• 한국미생물·생명공학회지
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• 제20권1호
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• pp.1-5
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• 1992

유백피 추출물의 항세균 효과를 측정하기 위해 각각 다른 농도의 추출물의 함유한 배지에서 MIC를 측정하고, 증식에 미치는 영향을 조사한 결과는 다음과 같았다.이 추출물은 실험에서 사용된 세균 중 S.aureus, S.epidermidis, S.faecalis, B.subtilis, M.luteus와 같은 그람 양성균 및 E.coli, S.typhi, P.vulgaris, P.aeruginosa와 같은 그람 음성균에서 높았고, K.pneumoniae에 대해서는 항균력을 나타내지 않았다. 이 추출물 중의 항세균 효과를 가진 물질은 $121^{\circ}C$에서 20분간 멸균한 후에도 항세균 효과에는 별다른 변화가 없었다.

• BMB Reports
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• 제34권1호
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• pp.21-27
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• 2001

The structural stability of brain pyrydoxine-5'-phosphate (PNP) oxidase and the catalytic properties of the monomeric species were investigated. The unfolding of brain pyridoxine-5'-phosphate (PNP) oxidase by guanidine hydrochloride (GuHCl) was monitored by means of fluorescence and circular dichroism spectroscopy Reversible dissociation of the dimeric enzyme into subunits was attained by the addition of 2 M GuHCl. The perturbation of the secondary structure under the denaturation condition resulted in the release of the cofactor FMN. Separation of the processes of refolding and reassociation of the monomeric species was achieved by the immobilization method. Dimeric PNP oxidase was immobilized by the covalent attachment to Affi-gel 15 without any significant lass of its catalytic activity. Matrix-bound monomeric species were obtained from the reversible refolding processes. The matrix bound-monomer was found to be catalytically active, possessing only a slightly decreased specific activity when compared to the refolded dimeric enzyme. In addition, limited chymotrypsin digestion of the oxidase yields two fragments of 12 and 161 kDa with a concomitant increase of catalytic activity The catalytically active fragment was isolated by ion exchange chromatography and analyzed for association of two subunits using the FPLC gel filtration analysis. The retention time indicated that the catalytic fragment of 16 kDa behaves as a compact monomer. Taken together, these results are consistent with the hypothesis that the native quaternary structure of PNP oxidase is not a prerequisite for catalytic function, but it could play a role in the regulation.

• BMB Reports
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• 제30권1호
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• pp.60-65
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• 1997

Expression of human Cu.Zn-superoxide dismutase (SOD) with activity comparable to human erythrocyte enzyme was achieved in E. coli B21(DE3) by using the pET-17b expression vector containing a T7 promoter. Recombinant human SOD was found in the cytosol of disrupted bacterial cells and represented > 25% of the total bacterial proteins. The protein produced by the E. coli cells was purified using a combination of ammonium sulfate precipitation, Sephacryl S-100 gel filtration and DEAE-Sephacel ion exchange chromatography. The recombinant Cu,Zn-SOD and human erythrocyte enzyme were compared using dismutation activity, SDS-PAGE and immunoblotting analysis. The mass of the subunits was determined to be 15,809 by using a electrospray mass spectrometer. The copper specific chelator. diethyldithiocarbamate (DOC) reacted with the recombinant Cu,Zn-SOD. At $50{\mu}M$ and $100{\mu}M$ concentrations of DOC, the dismutation activity was not inhibited for one hour but gradually reduced after one hour. This result suggests that the reaction of DOC with the enzyme occurred in two distinct phases (phase I and phase II). During phase I of this reaction, one DOC reacted with the copper center, with retention of the dismutation activity while the second DOC displaced the copper, with a loss of activity in phase II.

• Journal of Microbiology and Biotechnology
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• 제8권6호
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• pp.568-574
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• 1998

Two types of chitosanases produced from Aspergillus fumigatus KH-94 were purified by ion exchange and gel permeation chromatography. Molecular weights of the enzymes are 22.5 kDa (chitosanase I) and 108 kDa (chitosanase II). pI, optimum pH, and temperature of chitosanase I are 7.3, 5.5, and 70-$80^{\circ}C$, respectively, and those of chitosanase II are 4.8, 4.5~5.5, and 50~$60^{\circ}C$, respectively. Activities of both chitosanases were increased by $Mn^{2+}$ but inhibited by $Cu^{2+}$ and $Hg^{2+}$ . Chitosanase I has endo-splitting activity that hydrolyzes chitopentaose, chitohexaose, and chitosan to chitobiose, chitotriose, and chitotetraose, whereas chitosanase II has exo-splitting activity that hydrolyzes chitobiose and chitosan to glucosamine. Chitosanase II was found to have transglycosylation activity also in the reaction of 2% more chitooligosaccharides as a substrate and at the initial reaction. The higher degree of deacetylation, the stronger activities of chitosanase Iand II toward chitosans. Both chitosanases could hydrolyze chitosan and glycol chitosan but not chitin, cellulose, and carboxymethyl cellulose. To produce higher degree of polymerization of chitooligosaccharides, chitosanase I was used and yielded 80% of recovery.

• Journal of Microbiology and Biotechnology
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• 제8권6호
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• pp.588-594
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• 1998

Lactic acid bacteria were isolated from Kimchi and screened for bacteriocin production. Strain SE1, identified as Lactobacillus curvatus sp., showed the strongest inhibitory activity against Lactobacillus delbrueckii subsp. delbrueckii. The bacteriocin was inactivated by amyloglucosidase, trypsin, or protease K treatment. However, it maintained its activity under heat treatment at $100^{\circ}C$ for 60 min. The production of the bacteriocin had a growth-related mode and decreased around the early-stationary phase. The optimum temperature for the growth of L. curvatus SE1 was $37^{\circ}C$; however, the optimum temperature for bacteriocin production was $30^{\circ}C$. The bacteriocin activity was decreased by treatment with methanol, butanol, acetone, or chloroform, however, it was not affected by treatment with ethanol, iso-propanol, or cyclohexane. The inhibitory activity of bacteriocin was stable over a wide range of pHs (2 to 11). The bacteriocin from L. curvatus SE1 killed the indicator strain by a bactericidal mode of action. The bacteriocin from L. curvatus SE1 was partially purified by ethanol precipitation and ion exchange chromatography. SDS-polyacrylamide gel electrophoresis was used to determine the molecular weight of the bacteriocin by the bacteriocin activity test. The apparent molecular mass of the bacteriocin produced by L. curvatus SE1 was about 14 kDa.

• Journal of Microbiology and Biotechnology
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• 제14권5호
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• pp.1014-1021
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• 2004

Two thermostable xylanases, designated XynA and XynB, were purified to homogeneity from the culture supernatant of Paenibacillus sp. DG-22 by ion-exchange and gel-filtration chromatography. The molecular masses of xylanases A and B were 20 and 30 kDa, respectively, as determined by SDS-PAGE, and their isoelectric points were 9.1 and 8.9, respectively. Both enzymes had similar pH and temperature optima (pH 5.0-6.5 and $70^{\circ}C$), but their stability at various temperatures differed. Xylanase B was comparatively more stable than xylanase A at higher temperatures. Xylanases A and B differed in their $K_m$ and $V_{max}$ values. XynA had a $K_m$ of 2.0 mg/ml and a $V_{max}$ of 2,553 U/mg, whereas XynB had a K_m$ of 1.2 mg/ml and a $V_{max}$, of 754 U/mg. Both enzymes were endo-acting, as revealed by their hydrolysis product profiles on birchwood xylan, but showed different modes of action. Xylotriose was the major product of XynA activity, whereas XynB produced mainly xylobiose. These enzymes utilized small oligosaccharides such as xylotriose and xylotetraose as substrates, but did not hydrolyzed xylobiose. The amino terminal sequences of XynA and XynB were determined. Xylanase A showed high similarity with low molecular mass xylanases of family 11.

• Journal of Microbiology and Biotechnology
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• 제19권8호
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• pp.818-822
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• 2009

A ${\beta}-1$,3-1,4-glucanase from the fungus Laetiporus sulphureus var. miniatus was purified as a single 26 kDa band by ammonium sulfate precipitation, HiTrap Q HP, and UNO Q ion-exchange chromatography, with a specific activity of 29 U/mg. The molecular mass of the native enzyme was 52 kDa as a dimer by gel filtration. ${\beta}-1$,3-1,4-Glucanase showed optimum activity at pH 4.0 and $75^{\circ}C$. The half-lives of the enzyme at $70^{\circ}C$ and $75^{\circ}C$ were 152 h and 22 h, respectively. The enzyme showed the highest activity for barley ${\beta}$-glucan as ${\beta}-1$,3-1,4-glucan among the tested polysaccharides and p-nitrophenyl-${\beta}$-D-glycosides with a $K_m$, of 0.67 mg/ml, a $k_{cat}$ of 13.5 $S^{-1}$ and a $k_{cat}/K_m$ of 20 mg/ml/s.

• Applied Biological Chemistry
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• 제37권4호
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• pp.243-247
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• 1994

한국산 대추로부터 조직의 연화에 작용하는 endo-polygalacturonase를 gel filtration과 이온교환크로마토그라피에 의하여 단일 단백질로 정제할 수 있었고, 수율은 약 6% 정도였다. 정제된 이 효소는 sodium dodecyl sulfate-polyacrylamide gel electrophoresis에 의하여 분자량이 약 19,000정도의 monomer로 확인되었고, 결정구조는 육각판상 모양을 나타내었다.

• Journal of Microbiology and Biotechnology
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• 제13권4호
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• pp.591-597
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• 2003

Human urokinase kringle domain, sharing homology with angiostatin kringles, has been shown to be an inhibitor of angiogenesis, which can be used for the treatment of cancer, rheumatoid arthritis, psoriasis, and retinopathy. Here, the expression of the kringle domain of urokinase (UK1) as a secreted protein in high levels is reported. UK1 was expressed in the methylotrophic yeast Pichia pastoris GS115 by fusion of the cDNA spanning from Ser47 to Lys135 to the secretion signal sequence of ${\alpha}-factor$ prepro-peptide. In a flask culture, the secreted UK1 reached about 1 g/l level after 120h of methanol induction and was purified to homogeneity by ion-exchange chromatography. Amino-terminal sequencing of the purified UK1 revealed that it was cleaved at the Ste13 signal cleavage site. The molecular mass of UK1 was determined to be 10,297.01 Da. It was also confirmed that the purified UK1 inhibited endothelial cell proliferation stimulated by basic fibroblast growth factor, vascular endothelial growth factor, or epidermal growth factor, in a dose-dependent manner. These results suggest that a P. pastoris sytem can be employed to obtain large amounts of soluble and active UK1.

• 한국수산과학회지
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• 제50권5호
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• pp.520-526
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• 2017

Carnosine was recently reported to protect against the DNA damage induced by oxidative stress. In this study, we investigated the protective effect of eel Anguilla japonica carnosine extracts prepared using different methods (heat treatment extracts, HTEs; ion exchange chromatography, IEC; ultrafiltration permeation, UFP) on leukocyte DNA damage using the comet assay. Human leukocytes were incubated with extracts of eel carnosine at concentrations (of 10, 50, $100{\mu}g/mL$), and then subjected to an oxidative stimulus [$200{\mu}M$ hydrogen peroxide ($H_2O_2$)]. Pretreatment of the cells for 30 min with carnosine significantly reduced the genotoxicity of $H_2O_2$ measured as DNA strand breaks. The protective effects of the three types of extract (HTE, IEC, and UFP) increased with concentration. At the highest concentration (100 g/mL). there were no statistical differences in oxidative damage between each extract treatment and PBS-treated negative controls. When leukocytes were incubated with carnosine for 30 min after exposure to $H_2O_2$. the protective ability of each extract changed. Therefore, eel carnosine inhibits the $H_2O_2$ induced damage to cellular DNA in human leukocytes, supporting the protective effect of this compound against oxidative damage.