Expression, isolation, and identification of an ethanol-resistant ethyl carbamate-degrading amidase from Agrobacterium tumefaciens d3

Abstract

Ethyl carbamate (EC), widely found in alcoholic beverages, has been revealed to be a probable carcinogen in humans. Urethanase (EC 3.5.1.75) is an effective enzyme for the degradation of EC; however, the previously identified urethanases exhibited insufficient acid and alcohol resistance. In this study, an enantioselective amidase (AmdA) screened from Agrobacterium tumefaciens d₃ exhibited urethanase activity with excellent alcohol resistance. AmdA was first overexpressed in Escherichia coli; however, the recombinant protein was primarily located in inclusion bodies, and thus, co-expression of molecular chaperones was used. The activity of AmdA increased 3.1 fold to 307 U/L, and the specific activity of urethanase with C-terminal His-tags reached 0.62 U/mg after purification through a Ni-NTA column. Subsequently, the enzymatic properties and kinetic constants of AmdA were investigated. The optimum temperature for AmdA was 55 °C, it showed the highest activity at pH 7.5, and the K_m was 0.964 mM. Moreover, after 1 h of heat treatment at 37 °C in a 5-20% (v/v) ethanol solution, the residual urethanase activity was higher than 91%, considerably more than that reported thus far.

Keywords: Agrobacterium tumefaciens d(3); AmdA; Amidase; Ethyl carbamate; Urethanase.