• Journal of Animal Science and Technology
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• v.46 no.1
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• pp.77-82
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• 2004

This study was carried out to establish the purification protocol of angiogenin(ANG) from bovine milk. The purification of ANG from bovine milk was performed by using cation chromatography, high-performance liquid chromatography and gel-filtration. We obtained the ANG protein have the molecular weight of about 14 kD by SDS-PAGE analysis. This protein was confirmed as ANG by Nlb-terminal sequence analysis of the first 15 amino acids. Identified amino acids revealed the protein to be identical to that previously reported for bovine ANG.

• Korean Journal of Food Science and Technology
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• v.29 no.2
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• pp.369-375
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• 1997

We have isolated an anticoagulant polysaccharide from the alkali extracts of Coriolus versicolor. The anticoagulant polysaccharide was purified through a gradual ethanol precipitation and three concecutive chromatography of DEAE-Toyopearl 650C, Sephadex G-100, and Sepharose CL-6B by measuring activated partial thromboplastin time (aPTT). The anticoagulant polysaccharide showed the homogenecity on HPLC using a gel permeation column and had about $7.2{\times}10^{5}$ molecular weight. The polysaccharide consisted of fucose, glucose, and galactose in a molar ratio of 1.0:0.2:0.2:0.1, and also compromised 19.32% of sulfate at its constituent sugars. The polysaccharide showed the two typical bands of C-O-S $(823\;cm^{-1})$ and S=O $(1257\;cm^{-1})$ in the IR spectroscopy. The sulfated polysaccharide (CV-40-Va-1) inhibited the blood coagulation via the intrinsic pathway like heparin whose activity produced a concentration dependent effect in aPTT and thrombin time (TT).

• Journal of Microbiology and Biotechnology
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• v.10 no.6
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• pp.805-810
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• 2000

An antifungal protein antagonistic to the rice blast fungus, Pyricularia oryzae was purified from Paenibacillus macerans PM-1 by ammonium sulfate fractionation, Q Sepharose Fast Flow column chromatography, Phenyl Sepharose CL-4B column chromatography and Superose 12 gen filtration. An apparent molecular mass of the purified antifungal protein was determined as 8 kDa by SDS-PAGE and 9 kDa by analytical gel filtration, respectively, suggesting that the purified protein is a monomer. The antifungal protein was stable at pH range from 7-12 and up to $100^{\circ}C$. The protein was also stable at 0.1-1% Tween 20 and Triton X-100. The N-terminal amino acid sequence of the antifungal protein was Thr-Glu-Leu-Pro-Leu-Gly-Ile-Val-Met-Asp-Lys-Tyr-Thr-Asp-Ala-Phe-Lys-Phe-Asp-Met-Phe. Comparison of the determined sequence with other peptide and DNA sequences did not reveal homology at all. Therefore, the purified antifungal protein was speculated to be a novel protein. The condidial germination in vitro of P. oryzae KJ301:93-39 by the purified protein ($5.9{\mu} g/ml$) was limited to $9{\pm}3.2%$ only, compared with $69{\pm}2.4%$ of the control. Ungerminated conidia were swollen at basa and mid cell by the purified protein. In vivo bioassay for inhibition of conidial germination of P. oryzae KJ 301, one of the most predominating racesin Korea. the purified protein ($5.9{\mu} g/ml$)strongly inhibited the conidial germination. The conidia, even though germinated, could not develop any further to produce appressoria efficiently.

• The Korean Journal of Mycology
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• v.27 no.2 s.89
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• pp.100-106
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• 1999

The polysaccharides, intracellular and extracellular, extracted from the liquid culture of the Agrocybe cylindracea were purified and characterized. The mycellial cellular productivity of Agrocybe cylindracea was proved to be almost 2 folds in the shaking culture compared to the standing culture. These polysaccharides were purified by the DEAE-cellulose ion exchange chromatography and the Sepharose 2B size exclusive gel filteration. The two purified fractions of extracellular polysaccharides, ACEPDG and ACEPAG, contained 75.8% and 65.4% total sugar respectively. The total sugar content of ACIPDG and ACIPAG, the two purified fractions of intracellular polysaccharides, were 89.2% and 54.2% respectively. The molecular weights range of all the substances were estimated to be above 100,000, from 300KDa of ACEPDG to 600 KDa of ACIPAG. The results of sugar analysis by HPLC showed that the sugar part of ACEPDG was consisted of glucose and inositol. The ACIPDG, ACEPAG and ACIPAG contained three kinds of monosaccharides, glucose, fructose and inositol.

• The Korean Journal of Mycology
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• v.29 no.1
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• pp.22-27
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• 2001

This study was carried out to get the basic information for the submerged culture and analyze the biochemical components of Naematoloma sublateritium mycelia. The optimal temperature, pH, agitation speed and cultural time for the mycelial growth of Naematoloma sublateritium were $25^{\circ}C$, 5.5, 150rpm and 20 days, respectively. The proximate composition of mycelia was as follows; carbohydrate 55.8% (total sugar 48.7%), crude protein 22.4%, fat 4.1 % and ash 4.7% respectively. Among the free amino acid contents, phenylalanine, alanine and lysine were predominant component. The linoleic acid and palmitic acid were found to be the highest among the free fatty acids. The biopolymer extracts of mycelia was identified to be protein-bounded polysaccharide by color reaction and sepharose CL-4B gel chromatography.

• Journal of the Korean Society of Food Science and Nutrition
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• v.25 no.4
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• pp.687-694
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• 1996

Myrosinase was purified from Korean mustard seed(Brassica juncea) by a sequential process of DEAE-cellulose, concanavalin A-sepharose, and Superose 6 chromatography. The molecular weight of puri-fied myrosinase(II-2) determined by SDS-polyacrylamide electrophoresis was 67KD. About a 248-fold purification for myrosinase II-2 was obtained after Superose 6 chromatography. Optimum pH of the myrosinase was 7.0 and optimum temperature of the enzyme was $3^{\circ}C.$ The enzyme was stable at pH 7.0, and below $30^{\circ}C.$ Cu, Hg and Fe ion significantly inhibited the enzyme activity, but ascorbic acid enhanced, resulting in a maximum activity by 1mM ascorbic acid. Among tile ascorbic acid ana-logues, dehydroascorbic acid inhibited the enzyme activity, whereas others showed a little effect. Reducing agents such as 2-mercaptoethanol and dithiothreitol inhibited the enzyme activity, but the reducing agents with ascorbic acid was enhanced enzyme activity.

• The Korean Journal of Food And Nutrition
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• v.9 no.4
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• pp.404-408
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• 1996

One of the major objectives of the food industry is the enrichment of the functional properties and nutritional value of soybean protein. To attain this goal, an expression system of cDNA encoding native and protein-engineered soybean proteins in a microorganism must be developed and the function then ability of self-assembly and the functionalities of the expressed proteins should be evaluated before the modified genes are transfered to soybean plants. The pro-$\beta$-conglycinin synthesized in E. coli BL21(DE3) comprised approximately 20% of the total bacterial proteins and the expressed protein are formed soluble and trimer such as native protein in E. coli cells. The highly expressed protein was purified to homogeneity by salt precipitation with 20~40$ Ammonium sulfate ion-exchange chromatography with Q-Sepharose and hydrophobic column chromatography with Butyltoyopearl. Therefore, we concluded that the high-level expression system of $\beta$-conglycinin cDNA was established and a relatively simple and rapid method for purifying pro-$\beta$-conglicinin was also developed.

• Journal of Microbiology and Biotechnology
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• v.14 no.6
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• pp.1134-1141
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• 2004

A bacterial strain capable of hydrolyzing sulfate ester bonds in p-nitrophenyl sulfate and agar was isolated from the Southeast coast of Korea. The isolated strain (AS6330) is aerobic, Gram-negative, rod-shaped, and motile. Octadecanoic acid was the major cellular fatty acid in the isolate. An almost complete 16S rDNA sequence of the isolate was determined and the sequence similarity of the 16S rDNA with those of known Sphingomonas spp. was found to be at most $96.4\%$, implying that the isolate was a new Sphingomonas species. The organism was grown optimally at NaCl concentration of $1.5-3.5\%$. Optimum culture conditions were determined to be $30^{\circ}C$ and pH 7.0 for 48 h fermentation using a laboratory fermentor under constant culture conditions. Partially purified arylsulfatase through Q-Sepharose and phenyl­Sepharose chromatographies catalyzed hydrolysis of sulfate ester bonds in agar, and $97\%$ of sulfates in agar were removed after 4 h reaction at $45^{\circ}C$ and pH 7.0. The arylsulfatase from the isolated bacterium might be useful for the removal of sulfate groups in agar.

• Journal of Microbiology and Biotechnology
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• v.15 no.4
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• pp.792-799
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• 2005

Two novel $\beta$-glucuronidases, which metabolize glycyrrhizin (GL) to 18$\beta$-glycyrrhetinic acid-3-O-$\beta$-D-glucuronide (GAMG), were purified from Streptococcus LJ-22 isolated from human intestinal microflora. $\beta$-Glucuronidases I and II were purified to apparent homogeneity, using a combination of ammonium sulfate fractionation, butyl toyopearl, Q-Sepharose, hydroxyapatite Ultrogel, and GL-attached Sepharose column chromatographies, with the final specific activities of 137 and 190 nmole/min/mg, respectively. The molecular sizes of both $\beta$-glucuronidases were found to be 140 kDa by gel filtration, and they consisted of two identical subunits (M.W. 67 kDa by SDS-PAGE). $\beta$-Glucuronidases I and II showed optimal activity at pH 7.0 and pH 6.5, respectively. Both purified enzymes were potently inhibited by $Cu^{2+}$ and PCMS, and had maximum activity on glycyrrhizin, but did not hydrolyze p-nitrophenyl-$\beta$-glucuronides, baicalin, or GAMG These findings suggest that the biochemical properties and substrate specificities of these enzymes are different from those of the previously purified $\beta$-glucuronidases. This is the first reported purification of sugar (not aglycone)-recognizing $\beta$-glucuronidases from intestinal bacteria.

• Journal of Microbiology and Biotechnology
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• v.16 no.3
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• pp.408-413
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• 2006

A strain, P821, with phospholipase D activity was isolated from soil and identified as a Streptomyces species. The phospholipase D enzyme was purified from a culture broth of the isolated strain using ammonium sulfate precipitation and DEAE-Sepharose, phenyl-Sepharose, and Superose 12 HR column chromatographies. The purified enzyme exhibited an optimum temperature and pH of $55^{\circ}C$ and 6.0, respectively, in the hydrolysis of phosphatidylcholine and remained stable up to $60^{\circ}C$ within a pH range of 3.5-8.0. The enzyme also catalyzed a transphosphatidylation reaction to produce phosphatidylserine with phosphatidylcholine and serine substrates. The optimum conditions for the transphosphatidylation were $30^{\circ}C$ and pH 5.0, indicating quite different optimum conditions for the hydrolysis and transphosphatidylation reactions. The gene encoding the enzyme was cloned by Southern hybridization and colony hybridization using a DNA probe designed from the conserved regions of other known phospholipase D enzymes. The resulting amino acid sequence was most similar to that of the PLD enzyme from Streptomyces halstedii (89.5%). Therefore, the enzyme was confirmed to be a phospholipase D with potential use in the production of phosphatidylserine.