More than 20 fungi derived from marine sponge Siphonochalina sp. were screened for urease activity. Penicillium steckii S4-4 showed the highest activity and, thus, was selected for further investigation. Both NiSO4·6H2O and urea were added to examining the effect of Ni2+ and urea on the urease production of Penicillium steckii S4-4. As exhibited in Fig. 1, urease activity per milligram mycelium reached a climax when NiSO4·6H2O concentration was above 0.006% and the urease activity was enhanced along with the increased concentration of urea. However, the fungus cultivated in broth with 0.4% urea yielded the maximal biomass and thus showed the highest total urease activity (data not shown). As a result, it was found that 0.006% NiSO4·6H2O and 0.4% urea could increase urease production approximately 2.5 fold.
The protein quantification and urease activity of each purification fraction were assayed and are summarized in Table 1. The final purification recovered 17.54% of the original urease activity and reached a final specific activity of 1542.2 U mg protein-1 which was 61.4-fold of the crude extract activity. The 40-50% saturation fraction of ammonium sulfate precipitation showed the highest specific activity (62.36 U mg-1), followed by ion-exchange column chromatography where two peaks arose and the urease was eluted as a single peak in fraction 25 with 0.4 mol L-1 NaCl (Fig. 2a). During the last step, the elution profile (Fig. 2b) showed four peaks of proteins, and urease activity displayed a maximum at fraction 24 with a single peak.
Fraction Specific activity (U mg−1) Total activity (units) Total protein (mg) Purification (fold) Yield (%) Crude extract 25.12 6596.01 262.58 1 100 Crude enzyme solution 62.36 4946.40 79.32 2.48 75 Hitrap-DEAE fraction 272.09 2345.42 8.62 10.83 35.56 Superdex-200 fraction 1542.20 1156.65 0.75 61.40 17.54
Table 1. Purification scheme of urease from P. steckii S4-4
Figure 2. Chromatography of P. steckii S4-4 urease through a a Hitrap DEAE FF column and b a Superdex 200 10/300 GL column. Absorbance at 280 nm (filled circle), Urease specific activity (open triangle), and NaCl concentration (dashed line) are interpreted
As native-PAGE shows in Fig. 3a, a purple-red colored band homogeneously occurred in each lane. Likewise, the number of protein bands decreased during the processing of separation and only one single protein band with a molecular weight of 47 kDa was observed in the Superdex-200 fraction via SDS-PAGE (Fig. 3b). The relative native molecular mass of P. steckii S4-4 urease was estimated to be approximately 183 kDa by gel filtration (Fig. 4).
Figure 3. PAGE of P. steckii S4-4 urease. a Native-PAGE of various purification steps fractions that were stained for urease activity. b Each fraction was analyzed by SDS-PAGE and compositions were revealed by Coomassie brilliant blue G-250 stain. Lane M, protein molecule weight marker; lane 1, crude extract; lane 2, crude enzyme solution; lane 3, Hitrap-DEAE fraction; lane 4, Superdex-200 fraction
Figure 4. Calibration curve of ovalbumin (43 kDa), bovine serum albumin (66 kDa), aldolase (158 kDa), β-amylase (200 kDa), ferritin (440 kDa) and thyroglobulin (669 kDa) using Superdex 200 10/300 GL column. Standard proteins are marked as solid spheres while urease from P. steckii S4-4 is labelled as hollow square
The N-terminal amino acid sequence of the purified 47 kDa monomer was (M) GPVLKKTKAAAV. The best alignment for P. steckii S4-4 urease was completed when a two amino acid gap was placed at the third and fourth residue position and a five amino acid gap appeared at the sixth to tenth position (Fig. 5). Overall, ureases from eukaryotic organisms showed a higher affinity to P. steckii S4-4 urease relative to other prokaryotic sources. The urease sequence with the greatest similarity to the P. steckii S4-4 urease was from Ectocarpus siliculosus, a kind of marine alga.
As demonstrated in Fig. 6, the purified urease exhibited an estimated Km and Vmax of 7.3 mmol L-1 and 1.8 mmol urea min-1 mg protein-1, respectively, at 55 ℃, pH 8.5 and salinity 10%. The effects of temperature, pH and salinity on urease activity were evaluated using the Jack bean urease as a control (Fig. 7). It was found that the optimal temperature, pH and salinity for the urease from P. steckii S4-4 were 55 ℃, 8.5 and 10%, respectively, while the Jack bean ureases showed the highest activity at 35 ℃, pH 7.0 and 5% salinity. The Jack bean urease was stable and retained more than 80% of its maximal activity at a temperature lower than 40 ℃ (Fig. 7b), pH 6.0-8.5 (Fig. 7d) or salinity gradient of 0-10% (Fig. 7f). In contrast, the marine urease from P. steckii S4-4 exhibited higher thermostability, broader pH resistance and stronger salinity tolerance. More than 80% of its maximal activity was maintained at temperatures up to 60 ℃, pH 5.5-10.0 or 0-25% salinity (Fig. 7b, d, f).
Figure 6. Effect of substrate concentration on P. steckii S4-4 urease activity. Inset shows a Lineweaver-Burk plot of the purified urease. Data are presented as the mean ± standard error (n = 3)
Figure 7. Effects of temperature, pH and salinity on urease activity of P. steckii S4-4 (filled circle) and Jack bean (filled triangle). Activity of urease at a temperature range of 25-70 ℃ (a), various pH condition of 4.0-10.0 (c) and a salinity concentration gradient of 0-25% (e) as well as the thermostability (b), pH resistance (d) and salt tolerance (f) of urease were determined, respectively. Data are presented as the mean ± standard error (n = 3)