Aspergillus oryzae acetamidase catalyzes degradation of ethyl carbamate.

Urethanase (EC 3.5.1.75) catalyzes the hydrolysis of ethyl carbamate (EC) to ethanol, carbon dioxide, and ammonia. From our recent study, we expected that an acetamidase encoded by amdS of Aspergillus oryzae may catalyze the degradation of EC because it is homologous with a Candida parapsilosis urethanase (CPUTNase) recently identified. Urethanase is a prospective candidate to reduce EC in alcoholic beverages, but knowledge of this enzyme is very limited. Recombinant AmdS was expressed to study its enzymatic properties. Purified AmdS was identified as a homo-tetramer consisting of four 60 kDa units and exhibited urethanase activity. In a 20% ethanol solution, AmdS had 65% activity compared with a solution without ethanol. Residual activity after 18 h indicated that AmdS was stable in 0%-40% ethanol solutions. The optimum temperature of AmdS was 40 °C. This enzyme showed urethanase activity at pH 6.4-9.6 and exhibited its highest activity at pH 9.6. The Km value of AmdS for EC was 8.2 mM, similar to the Km value (7.6 mM) of CPUTNase. AmdS showed activity not only for EC and acetamide but also other amide compounds. In this study, we investigated the enzymatic properties of AmdS that was identified as acetamidase and showed that an amidase can be an enzymatic candidate that degrades EC.

New urethanase from the yeast Candida parapsilosis.

Urethanase (EC 3.5.1.75) is an effective enzyme for removing ethyl carbamate (EC) present in alcoholic beverages. However, urethanase is not well studied and has not yet been developed for practical use. In this study, we report a new urethanase (CPUTNase) from the yeast Candida parapsilosis. Because C. parapsilosis can assimilate EC as its sole nitrogen source, the enzyme was extracted from yeast cells and purified using ion-exchange chromatography. The CPUTNase was estimated as a homotetramer comprising four units of a 61.7 kDa protein. In a 20% ethanol solution, CPUTNase had 73% activity compared with a solution without ethanol. Residual activity after 18 h indicated that CPUTNase was stable in 0%-40% ethanol solutions. The optimum temperature of CPUTNase was 43°C. This enzyme showed urethanase activity at pH 5.5-10.0 and exhibited its highest activity at pH 10. The gene of CPUTNase was identified, and a recombinant enzyme was expressed in the yeast Saccharomyces cerevisiae. Characteristics of recombinant CPUTNase were identical to the native enzyme. The putative amino acid sequence indicated that CPUTNase was an amidase family protein. Further, substrate specificity supported this sequence analysis because CPUTNase showed higher activities toward amide compounds. These results suggest that amidase could be a candidate for urethanase. We discovered a new enzyme and investigated its enzymatic characteristics, sequence, and recombinant CPUTNase expression. These results contribute to a further understanding of urethanase.