• Food Engineering Progress
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• v.21 no.2
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• pp.103-109
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• 2017

Alginate lyase from Streptomyces violaceoruber was purified by DEAE sephacel chromatography and SP sepharose chromatography. The specific activity of the purified enzyme was 14.6 units/mg protein, representing a 40.6-fold purification of the crude extract. The final preparation thus obtained showed a single band on Tricine-SDS polyacrylamide gel electrophoresis whose molecular weight was determined to be 23.3 kDa. The polyMG block of sodium alginate was hydrolyzed by the purified alginate lyase and then separated by activated carbon column chromatography and bio gel P-2 gel filtration. The main hydrolysates were composed of hetero type M/G-oligosaccharides with the degrees of polymerization (D.P.) being 6 and 8. To investigate the effects of hetero type M/G-oligosaccharides from the sodium alginate on the growth of some intestinal bacteria, cells were cultivated individually on the modified-MRS medium containing D.P. 6 and 8 M/G-oligosaccharides. B. longumgrew 4.25-fold and 6.44-fold more effectively by the treatment of D.P. 6 and 8 M/G-oligosaccharides compared with those of standard MRS medium. In addition, B. bifidumgrew 3.3-fold and 5.4-fold more effectively by the treatment of D.P. 6 and 8 M/G-oligosaccharides. In conclusion, D.P. 8 was more effective than D.P. 6 hetero M/G-oligosaccharides as regards the growth of Bifidobacteriumspp. and Lactobacillus spp.

• Journal of Life Science
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• v.10 no.1
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• pp.1-5
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• 2000

Pectinase was isolated from culture medium of Bacillus sp. BS-214 and purified 105-fold with 3.4% yield by ammonium sulfate precipitation, gel filteration using Sephadex G-75 and DEAE-cellulose followed by gel filteration through Sephadex G-100. The molecular weight of the purified enzyme was estimated to be about 43 kDa on SDS-PAGE and by gel filtration, indicating that the enzyme is a monomer. the optium pH and temperature of the enzyme were 9.0 and 55$^{\circ}C$, respectively. the enzyme was stable at 60$^{\circ}C$ for 30min and in a pH range from 7.5 to 10.5 for 12 h ant 4$^{\circ}C$. The enzyme activity was highly enhanced by Ca2+, and also K+, Li+ and Na+showed a positive effect, while stongly inhibited by Zn2+ and Hg2+.

• Preventive Nutrition and Food Science
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• v.7 no.3
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• pp.310-316
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• 2002

Extracellular cyclodextrin glucanotransferase (CGTase) from Paenibacillus sp. JK-12 was purified through sev-eral purification steps consisting of ammonium sulfate precipitation and chromatographies on DEAE-sephadex A-50 and Mono QIM HR5/5. The purified CGTase exhibited a single band on SDS-PAGE and was estimated to be approximately 82 kDa. The isoelectric point of the enzyme was 7.2 as determined by isoelectric focusing. The CGTase from Paenibacillus sp. JK-12 had a transglucosylation activity at the C-2 position of L-ascorbic acid. The optimum pH and temperature for the CGTase activity were 8.0 and 5$0^{\circ}C$, respectively. The enzyme activity was stable from pH 6.0 to 9.() and at temperatures up to 55$^{\circ}C$ at pB 8.0, having 80% residual activity. The activity of the CGTase was strongly resistant to metals such as A $g^{+}$ and $Ba^{2+}$ but slightly inhibited by H $g^{+}$, N $i^{2+}$ and $Mg^{2+}$. The enzymeproduced $\alpha$ -cyclodextrin ($\alpha$-CD) and $\beta$-CD as the main products from starch, but not ${\gamma}$-CD.X>-CD.

• KSBB Journal
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• v.22 no.1
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• pp.53-57
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• 2007

We have studied biochemical characterization of lectin purified from kidney bean seedling through PBS extraction, $(NH_4)_2SO_4$ precipitation, and Sephadex G-100 affinity chromatography. The lectin was agglutinated by rabbit erythrocytes. This lectin analyzed by SDS-PAGE is a tetramer composed of two subunits with molecular weights of 46 and 44 kDa. The optimal temperature and thermal stability of the lectin was 30$^{\circ}C$ and 40-80$^{\circ}C$, respectively. The maximal pH of this lectin was pH 8.2.

• BMB Reports
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• v.36 no.5
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• pp.514-519
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• 2003

The ribonucleotide reductase (RR) 2 gene of the HSV-2 strain G was cloned, sequenced, and expressed in an E. coli cell. The RR2 gene was located on the PstI 2.4 kb fragment, which was cloned and sequenced. The ORF of the gene was 1,011 bp and its termination codon was TAG; also, the CATATAA sequence was present in the promoter of the RR2 gene. A Poly A signal sequence (AATAAA) was found in the 3'-noncoding region. The RR2 proteins that were produced in the E. coli and Vero cells were confirmed using a Western blot analysis. SDS-PAGE revealed that the molecular weights of the fusion-RR2 that was produced in the E. coli cells were approximately 24 kDa and 38 kDa in the Vero cells. The RR2 proteins were soluble. The differences in the molecular weights might be due to modifications in the Vero cells.

• Microbiology and Biotechnology Letters
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• v.17 no.5
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• pp.494-498
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• 1989

Hybridoma cell lines, which secrete monoclonal antibodies (mAbs) against the surface antigens of Cryptosporidium parvum Sporozoites, were produced by fusing spleen cells of C. parvum Sporozoite-immunized mice with P3-X63-Ag8 myeloma cells. Two cloned antibody-secreting cell lines, Kor1 and Ea2, were established and produced IgG1 and IgG2a antibodies, respectively. Percoll-purified sporozoites were solubilized and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Western blot assay demonstrates that an antigen of 20-kDa was bound by monoclonals. By indirect immunofluorescence microscopy, mAb exhibited uniform binding to the sporozoite surface.

• Journal of Microbiology and Biotechnology
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• v.19 no.9
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• pp.982-986
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• 2009

For the production of ghost bacteria vaccine to prevent the streptococcal disease in aquaculture fish species, a double cassettes vector was constructed and cloned in Escherichia coli DH5${\alpha}$. Ghost bacteria vaccine production from Escherichia coli DH5${\alpha}$/pHCE-InaN-GAPDH-Ghost 37 SDM (SIG) was maximized at a glucose concentration of 1 g/l, agitation of 300 rpm, and aeration of 1 vvm. The maximal efficiency of ghost bacteria formation was obtained at the mid-exponential phase ($OD_{600}=2.0$) with the concentration of 0.77 g/l for SIG. The molecular mass of GAPDH was detected at 67 kDa with the insoluble fraction, by SDS-PAGE and Western blot. The protective efficacy of ghost bacteria vaccine was evaluated by challenge test using olive flounder. The cumulative mortalities of the positive control, formalin-killed cell (FKC) vaccine, and SIG vaccine immunized groups were 91%, 74%, and 57%, respectively. These results suggest that SIG vaccine showed efficacy as a vaccine and had a higher potential to induce protective antibodies than did FKC vaccine.

• Archives of Pharmacal Research
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• v.28 no.6
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• pp.667-674
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• 2005

The leaves of Persimmon (Diospyros kaki L.) has long been used for tea in Korea since it was thought to be effective against hypertension. An anticoagulant fraction was purified through gel filtration G-100, hydrophobic, gel filtration G-150, and FPLC, Phenyl superpose column chromatographies. The purified fraction was homogenous and its Mr was estimated 10,000 Da by gel filtration and SDS-PAGE. The purified fraction was sensitive to treatment of subtilisin B, but not to heat and its activity was not changed after periodate oxidation, indicating that the activity was not due to carbohydrates. It delayed thrombin time (TT), activated partial thromboplastin time (APTT), and prothrombin time (PT) using human plasma. TT was more sensitive than APTT and PT, suggesting that the anticoagulant activity may be caused by a degradation or a defect of fibrin or thrombin. It did not cause the hydrolysis of fibrin after incubation. However, it inhibited thrombin-catalyzed fibrin formation with a competitive inhibition pattern. These results indicate that it may be an antithrombotic agent and that it is bound to fibrinogen binding sites of thrombin.

• BMB Reports
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• v.35 no.6
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• pp.576-582
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• 2002

Collagenase from the internal organs of a mackerel was purified using acetone precipitation, ion-exchange chromatography on a DEAE-Sephadex A-50, gel filtration chromatography on a Sephadex G-100, ion-exchange chromatography on DEAE-Sephacel, and gel filtration chromatography on a Sephadex G-75 column. The molecular mass of the purified enzyme was estimated to be 14.8 kDa by gel filtration and SDS-PAGE. The purification and yield were 39.5-fold and 0.1% when compared to those in the starting-crude extract. The optimum pH and temperature for the enzyme activity were around pH 7.5 and $55^{\circ}C$, respectively. The $K_m$ and $V_{max}$ of the enzyme for collagen Type I were approximately 1.1 mM and 2,343 U, respectively. The purified enzyme was strongly inhibited by $Hg^{2+}$, $Zn^{2+}$, PMSF, TLCK, and the soybean-trypsin inhibitor.

• BMB Reports
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• v.28 no.1
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• pp.33-39
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• 1995

The Gelatinases (typeIV collagenases) are metalloproteinases that may play an important role in tumor invasion and metastasis. Progelatinase A was purified from a conditioned medium of T98G human glioblastoma cells. TIMP-2 complexed progelatinase A and free progelatinase A were separated by heparin affinity HPLC. The final product was homogeneous on SDS-PAGE, with a molecular weight of 64,000 daltons without reduction. TIMP-2 and free progelatinase A were separated from TIMP-2 complexed progelatinase A by reverse-phase HPLC in the presence of trifluoroacetic acid. TIMP-2 complexed progelatinase A was resistant to activation by p-aminophenyl mercuric acetate (APMA), and showed less than 20% of the activity of the TIMP-2 free active enzyme. TIMP-2 free progelatinase A was easily activated to the mature form with a molecular weight of 57,000 daltons by APMA and showed high activity compared to the TIMP-2 complexed enzyme.