Study On Urethanase From Penicillium Variabile

Ethyl carbamate, proven to be type2A carcinogen, widely exists in fermented foods andbeverages and attracts much attention. Urethanase can rapidly and specifically remove ethylcarbamate and therefore reduce its concentration in foods and beverages. In this study, aurethanase-producing strain was screened and identified, followed by optimization offermentation conditions, separation and purification of the enzyme, study of the enzymaticproperties, and finally the enzyme was preliminarily used to reduce ethyl carbamate in ricewine.A strain with urethanase activity was isolated from mouse gastrointestine. Bycombination of morphological characterization of the colony, hyphae, and spore and thesequence analysis of its rDNA ITS, the strain was determined as Penicillium Variabile andnamed as P. variabile JN-A525.The fermentation process for P.variabile JN-A525was optimized by single factoroptimization followed by orthogonal tests. The optimum medium is composed of glucose20g/L, beef extract10g/L, KH2PO45g/L, NaCl1g/L and KCl5g/L. The optimumfermentation conditions is at32℃, inoculum volume of3%, the optimum volume of70/250(mL) in flask and the rotation speed of100r/min.After sequential use of ultrasonication, polyethylene glycol20000concentrating andSuperdex G-200gel filtration chromatography, the purified urethanase was used forenzymatic property study. The optimum temperature and pH of urethanase from P. Variabileare of50℃and6.0, respectively. The enzyme is stable within40-50℃and pH range of7.0-9.0. The Minchaeli constant (Km) and maximum rate (Vm) of urethanase were27.2mmol/L and156.25μmol/min, respectively. The enzyme exhibits ethanol-tolerant. Undersimulated conditions, urethanase shows relatively strong catalytic ability to urea and EC.Meanwhile, it cannot catalyze the decomposition of amino acids and other esters.By matrix-assisted laser desorption/ionization time of flight mass spectrometry,glucosamine-6-phosphate deaminase and6-phosphogluconate dehydrogenase get highestscore with urethanase, which are of75and74, respectively. N-terminal sequencing informsthat N-terminal amino acid sequence of urethanase is GTNTADNDAA.The removing of ethyl carbamate from Chinese rice wine by urethanase was studied byGC-MS. The result showed that it can apparently remove ethyl carbamate without the changeof flavor substances in rice wine.