Expression of an Acid Urease with Urethanase Activ

Authors: Xiaofeng Liu Qian Zhang Nandi Zhou Yaping Tian

Publish Date: 2017/02/14

Volume: 59, Issue: 2-3, Pages: 84-97

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Abstract

Urea in alcoholic beverage is a precursor of ethyl carbamate EC which is carcinogenic Enzymatic elimination of urea has attracted much research interest Acid urease with good tolerance toward ethanol and acid is ideal enzyme for such applications In the present work the structural genes of urease from Providencia rettgeri JNB815 ureABC were efficiently expressed in E coli BL21DE3 in an active form apourease exhibiting both urease and urethanase hydrolyze EC activities The specific activities of the purified apourease were comparatively low which were 21 U/mg for urease and 06 U/mg for urethanase respectively However apourease exhibited good resistance toward ethanol and acidic conditions The relative activities of urease and urethanase remained over 80 in the buffers within pH 4–7 And the recoveries of both urease and urethanase activities were more than 50 in 5–25 ethanol solution Apourease was utilized to eliminate urea in wine and the residual urea in model wine was less than 50 after treatment with apourease for 30 h Then 3D structure of UreC was predicted and it was docked with urea and EC respectively The docking result revealed that three hydrogen bonds were formed between urea and amino acid residues in the active site of urease whereas only one hydrogen bond can be formed between EC and the active center Moreover EC exhibited greater steric hindrance than urea when combined with the active site Due to the low specific activities of apourease both structural genes and accessory genes of urease were coexpressed in E coli BL21DE3 The holoenzyme was expressed as inclusion body After renaturation and purification the specific activities of urease and urethanase reached 107 and 38 U/mg which were 562fold and 633fold of those of apourease respectively Therefore accessory subunits of urease play an important role in enhancing urease and urethanase activitiesThis work was supported by the National Natural Science Foundation of China No 31271860 the Joint Innovation Fund of Jiangsu Province BY201402322 the Program for New Century Excellent Talents in University NCET120878 and the Fundamental Research Funds for the Central Universities JUSRP51402A There were no financial/commercial conflicts of interest in this article

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