Unravelling biochemical and structural features of Bacillus licheniformis GH5 mannanase using site-directed mutagenesis and high-resolution protein crystallography studies.

Glycoside hydrolase family 5 (GH5) encompasses enzymes with several different activities, including endo-1,4-β-mannosidases. These enzymes are involved in mannan degradation, and have a number of biotechnological applications, such as mannooligosaccharide prebiotics production, stain removal and dyes decolorization, to name a few. Despite the importance of GH5 enzymes, only a few members of subfamily 7 were structurally characterized.

Two-domain GH30 xylanase from human gut microbiota as a tool for enzymatic production of xylooligosaccharides: Crystallographic structure and a synergy with GH11 xylosidase.

Production of value-added compounds and sustainable materials from agro-industrial residues is essential for better waste management and building of circular economy. This includes valorization of hemicellulosic fraction of plant biomass, the second most abundant biopolymer from plant cell walls, aiming to produce prebiotic oligosaccharides, widely explored in food and feed industries. In this work, we conducted biochemical and biophysical characterization of a prokaryotic two-domain R.

Molecular mechanism of cellulose depolymerization by the two-domain BlCel9A enzyme from the glycoside hydrolase family 9.

Carbohydrate-active enzymes from the glycoside hydrolase family 9 (GH9) play a key role in processing lignocellulosic biomass. Although the structural features of some GH9 enzymes are known, the molecular mechanisms that drive their interactions with cellulosic substrates remain unclear. To investigate the molecular mechanisms that the two-domain Bacillus licheniformis BlCel9A enzyme utilizes to depolymerize cellulosic substrates, we used a combination of biochemical assays, X-ray crystallography, small-angle X-ray scattering, and molecular dynamics simulations.

SAXSMoW 3.0: New advances in the determination of the molecular weight of proteins in dilute solutions from SAXS intensity data on a relative scale.

SAXSMoW (SAXS Molecular Weight) is an online platform widely used over the past few years for determination of molecular weights of proteins in dilute solutions. The scattering intensity retrieved from small-angle X-ray scattering (SAXS) raw data is the sole input to SAXSMoW for determination of molecular weights of proteins in liquid. The current updated SAXSMoW version 3.

Xyloglucan processing machinery in Xanthomonas pathogens and its role in the transcriptional activation of virulence factors.

Xyloglucans are highly substituted and recalcitrant polysaccharides found in the primary cell walls of vascular plants, acting as a barrier against pathogens. Here, we reveal that the diverse and economically relevant Xanthomonas bacteria are endowed with a xyloglucan depolymerization machinery that is linked to pathogenesis. Using the citrus canker pathogen as a model organism, we show that this system encompasses distinctive glycoside hydrolases, a modular xyloglucan acetylesterase and specific membrane transporters, demonstrating that plant-associated bacteria employ distinct molecular strategies from commensal gut bacteria to cope with xyloglucans.

Impact of cellulose properties on enzymatic degradation by bacterial GH48 enzymes: Structural and mechanistic insights from processive Bacillus licheniformis Cel48B cellulase.

Processive cellulases are highly efficient molecular engines involved in the cellulose breakdown process. However, the mechanism that processive bacterial enzymes utilize to recruit and retain cellulose strands in the catalytic site remains poorly understood. Here, integrated enzymatic assays, protein crystallography and computational approaches were combined to study the enzymatic properties of the processive BlCel48B cellulase from Bacillus licheniformis.

Light-stimulated T. thermophilus two-domain LPMO9H: Low-resolution SAXS model and synergy with cellulases.

Lytic polysaccharide monooxygenases (LPMOs), monocopper enzymes that oxidatively cleave recalcitrant polysaccharides, have important biotechnological applications. Thermothelomyces thermophilus is a rich source of biomass-active enzymes, including many members from auxiliary activities family 9 LPMOs. Here, we report biochemical and structural characterization of recombinant TtLPMO9H which oxidizes cellulose at the C1 and C4 positions and shows enhanced activity in light-driven catalysis assays.

Transformation of xylan into value-added biocommodities using Thermobacillus composti GH10 xylanase.

Enzymatic transformation of xylans into renewable fuels and value-added products is mediated by xylanases. Here we describe the biochemical and X-ray structural characterization of Thermobacillus composti GH10 xylanase (TcXyn10A) at 2.1 Å resolution aiming to unravel details of its recognition of glucurono- and arabinoxylan at a molecular level.

Low-resolution molecular shape, biochemical characterization and emulsification properties of a halotolerant esterase from Bacillus licheniformis.

Bacterial esterases are highly versatile enzymes, currently widely used in detergents, biosurfactants, bioemulsifiers and as biocatalysts in paper and food industries. Present work describes heterologous expression, purification, and biophysical and biochemical characterization of a halotolerant esterase from Bacillus licheniformis (BlEstA). BlEstA preferentially cleaves pNP-octanoate and both activity and stability of the enzyme increased in the presence of 2 M NaCl, and also with several organic solvents (ethanol, methanol and DMSO).

Enzymatic versatility and thermostability of a new aryl-alcohol oxidase from Thermothelomyces thermophilus M77.

Background Fungal aryl-alcohol oxidases (AAOx) are extracellular flavoenzymes that belong to glucose-methanol-choline oxidoreductase family and are responsible for the selective conversion of primary aromatic alcohols into aldehydes and aromatic aldehydes to their corresponding acids, with concomitant production of hydrogen peroxide (HO) as by-product. The HO can be provided to lignin degradation pathway, a biotechnological property explored in biofuel production. In the thermophilic fungus Thermothelomyces thermophilus (formerly Myceliophthora thermophila), just one AAOx was identified in the exo-proteome.

Biochemical characterization and low-resolution SAXS structure of two-domain endoglucanase BlCel9 from Bacillus licheniformis.

Lignocellulose feedstock constitutes the most abundant carbon source in the biosphere; however, its recalcitrance remains a challenge for microbial conversion into biofuel and bioproducts. Bacillus licheniformis is a microbial mesophilic bacterium capable of secreting a large number of glycoside hydrolase (GH) enzymes, including a glycoside hydrolase from GH family 9 (BlCel9). Here, we conducted biochemical and biophysical studies of recombinant BlCel9, and its low-resolution molecular shape was retrieved from small angle X-ray scattering (SAXS) data.

Biochemical characterization, low-resolution SAXS structure and an enzymatic cleavage pattern of BlCel48 from Bacillus licheniformis.

Economic sustainability of modern biochemical technologies for plant cell wall transformations in renewable fuels, green chemicals, and sustainable materials is considerably impacted by the elevated cost of enzymes. Therefore, there is a significant drive toward discovery and characterization of novel carbohydrate-active enzymes. Here, the BlCel48 cellulase from Bacillus licheniformis, a glycoside hydrolase family 48 member (GH48), was functionally and biochemically characterized.

Biochemical characterization and low-resolution SAXS structure of an exo-polygalacturonase from Bacillus licheniformis.

Among the structural polymers present in the plant cell wall, pectin is the main component of the middle lamella. This heterogeneous polysaccharide has an α-1,4 galacturonic acid backbone, which can be broken by the enzymatic action of pectinases, such as exo-polygalacturonases, that sequentially cleave pectin from the non-reducing ends, releasing mono or di-galacturonic acid residues. Constant demand for pectinases that better suit industrial requirements has motivated identification and characterization of novel enzymes from diverse sources.

Biochemical Characterization and Low-Resolution SAXS Molecular Envelope of GH1 β-Glycosidase from Saccharophagus degradans.

The marine bacteria Saccharophagus degradans (also known as Microbulbifer degradans), are rod-shaped and gram-negative motile γ-proteobacteria, capable of both degrading a variety of complex polysaccharides and fermenting monosaccharides into ethanol. In order to obtain insights into structure-function relationships of the enzymes, involved in these biochemical processes, we characterized a S. degradans β-glycosidase from glycoside hydrolase family 1 (SdBgl1B).

Molecular characterization of a family 5 glycoside hydrolase suggests an induced-fit enzymatic mechanism.

Glycoside hydrolases (GHs) play fundamental roles in the decomposition of lignocellulosic biomaterials. Here, we report the full-length structure of a cellulase from Bacillus licheniformis (BlCel5B), a member of the GH5 subfamily 4 that is entirely dependent on its two ancillary modules (Ig-like module and CBM46) for catalytic activity. Using X-ray crystallography, small-angle X-ray scattering and molecular dynamics simulations, we propose that the C-terminal CBM46 caps the distal N-terminal catalytic domain (CD) to establish a fully functional active site via a combination of large-scale multidomain conformational selection and induced-fit mechanisms.

Crystallization and preliminary X-ray diffraction analysis of a new xyloglucanase from Xanthomonas campestris pv. campestris.

Xyloglucanases (Xghs) are important enzymes involved in xyloglucan modification and degradation. Xanthomonas campestris pv. campestris (Xcc) is a phytopathogenic bacterium which produces a large number of glycosyl hydrolases (GH), but has only one family 74 GH (Xcc-Xgh).