• Journal of Microbiology and Biotechnology
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• v.14 no.4
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• pp.759-765
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• 2004

The NCS inhibited the activity of glucosyltransferase which was produced by Streptococcus mutans JC-2, and the rate of inhibition at $100\muM<$ and $200\muM$ were 74.0% and 99.8%, respectively. It was stable in alkali condition, but unstable in acid condition. It was also stable up to $80^{\circ}C$. The kinetic study of the inhibition by NCS was carried out by Lineweaver-Burk plot and Dixon plot. It was non-competitive inhibition, determined by the two plots and $K_i$ and $K_i$ values were $15\muM$ and $19.3\muM$ respectively. The NCS did not show cytotoxicity against human gingival cells at $K_i$ ($15\muM$, $150\muM$, $1,500\mu$ M) concentrations. It had less cytotoxicity than chlohexidin, which has usually been used as the agent of anticaries. To evaluate the industrial applicability of the NCS, human pluck tooth was used. The inhibitory rates of tooth calcification and calcium ion elution by the NCS were 41 % and 2.5 times, respectively. These results suggested that NCS from Bacillus sp. S-1013 is an efficient anticaries agent.

• Microbiology and Biotechnology Letters
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• v.18 no.4
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• pp.344-351
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• 1990

An alkalophilic microoganism producing a detergent-resistant alkaline protease was isolated from soil and identified as Baeiltus sp. The alkaline protease has been partially purified by ammonium sulfate fractionation, DEAE-Cellulose, CM-Cellulose and Sephdex G-100 column chromatography. The purified alkaline protease was highly active at pH 12-13 toward casein and stable at pH values from 6 to ll. The optimum temperature for the enzyme reaction was $55^{\circ}C$. The enzyme was completely inactivated by diisopropyl fluorophosphate (DFP) indicating that the enzyme was serine protease, but considerabiy stable in the presence of surface active agents.

• 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.

• Korean Journal of Food Science and Technology
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• v.23 no.3
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• pp.349-354
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• 1991

An alkaline amylase-producing Bacillus sp. GM8901 was isolated and some properties of crude enzyme extract were examined. The microbiological and biochemical characteristics of GM8901 were very similar to those of B. licheniformis. The optimal temperature and pH for the cell growth and amylase production were $50^{\circ}C$ and pH 10.5. The crude amylase extract showed that the optimal temperature and pH were $50{\sim}60^{\circ}C\;and\;pH\;10{\sim}12$, respectively, and that the activity of amylase was stable up to $50^{\circ}C$ and in the range of $pH\;3{\sim}12$.

• Journal of Microbiology and Biotechnology
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• v.9 no.6
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• pp.811-819
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• 1999

The ${\beta}-CGTase$ gene of alkalophilic Bacillus firmus var. alkalophilus was cloned into E. coli using $pZErO^{TM}-2$ as a vector. The cloned gene encoded a total of 710 amino acid residues consisting of 674 amino acids of the matured protein and 36 amino acids of the signal peptide, including 20 amino acids from the lacZ gene in the vector. Although the cloned ${\beta}-CGTase$ gene did not contain the promoter and start codons, it was expressed by the lac promoter and lacZ start codon in the $pZErO^{TM}$ vector. A comparison was made with the amino acid sequence and ten other CGTases from Bacillus sp. Also, ten highly conserved regions, which are important amino acid residues in catalysis of CGTase, were identified. The lac promoter used for expression of the ${\beta}-CGTase$ gene was induced constitutively in recombinant E. coli even without IPTG possibly because of a lack of the lacI gene in both host and vector, repressing the lacZ gene in the lac operon. Its expression was catabolically repressed by glucose, however, its repression was reduced by soluble starch, mainly because of the extremely high increase of the cAMP level. ${\beta}-CGTase$ can be overproduced in the recombinant E. coli by maintaining intracellular cAMP levels mostly through the intermittent feeding of glucose during cultivation.

• Microbiology and Biotechnology Letters
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• v.20 no.3
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• pp.271-279
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• 1992

Genomic DNA of Bacillus stearothemzophilus, which expressed alkalophilic and thermophilic xylanases, was partially digested with HindIII, cloned into pBR322, and subsequently transferred into the Escherichra coli HB101 cells. Three among 5, 000 transformants screened formed clear zones around their colonies. From the functional clones, three recombinant plasmids (pMG11, pMG12 and pMG13) had been isolated, and they were identified to carry the same 4 kb HindIII fragment originated from B. stearothemzophilus which was responsible for the xylanase activity. pMGl3, however, had the foreign DNA of opposite orientation compared to the other two recombinant plasmids. This recombinant plasmid gave much lower xylanase activity. B. stearothermophilus was observed to produce at least three xylanase activities as evidenced by the PAGE-xylan zymogram. The xylanase from E. coli HB101/pMG12 was judged to correspond to the largest among the three B. stearothermophilus xylanases observed in the zymogrom. The enzyme hydrolyzed xylooligosaccharides larger than xylotriose and degraded xylan to produce xylobiose and xylotriose as major products. The xylanase was considered to have trans-xylosidase activity, too.

• 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)

• 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.

• Journal of Microbiology and Biotechnology
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• v.22 no.1
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• pp.58-68
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• 2012

An investigation was conducted on the enhancement of production and purification of an oxidant and SDS-stable alkaline protease (BHAP) secreted by an alkalophilic Bacillus horikoshii, which was screened from the body fluid of a unique Korean polychaeta (Periserrula leucophryna) living in the tidal mud flats of Kwangwha Island in the Korean West Sea. A prominent effect on BHAP production was obtained by adding 2% maltose, 1% sodium citrate, 0.8% NaCl, and 0.6% sodium carbonate to the culturing medium. The optimal medium for BHAP production contained (g/l) SBM, 15; casein, 10; $K_2HPO_4$, 2; $KH_2PO_4$, 2; maltose, 20; sodium citrate, 10; $MgSO_4$, 0.06; NaCl, 8; and $Na_2CO_3$, 6. A protease yield of approximately 56,000 U/ml was achieved using the optimized medium, which is an increase of approximately 5.5-fold compared with the previous optimization (10,050 U/ml). The BHAP was homogenously purified 34-fold with an overall recovery of 34% and a specific activity of 223,090 U/mg protein using adsorption with Diaion HPA75, hydrophobic interaction chromatography (HIC) on Phenyl-Sepharose, and ion-exchange chromatography on a DEAE- and CM-Sepharose column. The purified BHAP was determined a homogeneous by SDS-PAGE, with an apparent molecular mass of 28 kDa, and it showed extreme stability towards organic solvents, SDS, and oxidizing agents. The $K_m$ and $k_{cat}$ values were 78.7 ${\mu}M$ and $217.4s^{-1}$ for N-succinyl-Ala-Ala-Pro-Phe-pNA at $37^{\circ}C$ and pH 9, respectively. The inhibition profile exhibited by PMSF suggested that the protease from B. horikoshii belongs to the family of serine proteases. The BHAP, which showed high stability against SDS and $H_2O_2$, has significance for industrial application, such as additives in detergent and feed industries.

• Journal of Microbiology and Biotechnology
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• v.12 no.5
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• pp.753-759
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• 2002

$\beta$-Cyclodextrin glucanotransferase ($\beta$-CGTase) was overproduced extracellularly using recombinant E. coli by transforming the plasmid pECGT harboring a secretive signal peptide. The $\beta$-CGTase gene of alkalophilic Bacillus firmus var alkalophilus was inserted into the high expression vector pET20b(+) containing a secretive pelB signal peptide, and then transformed into E. coli BL2l(DE3)pLysS. The optimum culture conditions fer the overproduction of $\beta$-CGTase were determined to be TB medium containing 0.5% (w/v) soluble starch at post-induction temperature of $25^{\circ}C$. A significant amount of $\beta$-CGTase, up to 5.83 U/ml, which was nine times higher than that in the parent strain B. firmus var. alkalophilus, was overproduced in the extracellular compartment. A pH-stat fed-batch cultivation of the recombinant E. coli was also performed to achieve the secretive overproduction of $\beta$-CGTase at a high cell density, resulting in production of up to 21.6 U/ml of $\beta$-CGTase.