• Preventive Nutrition and Food Science
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• v.19 no.3
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• pp.213-219
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• 2014

Bacillus pumilus 2.g isolated from gembus, an Indonesian fermented soybean cake, secretes several proteases that have strong fibrinolytic activities. A fibrinolytic enzyme with an apparent molecular weight of 20 kDa was purified from the culture supernatant of B. pumilus 2.g by sequential application of ammonium sulfate precipitation, ion-exchange chromatography, and hydrophobic chromatography. The partially purified enzyme was stable between pH 5 and pH 9 and temperature of less than $60^{\circ}C$. Fibrinolytic activity was increased by 5 mM $MgCl_2$ and 5 mM $CaCl_2$ but inhibited by 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM sodium dodecyl sulfate (SDS), and 1 mM ethylenediaminetetraacetic acid (EDTA). The partially purified enzyme quickly degraded the ${\alpha}$ and ${\beta}$ chains of fibrinogen but was unable to degrade the ${\gamma}$ chain.

• Journal of Microbiology and Biotechnology
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• v.5 no.4
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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.

• Journal of Microbiology and Biotechnology
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• v.9 no.3
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• pp.271-275
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• 1999

The intracellular superoxide dismutase (SOD) from Staphylococcus sciuri was isolated to homogeneity by continuous steps, including ammonium sulfate fractionation, DEAE-ion-exchange chromatography, gel filtration, and phenyl hydrophobic gel chromatography. Pure SOD had a specific activity of 4,625 U/mg and was purified 158-fold with a yield of 31 % from a cell free extract. The molecular weight of the purified SOD was determined to be approximately 35.5 kDa by gel filtration and the enzyme was also shown to be composed of dimeric subunits on denaturing SDS-PAGE. The enzyme activity remained stable at pH 5 to 11 and also to heat treatment of up to $50^{\circ}C$ at pH 7.8, with 80% relative activity. The enzyme was insensitive to cyanide, hydrogen peroxide, and azide, indicating that it is a manganese-containing SOD. The EPR spectrum showed the enzyme containing manganese as a cofactor.

• Journal of the Korean Wood Science and Technology
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• v.28 no.4
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• pp.29-40
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• 2000

Highly purified Cerrena unicolor laccase (benzenediol:oxygen oxidoreductase, EC 1.10.3.2) caused the demethoxylation of milled wood lignin and several lignin related substances. The constitutive form of the enzyme produced extracellularly by C. unicolor fermenter culture was isolated and purified by ion-exchange chromatography on the DEAE-Toyopearl column and by affinity chromatography on a ConA-Sepharose and Syringyl-AH-Sepharose 4B columns. The enzyme was further immobilized on functionalized porous glass (CPG) and keratin coated CPG. The demethylating activity was monitored both by estimation of released methanol and by detection of the level of methoxyl groups (also in some water miscible solvents) after incubation of lignin materials with laccase preparations (free and immobilized). The effects of the incubation time and temperature on the demethoxylating activity of immobilized laccase preparations were also studied.

• Animal cells and systems
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• v.16 no.6
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• pp.455-461
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• 2012

Amongst the various antimicrobial peptides, lysozyme plays a central role in initiating and maintaining the antibacterial defense response of insect. Here we propose the biosynthesis and refolding of recombinant lysozyme in Escherichia coli expressed in inclusion body form. The Agrius lysozyme gene was amplified using gene specific primers and then ligated into the pGEX-4T-1 vector, which contained the glutathione S-transferase (GST) gene as a fusion partner. A recombinant lysozyme was expressed in E. coli Rosetta cells using a pGEX-4T-1 expression vector, and the fusion protein was induced by ioporpyl-${\beta}$-D-thiogalactopyranoside (IPTG). The recombinant protein produced as an inclusion body was resolubilized in solubilization buffer, and the resultant solution was dialyzed in refolding buffer. After thrombin cleavage, the recombinant lysozyme was purified by ion exchange chromatography and reverse phase chromatography. The recombinant lysozyme was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and immunoreactivity against the anti-Agrius lysozyme was observed by western blot analysis of this protein. The recombinant lysozyme displayed antibacterial activity against Bacillus megaterium and Micrococcus luteus, which was confirmed by the inhibition zone assay.

• Journal of the Korean Society of Food Science and Nutrition
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• v.21 no.6
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• pp.725-730
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• 1992

The internal invertase was purified from cell free extract of Rhodosporidium toruloides IFO 0559-M-919 by acid precipitation, ion-exchange chromatography and gel filtration to the unique enzyme protein on disc electrophoresis. We have found out that molecular weight of purified internal invertase was 90,000 by gel filtration and the purified enzyme was protein with 4 homogeneous subunits appearing as single band of 22,000daltons on SDS-polyacrylamide gel electrophoresis.

• Microbiology and Biotechnology Letters
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• v.20 no.3
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• pp.249-256
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• 1992

Potato juice was found out to have a strong inhibition activity on the growth of Clostridium perf;nngens during work of foodstuffs for the improvement of human intestinal microflora. The anti-bacterial activity of the precipitated protein obtained from the potato juice in 70% ammonium sulfate solution was stable at the range of pH 4 to 10, whereas it was lost by a heat treatment at $60^{\circ}C$ for 10 min. The minimal inhibitory concentration of the precipitated protein on the growth of C1. Pefingens was about 0.2 mg/ml. The potato protein also suppressed the growth of C1. butyrincm and Eubacterium iimosum, while it showed a promoting effect for the growth of Bifdobacterium bifidum, Bif: animalis, Lactobacillus plantarum and Lact. acidophitus. The potato protein was further purified by CM-Sepharose ion exchange column chromatography, Sephadex G-150 gel filtration column chromatography and SDS-polyacrylarnide gel electrophoresis. The purified protein(kCp) was proved to be a glycoprotein by PAS staining and its molecular weight was about 38.7 kd.

• Microbiology and Biotechnology Letters
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• v.19 no.3
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• pp.209-214
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• 1991

Purification of the bacteriocin from Lactococcus sp. 1112-1 was achieved by successive column chromatography on CM-Sephadex C-25 and Sephadex G-50, starting from cell disruption broth. 16.2% of the initial activity was recovered after this purification step and it was shown 123-fold increase in purification. Purified bacteriocin was shown a single band on SDS-polyacrylamide gel electrophoresis. This substance was rather stable at heat treatment and alkaline pH relatively. The residual antimicrobial activity was 38% when the bacteriocin was treated by heat at $100^{\circ}C$ for 60 min. And 23% of the activity remained at pH 8.0 after standing for 48 hr. The amino acid composition of purified bacteriocin was made up 26 residues.

• Microbiology and Biotechnology Letters
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• v.24 no.4
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• pp.459-464
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• 1996

Exo-polysaccharide (BWS) obtained from submerged cultivation of Ganoderma lucidum mycelium was fractionated. Antitumor activity of their fractions was investigated in comparison with the mycelial polysaccharide fractions. Eight kinds (BWS-DN, BWS-DA, BWS-DN-GI, BWS- DA-GI, MWS-DN, MWS-DA, MWS-DN-GI and MWS-DA-GI) of polysaccharide fractions were obtained by DEAE-cellulose ion exchange chromatography and Sepharose CL-4B gel chromatography from BWS and MWS, which were isolated from culture fiuid and mycelial cell, respectively. The anticomplementary activities (ITCH$_{50}$%) of the exo-polysaccharide fractions showing 15% to 30% were lower than those of mycelial polysaccharide fractions showing 15% to 70%. The acidic fractions of BWS-DA and BWS-DA-GI fractionated from BWS, showed the highest activity of 30%. In the MTT assay, BWS-DN and MWS against mouse leukemia L1210 exhibited high inhibition ratio of 86 and 89%, respectively at the concentration of 600 $\mu$g/ml. High inhibition ratio of 50% (IC$_{50}$) was achieved for BWS, BWS-DA and MWS-DA fractions against human colon adenocarcinoma COLO-205 and for BWS-DA, BWS-DN and MWS-DN fractions against human leukemia HL-60 at the concentra- tion of 300 $\mu$g/ml among the six polysaccharide fractions, respectively.

• Journal of the Korean Society of Food Science and Nutrition
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• v.18 no.3
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• pp.279-286
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• 1989

The alkaline protease producing mold isolated from and identified as Aspergillus fumigatus. It was found that the production of alkaline protease reach to maximum was cultured for 3 days at $30^{\circ}C$. The enzyme was purified 86.13 fold and yield of the enzyme purification was 6.4%, The purification procedure include ammonium sulfate treatment, gelfiltration on Sephadex G-25, G-75, G-150 and DEAE-cellulose ion-exchange chromatography. When the purified enzyme was applied sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the molecular weight was estimated about 63000. This enzyme composed 17 amino acids and main amino acids of this enzyme were glycine and glutamic acid.