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The ethyl carbamate (EC) content of a wine after a given temperature-time storage was theoretically predicted from the potential concentration of ethyl carbamate (PEC), as determined via an accelerated EC formation test. Such information was used to decide whether an enzymatic treatment was needed to reduce the wine urea level before bottling/aging. To this end, 6 white, red, and rosé wines, manufactured in Italy as such or enriched with urea, were tested for their PEC content either before or after enzymatic treatment using a purified acid urease preparation derived from Lactobacillus fermentum. The treatment was severely affected by the total phenolic content (TP) of the wine, the estimated pseudo-first-order kinetic rate constant for NH3 formation reducing by a factor of approximately 2000 as the TP increased from 0 to 1.64 g L(-1) . Such a sensitivity to TP was by far greater than that pertaining to a killed cell-based enzyme preparation used previously. Urea hydrolysis was successful at reducing EC concentration in wines with low levels of TP and other EC precursors., (© 2016 Institute of Food Technologists®)

The adsorption capacity and immobilization rate of two Eupergit® supports for acid urease was studied by varying the ionic strength and enzyme preparation concentration in the immobilizing solution at pH 7. Eupergit® C250 L yielded a series of derivatives with enzyme loadings (Y(P/B)) ranging from 48 to 171 mg of bovine serum albumin equivalent (BSAE) per gram of dry support (ds). Use of drastic postimmobilization conditions at pH 9 for 3-9 days yielded a slight decrease (8-14%) in the initial activity of immobilized enzymes and a limited increase in the stabilization factor (1.1-1.5), as assessed by accelerated aging tests at 65 °C. Further storage tests at 4 °C in the wet state showed that the activity of several derivatives either stabilized or not was practically constant for as long as 547 days. Both free enzyme and immobilized acid urease derivatives exhibited a kinetic pattern of the Michaelis-Menten type. Using the Eadie-Hofstee diagram, the specific ammonia formation rate constant for free (k(cat)) or immobilized (k'(cat)) enzyme resulted to be little affected by immobilization (k(cat) ≈ k'(cat) ≈ 18.86 ± 0.34 IU/mg BSAE), whereas the apparent Michaelis constant for immobilized enzymes exhibited a statistically significant increase at P < 0.05 from the intrinsic value (2.55 ± 0.14 mM) for free enzyme to 5.38 ± 0.87 mM as Y(P/B) increased to 171 mg BSAE/g ds. By estimating the observable Thiele modulus (Φ(obs) ), the activity of the biocatalyst with the greatest enzyme loading at the lowest urea concentrations tested (0.833 mM) was reduced by a factor of about 2 due to internal diffusional limitations. By operating in the pseudofirst-order regime with immobilized derivatives at Y(P/B) about 126 mg BSAE/g ds, their activity after grinding was no more limited by intraparticle diffusion and approached the value for free enzyme., (Copyright © 2012 American Institute of Chemical Engineers (AIChE).)

A purified acid urease preparation was covalently immobilized onto either Eupergit C 250 L or glutaraldehyde-cross-linked chitosan-derivative beads (i.e., Chitopearls BCW-3003 and BCW-3010). The kinetics of urea degradation in two target Italian white (i.e., Grechetto and Sauvignon Blanc) wines, as well as in a model wine solution, by using the above Eupergit C 250 L-, BCW-3003-, or BCW-3010-based biocatalysts, was confirmed to be of the pseudofirst order with respect to the urea concentration in the liquid bulk and not limited by urea mass transfer. In Grechetto and Sauvignon Blanc wines, the corresponding kinetic rate constants were quite similar, being about 7, 18, or 17% of that observed for free enzyme in the model wine solution, respectively. Owing to their minor sensitivity to the phenolic content of the wines tested, the chitosan-based biocatalysts might be potentially employable in the make up of packed-bed cartridges to continuously remove urea from commercial wines.

In this work, a purified acid urease preparation was covalently immobilized on Eupergit C 250 L and stabilized with glycine. Its average activity was found to be 69 +/- 16% of the initial one after 34-day storage at 4 degrees C . The kinetics of urea degradation in a model wine solution by immobilized enzyme was confirmed to be of pseudo-first-order with respect to the urea concentration in the liquid bulk, its apparent pseudo-first-order kinetic rate constant (k(Ii)) being about one-fourth of that (k(If)) pertaining to the free enzyme. In the operating conditions tested, the reaction kinetics was estimated as unaffected by the contribution of the external film and intraparticle diffusion mass transfer resistances. Because in the presence of the high-inhibitory tannins extracted from grape seeds in the range of 3-620 g of GAE m(-3) the loss in k(Ii) was quite smaller than that in k(If), the biocatalyst tested here is likely to overcome the present limits to the application of free acid urease in wine treatment.

Unlabelled: The specific activity of a whole cell acid urease preparation was tested in five wines manufactured in the Apulia region of Italy in the 2003 vintage at both short and long treatment times, thus confirming the validity of the pseudo-first-order kinetic model to describe urea removal in real wines. The ratio between the experimental pseudo-first-order kinetic rate constant (kIe) for any real wine tested and that (kI) referred to a model wine solution having the same composition and pH reduced from about 0.21 to 0.02 as the overall content of phenolic compounds (P) increased from 109 to 853 g m-3 of gallic acid equivalent (GAE). The specific inhibitory effect of such compounds was explained by accounting for the equilibrium constant (KP) of the reaction of polyphenols with acid urease, which was found to be about 21 g of GAE m-3 for the real wines tested, whereas it ranged from about 16 to 45 g of GAE m-3 when the model wine solution was enriched with tannins extracted from grape seeds or skins, respectively. A sequential experimental procedure consisting of accelerated acid urease tests at high doses of enzyme followed by accelerated ethyl carbamate tests on the resulting acid urease treated wine was recommended to assess preliminarily the technoeconomic feasibility of the acid urease hydrolytic process for the wine of concern., Keywords: Acid urease; real and model wines; phenolics; pseudo-first-order kinetic rate constant; inhibitory effect; urea degradation kinetics.

The specific activity (piA) of a whole cell acid urease preparation was assessed in model wine solutions at different levels of malic (M) and lactic acids, metabisulfite, ethanol, and pH by performing a central composite design. M and then pH were found to be the most controlling variables, their effects being practically coincident but of opposite sign. For urea concentrations up to approximately 1 mol m(-3) the ammonium formation rate was assumed of the pseudo-first-order with respect to urea, this being confirmed by two independent validation tests performed at 20 degrees C for as long as 24 h. In the case of real wines the effective pseudo-first-order kinetic rate constants were found to be smaller than those pertaining to the model solutions having the same wine composition and pH by a factor varying from 10 to 1000, this affecting significantly the specific urease treatment costs per liter of wine treated.

Highlights: [•] The activity of several acid urease immobilised derivatives was modelled. [•] The urea diffusion and Michaelis-Menten kinetic model was used. [•] In short-time tests severely grounded biocatalysts behaved as free enzyme. [•] Whole bead activity was limited by urea diffusion in the pseudo-1st order regime. [•] An empirical rule was suggested to design stirred or unstirred bioreactors. [ABSTRACT FROM AUTHOR]

Abstract: In this work, a purified acid urease preparation was covalently immobilised onto porous chitosan beads of different size. The covalent binding method was found to be more efficient than the adsorption cross-linkage one whatever the glutaraldehyde-to-chitosan bead ratio (Y GA/CHI) used. At the optimal Y GA/CHI ratio of 0.625gg−1 , the specific activity (A Bi) of the biocatalysts decreased from circa 300 to 70IUg−1 wet support, as the bead average diameter (d P) increased from 0.14 to 2.2mm. Generally, A Bi reduced less than 5% after preservation in the wet form at 4°C for 150–170 days. Only the biocatalyst prepared using the Chitopearl BCW-3001 lost about 40% of its initial activity. The kinetics of urea degradation in a model wine solution using these biocatalysts was of the pseudo-first order with respect to the urea concentration in the liquid bulk, the apparent pseudo-first order kinetic rate constant (k Ii) ranging from about two thirds to one fifth of that (k If) pertaining to free acid urease. In the operating conditions tested, the reaction kinetics was estimated as unaffected by the contribution of the external film and intraparticle diffusion mass-transfer resistances. When the model wine solution was enriched with the high-inhibitory tannins extracted from grape seeds, at the maximum level tested (374±2gGAEm−3) k Ii reduced to no more than (58±9)% of k If, this proving quite a higher protective action against such compounds for the chitosan-based biocatalysts towards free or Eupergit® C 250 L-immobilised acid urease. [Copyright &y& Elsevier]