• Journal of Microbiology and Biotechnology
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• v.6 no.1
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• pp.26-29
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• 1996

The genes coding for the urease of alkalophilic Bacillus pasteurii have been previously cloned and recently sequenced. (You, J. H., B. H. Song, J. H. Kim, M. H. Lee, and S. D. Kim (1995) Molecules and Cells 5, 359-369.) The recombinant Bacillus pasteurii urease expressed in an E. coli HB101 strain was purified 31.2 fold by using combinations of anion-exchange and hydrophobic chromatography followed by Mono-Q chromatography on a FPLC. In spite of the presence of three discrete structural peptide genes in the Bacillus pasteurii urease gene cluster, only one or two enzyme subunits have been observed to date. Here we report for the first time that the recombinant Bacillus pasteurii urease expressed in a E. coli strain consists of three distinct subunits. One large subunit was estimated to be of $M_r$=65, 200 and the two small-subunit peptides are of $M_r$=14, 500 and $M_r$=13, 700, respectively.

• Journal of Microbiology and Biotechnology
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• v.4 no.2
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• pp.108-112
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• 1994

The genetic organization of the urease gene cluster from an alkalophilic Bacillus pasteurii was determined by subcloning and Tn5 transposon mutagenesis of a 10.7 kilobasepair cloned fragment. A region of DNA between 5.0 and 6.0 kb in length is necessary for urease activity. In vitro transcription-translation analysis of transposon insertion mutants of the cloned urease genes demonstrated that the major ($M_r$ 67,000) and minor ($M_r$ 20,000) structural peptides of urease are encoded at one end of the urease gene cluster and at least 3 additional polypeptides are encoded by adjacent DNA sequences.

• Microbiology and Biotechnology Letters
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• v.25 no.1
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• pp.30-36
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• 1997

To genetically breed powerful multifunctional antagonistic bacteria, the urease gene of alkalophilic Bacillus pasteurii was transferred into Bacillus subtilis YB-70 which had been selected as a powerful biocontrol agent against root-rotting fungus Fusarium solani. Urease gene was inserted into the HindIII site of pGB215-110 and designated pGU266. The plasmid pGU266 containing urease gene was introduced into the B. subtilis YB-70 by alkali cation transformation system and the urease gene was very stably expressed in the transformant of B. subtilis YB-70(pGU266). The optimal conditions for the transfomation were also evaluated. From the in vitro antibiosis tests against F. solani, the antifungal activity of B. subtilis YB-70 containing urease gene was much efficient than that of the non-transformed strain. Genetic improvement of B. subtilis YB-70 by transfer of urease gene for the efficient control seemed to be responsible for enhanced plant growth and biocontrol efficacy by combining its astibiotic action and ammonia producing ability.

• Microbiology and Biotechnology Letters
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• v.20 no.5
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• pp.519-526
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• 1992

The gene coding for urease of alkalophilic Bacillus pasteurii had been cloned in Escherichia coli previously. The urease protein was purified 63.1-fold by TEAE-cellulose, DEAE-Sephadex A-50, Sephadex G-150 and Sephadex G-200 chromatographies with a 7.3% yield from the sonicated fluid of the E. coli HB1Ol(pBUll) encoding B. pasteurii urease gene. The ureases of E. coli (pBUll) and B. pasteurii possessed as a $K_m$ for urea, 42.1 mM and 40.4 mM, respectively. They hydrolyzed urea with $V_{max}$ of 86.9$\mu$mol/min and 160$\mu$mol/min, respectively. Both ureases were composed with four subunits (Mrs 67,000) and a subunit (Mr 20,000). The molecular weight of both native enzymes was Mr 280,OOO$pm$10,000 determined by gel filtration chromatography and Coomassie blue staining of the subunits. The optimal reaction pH of both ureases were pH 7.5. The ureases were stabled in pH 5.5-10.5. The optimal reaction temperature of both ureases were $60^{\circ}C$, and the ureases were stable for an hour at $50^{\circ}C$, 40min at $60^{\circ}C$ and 10 min at $70^{\circ}C$ The activity of both enzymes were inhibited completely by $Ag^{2+}$, $Hg^{2+}$, $Zn^{2+}$, $Cu^{2+}$, and were inhibited 60% by CoH, 30% by $Fe^{2+}$ and 10% by $Pb^{2+}$. However it was increased by the addition of $Sn^{2+}$, $Mn^{2+}$, $Mg^{2+}$ at concentration of $1{\times}10^{-3}$M. Both ureases were inhibited completely by p-CMB and acetohydroxamic acid. The urease expressed in E. coli (pBU11) was inhibited 70% by SDS. The urease of B. pasteurii was inhibited 40% by hydroxyurea, whereas the recombinant urease of E. coli strain was inhibited 17%. Both enzymes were not inhibited by cyclohexanediaminetetraacetic acid (CDTA) and ethylendiaminetetraacetic acid (EDTA).

• Microbiology and Biotechnology Letters
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• v.26 no.2
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• pp.122-129
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• 1998

It were reported that antifungal mechanism of Enterobacter cloacae is a volatile ammonia that produced by the strain in soil, and the production of ammonia is related to the bacterial urease activity. A powerful bacterium SH14 against soil-borne pathogen Fusarium solani, which cause root rot of many important crops, was selected from a ginseng pathogen suppressive soil. The strain SH14 was identified as Bacillus subtilis by cultural, biochemical, morphological method, and $API^{circledR}$ test. From several in vitro tests, the antifungal substance that is produced from B. subtilis SH14 was revealed as heat-stable and low-molecular weight antibiotic substance. In order to construct the multifunctional biocontrol agent, the urease gene of Bacillus pasteurii which can produce pathogenes-suppressive ammonia transferred into antifungal bacterium. First, a partial BamH I digestion fragment of plasmid pBU11 containing the alkalophilic B. pasteurii l1859 urease gene was inserted into the BamH I site of pEB203 and expressed in Escherichia coli JM109. The recombinant plasmid was designated as pGU366. The plasmid pGU366 containing urease gene was introduced into the B. subtilis SH14 with PEG-induced protoplast transformation (PIP) method. The urease gene was very stably expressed in the transformant of B. subtilis SH14. Also, the optimal conditions for transformation were established and the highest transformation frequency was obtained by treatment of lysozyme for 90 min, and then addition of 1.5 ${mu}g$/ml DNA and 40% PEG4000. From the in vitro antifungal test against F. solani, antifungal activity of B. subtilis SH14(pGu366) containing urease gene was much higher than that of the host strain. Genetical development of B. subtilis SH14 by transfer of urease gene can be responsible for enhanced biocontrol efficacy with its antibiotic action.