• Journal of Microbiology and Biotechnology
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• v.14 no.6
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• pp.1142-1149
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• 2004

Streptococcus mutans has the capacity of inducing dental caries. Thus, to develop a novel way of preventing dental caries, a cell wall hydrolase-producing strain was isolated and its characteristics were investigated. Among 200 alkalophilic strains isolated from soil, 8 strains exhibited lytic activities against Streptococcus mutans. However, strain YU5215 with the highest cell wall hydrolase activity was selected for further study. Strain YU5215 was identified as a novel strain of Bacillus based on analyzing its 16S rDNA sequence and Bergey's Manual of Systematic Bacteriology, and thus designated as Bacillus mutanolyticus YU5215. The optimal conditions for the production of the cell wall hydrolase from Bacillus mutanolyticus YU5215 consisted of glucose ($0.8\%$), yeast extract ($1.2\%$), polypeptone ($0.5\%$), $K_{2}HPO_{4}\;(0.1\%$), $MgSO_{4}{\cdot}7H_{2}O$ ($0.02\%$), and $Na_{2}CO_{3}\;(1.0\%$) at pH 10.0. Bacillus mutanolyticus YU5215 was cultured at 30^{circ}C for 72 h to produce the cell wall hydrolase, which was then purified by acetone precipitation and CM-agarose column chromatography. The molecular weight of the lytic enzyme was determined as 22,700 Da by SDS-PAGE. When the cell wall peptidoglycan of Streptococcus mutans was digested with the lytic enzyme, no increase in the reducing sugars was observed, while the free amino acids increased, indicating that the lytic enzyme had an endopeptidase-like property. The amino terminus of the cell wall peptidoglycan digested by the lytic enzyme was determined as a glutamic acid, while the lytic site of the lytic enzyme in the Streptococcus mutans peptidoglycan was identified as the peptide linkage of L-Ala and D-Glu.

• Journal of Microbiology and Biotechnology
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• v.11 no.3
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• pp.508-514
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• 2001

A stuructural gene (ycl) encoding novel yeast cell wall hydrolase, YCL, was cloned from alkalophilic Bacillus alcalophilus subsp. YB380 by PCR, and transformed into E. coli JM83. Based on the N-terminal and internal amino acid sequences of the enzyme, primers were designed for PCr. The positive clone that harbors 1.8 kb of the yeast cell wall hydrolase gene was selected by the colony hybridization method with a PCR fragment as a probe. According to the computer analysis, this gene contained a 400-base-paired N-terminal domain of the enzyme. Based on nucletide homology of the cloned gene, a 850 bp fragment was amplified and the C-terminal domain of the enzyme was sequenced. With a combination of the two sequences, a full nucleotide sequence for YCL was obtained. This gene, ycl, consisted of 1,297 nucleotides with 27 nucleotides with 27 amino acids of signal sequence, 83 redundant amino acids of prosequence, and 265 amino acids of the mature protein. This gene was then cloned into the pJH27 shuttle vector and transformed into the Bacillus subtilis DB104 to express the enzyme. It was confirmed that the expressed cell wall hydrolase that was produced by Bacillus subtilis DB104 was the same as that of the donor strain, by Western blot using polyclonal antibody (IgY) prepared from White Leghorn hen. Purified yeast cell wall hydrolase and expressed recombinant protein showed a single band at the same position in the Western blot analysis.

• Food Engineering Progress
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• v.14 no.3
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• pp.263-270
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• 2010

An alkalophilic microorganism named DK-2386, which produces xylanase, was isolated from soil of Taejo-mountain, Cheonan-si, Chungnam, Korea. The isolated strain was characterized as Gram-positive, with size of 0.4${\times}$2.5 ${\mu}$m, spore forming, anaerobic, catalase positive, possessed with hydrolysis abilities of casein, starch, sodium carboxy methyl cellulose, and xylan, reduction of nitrate to nitrite, resistant against lysozyme, urease positive, and motility positive. The color of culture broth was reddish yellow. The strain DK-2386 was identified as Bacillus agaradhaerens by whole cell fatty-acid composition analysis and 16S rDNA sequence analysis. However, it was not identical to Bacillus agaradhaerens 40952 obtained from the Korean Culture Center of Microorganism in its colour of culture broth. Therefore, we have named the newly isolated strain as Bacillus agaradhaerens DK-2386.

• 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.

• Korean Journal of Microbiology
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• v.47 no.3
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• pp.194-199
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• 2011

An alkalophilic bacterium producing alkaline protease was isolated from waste water and solar saltern sample and identified as Bacillus pseudofirmus HS-54 based on morphological, biochemical characteristics as well as 16S-rRNA gene sequencing. The HS-54 protease was purified to homogeneity using ammonium sulfate precipitation, DEAE cellulose column chromatography, and sephadex G-100 gel filtration with a 4.0 purification fold. The molecular mass of the purified enzyme was estimated by SDS-PAGE to be 27 kDa. The optimal pH and temperature for the purified protease activity were 10.0 and $50^{\circ}C$, respectively. The purified enzyme was relatively stable at the pH range of 6.0-11.0 and at the temperature below $50^{\circ}C$. This enzyme was activated by $Ca^{2+}$ and $Mg^{2+}$ and inhibited by $Hg^{2+}$, $Cu^{2+}$, $Zn^{2+}$, $Al^{3+}$, $Ag^{2+}$. And this enzyme was strongly inhibited by PMSF, suggesting that it belongs to the serine protease superfamily.

• Microbiology and Biotechnology Letters
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• v.22 no.1
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• pp.59-64
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• 1994

The production and the characteristics of protease produced by Bacillus sp. SH-8 and Bacillus sp. SH-8M were investigated under the different pH conditions. Bacillus sp. SH-8 and Bacillus sp. SH-8M showed the maximum activity of protease at 60 hours(70 units/ml) AND 96 houre(50 units/ml) cultivation, respectively, under the alkaline condition(pH 10.2). However, Bacillus sp. SH-8M exhibited the maximum activities in 8 days cultivation at pH 6.9 and in 6 days cultivation at pH 7.7 Bacillus sp. SH-8M showed the protease activity at the pH change from alkaline to neutral condition, whereas Bacillus sp. SH-8 did not. In addition. all the enzymatic characteristics of protease produced by Bacillus sp. SH-8 and Bacillus sp. SH-8M were similar with the regardless of different pH conditions.

• Korean Journal of Food Science and Technology
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• v.23 no.4
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• pp.503-509
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• 1991

Action of a cyclodextrin glycosyltransferase (CGTase) produced from alkalophilic Bacillus sp. No.4 was studied in a solution containing starch and sucrose to prepare glycosyl sucrose syrup with good sweetness and antidecaying properties of teeth. In the initial stage of the reaction the CGTase produced cyclodextrin, however, the cyclodextrin disappeared and glycosyl sucrose was formed with the lapse of reaction time. The best proportion of sucrose to starch for prodution of glycosyl sucrose was about 1 : 1. The optimum pH and temperature of the coupling reaction was pH 6.0 and $60^{\circ}C$, respectively. Main composition of glycosyl sucrose syrup prepared with 20% starch and 20% sucrose was sucrose 18%, glucosyl sucrose ($G_{2}F$) 15.3% and maltosyl sucorse ($G_{3}F$) 11.3%. And glucose, maltose and maltotriose were produced very little. Smaller amounts of acid and insoluble glucan were formed in the syrup by Streptococcus mtans OMZ176 than in the sucrose. Therefore, the prepared glycosyl sucrose sucrose syrup is expected to prevent teeth from decaying.

• Proceedings of the Microbiological Society of Korea Conference
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• 2005.05a
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• pp.202.2-202
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• 2005

• Proceedings of the Korean Society for Food Science of Animal Resources Conference
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• 2000.06a
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• pp.92-92
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• 2000

• Microbiology and Biotechnology Letters
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• v.14 no.6
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• pp.447-453
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• 1986

The culture filtrate of thermophilic alkalophilic Bacillus K17 strain contained two types of xylanases were purified by ammonium sulfate fractionation, DEAD-Sephadex A-50 column chromatography, CM-Sephadex C-50 column chromatography and Sephadex G-100 gel filtration. The purified enzymes were found to be homogeneous by sodium dodecyl sulfate and disc polyacrylamide gel electrophoresis. Xylanase I and II were characterized with respect to molecular weight, optimal temperature and pH, thermal and pH stability, and Michaelis constant. Xylanase II was more active and stable, and showed greater substrate affinity and molecular weight than xylanase I. The activities of xylanases I and II were inhibited by Cu$^{++}$, Ag$^+$, Hg$^{++}$ and Fe$^{++}$. Xylanase I hydrolyzed xylan to yield xylobiose and higher amount of xylooligosaccharides, but xylanase II produced xylose other than xylobiose and xylooligosacchrides.