GH11 xylanase from Aspergillus tamarii Kita: Purification by one-step chromatography and xylooligosaccharides hydrolysis monitored in real-time by mass spectrometry.

The present study describes the one-step purification and biochemical characterization of an endo-1,4-ß-xylanase from Aspergillus tamarii Kita. Extracellular xylanase was purified to homogeneity 7.43-fold through CM-cellulose. Enzyme molecular weight and pI were estimated to be 19.5kDa and 8.5, respectively. The highest activity of the xylanase was obtained at 60°C and it was active over a broad pH range (4.0-9.0), with maximal activity at pH 5.5. The enzyme was thermostable at 50°C, retaining more than 70% of its initial activity for 480min. The K0.5 and Vmax values on beechwood xylan were 8.13mg/mL and 1,330.20µmol/min/mg of protein, respectively. The ions Ba2+ and Ni2+, and the compounds ß-mercaptoethanol and DTT enhanced xylanase activity, while the heavy metals (Co2+, Cu2+, Hg+, Pb2+ and Zn2+) strongly inhibited the enzyme, at 5mM. Enzymatic hydrolysis of xylooligosaccharides monitored in real-time by mass spectrometer showed that the shortest xylooligosaccharide more efficiently hydrolyzed by A. tamarii Kita xylanase corresponded to xylopentaose. In agreement, HPLC analyzes did not detect xylopentaose among the hydrolysis products of xylan. Therefore, this novel GH11 endo-xylanase displays a series of physicochemical properties favorable to its application in the food, feed, pharmaceutical and paper industries.

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.

Effect of crotapotin on the biological activity of Asp49 and Lys49 phospholipases A(2) from Bothrops snake venoms.

Myonecrosis, in addition to edema and other biological manifestations, are conspicuous effects of Bothrops snake venoms, some of them caused by phospholipases A(2) (PLA(2)s). Asp49-PLA(2)s are catalytically active, whereas Lys49-PLA(2)s, although highly toxic, have little or no enzymatic activity upon artificial substrates, due to a substitution of lysine for aspartic acid at position 49. Crotapotin (CA), the acidic counterpart of crotoxin PLA(2) (CB), is a PLA(2)-like protein from Crotalus durissus terrificus snake venom, and is considered a chaperone protein for CB, able to increase its lethality about ten fold, but to inhibit the formation of the rat paw edema induced by carrageenin and by snake venoms. In this study, we demonstrate that CA significantly inhibits the edema induced by BthTX-I (23% inhibition), BthTX-II (27%), PrTX-I (25%), PrTX-III (35%) and MjTX-II (10%) on the mouse paw. CK levels evoked by isolated Asp49 or Lys49-PLA(2)s were reduced by 40% to 54% in the presence of CA and, in all cases, the membrane damaging activity of the toxins was also reduced. Circular dichroism spectra of the PLA(2)s in the presence and absence of CA showed that there was not any detectable secondary structural modification due to association between CA and the myotoxins. However, Fourier Transformed Infrared (FT-IR) analysis indicated that ionic and hydrophobic contacts contributed to stabilize this interaction.