The Screening,Characterization And Application Of Urease From Sponge-Derived Fungi

Urease,catalyzing the hydrolysis of urea,has extensive applications in the field of medicine,food industry and environmental sciences.The finding of novel ureases with superior performance has been one of the research trends.The current reported ureases are mainly from terrestrial organisms and the researches of marine-derived ureases are still very limited.Therefore,to determine if the marine-derived ureases have different characteristics from the terrestrial origins,microbes were isolated from the sponge Siphonochalina sp.in the South China Sea and Pennicillium steckii S4-4with urease activity was selected for the following investigations in this study.The urease production of P.steckii S4-4 reached maximum of 21.5U/mg mycelia after a 96-h fermentation with 0.006%NiSO₄·6H₂O and 0.4%urea in the broth.The specific activity of the crude extract of the urease was optimized to 25.12 U/mg protein when prepared by liquid nitrogen grinding combined with ultrasonic crushing.The crude extract from P.steckii S4-4 was purified 61.4-fold to homogeneity with a recovery of 17.54%and exhibited a maximum specific activity of 1542.2 U/mg after prepared successively by ammonium sulfate precipitation,Hitrap DEAE anion exchange chromatography and Superdex200 gel filtration chromatography.Only one species of urease was detected by nondenaturing PAGE with specific enzyme activity staining.The molecular weight of the enzyme was 183 kDa estimated from gel filtration and a single subunit was detected with a molecular mass of 47 kDa in SDS-PAGE stained with coomassie blue,indicating it was a tetramer formed as?₄.The N-terminal amino acid sequence of the urease was arranged as GPVLKKTKAAAV with the highest homology to the marine algae Ectocarpus siliculosus.The urease exhibited a K_m of 7.3 mM and a V_max of1.8 mM/min/mg.The optimum temperature,pH and salinity for the urease activity were 55?,8.5 and 10%,respectively.The enzyme retained more than 80%of its maximum specific activity after incubating at 25-60?for30 min,pH 5.5-10.0 or 0-25%salinity for 6 h.Compared with the terrestrial Jack bean urease,the marine urease showed higher thermostability,alkaline preference and salinity tolerance.The urease was immobilized by sodium alginate,polyvinyl alcohol and gelatin separately followed by the evaluation of the immobilization effects and the enzyme properties.Urease immobilized by alginate-CaCl₂ with an immobilization rate of 61.47%was selected.Comparing to the soluble urease with optimum temperature 55?and pH 8.5,the immobilized urease increased to 70?,pH 9.5,respectively.More than 90%of its maximal activity was detected after incubating at 35-85?for 30 min or at pH 7-10.5for 6 hours.Furthermore,its half-life exceeded three months when kept at4?,suggesting the improvements on thermostability and alkali resistance after immobilization.In the?2 cm×20 cm column loaded with the immobilized urease,90.25%decomposition rate of urea was achieved at70?,with a flow rate of 19 mL/min,and the efficiency remained constant for 35 days.The half-life of the immobilized enzyme was 65 days and the corresponding degradation rate of urease could be maintained at 50%.So,the immobilized marine urease presents a considerable alternative technique for the urea wastewater treatment.