Functional characterization and synergic action of fungal xylanase and arabinofuranosidase for production of xylooligosaccharides.

Plant cell wall degrading enzymes are key technological components in biomass bioconversion platforms for lignocellulosic materials transformation. Cost effective production of enzymes and identification of efficient degradation routes are two economic bottlenecks that currently limit the use of renewable feedstocks through an environmental friendly pathway. The present study describes the hypersecretion of an endo-xylanase (GH11) and an arabinofuranosidase (GH54) by a fungal expression system with potential biotechnological application, along with comprehensive characterization of both enzymes, including spectrometric analysis of thermal denaturation, biochemical characterization and mode of action description. The synergistic effect of these enzymes on natural substrates such as sugarcane bagasse, demonstrated the biotechnological potential of using GH11 and GH54 for production of probiotic xylooligosaccharides from plant biomass. Our findings shed light on enzymatic mechanisms for xylooligosaccharide production, as well as provide basis for further studies for the development of novel enzymatic routes for use in biomass-to-bioethanol applications.

Characterization and mRNA expression analysis of PI31, an endogenous proteasome inhibitor from Schistosoma mansoni.

The proline-rich inhibitor of 31 kDa (PI31) is highly conserved through metazoan evolution, and its activity in the proteasome inhibition is well-established although the precise mechanism of inhibition is unclear. The coding DNA sequence of Schistosoma mansoni PI31 (SmPI31) was cloned, and the recombinant protein was expressed in bacterial system. The correct amino acid sequence was confirmed by mass spectrometry and circular dichroism suggests that SmPI31 contains both α-helix and non-structured regions. Inhibition assays, using the Suc-Leu-Leu-Val-Tyr-4-MCA substrate for proteasome degradation, showed that the S. mansoni proteasome may be regulated by the inhibitory activity of SmPI31. A gene expression assay using qRT-PCR at various stages during the S. mansoni life cycle has shown that SmPI31 transcripts are expressed in all studied stages, suggesting that PI31 plays an important role during the developmental processes of the parasite. In this study first evidence is presented that PI31 has a conserved structure and plays a role as proteasome inhibitor in adult worms and it is expressed through life cycle.

Calcium-independent membrane damage by venom phospholipases A2.

Many snake venom phospholipase A(2)s (vPLA(2)s) present biological effects that are independent of hydrolytic activity. Here we review the evidence for the calcium-independent membrane damaging activity of vPLA(2)s, the possible relevance of this activity on their biological effects, and models for the mechanism of membrane permeabilization by these proteins.

A predominant role for hydrogen bonding in the stability of the homodimer of bothropstoxin-I, A lysine 49-phospholipase A2.

Bothropstoxin-I (BthTx-I) is a homodimeric Lys49-phospholipase A(2) isolated from Bothrops jararacussu venom which damages liposome membranes via a Ca(2+)-independent mechanism. The Glu12/Trp77/Lys80 triad at the dimer interface forms extensive intermolecular hydrogen bonds and hydrophobic contacts, and equilibrium chemical denaturation was used to evaluate the effect on homodimer stability of site-directed mutagenesis of these residues. Changes in the intrinsic fluorescence anisotropy and farUV circular dichroism signals were analyzed using a two-step unfolding model of the BthTx-I dimer to estimate the Gibbs free energy changes of transitions between the dimer and native monomer and between the native and denatured monomers. Whereas the Trp77His, Trp77Gln and Glu12Gln mutants showed native-like dimer stabilities, the Trp77Phe, Lys80Met and Lys80Gly mutants showed significantly reduced K(d) values. A reduced dimer stability is correlated with a decrease in the Ca(2+)-independent membrane damaging activity as monitored by the release of a liposome entrapped fluorescent marker. Although the membrane damaging activity of the monomer is fivefold less than the dimer, the myotoxic activity was unaffected, indicating that these two effects are not correlated. These data suggest that the BthTx-I dimer is predominantly stabilized by hydrogen bonding interactions, and highlight the importance of the homodimeric form for efficient Ca(2+)-independent membrane damage.

Crystallization and preliminary X-ray crystallographic studies of the mesophilic xylanase A from Bacillus subtilis 1A1.

Xylanases have been the focus of research owing to their industrial potential in animal feed production, food processing and pulp and paper processes. In order to obtain insight into the structural stability of family 11 xylanases, the mesophilic family 11 xylanase (beta-1,4-xylan xylanohydrolase; EC 3.2.1.8) from Bacillus subtilis 1A1 has been crystallized and diffraction data have been collected to 1.7 A. The crystals belong to the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 50.93, b = 70.50, c = 40.05 A. The structure has been determined by molecular replacement, resulting in a crystallographic residual of 36.4% after rigid-body refinement.

Tertiary structural changes of the alpha-hemolysin from Staphylococcus aureus on association with liposome membranes.

The interaction of alpha-hemolysin (also called alpha-toxin) from Staphylococcus aureus with mixed egg-yolk phosphatidylcholine/cholesterol liposomes has been investigated using the intrinsic tryptophan fluorescence emission (ITFE) signal. The ITFE intensity of alpha-hemolysin, which was obtained using a novel purification protocol, showed a triphasic increase on incubation with liposomes at low protein/lipid ratios. The first, rapid phase results in an increase in ITFE of 10%, which reflects rapid conformation changes in the alpha-hemolysin on association with the liposome membrane. The second phase of the ITFE increase is associated with a red shift from 334 to 339 nm in the maximum emission wavelength, suggesting the transition to a partially unfolded intermediate in the oligomerization process. The third phase of the ITFE intensity change demonstrates a temporal correlation with the appearance of SDS-stable oligomers. The results demonstrate the feasibility of identification of intermediate protein conformations in complex membrane-associated processes by manipulation of the liposomal membrane composition.