The Isolation Of Acid Urease And Urethanase Producing Strain And The Enzymatic Characterization

Ethyl Carbamate(EC), classified as a2A group carcinogen, is widely present in thefermented foods. As a result of the carcinogenic and genotoxic properties, the high level ofEC in Chinese rice wine has caused much attentions among the public. Therefore, largenumber of methods and strategis concerning the EC issues has been proposed and conductedby researchers and institutions. As for all the methods, the enzymatic method characterized asefficiency and high safety is viewed as the best method for the reduction of EC.The research include the followings: the isolation of a strain capable of catalyzing ureaand EC in acid solutions, the optimization of fermentation medium, the purification andcharacterization of enzyme from Providencia sp. JNB815, the effect of enzyme on the ureaand EC degradation in Chinese rice wine, the accesss and identification of the urease genes.(1) Using mouse testines as the source of the strains, a strain capable of catalyzing ureaand EC is isolated with special medium, and is identified as Providencia sp. by analyzingthe16S rDNA sequence and conducting the physiology and chemistry experiment. Theisolated strain, named as Providencia sp. JNB815, was preserved in CGMCC.(2) The fermentation condition was optimized based on single factor experiment. Theoptimal medium was identified as follows: glucose30g·L-1, yeast extract5g·L-1, peptone10g·L-1, beef extract5g·L-1, KH2PO42g·L-1, urea8g·L-1, NaAc2g·L-1, NaCl5g·L-1, MnSO40.02mmol·L-1, NiSO40.08mmol·L-1, pH4.5, Cultivation16h. Under the optimal condition,the acid urethanase activity was increased from0.32U mL-1to0.87U mL-1and acid ureaseactivity from0.81U·mL-1to2.28U·mL-1.(3) The enzymes were primarily purified with the method of (NH4)2SO4precipitationDEAE anion exchange chromatograthy and Superdex gel filtration. The enzymes specificactivity increase to5.6folds compared with the crude enzymes, and the activity recovery is3.3%. The substrate specificity was investigated with many structural analogues serving assubstrate. The purified enzymes showed catalytic activity to urea and EC specially.(4) The purified urease and urethanase showed maxium activity at pH4.5and35oC. It isstable within pH4.0-7.0and30-55oC. The Michaelis-Menten constant (Km) of the enzymesfor urea and EC were7.9mmol·L-1and138mmol·L-1. The maximum reaction (Vmax) were2.09mmol·L-1·min-1and1.49mmol·L-1·min-1.70%activity still remains while it catalyze thehydrolysis of urea and EC in modeling wine system. The results revealed that enzymes have apotential application prospect in the removal of urea and EC in Chinese rice wine.(5) The effect of the enzyme process on the urea and EC degradation in Chinese ricewine is evaluated. The experiment showed that enzymes can effectively decompose the ureaand EC in Chinese rice wine and had little effect on the flavor components.(6) Part of the UreC sequence is accessed and identified with the PCR of degeneratedprimers. The complete urease gene was obtained with different PCR strategies. Analyzed withVector NTI software and NCBI Blastx, the urease gene proved to have7open reading framedesignated as UreA UreB UreC UreE UreF UreG UreD.