• 한국미생물·생명공학회지
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• 제23권6호
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• pp.671-677
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• 1995

An extracellular enzyme showing lytic activity on E. coli peptidoglycan had been isolated from Bacillus sp. BL-29. The lytic enzyme was purified to homogeneity by ion-exchange chromatography and gel filtration, with a recovery of 5%. The enzyme was monomeric and had an estimated molecular weight of 31,000 Da. The mode of action of the purified enzyme was also investigated. When the purified lytic enzyme was incubated with cell wall peptidoglycan, N-terminal amino groups were released without the release of reducing groups. The N-terminal amino acid released was identified as dinitrophenylalanine (DNP-alanine) by analysis of terminal amino acid by dinitrophenylation method. This result suggests that the lytic enzyme should be a kind of N-acetylmura-myl-L-alanine amidase.

• Journal of Microbiology and Biotechnology
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• 제16권10호
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• pp.1613-1621
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• 2006

Isolation and identification of algal lytic bacteria were carried out. Nine strains of algal lytic bacteria were isolated by the double-layer method using Anabaena flos-aquae as a sole nutrient. The isolate, AFK-13, showing the highest algal lytic activity was identified as Sinorhizobium kostiense based on the l6S rDNA sequence. The algal lytic experiments of the culture supernatants of AFK-13 demonstrated that the bacterial cell growth reached a maximum at 36-h culture, but the supernatant of 72-h culture exhibited the highest activity. Components among the extracellular products in the crude enzyme of the supernatant from S. kostiense AFK-13 culture were responsible for degradation of cell walls of Anabaena flos-aquae. Algal lytic assay tests of the culture supernatants suggest that the main substances for algal lytic activity could be proteinaceous. The activity of glucosidase was observed highly by polysaccharolytic analysis using the crude enzyme from S. kostiense AFK-13, whereas activities of galactosidase, mannosidase, rhamnosidase, and arabinosidase were also detected in low levels. The molecular weights (MW) of ${\alpha}-\;and\;{\beta}$-glucosidases were estimated to be approximately 50-100 kDa by the ultrafiltration method.

• Journal of Microbiology and Biotechnology
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• 제17권5호
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• pp.745-752
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• 2007

A $\beta$-glucosidase from the algal lytic bacterium Sinorhizobium kostiense AFK-13, grown in complex media containing cellobiose, was purified to homogeneity by successive ammonium sulfate precipitation, and anion-exchange and gel-filtration chromatographies. The enzyme was shown to be a monomeric protein with an apparent molecular mass of 52 kDa and isoelectric point of approximately 5.4. It was optimally active at pH 6.0 and $40^{\circ}C$ and possessed a specific activity of 260.4 U/mg of protein against $4-nitrophenyl-\beta-D-glucopyranoside$(pNPG). A temperature-stability analysis demonstrated that the enzyme was unstable at $50^{\circ}C$ and above. The enzyme did not require divalent cations for activity, and its activity was significantly suppressed by $Hg^{+2}\;and\;Ag^+$, whereas sodium dodecyl sulfate(SDS) and Triton X-100 moderately inhibited the enzyme to under 70% of its initial activity. In an algal lytic activity analysis, the growth of cyanobacteria, such as Anabaena flos-aquae, A. cylindrica, A. macrospora, Oscillatoria sancta, and Microcystis aeruginosa, was strongly inhibited by a treatment of 20 ppm/disc or 30 ppm/disc concentration of the enzyme.

• 한국미생물·생명공학회지
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• 제21권5호
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• pp.453-460
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• 1993

The extracellular bacteriolytic enzyme from alkalophilic Bacillus sp. YJ-451 was endopeptidase which hydrolyzes the peptide bond at the amino group of D-glutamic acid in the peptidoglycan. Protoplast transfomation system of B. subtilis by the lytic enzyme that differs, in mechanisms, from lysozyme which was used to transformation of B. subtilis was investigated. High protoplast yield was obtained from cells cultured in PAB at the late logarithmic growth phase.

• Journal of Microbiology and Biotechnology
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• 제11권6호
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• pp.957-963
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• 2001

Bacillus subtilis YL-1004 was isolated from soil for the development of agents to control dental caries. This strain produced an extracellular lytic enzyme that hydrolyzed the Streptococcus mutans cell wall. The lytic enzyme was purified to homogeneity by affinity chromatography and gel permeation chromatography to give a single band on SDS-PAGE and non-denaturing polyacrylamide gel electrophoresis. The molecular weight of the enzyme was deduced from SDS-PAGE and gel chromatography to be 38 kDa and the PI to be 4.3 from isoelectric focusing. Sirty $\%$ of its lytic activity remained after incubation at $50^{\circ}C$ for 30 min, and its optimal temperature was $37^{\circ}C$ . The enzyme showed its highest activity at pH 8.0 and was stable at pHs ranging from 4.0 to 9.0. Treatment with several modifiers showed that a cysteine residue was involved in the active site of the enzyme. This lytic enzyme from Bacillus subtilis YL-1004 exhibited specificity towards Streptococci and also showed autolytic activity on Bacillus subtilis YL-1004.

• 미생물학회지
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• 제37권1호
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• pp.15-20
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• 2001

Aspergillus sp. HCLF-4가 생성하는 세균 세포벽 분해효소의 특성을 규명하였다. 본 세포벽 분해 효소는 Anabaena cylindrica 세포벽 분해능을 보였다. 이 세포벽 분해 효소는 Aspergillus sp. HCLF-4를 기질성분으로 0.05% heat killed Micrococcus luteus가 포함된 PDB 배지에 키웠을 때 생성되는 inducible enzyme으로 분자량은 약 14.3 kDa 이었다. 본 세포벽 분해효소는 pH 3.0-4.0, 온도 $30^{\circ}C$ 조건에서 최고의 활성을 보였고 $Mg^{2+}$와, $Mn^{2+}$의 2가 이온에서 분해 효소의 활성이 촉진되었다. 반면, 1가 양이온 $Na^{+}$와 $Li^{+}$, 2강 양이온 $Ca^{2+}$와 $Cu^{2+}$, 3가 양이온 $Fe^{3+}$에서는 활성이 억제되었으며 EDTA와 PMSF 또한 분해 효소의 활성을 억제 시켰다. 이 효소는 N-acetylmuramyl-L-amidase 또는 endopeptidase와 같은 활성을 보였다.

• 미생물학회지
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• 제35권3호
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• pp.231-236
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• 1999

Penicillium oxalicum 으로부터 세포외로 분비되는 Anabaena cylindrica 분해효소의 분자량은 renaturation SDS-PAGE에서 약 22kDa 으로 확인되었으며, 분해 효소의 농축은 ultrafiltration cut off fraction 중 30-10 kDa 구간에서 수획하였다. 최적 활성조건의 측정 결과 적정 pH는 3.5-4.0, 적정반응 온도는 $20^{\circ}C$, 그리고 온도 안정성은 $4^{\circ}C$ 이하에서 100% 이상, 20-$90^{\circ}C$ 범위에서는 50% 이상의 활성을 나타내었다. 금속이온 및 효소안정제의 영향에서는 $Na^+$,$K^+$, $Ba^(2+)$, $Mg^(2+)$, $Mn^(2+)$의 양이온과 BSA는 효소의 활성을 촉진시키는 반면, $Ca^(2+)$, $Cu^(2+)$의 양이온과 EDTA, PMSF 는 효소의 활성을 억제하는 작용을 하였다. 이러한 금속이온과 안정제의 영향에서 1가, 2가 양이온에 의해 활성이 증가하고, $Fe^(3+)$, $Ca^(2+)$, $Cu^(2+)$의 양이온에 의해서는 활성이 감소하는 결과는 대부분의 세포벽 분해효소가 갖는 특성과 유사한 결과였다. 분해효소는 A. cylindrica 과 Micrococcus. luteus 의 세포벽을 기질로 사용한 효소의 활성 반응에서 반응 시작 후 1시간에서 5시간 사이에 반응 산물로 환원당의 양이 급격히 증가하였다.

• 미생물학회지
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• 제36권1호
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• pp.14-19
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• 2000

Penicillium oxalicum(HCLF-34)의 세포외 분비효소로부터 ultrafilration, gel filtration chromatograph와 anion exchange chromatography법을 이용하여 남조세균(Anabaena cylindrica) 분해효소를 분리하였다. 이 효소의 분자량은 약 22 kDa이며, renaturation SDS-PAGE에서 monomer로서 남조세균 분해 활성을 갖는다. 아미노산 서열은 N-말단부터$NH_(2)$-Glu-Ser-Tyr-Ser-Ser-Asn-Ala-Ala-Gly-Ala-Val-Leu-Ile---, 13개의 아미노산을 분석하였으며, 분석된 아미노산의 homology를 조사한 결과 aspergillopepsin II precursor(acid protease A)와 13개의 아미노산 중 11개(84%)의 유사도를 나타내었고, acid proteinase EapC precursor과 13개 중 10개(81%)의 유사도를 나타내었다.

• Journal of Microbiology and Biotechnology
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• 제4권2호
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• pp.134-140
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• 1994

An antagonistic bacterium Pseudomonas stutzeri YPL-1 liberated extracellular chitinase and $\beta$-1,3-glucanase which are key enzymes in the decomposition of fungal hyphal walls. The lytic enzymes caused abnormal swelling and retreating at the hyphal tips of plant pathogenic fungus Fusarium solani in a dual culture. Scanning electron microscopy revealed the hyphal degradation of F. solani in the regions interacting with P. stutzeri YPL-1. The production of chitinase and properties of a crude preparation of the enzyme from P. stutzeri YPL-1 were investigated. Peak of the chitinase activity was detected after 4 hr of cultivation. The enzyme had optimum temperature and pH of 50$^{\circ}C$ and pH 5.3, respectively. The enzyme was stable in the pH range of 3.5 to 6.0 up to 50$^{\circ}C$. The enzyme was significantly inhibited by metal compounds such as $HgCl_2$, but was stimulated by $CoCl_2$. P. stutzeri YPL-1 produced high levels of the enzyme after 84 hr of incubation. Among the tested carbon sources, chitin was the most effective for the enzyme production, at the concentration level of 3%. As a source of nitrogen, peptone was the best for the enzyme production, at the concentration level of 4%. The maximum amount of enzyme was produced by cultivating the bacterium at a medium of initial pH 6.8.

• Journal of Microbiology and Biotechnology
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• 제5권4호
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• pp.206-212
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• 1995

A strain BL-29, which produces a extracellular lytic enzyme on E. coli was isolated from the soil. The strain was identified as belonging to the genus Bacillus sp. The lytic enzyme was purified to homogeneity by ion exchange chromatography and gel filtration. Specific activity of the purified enzyme was 28, 850 U/mg protein and yield of the enzyme was 5$%$. The purified enzyme showed a single band on SDS-PAGE and its molecular weight was estimated to be 31, 000 by SDS-polyacrylamide gel electrophoresis and gel filtration column chromatography. The optimum temperature and pH were $55^{\circ}C$ and pH 10.0, respectively. The enzyme was stable at $45^{\circ}C$ but enzyme activity was reduced by up to 50$%$ when the temperature was raised to $55^{\circ}C$ for 15 min. Stable range of pH was from 5.0 to 11.0. but Enzyme activity was inhibited by lead-acetate, mercuric chloride, ethylene glycol-bis-[$\beta$-aminoethyl ether]-N, N, $N^1, $N^1$-tetraacetic acid (EGTA), and ethylenediamine tetraacetic acid (EDTA), but not affected considerably by treatment with other chemical reagents.