The white-rot fungus Trametes trogii excretes a main laccase showing a molecular mass of 70 kDa, acidic isoelectric point and N-terminal sequence homologous to that of several phenol oxidases. The purified enzyme oxidizes a number of phenolic and non-phenolic compounds; recalcitrant molecules may be converted into substrates by introducing, in the correct position, o- or p-orienting ring-activating groups.
Background: Fungal laccases are useful enzymes for industrial applications; they exhibit broad substrate specificity and thus are able to oxidize a variety of xenobiotic compounds including chlorinated phenolics, synthetic dyes, pesticides and polycyclic aromatic hydrocarbons. Unfortunately, the biotechnological exploitation of laccases can be hampered by the difficulties concerning the enzyme production by the native hosts.
The influence of potential inhibitors, naturally present in wine, on the activity of stem bromelain was investigated in order to evaluate the applicability of this enzyme for protein stabilization in white wine. Bromelain proteolytic activity was tested against a synthetic substrate (Bz-Phe-Val-Arg-pNA) in a model wine system after adding ethanol, sulfur dioxide (SO(2)), skin, seed, and gallic and ellagic tannins at the average range of their concentration in wine. All the inhibitors of stem bromelain activity tested turned out to be reversible. Ethanol was a competitive inhibitor with a rather limited effect. Gallic and ellagic tannins have no inhibitory effect on stem bromelain activity, while both seed and skin tannins were uncompetitive inhibitors. The strongest inhibition effect was revealed for sulfur dioxide, which was a mixed-type inhibitor for the enzyme activity. This study provides useful information relative to a future biotechnological application of stem bromelain in winemaking.
a b s t r a c tBromelain from pineapple stem has been studied in unexplored model wine buffer (pH 3.2 tartaric acid and ethanol) in order to evaluate its applicability for white wine protein stabilization.Bromelain proteolytic activity was studied against several synthetic peptide substrates in McIlvaine buffer covering a wide pH range. Among different synthetic substrates, Bz-Phe-Val-Arg-pNA turned out to be the most suitable one to test bromelain activity at the average minimum pH value of wine (3.2). The protease activity was significantly increased by the presence of 5 mM cysteine while no influence was exerted by EDTA. The inhibiting effect of ethanol was investigated in the range 0-25% (v/v), resulting in being rather limited at the average minimum concentration found in wine (10% v/v).The kinetic parameters were estimated in both McIlvaine (as reference) and model wine buffers. This study showed that stem bromelain worked well at the average minimum pH value of wine, and had a significantly higher activity in model wine buffer (K a increased of 42%). These results are the basis for suggesting a future biotechnological application of this protease in winemaking.
A comparative study has been performed on five native laccases purified from the three basidiomycete fungi Pleurotus ostreatus, Rigidoporus lignosus, and Trametes trogii to relate their different catalytic capacities to their structural properties. Spectroscopic absorption features and EPR spectra at various pH values of the five enzymes are very similar and typical of the blue oxidases. The analysis of the dependence of kinetic parameters on pH suggested that a histidine residue is involved in the binding of nonphenolic substrates, whereas both a histidine and an acidic residue may be involved in the binding of phenolic compounds. His and an Asp residue are indeed found at the bottom of a cavity which may be regarded as a suitable substrate channel for approaching to type 1 copper in the 3D homology models of the two laccases from Pleuorotus ostreatus (POXC and POXAlb) whose sequences are known.
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