Explore the vast range of titles available.

In this study, the anti-cancer and anti-inflammatory activities of PS14, a short peptide derived from the cellulase binding domain of pathogenic fungus, Aphanomyces invadans, have been evaluated, in vitro and in vivo. Bioinformatics analysis of PS14 revealed the physicochemical properties and the web-based predictions, which indicate that PS14 is non-toxic, and it has the potential to elicit anti-cancer and anti-inflammatory activities. These in silico results were experimentally validated through in vitro (L6 or Hep-2 cells) and in vivo (zebrafish embryo or larvae) models. Experimental results showed that PS14 is non-toxic in L6 cells and the zebrafish embryo, and it elicits an antitumor effect Hep-2 cells and zebrafish embryos. Anticancer activity assays, in terms of MTT, trypan blue and LDH assays, showed a dose-dependent inhibitory effect on cell proliferation. Moreover, in the epithelial cancer cells and zebrafish embryos, the peptide challenge (i) caused significant changes in the cytomorphology and induced apoptosis; (ii) triggered ROS generation; and (iii) showed a significant up-regulation of anti-cancer genes including BAX, Caspase 3, Caspase 9 and down-regulation of Bcl-2, in vitro. The anti-inflammatory activity of PS14 was observed in the cell-free in vitro assays for the inhibition of proteinase and lipoxygenase, and heat-induced hemolysis and hypotonicity-induced hemolysis. Together, this study has identified that PS14 has anti-cancer and anti-inflammatory activities, while being non-toxic, in vitro and in vivo. Future experiments can focus on the clinical or pharmacodynamics aspects of PS14.

Cellulase enzymes deconstruct recalcitrant cellulose into soluble sugars, making them a biocatalyst of biotechnological interest for use in the nascent lignocellulosic bioeconomy. Cellobiohydrolases (CBHs) are cellulases capable of liberating many sugar molecules in a processive manner without dissociating from the substrate. Within the complete processive cycle of CBHs, dissociation from the cellulose substrate is rate limiting, but the molecular mechanism of this step is unknown. Here, we present a direct comparison of potential molecular mechanisms for dissociation via Hamiltonian replica exchange molecular dynamics of the model fungal CBH, Trichoderma reesei Cel7A. Computational rate estimates indicate that stepwise cellulose dethreading from the binding tunnel is 4 orders of magnitude faster than a clamshell mechanism, in which the substrate-enclosing loops open and release the substrate without reversing.We also present the crystal structure of a disulfide variant that covalently links substrate-enclosing loops on either side of the substrate-binding tunnel, which constitutes a CBH that can only dissociate via stepwise dethreading. Biochemical measurements indicate that this variant has a dissociation rate constant essentially equivalent to the wild type, implying that dethreading is likely the predominant mechanism for dissociation.

Enzyme reactions, both in Nature and technical applications, commonly occur at the interface of immiscible phases. Nevertheless, stringent descriptions of interfacial enzyme catalysis remain sparse, and this is partly due to a shortage of coherent experimental data to guide and assess such work. In this work, we produced and kinetically characterized 83 cellulases, which revealed a conspicuous linear free energy relationship (LFER) between the substrate binding strength and the activation barrier. The scaling occurred despite the investigated enzymes being structurally and mechanistically diverse. We suggest that the scaling reflects basic physical restrictions of the hydrolytic process and that evolutionary selection has condensed cellulase phenotypes near the line. One consequence of the LFER is that the activity of a cellulase can be estimated from its substrate binding strength, irrespectively of structural and mechanistic details, and this appears promising for in silico selection and design within this industrially important group of enzymes. Enzyme reactions at interfaces are common in both Nature and industrial applications but no general kinetic framework exists for interfacial enzymes. Here, the authors kinetically characterize 83 cellulases and identify a scaling relationship between ligand binding strength and maximal turnover, a so-called linear free energy relationship, which may help rationalize cellulolytic mechanisms and guide the selection of technical enzymes.

Enzymatic depolymerization of recalcitrant polysaccharides plays a key role in accessing the renewable energy stored within lignocellulosic biomass, and natural biodiversities may be explored to discover microbial enzymes that have evolved to conquer this task in various environments. Here, a metagenome from a thermophilic microbial community was mined to yield a novel, thermostable cellulase, named mgCel6A, with activity on an industrial cellulosic substrate (sulfite-pulped Norway spruce) and a glucomannanase side activity. The enzyme consists of a glycoside hydrolase family 6 catalytic domain (GH6) and a family 2 carbohydrate binding module (CBM2) that are connected by a linker rich in prolines and threonines. MgCel6A exhibited maximum activity at 85°C and pH 5.0 on carboxymethyl cellulose (CMC), but in prolonged incubations with the industrial substrate, the highest yields were obtained at 60°C, pH 6.0. Differential scanning calorimetry (DSC) indicated a Tm(app) of 76°C. Both functional data and the crystal structure, solved at 1.88 Å resolution, indicate that mgCel6A is an endoglucanase. Comparative studies with a truncated variant of the enzyme showed that the CBM increases substrate binding, while not affecting thermal stability. Importantly, at higher substrate concentrations the full-length enzyme was outperformed by the catalytic domain alone, underpinning previous suggestions that CBMs may be less useful in high-consistency bioprocessing.

The assumption that cellulose degradation and assimilation can only be carried out by heterotrophic organisms was shattered in 2012 when it was discovered that the unicellular green alga, Chlamydomonas reinhardtii (Cr), can utilize cellulose for growth under CO2-limiting conditions. Publications of genomes/transcriptomes of the colonial microalgae, Gonium pectorale (Gp) and Volvox carteri (Vc), between 2010–2016 prompted us to look for cellulase genes in these algae and to compare them to cellulases from bacteria, fungi, lower/higher plants, and invertebrate metazoans. Interestingly, algal catalytic domains (CDs), belonging to the family GH9, clustered separately and showed the highest (33–42%) and lowest (17–36%) sequence identity with respect to cellulases from invertebrate metazoans and bacteria, respectively, whereas the identity with cellulases from plants was only 27–33%. Based on comparative multiple alignments and homology models, the domain arrangement and active-site architecture of algal cellulases are described in detail. It was found that all algal cellulases are modular, consisting of putative novel cysteine-rich carbohydrate-binding modules (CBMs) and proline/serine-(PS) rich linkers. Two genes were found to encode a protein with a putative Ig-like domain and a cellulase with an unknown domain, respectively. A feature observed in one cellulase homolog from Gp and shared by a spinach cellulase is the existence of two CDs separated by linkers and with a C-terminal CBM. Dockerin and Fn-3-like domains, typically found in bacterial cellulases, are absent in algal enzymes. The targeted gene expression analysis shows that two Gp cellulases consisting, respectively, of a single and two CDs were upregulated upon filter paper addition to the medium.

PcMulGH9, a novel glycoside hydrolase family 9 (GH9) from Paenibacillus curdlanolyticus B-6, was successfully expressed in Escherichia coli. It is composed of a catalytic domain of GH9, two domains of carbohydrate-binding module family 3 (CBM3) and two domains of fibronectin type 3 (Fn3). The PcMulGH9 enzyme showed broad activity towards the β-1,4 glycosidic linkages of cellulose, mannan and xylan, including cellulose and xylan contained in lignocellulosic biomass, which is rarely found in GH9. The enzyme hydrolysed substrates with bifunctional endo-/exotypes cellulase, mannanase and xylanase activities, but predominantly exhibited exo-activities. This enzyme released cellobiose as a major product from cellohexaose, while mannotriose and xylotriose were major hydrolysis products from mannohexaose and xylohexaose, respectively. Moreover, PcMulGH9 could hydrolyse untreated corn hull and rice straw into xylo- and cello-oligosaccharides. Enzyme kinetics, site-directed mutagenesis and molecular docking revealed that Met394, located at the binding subsite + 2, was involved in broad substrate specificity of PcMulGH9 enzyme. This study offers new knowledge of the multifunctional cellulase/mannanase/xylanase in GH9. The PcMulGH9 enzyme showed a novel function of GH9, which increases its potential for saccharification of lignocellulosic biomass into value-added products, especially oligosaccharides.

Enzymatic degradation of abundant renewable polysaccharides such as cellulose and starch is a field that has the attention of both the industrial and scientific community. Most of the polysaccharide degrading enzymes are classified into several glycoside hydrolase families. They are often organized in a modular manner which includes a catalytic domain connected to one or more carbohydrate-binding modules. The carbohydrate-binding modules (CBM) have been shown to increase the proximity of the enzyme to its substrate, especially for insoluble substrates. Therefore, these modules are considered to enhance enzymatic hydrolysis. These properties have played an important role in many biotechnological applications with the aim to improve the efficiency of polysaccharide degradation. The domain organization of glycoside hydrolases (GHs) equipped with one or more CBM does vary within organisms. This review comprehensively highlights the presence of CBM as ancillary modules and explores the diversity of GHs carrying one or more of these modules that actively act either on cellulose or starch. Special emphasis is given to the cellulase and amylase distribution within the filamentous microorganisms from the genera of Streptomyces and Aspergillus that are well known to have a great capacity for secreting a wide range of these polysaccharide degrading enzyme. The potential of the CBM and other ancillary domains for the design of improved polysaccharide decomposing enzymes is discussed.Enzymatic degradation of abundant renewable polysaccharides such as cellulose and starch is a field that has the attention of both the industrial and scientific community. Most of the polysaccharide degrading enzymes are classified into several glycoside hydrolase families. They are often organized in a modular manner which includes a catalytic domain connected to one or more carbohydrate-binding modules. The carbohydrate-binding modules (CBM) have been shown to increase the proximity of the enzyme to its substrate, especially for insoluble substrates. Therefore, these modules are considered to enhance enzymatic hydrolysis. These properties have played an important role in many biotechnological applications with the aim to improve the efficiency of polysaccharide degradation. The domain organization of glycoside hydrolases (GHs) equipped with one or more CBM does vary within organisms. This review comprehensively highlights the presence of CBM as ancillary modules and explores the diversity of GHs carrying one or more of these modules that actively act either on cellulose or starch. Special emphasis is given to the cellulase and amylase distribution within the filamentous microorganisms from the genera of Streptomyces and Aspergillus that are well known to have a great capacity for secreting a wide range of these polysaccharide degrading enzyme. The potential of the CBM and other ancillary domains for the design of improved polysaccharide decomposing enzymes is discussed.

Plant cell-wall polysaccharides represent a vast source of food in nature. To depolymerize polysaccharides to soluble sugars, many organisms use multifunctional enzyme mixtures consisting of glycoside hydrolases, lytic polysaccharide mono-oxygenases, polysaccharide lyases, and carbohydrate esterases, as well as accessory, redox-active enzymes for lignin depolymerization. Many of these enzymes that degrade lignocellulose are multimodular with carbohydrate-binding modules (CBMs) and catalytic domains connected by flexible, glycosylated linkers. These linkers have long been thought to simply serve as a tether between structured domains or to act in an inchworm-like fashion during catalytic action. To examine linker function, we performed molecular dynamics (MD) simulations of the Trichoderma reesei Family 6 and Family 7 cellobiohydrolases (TrCel6A and TrCel7A, respectively) bound to cellulose. During these simulations, the glycosylated linkers bind directly to cellulose, suggesting a previously unknown role in enzyme action. The prediction from the MD simulations was examined experimentally by measuring the binding affinity of the Cel7A CBM and the natively glycosylated Cel7A CBM-linker. On crystalline cellulose, the glycosylated linker enhances the binding affinity over the CBM alone by an order of magnitude. The MD simulations before and after binding of the linker also suggest that the bound linker may affect enzyme action due to significant damping in the enzyme fluctuations. Together, these results suggest that glycosylated linkers in carbohydrate-active enzymes, which are intrinsically disordered proteins in solution, aid in dynamic binding during the enzymatic deconstruction of plant cell walls.

In ectomycorrhiza, root ingress and colonization of the apoplast by colonizing hyphae is thought to rely mainly on the mechanical force that results from hyphal tip growth, but this could be enhanced by secretion of cell-wall-degrading enzymes, which have not yet been identified. The sole cellulose-binding module (CBM1) encoded in the genome of the ectomycorrhizal Laccaria bicolor is linked to a glycoside hydrolase family 5 (GH5) endoglucanase, LbGH5-CBM1. Here, we characterize LbGH5-CBM1 gene expression and the biochemical properties of its protein product. We also immunolocalized LbGH5-CBM1 by immunofluorescence confocal microscopy in poplar ectomycorrhiza. We show that LbGH5-CBM1 expression is substantially induced in ectomycorrhiza, and RNAi mutants with a decreased LbGH5-CBM1 expression have a lower ability to form ectomycorrhiza, suggesting a key role in symbiosis. Recombinant LbGH5-CBM1 displays its highest activity towards cellulose and galactomannans, but no activity toward L. bicolor cell walls. In situ localization of LbGH5-CBM1 in ectomycorrhiza reveals that the endoglucanase accumulates at the periphery of hyphae forming the Hartig net and the mantle. Our data suggest that the symbiosis-induced endoglucanase LbGH5-CBM1 is an enzymatic effector involved in cell wall remodeling during formation of the Hartig net and is an important determinant for successful symbiotic colonization.

Components of modular cellulases, type-A cellulose-binding modules (CBMs) bind to crystalline cellulose and enhance enzyme effectiveness, but structural details of the interaction are uncertain. We analyzed cellulose binding by EXLX1, a bacterial expansin with ability to loosen plant cell walls and whose domain D2 has type-A CBM characteristics. EXLX1 strongly binds to crystalline cellulose via D2, whereas its affinity for soluble cellooligosaccharides is weak. Calorimetry indicated cellulose binding was largely entropically driven. We solved the crystal structures of EXLX1 complexed with cellulose-like oligosaccharides to find that EXLX1 binds the ligands through hydrophobic interactions of three linearly arranged aromatic residues in D2. The crystal structures revealed a unique form of ligand-mediated dimerization, with the oligosaccharide sandwiched between two D2 domains in opposite polarity. This report clarifies the molecular target of expansin and the specific molecular interactions of a type-A CBM with cellulose.