Molecular Engineering of Bacillus paralicheniformis Acid Urease To Degrade Urea and Ethyl Carbamate in Model Chinese Rice Wine

Abstract

Bacillus paralicheniformis urease (BpUrease) has been shown to be a promising biocatalyst for degrading the carcinogenic chemical ethyl carbamate (EC or urethane) in rice wine. However, low EC affinity and catalytic efficiency limit the practical application of BpUrease. In this study, we improved the EC degradation capability of BpUrease by site-saturation mutagenesis (SSM). The best variant L253P/L287N showed a 49% increase in EC affinity, 1027% increase in catalytic efficiency (k cat/K m), and 583% increase in half-life (t 1/2) at 70 °C. Homology modeling analysis suggest that mutation of Leu253 to Pro increased the BpUrease EC specificity by affecting the interaction between Arg339 with the catalytic residue His323, while Leu287Asn mutation benefits EC specificity and affinity by changing the interaction networks among the residues in the catalytic pocket. Our results show that the L253P/L287N variant efficiently degraded urea and EC in a model rice wine, making it a good candidate for practical application in the food industry.

Genetic Engineering Production of Ethyl Carbamate Hydrolase and Its Application in Degrading Ethyl Carbamate in Chinese Liquor

“…The highest content of flavor substances in both samples was ethyl hexanoate, followed by ethyl caprylate. The content of ethyl hexanoate exceeds 200 mg/L, indicating the high quality of strong-aroma liquor [ 43 ]. Compared with the non-treated sample, the contents of ethyl caproate, ethyl acetate, ethyl valerate, and furfural in the ECH-treated sample were reduced by 108.1 mg/L (from 490.58 ± 16.74 mg/L to 382.48 ± 5.27 mg/L), 6.4 mg/L (from 13.63 ± 1.10 mg/L to 7.23 ± 0.91 mg/L), 8.67 mg/L (from 22.18 ± 2.21 mg/L to 13.51 ± 1.26 mg/L), and 5.36 mg/L (from 13.92 ± 0.25 mg/L to 8.56 ± 0.25 mg/L), respectively.…”

Occurrence of Ethyl Carbamate in Foods and Beverages: Review of the Formation Mechanisms, Advances in Analytical Methods, and Mitigation Strategies

“…Use of urethanase to directly degrade EC has been proposed, and ongoing experiments are being conducted to determine how well this treatment can scale up. Urethanase can reduce EC in rice wine and soy sauce by 15 to 50% (93,128,211). An alternative approach to direct addition of a purified enzyme is inoculation of beverages with microorganisms possessing degradation activity similar to that of the purified enzymes.…”

Origin, Succession, and Control of Biotoxin in Wine

“…Without affecting the flavor of the wine, an appropriate reduction in temperature also helps reduce the EC content in the wine, which is a key adjustment point for EC control from a process perspective ( Hasnip et al, 2004 ). Some studies showed that acid urease catalyzed the decomposition of urea to ammonia and carbon dioxide, decreasing the content of an important precursor of EC in wine ( Cerreti et al, 2016 ; Liu et al, 2018 ; Yang et al, 2021 ). Since 1999, Europe has approved the use of acid urease extracted from fermented LAB in wine ( Cerreti et al, 2016 ).…”