Prokaryotic Expression Of Acid Urease From Providencia Rettgeri And Basic Analysis Of Protein Structure With Enzyme Activity

Ethyl carbamate（EC） was proved to be carcinogenic of multiple site and genotoxic. And many researches showed that EC found in alcoholic beverage and fermented foods endangered human’s health. With the mechanism of EC formation in fermented wines investigated, enzymatic elimination of EC was of great interest. Therefore, acid ureases with good tolerance towards ethanol and acid were focused on by researchers at home and abroad.In the present study, an acid urease from P. rettgeri JN-B815 was partially purified and crosslinked with genipin. It was turned out that when the concentration of genipin was 0.3% and the crosslinking time was 2.5 h, the amount of proteins crosslinked with genipin was maximum which was 118 mg. And after stored in 4 oC for 10 d, the relative urease activity of crosslinked enzyme aggregates of urease was over 60%.On the basis of urease complete gene sequence isolated by predecessor, the urease structural gene cluster ureABC was amplified to express in E. coli BL21（DE3） in a bioactive form. After optimization with single factor test, it was suggested that the optimal conditions were as follows. The concentration of Ni2+ was 1.5 mmol·L^-1, and the induction temperature was 25 oC. While the concentration of IPTG was 0.5 mmol·L^-1 and the induction time was 10 h. After optimization, urease activity of urease apoprotein expressed by ureABC reached 0.69 U·m L^-1 and urethanase activity was 0.29 U·mL^-1. The specific activity of urease and urethanase was 2.10 U·mg^-1 and 0.60 U·mg^-1 respectively after purified with Ni-NTA chromatography. The purification folds of urease and urethanase were 1.75 and 1.33, respectively. And it was confirmed that urease from P. rettgeri JN-B815 was a bioenzyme with urethanase activity.The recombinant urease apoprotein exhibited good stability in ethanol solution and acidic conditions. The relative activity of urease and urethanase remained over 80% in buffers of pH 4-7 and which was more than 50% in 5%-25% ethanol solutions. Additionally, the residual of urea in model wine was less than 50% after treated with urease apoprotein for 30 h.Homologous modeling was used to predict the 3D structure of UreC from P.rettgeri JN-B815 urease. And substrates of urea and urethane were docked with the predicted structure, respectively. The docking result showed that three hydrogen bonds were formed by urea and ammino acid residues in the center of urease active site. However, there was only one hydrogen bond between urethane and the ammino acid residues. Otherwise, urethane exhibited greater steric hindrance than urea when combined with urease active site.Due to low activities of urease apoprotein, another recombinant plasmid was constructed to co-express of structural gene ureABC and accessory gene ureEFGD. However, it was expressed as urease inclusion bodies. After naturation and purification, the specific activity of urease and urethanase was 11.80 U·mg^-1 and 3.80 U·mg^-1, respectively. Either of them enchanced 5.62 folds and 6.33 folds, respectively. The response surface methodology was used to study the influence of fermentation conditions on the structure of urease inclusion bodies. It came out that the optimal conditions were that induction temperature was 21.3 °C, IPTG concentration was 0.75 mmol·L^-1, and induction time was 12.5 h.The second structure prediction of urease was a little different from refolded urease which was measured by circular dichroism spectra. The refolded urease was consisted of 37.2% α helix, 20.1% β sheet and 42.7% random coli.