scholarly journals Characterization of Thermobifida fusca Cutinase-Carbohydrate-Binding Module Fusion Proteins and Their Potential Application in Bioscouring

2010 ◽  
Vol 76 (20) ◽  
pp. 6870-6876 ◽  
Author(s):  
Yao Zhang ◽  
Sheng Chen ◽  
Meng Xu ◽  
Artur Cavoco-Paulo ◽  
Jing Wu ◽  
...  
Keyword(s):  
Cotton Fiber ◽  
Potential Application ◽  
Fusion Proteins ◽  
Textile Industry ◽  
Enzyme Characterization ◽  
Binding Activity ◽  
Thermobifida Fusca ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Modules ◽  
Fusion Enzymes

ABSTRACT Cutinase from Thermobifida fusca is thermally stable and has potential application in the bioscouring of cotton in the textile industry. In the present study, the carbohydrate-binding modules (CBMs) from T. fusca cellulase Cel6A (CBMCel6A) and Cellulomonas fimi cellulase CenA (CBMCenA) were fused, separately, to the carboxyl terminus of T. fusca cutinase. Both fusion enzymes, cutinase-CBMCel6A and cutinase-CBMCenA, were expressed in Escherichia coli and purified to homogeneity. Enzyme characterization showed that both displayed similar catalytic properties and pH stabilities in response to T. fusca cutinase. In addition, both fusion proteins displayed an activity half-life of 53 h at their optimal temperature of 50�C. Compared to T. fusca cutinase, in the absence of pectinase, the binding activity on cotton fiber was enhanced by 2% for cutinase-CBMCel6A and by 28% for cutinase-CBMCenA, whereas in the presence of pectinase, the binding activity was enhanced by 40% for the former and 45% for the latter. Notably, a dramatic increase of up to 3-fold was observed in the amount of released fatty acids from cotton fiber by both cutinase-CBM fusion proteins when acting in concert with pectinase. This is the first report of improving the scouring efficiency of cutinase by fusing it with CBM. The improvement in activity and the strong synergistic effect between the fusion proteins and pectinase suggest that they may have better applications in textile bioscouring than the native cutinase.

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.

Effects of Thermobifida fusca cutinase-carbohydrate-binding module fusion proteins on cotton bioscouring

2011 ◽  
Vol 16 (4) ◽  
pp. 645-653 ◽  
Author(s):  
Yao Zhang ◽  
Sheng Chen ◽  
Miao He ◽  
Jing Wu ◽  
Jian Chen ◽  
...  
Keyword(s):  
Fusion Proteins ◽  
Thermobifida Fusca ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module

scholarly journals Minimalistic Cellulosome of the Butanologenic Bacterium Clostridium saccharoperbutylacetonicum

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Bosmat Levi Hevroni ◽  
Sarah Moraïs ◽  
Yonit Ben-David ◽  
Ely Morag ◽  
Edward A. Bayer
Keyword(s):  
Building Blocks ◽  
Binding Activity ◽  
Cellulosic Biomass ◽  
Enzyme Linked Immunosorbent Assay ◽  
Carbohydrate Binding ◽  
Binding Properties ◽  
Content Type ◽  
Affinity Electrophoresis ◽  
Carbohydrate Binding Modules

ABSTRACT Clostridium saccharoperbutylacetonicum is a mesophilic, anaerobic, butanol-producing bacterium, originally isolated from soil. It was recently reported that C. saccharoperbutylacetonicum possesses multiple cellulosomal elements and would potentially form the smallest cellulosome known in nature. Its genome contains only eight dockerin-bearing enzymes, and its unique scaffoldin bears two cohesins (Cohs), three X2 modules, and two carbohydrate-binding modules (CBMs). In this study, all of the cellulosome-related modules were cloned, expressed, and purified. The recombinant cohesins, dockerins, and CBMs were tested for binding activity using enzyme-linked immunosorbent assay (ELISA)-based techniques. All the enzymes were tested for their comparative enzymatic activity on seven different cellulosic and hemicellulosic substrates, thus revealing four cellulases, a xylanase, a mannanase, a xyloglucanase, and a lichenase. All dockerin-containing enzymes interacted similarly with the second cohesin (Coh2) module, whereas Coh1 was more restricted in its interaction pattern. In addition, the polysaccharide-binding properties of the CBMs within the scaffoldin were examined by two complementary assays, affinity electrophoresis and affinity pulldown. The scaffoldin of C. saccharoperbutylacetonicum exhibited high affinity for cellulosic and hemicellulosic substrates, specifically to microcrystalline cellulose and xyloglucan. Evidence that supports substrate-dependent in vivo secretion of cellulosomes is presented. The results of our analyses contribute to a better understanding of simple cellulosome systems by identifying the key players in this minimalistic system and the binding pattern of its cohesin-dockerin interaction. The knowledge gained by our study will assist further exploration of similar minimalistic cellulosomes and will contribute to the significance of specific sets of defined cellulosomal enzymes in the degradation of cellulosic biomass. IMPORTANCE Cellulosome-producing bacteria are considered among the most important bacteria in both mesophilic and thermophilic environments, owing to their capacity to deconstruct recalcitrant plant-derived polysaccharides (and notably cellulose) into soluble saccharides for subsequent processing. In many ecosystems, the cellulosome-producing bacteria are particularly effective “first responders.” The massive amounts of sugars produced are potentially amenable in industrial settings to further fermentation by appropriate microbes to biofuels, notably ethanol and butanol. Among the solvent-producing bacteria, Clostridium saccharoperbutylacetonicum has the smallest cellulosome system known thus far. The importance of investigating the building blocks of such a small, multifunctional nanomachine is crucial to understanding the fundamental activities of this efficient enzymatic complex.

scholarly journals Improved lignocellulose degradation efficiency by fusion of β-glucosidase, exoglucanase, and carbohydrate-binding module from Caldicellulosiruptor saccharolyticus

BioResources ◽  
2019 ◽  
Vol 14 (3) ◽  
pp. 6767-6780 ◽  
Author(s):  
Jilin Xia ◽  
Yu Yu ◽  
Huimin Chen ◽  
Jia Zhou ◽  
Zhongbiao Tan ◽  
...  
Keyword(s):  
Rice Straw ◽  
Degradation Efficiency ◽  
Cellulosic Biomass ◽  
Lignocellulose Degradation ◽  
Carbohydrate Binding ◽  
Optimal Temperature ◽  
Caldicellulosiruptor Saccharolyticus ◽  
Carbohydrate Binding Modules ◽  
Fusion Enzymes ◽  
Optimal Ph

Bifunctional cellulases with β-glucosidase (Bgl1), exoglucanase (Exo5), and carbohydrate-binding modules (CBMs) from Caldicellulosiruptor saccharolyticus were fused to yield several recombinant plasmids, Bgl1-CBM-Exo5, Bgl1-2CBM-Exo5, and Bgl1-3CBM-Exo5. The fused enzymes possessed both β-glucosidase and exoglucanase activities and were used to improve the degradation efficiency of lignocellulosic biomass. The optimal temperature of Bgl1-3CBM-Exo5 was 70 °C, which was the same as Bgl1, and the optimal temperature of the other two enzymes was 80 °C, which was the same as Exo5. The optimal pH of fused enzymes was 4 to 5, the same as Exo5, but the optimal pH of Bgl1 was 5.5. Compared with Bgl1-CBM-Exo5 and Bgl1-2CBM-Exo5, the hydrolysis efficiency of Bgl1-3CBM-Exo5 on sodium carboxymethyl cellulose (CMC-Na) was increased by 67% and 50%, respectively. The activities of these enzymes on CMC-Na were increased by 128 to 192% when 10 mM MnCl2 was added. Filter paper, microcrystalline cellulose (MCC), steam-pretreated rice straw, rice straw, and wheat straw were efficiently degraded by these fused enzymes. Specific activities of the fusion enzymes on MCC reached 34.4 to 76.4 U/μmol. The results indicated that bifunctional cellulases fused with CBMs were functional on cellulosic biomass, and CBMs contributed to further deconstruction of MCC and other natural substrates.

Designing Fusion Proteins with Carbohydrate-Binding Modules Having Affinity to Enzymatically Gellable Carboxymethylcellulose Derivative Hydrogel

2014 ◽  
Vol 47 (11) ◽  
pp. 835-840 ◽  
Author(s):  
Tomoaki Ashida ◽  
Yoshihiro Ojima ◽  
Shinji Sakai ◽  
Makiko Sakka ◽  
Kazuo Sakka ◽  
...  
Keyword(s):  
Fusion Proteins ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Modules

Genomic analysis of C-type lectins

2002 ◽  
Vol 69 ◽  
pp. 59-72 ◽  
Author(s):  
Kurt Drickamer ◽  
Andrew J. Fadden
Keyword(s):  
Biological Effects ◽  
Cell Signalling ◽  
Genomic Analysis ◽  
Binding Activity ◽  
Immunoglobulin Superfamily ◽  
Carbohydrate Binding ◽  
Carbohydrate Recognition ◽  
Calcium Dependent ◽  
Binding Domains ◽  
Carbohydrate Recognition Domains

Many biological effects of complex carbohydrates are mediated by lectins that contain discrete carbohydrate-recognition domains. At least seven structurally distinct families of carbohydrate-recognition domains are found in lectins that are involved in intracellular trafficking, cell adhesion, cell–cell signalling, glycoprotein turnover and innate immunity. Genome-wide analysis of potential carbohydrate-binding domains is now possible. Two classes of intracellular lectins involved in glycoprotein trafficking are present in yeast, model invertebrates and vertebrates, and two other classes are present in vertebrates only. At the cell surface, calcium-dependent (C-type) lectins and galectins are found in model invertebrates and vertebrates, but not in yeast; immunoglobulin superfamily (I-type) lectins are only found in vertebrates. The evolutionary appearance of different classes of sugar-binding protein modules parallels a development towards more complex oligosaccharides that provide increased opportunities for specific recognition phenomena. An overall picture of the lectins present in humans can now be proposed. Based on our knowledge of the structures of several of the C-type carbohydrate-recognition domains, it is possible to suggest ligand-binding activity that may be associated with novel C-type lectin-like domains identified in a systematic screen of the human genome. Further analysis of the sequences of proteins containing these domains can be used as a basis for proposing potential biological functions.

scholarly journals Biochemical and Initial Structural Characterization of the Monocot Chimeric Jacalin OsJAC1

2021 ◽  
Vol 22 (11) ◽  
pp. 5639
Author(s):  
Nikolai Huwa ◽  
Oliver H. Weiergräber ◽  
Christian Kirsch ◽  
Ulrich Schaffrath ◽  
Thomas Classen
Keyword(s):  
Physiological Role ◽  
Basic Function ◽  
Binding Activity ◽  
Carbohydrate Binding ◽  
Lectin Domain ◽  
Reducing Conditions ◽  
Transition Points ◽  
The Individual ◽  
High Yields ◽  
Dirigent Domain

The monocot chimeric jacalin OsJAC1 from Oryza sativa consists of a dirigent and a jacalin-related lectin domain. The corresponding gene is expressed in response to different abiotic and biotic stimuli. However, there is a lack of knowledge about the basic function of the individual domains and their contribution to the physiological role of the entire protein. In this study, we have established a heterologous expression in Escherichia coli with high yields for the full-length protein OsJAC1 as well as its individual domains. Our findings showed that the secondary structure of both domains is dominated by β-strand elements. Under reducing conditions, the native protein displayed clearly visible transition points of thermal unfolding at 59 and 85 °C, which could be attributed to the lectin and the dirigent domain, respectively. Our study identified a single carbohydrate-binding site for each domain with different specificities towards mannose and glucose (jacalin domain), and galactose moieties (dirigent domain), respectively. The recognition of different carbohydrates might explain the ability of OsJAC1 to respond to different abiotic and biotic factors. This is the first report of specific carbohydrate-binding activity of a DIR domain, shedding new light on its function in the context of this monocot chimeric jacalin.

scholarly journals Functional diversity of three tandem C-terminal carbohydrate-binding modules of a β-mannanase

2021 ◽  
pp. 100638
Author(s):  
Marie Sofie Møller ◽  
Souad El Bouaballati ◽  
Bernard Henrissat ◽  
Birte Svensson
Keyword(s):  
Functional Diversity ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Modules

scholarly journals Integrated Counts of Carbohydrate-Active Protein Domains as Metabolic Readouts to Distinguish Probiotic Biology and Human Fecal Metagenomes

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Hong-Hsing Liu ◽  
Yu-Chen Lin ◽  
Chen-Shuan Chung ◽  
Kevin Liu ◽  
Ya-Hui Chang ◽  
...  
Keyword(s):  
Markov Models ◽  
Protein Domains ◽  
Degradation Pathway ◽  
The Other ◽  
Carbohydrate Binding ◽  
Gram Stain ◽  
Active Protein ◽  
Metabolic Potential ◽  
Independent Validation ◽  
Carbohydrate Binding Modules

AbstractBowel microbiota is a “metaorgan” of metabolisms on which quantitative readouts must be performed before interventions can be introduced and evaluated. The study of the effects of probiotic Clostridium butyricum MIYAIRI 588 (CBM588) on intestine transplantees indicated an increased percentage of the “other glycan degradation” pathway in 16S-rRNA-inferred metagenomes. To verify the prediction, a scoring system of carbohydrate metabolisms derived from shotgun metagenomes was developed using hidden Markov models. A significant correlation (R = 0.9, p < 0.015) between both modalities was demonstrated. An independent validation revealed a strong complementarity (R = −0.97, p < 0.002) between the scores and the abundance of “glycogen degradation” in bacteria communities. On applying the system to bacteria genomes, CBM588 had only 1 match and ranked higher than the other 8 bacteria evaluated. The gram-stain properties were significantly correlated to the scores (p < 5 × 10−4). The distributions of the scored protein domains indicated that CBM588 had a considerably higher (p < 10−5) proportion of carbohydrate-binding modules than other bacteria, which suggested the superior ability of CBM588 to access carbohydrates as a metabolic driver to the bowel microbiome. These results demonstrated the use of integrated counts of protein domains as a feasible readout for metabolic potential within bacteria genomes and human metagenomes.

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