Purification, Analysis Of One Acid Urease And Identification Of Producing Strain

Since some kinds of acid urease can exert catalytic activity in low pH range and tolerate to ethanol, it bears the potential to be applied to wine-making industry, especially in Chinese rice wine. The objects of this paper were to identify a strain with acid urease catalytic activity, named JN_R12, and based on the results of its phenotypic, physiological-biochemical properties, 16S rDNA sequence similarity and phylogenetic analysis by using 16S rDNA sequencing, the urease family proteins were searched and aligned. The primary structure of JN_R12 was explored as well.With the results of both phylogenetic and genetic identification, JN_R12 was homologous to Enterohemorrhagic E.coli (EHEC) O157:H7 str. Sakai. Urea elimination ratio of centrifugal cells in simulation wine reached almost 100% within 24 hours. When applying its urease to Chinese rice wine, up to 65.2% of the urea was eliminated after 24 h incubation.The alignments of amino acids and nucleotide sequences showed a great conservation among bacterias of the same family. Distribution of E.coli JN_R12 urease was researched, and there basically existed an ure operon with multiple subunits, but somehow a little different from those urease operon reported.The conditions for exacting crude enzyme were improved. The crude enzyme was purified by using Ultrogel AcA 34, DEAE 52 and Hitrap Q with a final yield and purification-fold of 15.88% and 7.28, respectively. One subunit of preliminarily purified urease was identified by peptide mass fingerprinting analysis after SDS-PAGE electrophoresis, leading to the conclusion that the 69-kDa urease subunit had great similarity with the urease protein (gi 69936847) from Paracoccus denitrificans PD1222, and was identified as a novel urease protein.