Development of novel economical methodologies for zymographic analysis of purified xylano-pectinolytic enzymes using agrowaste-based substrates.

In this study, zymographic analysis for xylanase and pectinase enzymes has been carried out using agrowaste residues, wheat bran and citrus peel as well as their extracts. Isozymic forms of xylanase as well as pectinase enzyme displayed comparable zymographic bands onto agar petriplates containing either commercial substrates (xylan and pectin), agrowaste-based substrates (wheat bran and citrus peel), or polysaccharides extracted from these agrowastes (crude xylan and pectin extracted from wheat bran and citrus peel, respectively), indicating the fact that agro residues and their extracts can be utilized as a substitute of cost-intensive commercial substrates, xylan and pectin for zymographic analysis. This is the first report revealing the zymographic analysis of xylano-pectinolytic enzymes using agro-based solid residues particles or polysaccharides extracted from agro-based residues.

Lytic xylan oxidases from wood-decay fungi unlock biomass degradation.

Wood biomass is the most abundant feedstock envisioned for the development of modern biorefineries. However, the cost-effective conversion of this form of biomass into commodity products is limited by its resistance to enzymatic degradation. Here we describe a new family of fungal lytic polysaccharide monooxygenases (LPMOs) prevalent among white-rot and brown-rot basidiomycetes that is active on xylans-a recalcitrant polysaccharide abundant in wood biomass. Two AA14 LPMO members from the white-rot fungus Pycnoporus coccineus substantially increase the efficiency of wood saccharification through oxidative cleavage of highly refractory xylan-coated cellulose fibers. The discovery of this unique enzyme activity advances our knowledge on the degradation of woody biomass in nature and offers an innovative solution for improving enzyme cocktails for biorefinery applications.

A molecular modeling approach to understand the structure and conformation relationship of (GlcpA)Xylan.

The structure and conformation relationships of a heteropolysaccharide (GlcpA)Xylan in terms of various molecular weights, Xylp/GlcpA ratio and the distribution of GlcpA along xylan chain were investigated using computer modeling. The adiabatic contour maps of xylobiose, XylpXylp(GlcpA) and (GlcpA)XylpXylp(GlcpA) indicated that the insertion of the side group (GlcpA) influenced the accessible conformational space of xylobiose molecule. RIS-Metropolis Monte Carlo method indicated that insertion of GlcpA side chain induced a lowering effect of the calculated chain extension at low GlcpA:Xylp ratio (GlcpA:Xylp = 1:3). The chain, however, became extended when the ratio of GlcpA:Xylp above 2/3. It was also shown that the spatial extension of the polymer chains was dependent on the distribution of side chain: the random distribution demonstrated the most flexible structure compared to block and alternative distribution. The present studies provide a unique insight into the dependence of both side chain ratio and distribution on the stiffness and flexibility of various (GlcpA)Xylan molecules.

Xylooligosaccharides: an economical prebiotic from agroresidues and their health benefits.

Oligosaccharides and dietary fibres are non-digestible food ingredients that preferentially stimulate the growth of prebiotic Bifidobacterium and other lactic acid bacteria in the gastro-intestinal tract. Xylooligosaccharides (XOS) provide a plethora of health benefits and can be incorporated into several functional foods. In the recent times, there has been an over emphasis on the microbial conversion of agroresidues into various value added products. Xylan, the major hemicellulosic component of lignocellulosic materials (LCMs), represents an important structural component of plant biomass in agricultural residues and could be a potent bioresource for XOS. On an industrial scale, XOS can be produced by chemical, enzymatic or chemo-enzymatic hydrolysis of LCMs. Chemical methods generate XOS with a broad degree of polymerization (DP), while enzymatic processes will be beneficial for the manufacture of food grade and pharmaceutically important XOS. Xylooligomers exert several health benefits, and therefore, have been considered to provide relief from several ailments. This review provides a brief on production, purification and structural characterization of XOS and their health benefits.

Influence of twin-screw extrusion on soluble arabinoxylans and corn fiber gum from corn fiber.

BACKGROUND: The effect of feed moisture content and screw speed in the extrusion process with and without chemical pretreatment of corn fiber was investigated. Different chemical pretreatment methods (NaOH and H2 SO4 solution) were compared. The improvement of reducing sugar, soluble arabinoxylans (SAX) content and the yield of corn fiber gum was measured. RESULTS: A high reducing sugar content was obtained in the filtrate fraction from the extruded destarched corn fiber (EDCF) with H2SO4 pretreatment. Feed moisture content most effectively improved both reducing sugar and SAX content of filtrate. Increasing feed moisture content and screw speed resulted in a higher SAX content in the filtrate of the EDCF with NaOH pretreatment. The SAX content of the residual solid from the EDCF with NaOH pretreatment was higher compared to H2SO4 pretreated and unpretreated samples and significantly increased with decreasing feed moisture content. The screw speed did not have a major impact after enzyme hydrolysis. The yield of corn fiber gum was increased by 12% using NaOH pretreatment combined with extrusion process as compared to the destarched corn fiber. CONCLUSION: The results show the great potential of the extrusion process as an effective pretreatment for disruption the lignocelluloses of corn fiber, leading to conversion of cellulose to glucose and hemicelluloses to SAX and isolation of corn fiber gum.

Structural basis for carbohydrate-binding specificity--a comparative assessment of two engineered carbohydrate-binding modules.

Detection, immobilization and purification of carbohydrates can be done using molecular probes that specifically bind to targeted carbohydrate epitopes. Carbohydrate-binding modules (CBMs) are discrete parts of carbohydrate-hydrolyzing enzymes that can be engineered to bind and detect specifically a number of carbohydrates. Design and engineering of CBMs have benefited greatly from structural studies that have helped us to decipher the basis for specificity in carbohydrate-protein interactions. However, more studies are needed to predict which modifications in a CBM would generate probes with predetermined binding properties. In this report, we present the crystal structures of two highly related engineered CBMs with different binding specificity profiles: X-2, which is specific for xylans and the L110F mutant of X-2, which binds xyloglucans and ß-glucans in addition to xylans. The structures of the modules were solved both in the apo form and complexed with oligomers of xylose, as well as with an oligomer of glucose in the case of X-2 L110F. The mutation, leucine to phenylalanine, converting the specific module into a cross-reactive one, introduces a crucial hydrogen-π interaction that allows the mutant to retain glucan-based ligands. The cross-reactivity of X-2 L110F is furthermore made possible by the plasticity of the protein, in particular, of residue R142, which permits accommodation of an extra hydroxymethyl group present in cellopentaose and not xylopentaose. Altogether, this study shows, in structural detail, altered protein-carbohydrate interactions that have high impact on the binding properties of a carbohydrate probe but are introduced through simple mutagenesis.

A novel xyloglucan film-based biosensor for toxicity assessment of ricin in castor seed meal.

Oil from the seed of the castor plant (Ricinus communis L.) is an important commodity for a number of industries, ranging from pharmaceuticals to renewable energy resources. However, the seed and subsequent seed meal contain ricin (RCA60), a potent cytotoxin, making it an unusable product for animal feed. In order to investigate the efficiency of reducing the toxicity of the seed meal, a biosensor is proposed by exploring the lectin-carbohydrate binding. A gold electrode was assembled with a film of Xyloglucan (XG) extracted from Hymenaea courbaril L. The analytical response to RCA60 was obtained using a polyclonal antibody against RCA60 conjugated to peroxidase. The current responses were generated by reaction with H2O2 and amplified with hydroquinone as chemical mediator. Voltammetric studies showed that the XG film was tightly bound to the gold electrode. This biosensor allows discriminate lectins in native and denatured forms. The limit of detection of native RCA60 was 2.1 µg mL(-1). This proposed biosensor showed to be a potential and accurate method for toxicity assessment of the ricin in castor seed meal by simple polysaccharide film-electrode strategy.