US EPA

7423084 

Journal Article 

Review 

Expression of an Acid Urease with Urethanase Activity in E. coli and Analysis of Urease Gene 

Liu, X; Zhang, Q; Zhou, N; Tian, Y; , 

2017 

1 

Molecular Biotechnology
ISSN: 1073-6085
EISSN: 1559-0305 

HUMANA PRESS INC 

TOTOWA 

59 

2-3 

84-97 

English 

28197768 

10.1007/s12033-017-9994-x 

WOS:000397823400004 

http://link.springer.com/10.1007/s12033-017-9994-x
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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 JN-B815, ureABC were efficiently expressed in E. coli BL21(DE3) 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 2.1 U/mg for urease and 0.6 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 co-expressed in E. coli BL21(DE3). The holoenzyme was expressed as inclusion body. After renaturation and purification, the specific activities of urease and urethanase reached 10.7 and 3.8 U/mg, which were 5.62-fold and 6.33-fold of those of apourease, respectively. Therefore, accessory subunits of urease play an important role in enhancing urease and urethanase activities. 

Apourease; Co-expression; Enzymatic properties; Inclusion body; Molecular docking; Providencia rettgeri; Urease; Urethanase; Alcoholic beverages; Beverages; Escherichia coli; Ethanol; Genes; Hydrogen bonds; Metabolism; Purification; Urea; Apourease; Co-expression; Enzymatic properties; Inclusion bodies; Molecular docking; Providencia rettgeri; Urease; Urethanase; Gene expression; alcohol; bacterial enzyme; holoenzyme; hydrogen; unclassified drug; urea; urease; urethanase; amidase; bacterial protein; holoenzyme; urease; urethanase; cell inclusion; controlled study; enzyme active site; enzyme activity; Escherichia coli; hydrogen bond; molecular docking; nonhuman; pH; protein expression; Providencia rettgeri; renaturation; Review; wine; analysis; chemistry; enzyme stability; enzymology; Escherichia coli; genetics; metabolism; molecular cloning; Providencia; Amidohydrolases; Bacterial Proteins; Cloning, Molecular; Enzyme Stability; Escherichia coli; Holoenzymes; Hydrogen-Ion Concentration; Molecular Docking Simulation; Providencia; Urea; Urease; Wine