Mining, Cloning, and Bioinformatic Analysis of Amidase Genes from Three Brewing Microorganisms

Abstract

Ethyl carbamate (EC), a Group 2A carcinogen, is unavoidably formed during fermentation and poses a persistent safety concern in alcoholic beverages and fermented foods. Although enzymatic degradation offers a mild and substrate-specific strategy, EC hydrolases suitable for brewing environments remain scarce, and heterologous expression of known candidates often results in inactive inclusion bodies. In this study, we mined amidase genes from three brewing-associated microorganisms—Bacillus velezensis, Clavispora lusitaniae, and Saccharomyces cerevisiae—based on the conserved GGSSGG motif of the AS-family amidases. Three full-length genes, amiE (1458 bp), amdA (1632 bp), and amd08 (1665 bp), were successfully cloned into pET-28a vectors. Prokaryotic expression revealed that AmiE (52.63 kDa) and AmdA (60.76 kDa) were predominantly deposited as insoluble inclusion bodies, while Amd08 (61.39 kDa) was not detected due to apparent gene silencing. To circumvent these expression barriers, we performed comprehensive bioinformatic analyses. All three proteins were predicted as stable, hydrophilic molecules with theoretical pI values of 5.12–5.47 and negative GRAVY indices. Secondary structures were dominated by α-helices and random coils, and homology modeling confirmed the presence of intact GGSSGG motifs and Lys-Ser-Ser catalytic triads in each protein. Multidimensional model validation further supported their stereochemical reliability. Collectively, this study enriches the genetic reservoir of brewing-origin EC hydrolases and provides essential molecular and structural foundations for future solubility engineering and enzyme preparation development.