Fungal xylanases-mediated synthesis of silver nanoparticles for catalytic and biomedical applications.

Green synthesis of nanoparticles has fuelled the use of biomaterials to synthesise a variety of metallic nanoparticles. The current study investigates the use of xylanases of Aspergillus niger L3 (NEA) and Trichoderma longibrachiatum L2 (TEA) to synthesise silver nanoparticles (AgNPs). Characterisation of AgNPs was carried out using UV-Vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), and transmission electron microscopy, while their effectiveness as antimicrobial, antioxidant, catalytic, anticoagulant, and thrombolytic agents were determined. The colloidal AgNPs was brownish with surface plasmon resonance at 402.5 and 410 nm for NEA-AgNPs and TEA-AgNPs, respectively; while FTIR indicated that protein molecules were responsible for the capping and stabilisation of the nanoparticles. The spherical nanoparticles had size of 15.21-77.49 nm. The nanoparticles significantly inhibited the growth of tested bacteria (63.20-88.10%) and fungi (82.20-86.10%), and also scavenged DPPH (37.48-79.42%) and hydrogen peroxide (20.50-96.50%). In addition, the AgNPs degraded malachite green (78.97%) and methylene blue (25.30%). Furthermore, the AgNPs displayed excellent anticoagulant and thrombolytic activities using human blood. This study has demonstrated the potential of xylanases to synthesise AgNPs which is to the best of our knowledge the first record of such. The present study underscores the relevance of xylanases in nanobiotechnology.

α-Xylosidase plays essential roles in xyloglucan remodelling, maintenance of cell wall integrity, and seed germination in Arabidopsis thaliana.

Regulation and maintenance of cell wall physical properties are crucial for plant growth and environmental response. In the germination process, hypocotyl cell expansion and endosperm weakening are prerequisites for dicot seeds to complete germination. We have identified the Arabidopsis mutant thermoinhibition-resistant germination 1 (trg1), which has reduced seed dormancy and insensitivity to unfavourable conditions for germination owing to a loss-of-function mutation of TRG1/XYL1, which encodes an α-xylosidase. Compared to those of wild type, the elongating stem of trg1 showed significantly lower viscoelasticity, and the fruit epidermal cells were longitudinally shorter and horizontally enlarged. Actively growing tissues of trg1 over-accumulated free xyloglucan oligosaccharides (XGOs), and the seed cell wall had xyloglucan with a greatly reduced molecular weight. These observations suggest that XGOs reduce xyloglucan size by serving as an acceptor in transglycosylation and eventually enhancing cell wall loosening. TRG1/XYL1 gene expression was abundant in growing wild-type organs and tissues but relatively low in cells at most actively elongating part of the tissues, suggesting that α-xylosidase contributes to maintaining the mechanical integrity of the primary cell wall in the growing and pre-growing tissues. In germinating seeds of trg1, expression of genes encoding specific abscisic acid and gibberellin metabolism enzymes was altered in accordance with the aberrant germination phenotype. Thus, cell wall integrity could affect seed germination not only directly through the physical properties of the cell wall but also indirectly through the regulation of hormone gene expression.

Opposing influences by subsite -1 and subsite +1 residues on relative xylopyranosidase/arabinofuranosidase activities of bifunctional ß-D-xylosidase/α-L-arabinofuranosidase.

Conformational inversion occurs 7-8kcal/mol more readily in furanoses than pyranoses. This difference is exploited here to probe for active-site residues involved in distorting pyranosyl substrate toward reactivity. Spontaneous glycoside hydrolysis rates are ordered 4-nitrophenyl-α-l-arabinofuranoside (4NPA)>4-nitrophenyl-ß-d-xylopyranoside (4NPX)>xylobiose (X2). The bifunctional ß-d-xylosidase/α-l-arabinofuranosidase exhibits the opposite order of reactivity, illustrating that the enzyme is well equipped in using pyranosyl groups of natural substrate X2 in facilitating glycoside hydrolysis. Probing the roles of all 17 active-site residues by single-site mutation to alanine and by changing both moieties of substrate demonstrates that the mutations of subsite -1 residues decrease the ratio k(cat)(4NPX/4NPA), suggesting that the native residues support pyranosyl substrate distortion, whereas the mutations of subsite +1 and the subsite -1/+1 interface residues increase the ratio k(cat)(4NPX/4NPA), suggesting that the native residues support other factors, such as C1 migration and protonation of the leaving group. Alanine mutations of subsite -1 residues raise k(cat)(X2/4NPX) and alanine mutations of subsite +1 and interface residues lower k(cat)(X2/4NPX). We propose that pyranosyl substrate distortion is supported entirely by native residues of subsite -1. Other factors leading to the transition state are supported entirely by native residues of subsite +1 and interface residues.

Gene disruption of an arabinofuranosidase/beta-xylosidase precursor decreases Sclerotinia sclerotiorum virulence on canola tissue.

Although Sclerotinia sclerotiorum (Lib.) de Bary has been studied extensively, there are still aspects of this important phytopathogen's ability to cause disease in susceptible plants that remain unclear. A recent comprehensive proteome-level investigation of this fungus identified a number of proteins whose functions in disease initiation and progression have not been clearly established. Included among these proteins was an arabinofuranosidase/beta-xylosidase precursor whose role as a potential virulence factor had not been investigated previously. This article describes the generation of gene-disrupted mutant S. sclerotiorum unable to produce this arabinofuranosidase/beta-xylosidase precursor as well as the comparison of the virulence of this mutant with that of wild-type mycelia on susceptible canola leaves and stems. At all time points tested, the degree of necrosis was observed to be significantly greater on the plant tissue inoculated with wild-type mycelia. To our knowledge, this is the first report that clearly demonstrates that this arabinofuranosidase/beta-xylosidase precursor is a virulence factor for S. sclerotiorum.

AtBXL1 encodes a bifunctional beta-D-xylosidase/alpha-L-arabinofuranosidase required for pectic arabinan modification in Arabidopsis mucilage secretory cells.

Following pollination, the epidermal cells of the Arabidopsis (Arabidopsis thaliana) ovule undergo a complex differentiation process that includes the synthesis and polar secretion of pectinaceous mucilage followed by the production of a secondary cell wall. Wetting of mature seeds leads to the rapid bursting of these mucilage secretory cells to release a hydrophilic gel that surrounds the seed and is believed to aid in seed hydration and germination. A novel mutant is identified where mucilage release is both patchy and slow and whose seeds display delayed germination. While developmental analysis of mutant seeds reveals no change in mucilage secretory cell morphology, changes in monosaccharide quantities are detected, suggesting the mucilage release defect results from altered mucilage composition. Plasmid rescue and cloning of the mutant locus revealed a T-DNA insertion in AtBXL1, which encodes a putative bifunctional beta-d-xylosidase/alpha-l-arabinofuranosidase that has been implicated as a beta-d-xylosidase acting during vascular development. Chemical and immunological analyses of mucilage extracted from bxl1 mutant seeds and antibody staining of developing seed coats reveal an increase in (1-->5)-linked arabinans, suggesting that BXL1 is acting as an alpha-l-arabinofuranosidase in the seed coat. This implication is supported by the ability to rescue mucilage release through treatment of bxl1 seeds with exogenous alpha-l-arabinofuranosidases. Together, these results suggest that trimming of rhamnogalacturonan I arabinan side chains is required for correct mucilage release and reveal a new role for BXL1 as an alpha-l-arabinofuranosidase acting in seed coat development.

Role of tfxE, but not tfxG, in trifolitoxin resistance.

Eight genes, tfxABCDEFG and tfuA, confer production of trifolitoxin (TFX), a ribosomally synthesized, posttranslationally modified peptide antibiotic, in TFX-sensitive alpha-proteobacteria. An in-frame deletion in tfxE significantly reduced a strain's resistance to TFX in comparison to that of an otherwise identical construct containing wild-type tfxE. The deletion of tfxG had no effect on TFX resistance. Nevertheless, RNase protection assays showed that tfxE and tfxG are transcribed, showing that the tfxDEFG mRNA was produced on the same transcript. Examination of the role of tfxG in TFX production showed that the tfxG mutant expressed slightly less TFX activity and produced only one TFX isomer while four are produced by the wild-type strain. Thus, tfxE plays an important role in TFX resistance while tfxG is important in optimal TFX production through the production of TFX isomers.

Glycosylation by Pichia pastoris decreases the affinity of a family 2a carbohydrate-binding module from Cellulomonas fimi: a functional and mutational analysis.

When produced by Pichia pastoris, three of the five Asn-Xaa-Ser/Thr sequences (corresponding to Asn-24, Asn-73 and Asn-87) in the carbohydrate-binding module CBM2a of xylanase 10A from Cellulomonas fimi are glycosylated. The glycans are of the high-mannose type, ranging in size from GlcNAc(2)Man(8) to GlcNAc(2)Man(14). The N-linked glycans block the binding of CBM2a to cellulose. Analysis of mutants of CBM2a shows that glycans on Asn-24 decrease the association constant (K(a)) for the binding of CBM2a to bacterial microcrystalline cellulose approx. 10-fold, whereas glycans on Asn-87 destroy binding. The K(a) of a mutant of CBM2a lacking all three N-linked glycosylation sites is the same when the polypeptide is produced by either Escherichia coli or P. pastoris and is approx. half that of wild-type CBM2a produced by E. coli.

Structural and functional properties of low molecular weight endo-1,4-beta-xylanases.

There are currently four crystal structures of low molecular weight endo-1,4-beta-xylanases (E.C.3.2.1.8), i.e. family G/11 xylanases, available at the Brookhaven Data Bank: 2 xylanases from Trichoderma reesei (Törrönen et al., 1994; Törrönen and Rouvinen, 1995) and one from Bacillus circulans and another from Trichoderma harzianum (Campbell et al., 1993). They consist of two beta-sheets and one alpha-helix and have been described to resemble a partly-closed right hand. The catalytic residues are two conserved glutamate residues, which are located opposite to each other in an open active site cleft. The catalytic mechanism is thought to resemble that of the widely-studied enzyme lysozyme. The role of one glutamate is to act as an acid/base catalyst whereas the other is a nucleophile and stabilizes the reaction intermediate. Complex structures of partly-bound xylotetraose in mutated XYN from Bacillus circulans (Wakarchuck et al., 1994a) and three recently-obtained structures of XYNII from Trichoderma reesei with epoxyalkyl-xylose derivatives (Havukainen et al., 1996) have provided important information on substrate binding. Family G/11 xylanases show clear amino acid homology and thus have a common fold. However, variations in their functional properties, such as catalytic activity, substrate cleaving patterns, pH optima and thermostabilities, exist.

Do the non-catalytic polysaccharide-binding domains and linker regions enhance the biobleaching properties of modular xylanases?

Xylanase A (XylA) from Pseudomonas fluorescens subsp. cellulosa consists of an N-terminal non-catalytic cellulose-binding domain joined to a functionally independent C-terminal catalytic domain by a sequence rich in serine residues. Xylanase D (XylD) from Cellulomonas fimi also exhibits a modular structure comprising an N-terminal catalytic domain linked to an internal non-catalytic xylan-binding domain and a C-terminal cellulose-binding domain. To determine the importance of the non-catalytic polysaccharide-binding domains and linker sequences of XylA and XylD in relation to their capacity to hydrolyse pulp xylan and enhance bleachability, purified full-length and modified derivatives of both enzymes were incubated with a hardwood kraft pulp. Deletion of the cellulose-binding domain or linker region from XylA decreased the activity of the enzyme against pulp xylan, but had no significant effect on the capacity of the enzyme to facilitate delignification and reduce pulp kappa number. While full-length and truncated forms of XylD, lacking either the cellulose-binding or the cellulose- and xylan-binding domains, were equally effective in hydrolysing pulp xylan, enzyme derivatives containing a polysaccharide-binding domain were marginally more efficient in reducing pulp kappa number. The reduction in kappa number elicited by full-length and isolated catalytic domains of XylA and XylD was reflected in an increase in the brightness of paper handsheets derived from pretreated pulps. Thus, the polysaccharide-binding domains of XylA and XylD did not appear to confer any advantage in terms of the ability of the enzymes to improve pulp bleachability. However, XylA and XylD, which belong to different glycosyl hydrolase families, differed in their ability to hydrolyse pulp xylan and facilitate the delignification of kraft pulp.

The conserved noncatalytic 40-residue sequence in cellulases and hemicellulases from anaerobic fungi functions as a protein docking domain.

Two cDNAs, designated xynA and manA, encoding xylanase A (XYLA) and mannanase A (MANA), respectively, were isolated from a cDNA library derived from mRNA extracted from the anaerobic fungus, Piromyces. XYLA and MANA displayed properties typical of endo-beta 1,4-xylanases and mannanases, respectively. Neither enzyme hydrolyzed cellulosic substrates. The nucleotide sequences of xynA and manA revealed open reading frames of 1875 and 1818 base pairs, respectively, coding for proteins of M(r) 68,049 (XYLA) and 68,055 (MANA). The deduced primary structure of MANA revealed a 458-amino acid sequence that exhibited identity with Bacillus and Pseudomonas fluorescens subsp. cellulosa mannanases belonging to glycosyl hydrolase Family 26. A 40-residue reiterated sequence, which was homologous to duplicated noncatalytic domains previously observed in Neocallimastix patriciarum xylanase A and endoglucanase B, was located at the C terminus of MANA. XYLA contained two regions that exhibited sequence identity with the catalytic domains of glycosyl hydrolase Family 11 xylanases and were separated by a duplicated 40-residue sequence that exhibited strong homology to the C terminus of MANA. Analysis of truncated derivatives of MANA confirmed that the N-terminal 458-residue sequence constituted the catalytic domain, while the C-terminal domain was not essential for the retention of catalytic activity. Similar deletion analysis of XYLA showed that the C-terminal catalytic domain homologue exhibited catalytic activity, but the corresponding putative N-terminal catalytic domain did not function as a xylanase. Fusion of the reiterated noncatalytic 40-residue sequence conserved in XYLA and MANA to glutathione S-transferase, generated a hybrid protein that did not associate with cellulose, but bound to 97- and 116-kDa polypeptides that are components of the multienzyme cellulase-hemicellulase complexes of Piromyces and Neocallimastix patriciarum, respectively. The role of this domain in the assembly of the enzyme complex is discussed.

Purification and properties of an endo-1,4-beta-glucanase from Clostridium josui.

An enzyme active against carboxymethyl cellulose (CMC) was purified from the stationary-phase-culture supernatant of Clostridium josui grown in a medium containing ball-milled cellulose. The purification in the presence of 6 M urea yielded homogeneous enzyme after an approximately 50-fold increase in specific activity and a 13% yield. The enzyme had a molecular mass of 45 kilodaltons. The optimal temperature and pH of the enzyme against CMC were 60 degrees C and 6.8, respectively. The enzyme hydrolyzed cellotetraose, cellopentaose, and cellohexaose to cellobiose and cellotriose but did not hydrolyze cellobiose or cellotriose. A microcrystalline cellulose, Avicel, was also hydrolyzed significantly, but the extent of hydrolysis was remarkably less than that of CMC. On the basis of these results, the enzyme purified here is one of the endo-1,4-beta-glucanases. The N-terminal amino acid sequence of the enzyme is Tyr-Asp-Ala-Ser-Leu-Lys-Pro-Asn-Leu-Gln-Ile-Pro-Gln-Lys-Asn-Ile-Pro-Asn- Asn-Asp-Ala-Val-Asn-Ile-Lys.