Effective Ethyl Carbamate Prevention in Red Wines by Treatment with Immobilized Acid Urease.

Climate change poses several challenges in the wine industry, including increasing risks related to chemical food contaminants such as biogenic amines and ethyl carbamate (EC). In this work, we focused on urea removal in red wines by immobilized acid urease aiming at limiting EC formation during wine storage. By considering separable kinetics of catalyst deactivation and urea hydrolysis, it was possible to model the time course of urea removal in repeated uses in stirred batch reactors. Treatments based on immobilized urease of red wine enriched with 30 mg/L of urea allowed the reduction in the contaminant concentration to <5 mg/L. After 28.5 h of treatment, the observed urea level was reduced to about 0.5 mg/L, corresponding to a decrease in the potential ethyl carbamate (PEC) from 1662 µg/L to 93 µg/L, below the level of the non-enriched wine (187 µg/L). As a comparison, when treating the same wine with the free enzyme at maximum doses allowed by the EU law, urea and PEC levels decreased to only 12 mg/L and 415 µg/L respectively, after 600 h of treatment. These results show that, for red wines, urease immobilization is an effective strategy for urea removal and, thus, effective reduction in ethyl carbamate as a process contaminant. This study provides the scientific background for the future scaling-up of the process at an industrial level.

[Advances in microbial enzymatic elimination of ethyl carbamate in Chinese rice wine].

Ethyl carbamate (EC), a carcinogenic and teratogenic chemical that is widely distributed in various alcoholic beverages, has attracted much attention. Microbial enzymatic degradation of EC in rice wine is always efficient and attractive. In this review, we summarize the research progress and problems of microbial enzymatic elimination of EC in rice wine from three aspects: the mechanisms of EC formation in rice wine, the research progress of acid urease, and the research progress of urethanase. Then, we propose the corresponding strategies to solve the problems: screening new urethanase with satisfied enzyme properties, food-grade expression and directed evolution of the bifunctional Fe³âº-dependent acid urease and acid urease used in combination with urethanase to eliminate both urea and EC in rice wine.

Characteristics of refold acid urease immobilized covalently by graphene oxide-chitosan composite beads.

Bifunctional acid urease can not only remove urea from rice wine, but also effectively remove ethyl carbamate (EC), thereby reducing harmful substances in rice wine. Acid urease from Providencia rettgeri JN-B815 was expressed in Escherichia coli BL21(DE3) as an inclusion body. Subsequently, acid urease was refolded gradually using dilution-ultrafiltration and specific activity of 12.3 U mg-1 was obtained. The acid urease thus obtained was immobilized on graphene oxide/chitosan beads. The recovery of immobilized urease was 77% for the graphene oxide/chitosan composite when coupled for 8 h with 5% glutaraldehyde. Steady-state kinetic analysis showed that the optimum temperature for urease activity was 37°C. Time-dependent thermal inactivation studies showed that the stability of the immobilized urease improved at 37°C and the t1/2 (half-life) of decay in urease activity was 12 h, whereas it was 43 days for soluble and chitosan/graphene oxide immobilized urease at 4°C. A significant change in Km occurred during the immobilization; the Km for urea and EC in the free state were 9.14 mM and 386 mM, respectively, whereas they were 5.28 mM and 180 mM, respectively, in the immobilized state. The values of Vmax for immobilized EC and urea were 1.08 µmol min-1 and 1.16 µmol min-1, respectively, whereas they were 0.782 µmol min-1 and 0.931 µmol min-1, respectively, for soluble EC and urea. Furthermore, 90% of the original activity of graphene oxide/chitosan-urease beads were retained after the beads were used 10 times, indicating excellent reusability.

Advances in microbial enzymatic elimination of ethyl carbamate in Chinese rice wine / 生物工程学报

Ethyl carbamate (EC), a carcinogenic and teratogenic chemical that is widely distributed in various alcoholic beverages, has attracted much attention. Microbial enzymatic degradation of EC in rice wine is always efficient and attractive. In this review, we summarize the research progress and problems of microbial enzymatic elimination of EC in rice wine from three aspects: the mechanisms of EC formation in rice wine, the research progress of acid urease, and the research progress of urethanase. Then, we propose the corresponding strategies to solve the problems: screening new urethanase with satisfied enzyme properties, food-grade expression and directed evolution of the bifunctional Fe³⁺-dependent acid urease and acid urease used in combination with urethanase to eliminate both urea and EC in rice wine.

A Bacillus paralicheniformis Iron-Containing Urease Reduces Urea Concentrations in Rice Wine.

Urease, a nickel-containing metalloenzyme, was the first enzyme to be crystallized and has a prominent position in the history of biochemistry. In the present study, we identified a nickel urease gene cluster, ureABCEFGDH, in Bacillus paralicheniformis ATCC 9945a and characterized it in Escherichia coli Enzymatic assays demonstrate that this oxygen-stable urease is also an iron-containing acid urease. Heterologous expression assays of UreH suggest that this accessory protein is involved in the transmembrane transportation of nickel and iron ions. Moreover, this iron-containing acid urease has a potential application in the degradation of urea in rice wine. The present study not only enhances our understanding of the mechanism of activation of urease but also provides insight into the evolution of metalloenzymes.IMPORTANCE An iron-containing, oxygen-stable acid urease from B. paralicheniformis ATCC 9945a with good enzymatic properties was characterized. This acid urease shows activities toward both urea and ethyl carbamate. After digestion with 6 U/ml urease, approximately 92% of the urea in rice wine was removed, suggesting that this urease has great potential in the food industry.

Assessing the Potential Content of Ethyl Carbamate in White, Red, and Rosé Wines as a Key Factor for Pursuing Urea Degradation by Purified Acid Urease.

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.