• Korean Journal of Microbiology
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• v.37 no.4
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• pp.253-258
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• 2001

A Bacillus circulans KCTC3004 $\beta$-1,3-glucanase gene contained in a recombinant plasmid pLM460 derived from subcloning the original recombinant plasmid pLM530 was trasferred into a new shuttle vector plasmid pLMS1180 by ligating linearized DNAs of pLM460 and pUB110. B. subtilis RM125 and B. megaterium ATCC14945 transformed with pLMS1180 produced the $\beta$-1,3-glucanase substantially. Most of the enzyme was produced during the exponential growth period. The maxium activities of the $\beta$-1,3-glucanase produced by the Bacillus transformants were compared with that of the B. circulans gene donor strain. The B. subtilis RM125 (pLM1180) enzyme showed the activity 14 times higher than that of the gene donor cells, followed by the B. megaterium ATCC14945 (pLMS 1180) enzyme with activity 5 times higher than that of the gene donor cells. While E. coli secreted about 7% of the produced enzyme, B. subtilis excreted the enzyme into the medium wholly and B. megaterium about 97% of the total product. The SDS-PAGE of this enzyme produced in E. coli (pLMS1180), B subtilis (pLMS1180) or B. megaterium (pLMS1180) indicated a molecular weight of 38,000. The enzymes overproduced in three different host cells hydrolyzed laminarin to produce mainly laminaribiose, laminaritriose, and laminarioligosaccharides. The plasmid pLMS1180 was stable in B. megaterium, E. coli, but was unstable in B. subtilis.

• Journal of Microbiology
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• v.42 no.3
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• pp.205-210
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• 2004

The sequence coding for carboxymethylcellulase (CMCase, CelC) was isolated from the DNA of Salmonella typhimurium URl. Comparison between the deduced amino acid sequence of CelC (368 amino acid residues, Molecular mass 41 kDa) and that of the previously published CMCase revealed that this enzyme belongs to the cellulase family 8 and D. The protein was overproduced in Escherichia coli using T7 expression system, and its activity was confirmed by CMC-SDS-PAGE. When the overexpressed CelC protein was tested on cellulose-type substrates, the recombinant protein is able to degrade cellulose-type substrates, such as CM-cellulose, xylan, avicel, lichenan, and laminarin. Optimal temperature and pH for enzyme activity were found to be 50$^{\circ}C$ and pH 6.5, respectively.

• Journal of Microbiology and Biotechnology
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• v.14 no.6
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• pp.1295-1302
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• 2004

A gene encoding the mannanase of Bacillus subtilis WL-7, which had been isolated from Korean soybean paste, was cloned into Escherichia coli, and the gene product was purified from the culture filtrate of the recombinant E. coli. This mannanase gene, designated manA, consisted of 1,086 nucleotides, encoding a polypeptide of 362 amino acid residues. The deduced amino acid sequence was highly homologous to those of mannanases belonging to the glycosyl hydrolase family 26. The molecular mass of the purified mannanase was 38 kDa as estimated by SDS-PAGE. The enzyme had a pH optimum at 6.0 and a temperature optimum at $55^{\circ}C$. The enzyme was active on locust bean gum, konjak, guar gum, and lichenan, while it did not exhibit activity towards yeast mannan, laminarin, carboxymethylcellulose, $\beta$­glucan, xylan, and para-nitrophenyl-$\beta$-mannopyranoside.

• Microbiology and Biotechnology Letters
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• v.23 no.3
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• pp.304-310
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• 1995

A thermophilic bacterium producing the extracellular cellulase-free xylanase was isolated from soil and has been identified as Bacillus sp. The optimal growth temperature was 50$\circ$C and the optimal pH, 7.0. Under the optimal growth condition, maximal xylanase production was 2.2 units/ml in the flask culture. The enzyme production was induced by xylan and xylose, but was repressed by sucrose or trehalose. The partially purified xylanase was most active at 70$\circ$C. It was found that the enzyme was stable at 65$\circ$C for 10 hours with over 75% of the activity. The enzyme was most active at pH 7.0 and retained 90% of its maximum activity between pH 5.0 and pH 9.0 though Bacillus sp. was not grown on alkaline conditions (>pH 8.0). In addition, the activity of xylanase was over 60% at pH 10.0. At the ambient temperature, the enzyme was stable over a pH range of 5.0 to 9.0 for 10 h, indicating that the enzyme is thermostable and alkalotolerant. The activity of xylanase was completely inhibited by metal ions including Hg$^{2+}$ and Fe$^{2+}$, while EDTA, phenylmethylsulfonyl fluoride (PMSF), $\beta$-mercaptoethanol and SDS didn't affect its activity. The enzyme was also identified to exert no activity on carboxymethylcellulose, laminarin, galactomannan, and soluble starch.

• Journal of Applied Biological Chemistry
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• v.42 no.3
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• pp.107-112
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• 1999

A carboxymethyl-cellulase (CMCase) was purified from the culture supernatant of Bacillus sp. KD1014 by ultrafiltration, ammonium sulfate precipitation, and a series of chromatography on QAE-Sephadex A-50, hydroxylapatite and Sephadex G-75. The purified CMCase was a single protein of 32 kDa, showed an optimum activity at $60^{\circ}C$ and pH 6.0, and had a half-life of 23 min at $70^{\circ}C$. The enzyme activity was not influenced by metal ions such as $Mg^{2+},\;Fe^{3+},\;K^+,\;Zn^{2+}$, and $Cu^{2+}$ at a concentration of 1.0 mM, partially inhibited by $Mn^{2+}$ and $Ag^+$, and significantly inhibited by pentachlorophenol (PCP). The purified enzyme showed a 3.9-times higher activity on lichenan than on CMC, but hardly cleaved xylan, starch, avicel, laminarin, filter paper and levan. The results of activity staining of the purified enzyme separated by native and denaturing gel electrophoresis suggested that the CMCase might exist in dimeric, oligomeric or aggregated form as well as in monomeric form. The enzymatic cleavage products from cellotetraose indicated that the CMCase possessed transglycosylation activity.

• International Journal of Industrial Entomology and Biomaterials
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• v.44 no.2
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• pp.37-43
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• 2022

Silkworm food has been found to be effective for dementia, liver function, lowering blood sugar, and possesses antioxidant properties, which has been attracting attention as a health functional food. In this study, methods for enhancing the functionality of silkworms were explored and the production potential of high-functional silkworms was analyzed. For enhancing antioxidant activity, ascorbic acid, caffeic acid, laminarin, and glutathione were injected or fed to 5th instar silkworms, and the antioxidant activity of silkworm extract was comparatively analyzed. There was no significant change in polyphenol and flavonoid content, but it was confirmed that 2,2-diphenyl-1-picrylhydrazyl radical scavenging ability, superoxide dismutase-like activity, and reducing power were slightly increased after injection of ascorbic acid, caffeic acid, and glutathione. To confirm the increase in antioxidant efficacy through feeding, an inducer was mixed with sucrose and sprayed on mulberry leaves. As a result, the growth rate of silkworms improved and all indicators of antioxidant activity were improved in silkworms fed with ascorbic acid and glutathione. Considering these results, producing high-functional silkworms was deemed possible.

• International Journal of Industrial Entomology and Biomaterials
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• v.12 no.2
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• pp.57-61
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• 2006

Suppressor of cytokine signaling (SOCS) is known to be as a negative feedback regulator in Janus kinase signal transducer and activator of transcription signaling. Highly conserved SOCS box domain was cloned from a Korean malaria vector, Anopheles sinensis. Sequence analysis indicates that it has identity to Anopheles gambiae (96%), Aedes aegypti (94%), Drosophila melanogaster (78%), Mus musculus (72%) and Homo sapiens (72%), respectively. Tissue specificity RT-PCR demonstrated that the expression level of AsSOCS transcript was high at abdomen, midgut, and ovary, whereas developmental expression patterns showed that the level of AsSOCS was high at egg, early pupae, and adult female. On the other hand, RT-PCR analysis after bacterial challenge showed that SOCS mRNA was strongly induced in larvae. In addition, it was also induced by various immune elicitors such as lipoteicoic acid, CpG-DNA, and laminarin. It seems that AsSOCS, repressor of JAK-STAT pathway, is highly conserved in mosquito, and may play an important role in mosquito innate immune response.

• Applied Biological Chemistry
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• v.35 no.3
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• pp.165-169
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• 1992

Two types of $endo-{\beta}-1,3-glucanases$ were purified from green malt and their basic characteristics were studied. Molecular weights of glucanase I and glucanase II were estimated, by electrophoresis, to be 35,000 and 28,000, respectively. Purified glucanase I and II showed the highest activity at pH $5.0{\sim}7.0$ and $5.0{\sim}8.0$, respectively. The optimal temperature of purified glucanase I and II was $40^{\circ}C$. Purified glucanase I and glucanase II were stable at $40^{\circ}$ for 60 min and at $50^{\circ}$ for 30 min. All enzymes were inactivited by $AgNO_3$ and $HgCl_2$ while those were not activated by various compounds tried. Km values of glucanase I and II were 1.03 mg/ml, 1.20 mg/ml, respectively.

• IMMUNE NETWORK
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• v.4 no.2
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• pp.94-99
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• 2004

Background: We previously reported that ginsan, a polysaccharide extracted from Panax ginseng had an immunostimulatory activity such as mitogenic activity, activation of macrophages and killer cells, and production of a variety of cytokines which resulted in antitumor and antiseptic effects. We further purified $\alpha$-(1$\longrightarrow$6)-glucan and $\beta$-(2$\longrightarrow$6)-fructan from the ginsan with size exclusion and ion-exchange column chromatography successively. In this study, we performed the structure-based activity of ginsan by comparison with known polysacchrides such as $\beta$-glucan, curdlan, laminarin, levan, dextran, lentinan and OK-432. Methods: To investigate the immunostimulatory activity of several polysaccharide compounds, we investigated the stimulation of lymphocytes proliferation, the generation of activated killer cells and the secretion of nitrites from activated macrophages. Results: Of polysaccharides tested, curdlan and ginsan stimulated lymphocyte proliferation, suggesting that the molecular weight and composition of polysaccharide are dependent on the mitogenic activity. The production of nitric oxide was significantly increased in curdlan, levan, ginsan and its fraction, indicating that fructan has also capacity to activate macrophages and may devote to kill pathogens. In addition, the activation of macrophages was seemed to be independent of molecular weight of polysaccharide. The generation of AK cells was exhibited in order of curdlan, OK-432> F1, ginsan, F3> levan> etc. The AK activity may be dependent on molecular weight and composition of polysaccharides. Conclusion: Unfortunately, purified polysaccharide from ginsan were less active on immunostimulatory activity than mixed compounds of polysaccharides. From the viewpoint of structure and activity relationships, we found several characteristic features.

• Archives of Pharmacal Research
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• v.17 no.1
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• pp.26-30
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• 1994

Some ammonium oxalate soluble pectic fragments prepared from cultured cell wall of Ephycla distrahya elicited the accumulation of p-coumarocylamino acids (p-CAA) in E. distachya cultures while water soluble and alkali soluble fractions had no activity. Partial purification of the pectic fragments fraction using DEAE-cellulose chromatography afforded two active fractions (PS-I and PS-II) which were composed of mainly uronic acids (98-99 w/w %). They elicited the accumulation of p-CAA in an amount of 52-60 nmol per gram fresh weight of cultures. The acidic sugar compositions of PS-I and PS-II were found to be galacturonic acid and glucuronic acid by TLC analysis. They were supposed to act as endogenous elicitors of p-CAA accumulation. In order to investigate the effect of ethylene on p-CAA accumulation, Ethrel, which is known as ethylene generator, and ACC(1-aminocyclopropane-1-carboxylic acid), a direct precusor of ethylene biosynthesis, were added to the culture. However, they did not glycopeptide elicitor [(Con A-II)], either. Consequently, no relationships between ethylene and p-CAA accumulation were recognized. Several tentative elicitors were teted for their activity. Commercial yeast glucan, $CuCl_2$, laminarin and laminariheptaose had slight activity whereas ${\alpha}$-methylmannopyranoside and commercial yeast mannan had no elicitor activity. ${\alpha}$-methylmannopyranoside which has been known as a tentative inhibitor of glucan elicitor in Glycine max did not affect on the elicitor activity of Con A-II.