A Bacillus paralicheniformis Iron-Containing Urease Reduces Urea Concentrations in Rice Wine

Abstract

Urease, a nickel-containing metalloenzyme, was the first enzyme to be crystallized and has a prominent position in the history of biochemistry. In the present study, we identified a nickel urease gene cluster, ureABCEFGDH, in Bacillus paralicheniformis ATCC 9945a and characterized it in Escherichia coli. Enzymatic assays demonstrate that this oxygen-stable urease is also an iron-containing acid urease. Heterologous expression assays of UreH suggest that this accessory protein is involved in the transmembrane transportation of nickel and iron ions. Moreover, this iron-containing acid urease has a potential application in the degradation of urea in rice wine. The present study not only enhances our understanding of the mechanism of activation of urease but also provides insight into the evolution of metalloenzymes.IMPORTANCE An iron-containing, oxygen-stable acid urease from B. paralicheniformis ATCC 9945a with good enzymatic properties was characterized. This acid urease shows activities toward both urea and ethyl carbamate. After digestion with 6 U/ml urease, approximately 92% of the urea in rice wine was removed, suggesting that this urease has great potential in the food industry.

Construction of Synthetic Promoters by Assembling the Sigma Factor Binding −35 and −10 Boxes

“…To produce L‐asparaginase (EC 3.5.1.1, ASN), the asn gene (Gene Bank: EF382347.1) was amplified from the genome of E. coli BL21(DE3) with primers asn‐F and asn‐R and replaced the egfp of pCOLADuet‐P 21285 ‐ egfp to generate pCOLADuet‐P 21285 ‐ asn or inserted into pCOLADuet‐1 to make pCOLADuet‐ asn . To produce urease (EC 3.5.1.5), the urease gene cluster was amplified from the plasmid pRSFDuet‐ Bpure‐2 constructed in our previous work, with primers ure‐F and ure‐R and digested with Bgl II and Xho I before being ligated into equally treated plasmids pCOLADuet‐1 and pCOLADuet‐P 21285 ‐ egfp, to generate the pCOLADuet‐ urease and pCOLADuet‐P 21285 ‐ urease, respectively.…”

“…The mixture was incubated at 37 °C for 20 min and terminated by adding 200 µL of 10% trichloroacetic acid. After that, 200 µL of chromogenic reagent I (15 g phenol and 0.625 g sodium nitroprusside in 250 mL of ultrapure water) and 200 µL of chromogenic reagent II (13.125 g NaOH and 7.5 mL NaClO in 250 mL of ultrapure water) were added to the mixture and incubated for another 20 min before the absorbance at 625 nm was measured . One unit of enzyme activity was defined as the amount of enzyme required to hydrolyze 1 µmol urea (or L‐asparagine) in 1 min at 37 °C, pH 4.5 (for urease) or pH 7.5 (for L‐asparagine).…”

Marine urease with higher thermostability, pH and salinity tolerance from marine sponge-derived Penicillium steckii S4-4

“…Urease activity was measured by monitoring the rate of release of ammonium from urea. The concentration of ammonium in the reaction mixture was determined as indophenol produced by a phenol-hypochlorite reaction at 625 nm (Weatherburn 1967) and the main steps were executed as reported previously (Liu et al 2017a). Control assays included the detection of ammonium in the reaction mixture at time zero and when using boiled enzyme extract.…”

“…Analogously, the thermostability, pH stability and the tolerance to salinity of the urease were determined by contrasting the residual activity after pre-incubating the purified enzyme at different temperature for 30 min, pH at 4 °C for 6 h, or salinity gradients at 4 °C for 6 h, respectively. All tests were conducted in triplicate (Liu et al 2017a).…”

“…Urease (urea amidohydrolase, EC 3.5.1.5) as the first enzyme crystallized from Jack bean and the first enzymatic protein possessing nickel ions in the active site (Dixon et al 1975), hydrolyzes urea to yield ammonia and carbamate. To date, urease has been found widespread in organisms including archaea (Bhatnagar et al 1984), cyanobacteria (Carvajal et al 1982;Collier et al 1999;Palinska et al 2000;Rai 1989), bacteria (Benoit and Maier 2011;Cai and Ni 1996;Liu et al 2017a;Todd and Hausinger 1987;Turbett et al 1992), fungi (Geweely 2006;Jahns 1995;Lubbers et al 1996;Mirbod et al 2002), plants (Das et al 2002;El-Hefnawy et al 2014;Hirayama et al 2000;Menegassi et al 2008) and invertebrates (Mcdonald et al 1980). Urease has been well used in determining the concentration of blood urea in clinical diagnosis (Qin and Cabral 2002), removing the urea lest forming carcinogenic ethyl carbamate in alcoholic beverages (Yang et al 2015), detecting heavy metal ion pollutants in environmental protection (Preininger 1999;Wittekindt et al 1996), and eliminating urea in industrial wastewaters (George et al 1997).…”