• Microbiology and Biotechnology Letters
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• v.21 no.1
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• pp.18-22
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• 1993

An assay system by using antibody was adopted to screen the endoinulinase producingfungi due to its high specificity toward endoinulinase, To determine whether the affinity-purified rabbit serum, which were generated against the purified endoinulinase, can react only with the endoinulinase, rocket immunoelectrophoresis was performed. The results showed that the serum specifically reacts with endoinulinase but not with exoinulinase and other proteins in the culture media. Using this polyclonal antibody, a strain from 62 fungal colonies was selected and it secreted an endoinulinase in the culture media to the amount comparable to that of Aspergillus ficuum A Tee 16882 known as a high endoinulinase producer.

• KSBB Journal
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• v.14 no.5
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• pp.578-584
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• 1999

The lectin was purified through 0.15 M NaCl extraction, ammonium sulfate precipitation, sepharose 4B affinity chromatography and gel filtration using sephadex G-150 from the leaves of Visum album collected in Mt. Duk Yu. The final gel filtration step resulted in 11.64 folds purification with 0.14% of recovery yield. We also performed biochemical characterization of the purified Visum album lectin. HPLC analysis of lectin purified by gel filtration revealed a singel peak. The analysis of the purified lectin by SDS-PAGE showed a tetramer composed of two identical subunits with molecular weights of 32 and 30 kDa. The lectin was a glycoprotein containing 14.4% carbohydrate, which consist of glucose, fructose, arabinose and xylose, and the amino acids such as phenylalanine, lysine and tyrosine. The purified lectin agglutinated human red blood cell types with similar potency, but when tested against red blood cells from mouse, bovine, rabbit, chicken and porcine, significant difference in potency were observed. Hemaggluting activity was inhibited by D-galactose, D-mannose, D-lactose and D-raffinose, but not by D-glucose, D-glucosamine, D-mannosamine, L-fructose, D-xylose, D-arabinose, D-galacturonic acid, D-fructose, L-rhamnose and N-acetyl-D-galactosamine. The optimal pH and thermal stability of the purified lectin were pH 4.0-7.0 and 20-5$0^{\circ}C$, respectively.

• Microbiology and Biotechnology Letters
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• v.20 no.6
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• pp.655-662
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• 1992

Pectate lyase produced by recombinant strain containing pectate lyase gene from alkalitolerant Bacillus sp. YA-14 was succesively purified with 257.6 purification folds and a 10.2% yields by the affinity method, eM-cellulose column chromatography followed by gel filtration on Sephadex G-I00 column. The optimal pH and temperature for pectate lyase activity were 10.0 and $60^{\circ}C$, respectively. The enzyme was stable between pH 4.0 and 10.0, and up to $50^{\circ}C$. The molecular weight of this enzyme was estimated to be 43,000 daltons by SDS-PAGE. Amino acid analysis showed that the enzyme contained more polar and basic amino acids, especially serine, glycine and tyrosine, than that of various pectate lyase from other strains. The N-terminal amino acid sequence was Ala-Asp-Leu-Gly-His-Gln-Thr.

• Microbiology and Biotechnology Letters
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• v.18 no.6
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• pp.599-606
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• 1990

A chitinase-producing bacterium, Aeromonas salmonicida YA7-625, was isolated from domestic seashore muds. The preferable medium composition for the production of chitinase was as follows: colloidal chitin 1.26% (w/v), tryptone 2.95% (w/v), $MgSO_4-7H_20$ 0.15% (w/v) and $K_2HP0_4$, 0.15% (w/v) (pH 8.5). The highest enzyme production was observed after cultivation of 48 hours at 27OC. The chitinase of Aeromonas salmonicida YA7-625 was purified successively by ammonium sulfate precipitation, affinity adsorption, hydroxylapatite column chromatography and gel filtration. The optimal temperature and pH for the activity of purified chitinase were $50^{\circ}C$ and 7.0, respectively. The molecular weight of purified chitinase was ca. 200,000 daltons and apparent Km value of it was 1.276 mglml on colloidal chitin.

• BMB Reports
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• v.35 no.2
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• pp.228-235
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• 2002

Methionine aminopeptidase (MetAP) catalyzes the removal of an amino-terminal methionine from a newly synthesized polypeptide. The enzyme was purified to homogeneity from Bacillus stearothermophilus (KCTC 1752) by a procedure that involves heat precipitation and four sequential chromatographs (including DEAE-Sepharose ion exchange, hydroxylapatite, Ultrogel AcA 54 gel filtration, and Reactive red 120 dye affinity chromatography). The apparent molecular masses of the enzyme were 81,300 Da and 41,000 Da, as determined by gel filtration chromatography and sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), respectively. This indicates that the enzyme is comprised of two identical subunits. The MetAP specifically hydrolyzed the N-terminal residue of Met-Ala-Ser that was used as a substrate, and exhibited a strong preference for Met-Ala-Ser over Leu-Gly-Gly, Leu-Ser-Phe, and Leu-Leu-Tyr. The enzyme has an optimal pH at 8.0, an optimal temperature at $80^{\circ}C$, and pI at 4.1. The enzyme was heat-stable, as its activity remained unaltered when incubated at $80^{\circ}C$ for 45 min. The Km and Vmax values of the enzyme were 3.0mM and 1.7 mmol/min/mg, respectively. The B. stearothernmophilus MetAP was completely inactivated by EDTA and required $Co^{2+}$ ion(s) for activation, suggesting the metal dependence of this enzyme.

• Food Science of Animal Resources
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• v.18 no.4
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• pp.316-321
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• 1998

In this study, we carried out to isolate lactoferrin from Korean native cattle and Holstein cow by batch extraction, ion exchange chromatography, gel filtration, and affinity chromatography. The purity of the isolated lactoferrin was higher than that of lactoferrin purchased from Sigma, when determined by SDS-PAGE and HPLC analysis. Antibacterial activity of E. coli O111 by Korean native cattle lactoferrin was lower than that of Holstein lactoferrin. A minimal inhibitory concentration(MIC) of Korean native cattle lactoferrin and Holstein lactoferrin was 2.75 mg/ ml and 1.5 mg/ml respectively. The lactoferrin hydrolyzate of Korean native cattle exhibited antimicrobial activity at 0.25 mg/ml, whereas that of Holstein cow exhibited antimicrobial activity at 0.12 mg/ml. The antibacterial potency of the hydrolyzate was at least tenfold greater than that of undigeated lactoferrin with strains tested. The effect of hydrolyzate was bactericidal as indicated by rapid loss of viability of E. coli O111.

• Microbiology and Biotechnology Letters
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• v.23 no.4
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• pp.411-415
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• 1995

The dextranase (EC 3.2.1.11) produced by Aspergillus ustus GR-98 was purified by the following sequential methods; salting-out and dialysis, gel filtration on BIO-GEL P-100, ion exchange chromatography on DEAH-cellulose, affinity chromatography on hydroxyapatite, and preparative electrophoresis. Three active fractions, dextranases 1, 11 and 111, were isolated in electrophoretically pure states, and specific activities of the dextranases were 1,276, 1,154 and 1,125 units/mg, the degrees of yield were 9.0, 3.6 and 2.2%, having 145, 131.1 and 127.8 times as those of culture filtrate in degree of purification, respectively. The enzyme purity was confirmed by the PAGE, SDS-PAGE and get permeation-HPLC.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 1979.04a
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• pp.114.2-115
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• 1979

Horikoshi등이 호 alkali 성 미생물에 관한 연구에서 분리한 수종의 cellulolytic bacteria중에서 가장 강력한 균체의 효소를 생산하는 Aeromonas 속 세균의 cellulolytic 효소에 관한 연구 결과를 보고한다. 공업적으로 생산된 효소를 사용하여 효소작용의 최적조건을 측정하고 gel filtration, ion-exchange chromatography 및 affinity chromatography 로 cel-luplytic 효소를 분리정제하였다. 본 효소의 활성 최적 pH는 7.0~8.5로 alkaline 효소였으며 반응온도 5$0^{\circ}C$에서 가장 강한 활성을 보였다. 분리 정제과정에서 carboxymethyl cellulose (CMC)에 대하여 활성이 있는 단백질이 최소 8종이상 분리되었으며 이중 1개 효소는 CMC에 대해서는 극히 낮은 활성을 보였으나 결정성 기질인 Avicel 에는 강한 활성을 보였다. 본 연구의 결과를 Cellulomonas속 세균 및 Trichoderma속 곰팡이의 효소와 그 성질을 비교 검토하였다.

• Preventive Nutrition and Food Science
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• v.3 no.2
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• pp.152-156
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• 1998

A Fructan-degrading enzyme was partially purified from onion (Allium cepa)bulbs by a combination of ammonium sufate precipitation, concanavalin-A-Affinity chromatography, and ion-exchange and gel-filtration chromatography. The enzyme hydrolyzed sucrose more effectively than inulin and was identified as a $\beta$- fructofuranosidase (invertase). The optimum pH and temperature were pH 5.5 and 35$^{\circ}C$, respectively. The enzymehydrolyzed sucrose with a Km of 1.2mM . The soluble $\beta$-fructofuranosidase is likely glycoprotein based on its ability to bind the lectin concanavalin-A. The enzyme was heatlabie, with mose activity being lost at 5$0^{\circ}C$ in 1 hr of incubation. The onion $\beta$-fructofuranosidase was partially inhibited by ZnCl2 HgCl2 and CuSo4.

• Parasites, Hosts and Diseases
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• v.32 no.4
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• pp.231-242
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• 1994

Pnragonimus westermnni, the lung fluke, is known to migrate to the pulmonary tissue of mammalian hosts and causes pathological changes in the lungs. An acidic thiol-dependent proteinase with a molecular weight of approximately 20,000 daltons was purified to homogeneity using ion-exchange chromatography and gel filtration chromatography. On SDS-PAGE, the molecular weight of the enzyme was 17,500 daltons. Isoelectric point was 6.45. The enzyme was similar to the acidic cysteine proteinase of vertebrates in the properties of pH optimum, substrate specificity and inhibitor sensitivity. Enzymatic activity was stable at pH 5.5 for at least two days when stored at 4℃. The cysteine proteinase was capable of degrading collagen and hemoglobin. Sera of patients with paragonimiasis and mice infected with R westermani reacted in immunoblots with the partially purified proteinase. This result suggested that the cysteine proteinase of P. westermnni may play a role in migration in tissues, and in acquisition of nutrients by parasites from the host. It is also potentially an antigen for the serodiagnosis of paragonimiasis.