• Applied Biological Chemistry
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• v.40 no.6
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• pp.495-500
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• 1997

To isolate a gene for cyclodextrin glycosyltransferase (CGTase) from alkalophilic Bacillus sp. E1, polymerase chain reaction (PCR) amplification was carried out. Direct molecular cloning of 1.2 kbp fragment and partial nucleotide sequence analysis of the PCR amplified clone, pH12, showed close homology with CGTases from Bacillus species. To investigate the genomic structure of the gene, Southern blot analysis of genomic DNA was carried out with the clone pH12 as a molecular probe. It showed that 5.3 kbp XbaI fragment was hybridized with the probe pH12. To isolate a genomic clone, genomic DNA library was constructed and a genomic clone for CGTase, pCGTE1, was isolated. Nucleotide sequence analysis of the clone pCGTE1 revealed that BCGTE1 contained 2,109 bp open reading frame encoding a polypeptide of 703 amino acids and showed over 94.3% amino acid sequence homology with CGTase of ${\beta}-cyclodextrin$ producer, Bacillus sp. KC201.(Received October 7, 1997; accepted October 20, 1997)

• Korean Journal of Environmental Biology
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• v.17 no.2
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• pp.191-198
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• 1999

Enzyme-aided bleaching of softwood and hardwood kraft pulps by a xylanase preparation from an alkalophilic fungus Cephalospotium sp. RYM-202 was studied. Maximal solubilization of Pulp xylan was obtained at 5$0^{\circ}C$ in both kraft pulps. The optimum pH of the enzyme for the hydrolysis of pulp xylan was 8.0 and more than 90% of the maximal activity was detected at 9.0. The positive effects of xylanase pretreatment on bleachability of softwood and hardwood kraft pulps were observed. The kappa number of softwood and hardwood kraft pulps was decreased by 3.7 and 2.0 units, respectively. The pulp fibre integrity was not significantly affected by xylanase pretreatment when the physical properties of handsheets made from xylanase-treated pulps were compared with those of handsheets from untreated pulps. These results indicate that the alkaline xylanase of Cephalospotium sp. RYM-202 is well suitable for application in enzymatic prebleaching of softwood and hardwood kraft pulps under the alkaline conditions.

• Microbiology and Biotechnology Letters
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• v.20 no.4
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• pp.371-376
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• 1992

The growth and cultural characteristics of Bacillus sp. SH-8 and SH-8M were investigated at various pH conditions. Bacillus sp. SH-8 showed normal growth pattern above pH 9.0. However, with the pH adjusted below 7.7, 0.$D_{550}$ decreased rapidly with concomitant reduction in viable cell numbers. In contrast, Bacillus sp. SH-8M demonstrated growth capability at pH 7.7, but with slightly reduced growth rate at pH 6.9. Similar results were obtained when those two strains were cultivated on the solid medium. Both of them showed short rod shapes at pH 10.2. However, at pH 7.7 only Bacillus sp. SH-8 was observed to have elongated rod shape. Extracellular pH of both the strains, when cultured at initial pH of 10.2, reached to 9.0 after the incubation of 28 hours. At the initial pH of 9.0 and 9.6, the extracellular pH was reduced at the beginning of cultivation, but elevated after 12 hours. When cultured at initial pH of 6.9 and 7.7, extracelluar pH of Bacillus sp. SH-8M increased to 8.0 and 8.7, respectively, while that of Bacillus sp. SH8 remained constant pH 7.0. The highest sporulation rate of Bacillus sp. SH-8 and SH-8M was obtained at the initial pH of 10.2 and after the incubation of 3 days with the sporulation rate of 95% and 85%, respectively.

• Journal of agriculture & life science
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• v.45 no.6
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• pp.201-212
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• 2011

The alkaline protease gene was cloned from a halo-tolerant alkalophilic Bacillus clausii I-52 isolated from the heavily polluted tidal mud flat of West Sea in Inchon Korea, which produced a strong extracellular alkaline protease (BCAP). Based on the full genome sequence of Bacillus subtilis, PCR primers were designed to allow for the amplification and cloning of the intact pro-BCAP gene including promoter region. The full-length gene consists of 1,143 bp and encodes 381 amino acids, which includes 29 residues of a putative signal peptide and an additional 77-amino-acid propeptide at its N-terminus. The mature BCAP deduced from the nucleotide sequence consists of 275 amino acids with a N-terminal amino acid of Ala, and a relative molecular weight and pI value was 27698.7 Da and 6.3, respectively. The amino acid sequence shares the highest similarity (99%) to the nattokinase precursor from B. subtilis and subtilisin E precursor from B. subtilis BSn5. The substrate specificity indicated that the recombinant BCAP could hydrolyze efficiently the synthetic substrate, N-Suc-Ala-Ala-Pro-Phe-pNA,and did not hydrolyze the substrates with basic amino acids at the P1 site. The recombinant BCAP was strongly inhibited by typical serine protease inhibitor, PMSF, indicating that BCAP is a member of the serine proteases.

• The Korean Journal of Mycology
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• v.50 no.1
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• pp.65-73
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• 2022

This study aimed to isolate wild yeasts from seawaters and soils samples of the Haeundae and Mongdol beaches on the southern coast of Korea, and to characterize these unrecorded wild yeast strains. In total, 41 strains, representing 37 different species of wild yeast were isolated from 70 samples collected from the beaches. Among these, 14 strains were isolated from the alkalophilic medium of yeast extract-peptone-dextrose (YPD) medium (pH 9.0), and 27 strains were isolated concurrently on general YPD medium (pH 6.5). Among the 41 isolated wild yeast strains, Candida insectorum HUD 16-3(JSL-KSS-002) and Metschnikowia citriensis HUD 12-5(JSL-KSS-001) had not previously been recorded. We investigated the microbiological characteristics of these two unrecorded yeast strains and three other strains-, Cystobasidium lysinophilum JSC 52-2(JSL-GGU-019), Candida takata NMD 11-1(JSL-GGU-017) and Candida panamensis ASG 58M-2(JSL-GGU-018) from Jangseoncheon in Jellabuk-do and Jangtaesan in Deajeon city. All five previously unrecorded yeasts were oval and did not form spores. All strains grew well in YPD and yeast extract-malt extract media in a vitamin-free medium. Two strains, including C. insectorum HUD 16-3(JSL-KSS-002) grew well in a 15% NaCl-containing YPD medium. Three strains, including Cys. lysinophilum JSC52-2(JSL-GGU-019) assimilated lactose, and all strains assimilated starch.

• 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.