RESUMEN
Ethyl carbamate (EC) is mainly found in fermented foods and fermented alcoholic beverages, which could cause carcinogenic potential to humans. Reducing EC is one of the key research priorities to address security of fermented foods. Enzymatic degradation of EC with EC hydrolase in food is the most reliable and efficient method. However, poor tolerance to ethanol severely hinders application of EC hydrolase. In this study, the mutants of EC hydrolase were screened by diphasic high pressure molecular dynamic simulations (dHP-MD). The best variant with remarkable improvement in specific activity and was H68A/K70R/S325N, whose specific activity was approximately 3.42-fold higher than WT, and relative enzyme activity under 20% (v/v) was 5.02-fold higher than WT. Moreover, the triple mutant increased its stability by acquiring more hydration shell and forming extra hydrogen bonds. Furthermore, the ability of degrading EC of the immobilized triple mutant was both detected in mock wine and under certain reaction conditions. The stability of immobilized triple mutant and WT were both improved, and immobilized triple mutant degraded nearly twice as much EC as that of immobilized WT. Overall, dHP-MD was proved to effectively improve enzyme activity and ethanol tolerance for extent application at industrial scale.
RESUMEN
Ethyl carbamate (EC), classified as a Group 2A carcinogen, is most abundant in the fermented foods, such as Cachaca, Shaoxing wine, and Chinese liquor (baijiu). Although biodegradation can reduce its concentration, a high ethanol concentration and acidic environment often limit its degradation. In the present study, a novel ethyl carbamate hydrolase (ECH) with high specificity to EC was isolated from Acinetobacter calcoaceticus, and its enzymatic properties and EC degradability were investigated. ECH was immobilized to resist extreme environmental conditions, and the flavor substance changes were explored by gas chromatography-mass spectrometry (GC/MS). The specific enzymatic activity of ECH was 68.31 U/mg. Notably, ECH exhibited excellent thermal stability and tolerance to sodium chloride and high ethanol concentration (remaining at 40% activity in 60% (v/v) ethanol, 1 h). The treatment of immobilized ECH for 12 h decreased the EC concentration in liquor by 71.6 µg/L. Furthermore, the immobilized ECH exerted less effect on its activity and on the flavor substances, which could be easily filtrated during industrial production.