. 479-491 ◽  
Author(s):  
Vinay Khatri ◽  
Fatma Meddeb-Mouelhi ◽  
Marc Beauregard
Keyword(s):  
Cost Effectiveness ◽  
Enzymatic Hydrolysis ◽  
Rapid Method ◽  
Fluorescent Probes ◽  
Carbohydrate Binding ◽  
Wood Fibers ◽  
Carbohydrate Binding Modules ◽  
Improve Cost Effectiveness ◽  
Hydrolysis Of ◽  
Improve Cost

Modification of lignocellulosic polymers at the surface of wood fibers can easily be monitored using fluorescent probes (FTCM method). By applying FTCM, a simple and rapid method, biomass industries could substantially improve cost-effectiveness of production of biofuels and other lignocellulosic biomass-based products.

scholarly journals Ubiquitous Carbohydrate Binding Modules Decorate 936 Lactococcal Siphophage Virions

2019 ◽  
Author(s):  
Stephen Hayes ◽  
Jennifer Mahony ◽  
Renaud Vincentelli ◽  
Laurie Ramond ◽  
Arjen Nauta ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Bioinformatic Analysis ◽  
Infection Process ◽  
Ecological Niches ◽  
Carbohydrate Binding ◽  
Binding Assays ◽  
Protein Fusion ◽  
Carbohydrate Binding Modules ◽  
Structural Assembly ◽  
Green Fluorescent

Abstract: With the availability of an increasing number of 3D structures of bacteriophage components, combined with powerful in silico predictive tools, it has become possible to decipher the structural assembly and functionality of phage adhesion devices. In the current study, we examined 113 members of the 936 group of lactococcal siphophages, and identified a number of Carbohydrate Binding Modules (CBMs) in the neck passage structure and major tail protein, on top of evolved Dit proteins as recently reported by us. The binding ability of such CBM-containing proteins was assessed through the construction of green fluorescent protein fusion proteins and subsequent binding assays. Two CBMs, one from the phage tail and another from the neck, demonstrated definite binding to their phage-specific host. Bioinformatic analysis of the structural proteins of 936 phages reveals that they incorporate binding modules which exhibit structural homology to those found in other lactococcal phage groups and beyond, indicating that phages utilize common structural “bricks” to enhance host binding capabilities. The omnipresence of CBMs in Siphophages supports their beneficial role in the infection process, as they can be combined in various ways to form appendages with different shapes and functionalities, ensuring their success in host detection in their respective ecological niches.

Carbohydrate binding modules enhance cellulose enzymatic hydrolysis by increasing access of cellulases to the substrate

2019 ◽  
Vol 211 ◽  
pp. 57-68 ◽  
Author(s):  
A. Bernardes ◽  
V.O.A. Pellegrini ◽  
F. Curtolo ◽  
C.M. Camilo ◽  
B.L. Mello ◽  
...  
Keyword(s):  
Enzymatic Hydrolysis ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Modules

scholarly journals The most abundant cyst wall proteins ofAcanthamoeba castellaniiare cellulose-binding lectins from three gene families that localize to distinct structures in cyst walls

2018 ◽  
Author(s):  
Pamela Magistrado-Coxen ◽  
Yousuf Aqeel ◽  
Angelo Lopez ◽  
John R. Haserick ◽  
Breeanna R. Urbanowicz ◽  
...  
Keyword(s):  
Cyst Wall ◽  
Fluorescent Protein ◽  
Acanthamoeba Castellanii ◽  
Gene Families ◽  
Carbohydrate Binding ◽  
Structured Illumination ◽  
Structured Illumination Microscopy ◽  
Carbohydrate Binding Modules ◽  
Green Fluorescent ◽  
Cellulose Binding

AbstractAcanthamoeba castellanii, cause of keratitis and blindness, is an emerging pathogen because of its association with contact lens use. The cyst wall contributes to pathogenesis as cysts are resistant to sterilizing reagents in lens solutions and to antibiotics applied to the eye. Here we used structured illumination microscopy (SIM) and probes for glycopolymers to show that purified cyst walls ofA. castellaniiretain endocyst and ectocyst layers and conical structures (ostioles) that connect them. Mass spectrometry showed candidate cyst wall proteins (CWPs) are dominated by three families of lectins (named here Luke, Leo, and Jonah), because each binds to microcrystalline cellulose +/- chitin. Luke lectins contain two or three carbohydrate-binding modules (CBM49), which were first identified in a tomato cellulase. Leo lectins have two unique domains with eight Cys residues each (8-Cys) +/- a Thr-, Lys-, and His-rich spacer. Jonah lectins contain one or three choice-of-anchor A (CAA) domains previously of unknown function. Representative members of each family were tagged with green fluorescent protein (GFP) and expressed under their own promoters in transfected parasites. A representative Jonah lectin with one CAA domain is made early during encystation and localizes to the ectocyst layer. In contrast, Leo and Luke lectins are made later and localize to the endocyst layer and ostioles. Probes for CWPs (anti-GFP antibodies) and for glycopolymers (maltose-binding protein-fusions with CWPs) suggest Jonah lectin and the glycopolymers to which it binds are accessible in the ectocyst layer, while Luke and Leo lectins and their epitopes are mostly inaccessible in the ectocyst layer and ostioles. In summary, the most abundantA. castellaniiCWPs are three sets of lectins, which have conserved (CBM49s of Luke), newly characterized (CAA of Jonah), or unique carbohydrate-binding modules (8-Cys of Jonah).Author summaryFifty years ago, the cyst wall ofAcanthamoeba castellaniiwas shown to contain cellulose and have an ectocyst layer, an endocyst layer, and conical ostioles that attach them. The goals here were to identify abundant cyst wall proteins (CWPs) and begin to determine how the wall is assembled. We used wheat germ agglutinin to show cyst walls also contain chitin fibrils. When trophozoites are starved of nutrients, they become immotile and make CWPs and glycopolymers in dozens of small vesicles. The primordial cyst wall is composed of a single, thin layer containing cellulose, chitin, and an abundant CWP we called Jonah. The primordial wall also has small, flat ostioles that contain another abundant CWP we called Luke. Jonah (the best candidate for diagnostic antibodies) is accessible in the ectocyst layer of mature cyst walls, while Luke and a third abundant CWP we termed Leo are present but mostly inaccessible in the endocyst layer and ostioles. WhileA. castellaniicyst walls contain cellulose (like plants) and chitin (like fungi), the glycopolymers are made in vesicles rather than at the plasma membrane, and the CWPs (Luke, Leo, and Jonah lectins) are unique to the protist.

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