Comparative characterization of urease secreted by bacterial and fungal isolates from soil samples of farm fields

Abstract

ABSTRACT newlineThe aim of the study was to isolate the extracellular urease producing bacterial and fungal microbes from soil samples of farm fields. Bacterial isolate designated as BS-13 and fungal isolate designated as FS-13 were selected on the basis of qualitative and quantitative screening analysis for urease activity. The bacterial isolate BS-13 was Gram s positive and motile. Colony morphology of BS-13 was creamish white, flat and circular in form with undulated margins. This bacterial isolate BS-13 was positive for VP test, citrate utilization, casein hydrolysis, starch hydrolysis, oxidase and catalase activity but negative for indole and methyl red test. On the basis of biochemical tests and 16S rDNA sequence analysis, the BS-13 was identified as Bacillus species. The fungal isolate consisted of dense layer of brown-black conidial heads on white-yellow mycelia base. On the basis of colony morphology, LCB staining and ITS region sequencing, the fungus was identified as Aspergillus niger. A 1488 bps 16S rDNA and 898 bps ITS nucleotide sequence of BS-13 and FS-02 isolates was submitted to NCBI under the accession number KM668223 and KM461718, respectively. The temperature, pH, substrate concentration, and time required for optimum production of urease by Bacillus sp. was 35 °C, pH 8.0, 3 mM of urea, and 48 h, respectively. The optimum incubation time period, temperature, pH and substrate concentration for production of urease enzyme by Aspergillus niger was found to be at 120 h, 30 °C, pH 5.0 and 50 mM of urea, respectively. Urease production by Bacillus sp. and Aspergillus niger with their above mentioned optimized conditions showed a production of 2.52 U/ml and 3.06 U/ml, respectively. The urease activity of Bacillus was inhibited by 97%, and 50% in the presence of Zn2+ and Cu2+ salts, respectively. The urease activity of Bacillus was inhibited 60% to 68% with the addition of Co2+, Ca2+, Ni2+ and Fe3+ at concentration of 10 mM. The urease activity of Aspergillus was inhibited 66%, 58%, and 57% in the presence of Ca2+, Fe3+ and Co2+ salts, respectively at 10 mM concentration. The urease activity of Aspergillus was inhibited 20% to 25% with the addition of Zn2+, Cu2+, Ni2+ at concentration of 10 mM. From the phylogenetic analysis, it has been found that isolated strains Bacillus sp. and Aspergillus niger could be new strains of Bacillus and Aspergillus species. Urease produced by Bacillus sp. and Aspergillus niger was partially purified by ammonium sulphate precipitation with 60% saturation, and fold purification was 3.68 and 2.01 for newlinevii newlineBacillus and Aspergillus niger urease, respectively. The urease of free and immobilized state was compared as a function of thermal stability, pH, incubation time and storage stability. For both free and immobilized state of urease, optimal temperature was observed at 55 °C and 60 °C from Bacillus sp. and Aspergillus niger, respectively. Reaction time for immobilized enzyme assay was increased by 10 minutes with reference to free enzyme that was from 10 to 20 minutes. The immobilized state of urease showed an increase in optimal pH from pH 7.5 to 8.0 for maximum urease activity over free state of urease from Bacillus and Aspergillus niger. The immobilized state of enzyme showed 25% loss in activity, whereas free urease showed 50% loss in activity on storage of 30 days from both the microbial strains. newline