Response surface methodology (RSM) was applied to optimise laccase production by a new strain of Pycnoporus sanguineus, isolated from the Uruguayan forests, grown on bark shavings under semi-solidstate fermentation conditions. Enhancement of laccase production was studied, with a full 2 4 factorial design with two replicates and four central points. Operating under the optimal conditions obtained (pH 3.0, yeast extract 10 g L À1 , glucose 5 g L À1 and bark shavings 3 g), a laccase activity of nearly 3000 U L À1 was achieved. Subsequently, the effect of different potential inducers on laccase production (Cu +2 , Mn +2 and lignin) was tested using the one-factor-at-a-time approach. It was found that the addition of 0.5 mM Cu +2 into the cultures considerably increased laccase production and, thus, maximum activity values of about 16 000 U L À1 were achieved. Further, the optimised culture broth (containing mainly laccase) showed high capacity to decolourise different recalcitrant synthetic dyes such as Acid Red 88 (AR88), Reactive Black 5 (RB5) and Lanaset Grey G (LG). After selecting enzyme concentration and time of reaction, almost 70% of AR88 could be decolourised in the absence of redox mediators. However, mediators were necessary to bleach RB5 and LG. Methyl syringate was the most effective mediator for AR88 (reaching a decolouration degree over 90%) and RB5 (nearly 70%). Around 80% of LG could be decolourised in the presence of violuric acid. These promising results make this enzyme a very useful tool for developing wastewater treatments.
A thiol-β-cyclodextrin was synthesized by a simple and environmentally friendly three-step method comprising epoxy activation of β-cyclodextrin, thiosulfate-mediated oxirane opening, and further reduction of the S-alkyl thiosulfate to a thiol group. The final step was optimized by using thiopropyl-agarose, a solid phase reducing agent with many advantages over soluble ones. β-Cyclodextrin thiolation was confirmed by titration with a thiol-reactive reagent, NMR studies, and MALDI-TOF/TOF. Thiolated cyclodextrin had an average value of one thiol group per molecule. Thiol-β-cyclodextrin proved to be an excellent agent for controlling polyphenol oxidase activity. This copper-containing enzyme is responsible for browning in fruits and vegetables. Under the same conditions, thiol-β-cyclodextrin generated a reductive microenvironment that increased the antibrowning effect on Red Delicious apples compared to unmodified β-cyclodextrin.
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