• The Korean Journal of Mycology
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• v.50 no.1
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• pp.31-40
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• 2022

We aimed to produce a potent β-glucuronidase inhibitor from wild yeast that could inactivate toxic substances in the colon. Culture supernatants and cell-free extracts of non-pathogenic wild yeasts were prepared and their β-glucuronidase inhibitory activities were measured. Cell-free extract from Candida oleophila WP5-19-1 showed the highest β-glucuronidase inhibitory activity (49.0%). The β-glucuronidase inhibitor was maximally produced (IC₅₀ value; 8.4 mg) when C. oleophila WP5-19-1 was cultured in potato dextrose medium containing 5% dextrose (initial pH; 6.0) at 30℃ for 24 hours. β-glucuronidase inhibitor of C. oleophila WP5-19-1 was partially purified by trypsin hydrolysis, ultrafiltration (3 kDa), and Sephadex G-50 filtration. The partially purified β-glucuronidase inhibitor was stable from 30℃ to 60℃ and at pH 6.0 9.0, and showed residual inhibitory activity of about 80%.

• Korean Journal of Environmental Biology
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• v.39 no.4
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• pp.445-451
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• 2021

In this study, we described the production of an antibody to living modified organisms (LMOs) containing the gene encoding for 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) from Agrobacterium tumefaciens strain CP4 EPSPS provides resistance to the herbicide glyphosate (N- (phosphonomethyl) glycine). These LMOs were approved and have recently been used in the feed, food production, and processing industries in South Korea. Highly efficient monoclonal antibody (mAb) production is crucial for developing assays that enable the proper detection and quantification of the CP4 EPSPS protein in LMOs. This study describes the purification and characterization of recombinant CP4 EPSPS protein in E. coli BL21 (DE3) based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrixassisted laser desorption/ionization time-of-flight mass spectrometry. The production of mAbs was undertaken based on the standard operating procedure of Abclon, Inc.(South Korea), and the purity of the mAbs was assessed using SDS-PAGE. The following five mAb clones were produced: 2F2, 4B9, 6C11, 10A9, and 10G9. To verify the efficiency and specificity of the five developed mAbs, we performed Western blotting analysis using the LM (living modified) cotton crude extracts. All mAbs could detect the CP4 EPSPS protein in the LM cotton traits MON1445 and MON88913 with high specificity, but not in any other LM cottons or non-LM cottons. These data indicate that these five mAbs to CP4 EPSPS could be successfully used for the further development of antibody-based detection methods to target CP4 EPSPS protein in LMOs.

• Journal of Applied Biological Chemistry
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• v.55 no.3
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• pp.173-178
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• 2012

Sucrose-glucan glucosyltransferase (Gtf) is an important enzyme involved in the cavity formation process where insoluble glucan is synthesized. In this study, we purified Gtf from Streptcoccus mutans Ingbritt through ammonium sulfate precipitation, Sephadex G-150, CM-Sephadex, and DEAE-Sephadex column chromatographies. A 13-fold of purification was achieved with a total yield of 6.3%. The apparent molecular mass of the enzyme was determined to be 66 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal pH and temperature were established to be 6.0 and $40^{\circ}C$, respectively. The enzyme activity could be inhibited to 22-59% by 1 mM $Hg^{2+}$, $Cu^{2+}$ and $Al^{3+}$, and to 68% by 1 mM EDTA. It was also inhibited 40% by 2 mM xylitol and 35-45% by 0.05% soluble chitosan, glycol chitosan, and glycol chitin. This is the first report to reveal the inhibition effect of chitin derivatives on Gtf activity, which may be further applicable to develop gargles to overcome cavity.

• Journal of Life Science
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• v.8 no.5
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• pp.614-621
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• 1998

An extracellular inulinase from Penicillium sp. which isolated from soil sample was purified to a single protein th-rough ammonium sulfate fractionation, DEAE-Sephacel ion exchange chromatography and Toyopearl HW 65 F gel filtration. The temperature and pH for the enzyme reaction were around 6$0^{\circ}C$ and 4.0, respectively. The enzyme was stable at 3$0^{\circ}C$-5$0^{\circ}C$ and in the pH range of 4 to 5. $CuCl_2$, $HgCl_2$ and EDTA inhibited the enzyme activity strongly. By contrast, $MnCl_2$ and $CaCl_2$ activated the enzyme activity. The molecular weight of the purified enzyme was esti-mated to be 77,000 dalton by SDS-polyacrylamide gel electrophoresis. The Km values of the enzyme for inulin were calculated to be $2.2\times10^{-3}$M. TLC analysis suggested that purified enzyme is exo-type inulinase. The NH2-terminal amino acid sequences of the purified enzyme was determined to be $NH_2$-X-Glu-Ser-Tyr-Thr-Glu-Lys/Leu-Tyr-Arg-Pro.

• Food Science of Animal Resources
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• v.23 no.4
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• pp.327-332
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• 2003

This study was conducted to investigate the characteristics of strains which were isolated from market milk treated with ESL(extended shelf life) and conventional system, and to compare the microbiological quality of ESL milk with conventional milk. In order to characterize the isolated strains, purification, Gram staining, spore staining, catalase, oxidase, motility test, and identification by means of automatic identificator were performed. The results obtained are as follows: total 364 selected strains were analyzed in this study. Depending upon the isolated source, the number of strains from conventional milk was found to be Higher than ESL-milk. By means of grouping of total strains, Bacillus ssp. and Staphylococcus ssp. showed to be predominant. But most of strains were distributed with various groups except Lactobacillus ssp. When the isolates were compared with milk process methods, Enterococcus ssp. was detected much on market milk treated with LTLT pasteurization. Also, Pseudomonas ssp. was detected much on conventional milk treated with UHT pasteurization. By comparison with genus groups depending upon storage temperature of market milk, the higher milk storage temperature increased, the most frequency detected Bacillus ssp. increased. Also, Pseudomonas ssp. was detected most frequently at 10$^{\circ}C$ storage condition. Generally this genus derived from post-contamination during milk processing and related to the quality of market milk during chilled system. In conclusion, it was shown that ESL system reduced post-contamination during milk process, following the improvement of product quality and life cycle during the distribution of market milk.

• Journal of Life Science
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• v.26 no.3
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• pp.296-301
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• 2016

An antibacterial peptide from skin extract of the catfish Silurus asotus was purified and characterized. The acidified skin extract was put through a Sep-Pak C18 solid phase extraction cartridge using a stepwise gradient and divided into flow-through (F.T.), 10% methanol-elute (RM10), 60% methanolelute (RM60), and 100% methanol-elute (RM100) fractions. RM10, RM60, and RM 100 showed antimicrobial activity against Escherichia coli D31. On the other hand, the F.T. fraction did not show antimicrobial activity. Among the various fractions, RM 60 had the highest activity. RM 60 was partially purified on a cation exchange column (CM52) by a stepwise gradient. The ammonium acetate (pH 5.15) 0.02 M – 0.8 M fraction showed antimicrobial activity. Then an antimicrobial peptide was purified using a 0.6M fraction with strong antibacterial activity through a series of five C18 reversed-phase HPLC columns. For the characterization of the purified peptide, the molecular weight and amino acid sequence were analyzed by MALDI-TOF MS and Edman degradation. The molecular weight of this peptide was about 4182.1 [M+H]⁺. The amino acid sequence of this peptide was partially determined as follows: PALXXKARREAKVKF. These findings suggest that this peptide plays a significant role in the innate defense system of catfish skin.

• BMB Reports
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• v.36 no.6
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• pp.545-551
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• 2003

A cDNA of bovine brain glutamate dehydrogenase (GDH) was isolated from a cDNA library by recombinant PCR. The isolated cDNA has an open-reading frame of 1677 nucleotides, which codes for 559 amino acids. The expression of the recombinant bovine brain GDH enzyme was achieved in E. coli. BL21 (DE3) by using the pET-15b expression vector containing a T7 promoter. The recombinant GDH protein was also purified and characterized. The amino acid sequence was found 90% homologous to the human GDH. The molecular mass of the expressed GDH enzyme was estimated as 50 kDa by SDS-PAGE and Western blot using monoclonal antibodies against bovine brain GDH. The kinetic parameters of the expressed recombinant GDH enzymes were quite similar to those of the purified bovine brain GDH. The $K_m$ and $V_{max}$ values for $NAD^+$ were 0.1 mM and $1.08\;{\mu}mol/min/mg$, respectively. The catalytic activities of the recombinant GDH enzymes were inhibited by ATP in a concentration-dependent manner over the range of 10 - $100\;{\mu}M$, whereas, ADP increased the enzyme activity up to 2.3-fold. These results indicate that the recombinant-expressed bovine brain GDH that is produced has biochemical properties that are very similar to those of the purified GDH enzyme.

• Korean Journal of Microbiology
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• v.43 no.2
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• pp.83-90
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• 2007

Sphingomonas sp. KS 301, which was isolated from oil contaminated soil, was shown to have five different SODs (SODI, II, III, IV, V) which can be separated by DEAE-Sepharose chromatography, and SOD III was finally purified in this study by ammonium sulfate precipitation, DEAE-Sepharose chromatography, Superose 12 gel filtration and Uno-Q1 ion exchange chromatography. The molecular weight of SOD III was 23 kDa as determined by SDS-PAGE and the apparent molecular weight of the native enzyme was estimated to be approximately 71 kDa by Superose-12 gel filtration chromatography. These data suggest that the purified SOD consists of at least two subunits. The specific activity of the SOD III was higher than Mn type or Fe type SOD of Escherichia coli by 5 fold. To determine the type of SOD III, inhibitory effects of $NaN_{3},\;H_{2}O_{2},\;KCN$ were examined. 10 mM $NaN_{3}$ was able to inhibit 56% of the SOD III activity, which indicates that this SOD is Mn type. The optimum pH of the SOD III was 7.0 and the optimum temperature was $20^{\circ}C$. N-terminal amino acid sequence of purified SOD III was most similar to those of Psudomonase ovalis and Vibrio cholerae among bacteria.

• Korean Journal of Microbiology
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• v.39 no.3
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• pp.148-154
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• 2003

Moraxella sp. CK-l is known to inhibits the growth of Anabaena cylindrica, a cyanobacterium. It has been documented that the ability of this growth inhibition of Anabaena cylindrica was attributed to extracellular autolysin from Moraxella sp. CK-l. However, it remains to be elucidated identification and characterization of autolysin have yet been elucidated. In this study, we tried to purify and identify autolysin secreted from Moraxella sp. CK-l. Cells were grown in a complex liquid medium (BGC-11) and culture supernatants were collected, followed by ammonium sulfate fractionation. Fractions were further separated with anion exchange column, Mono-Q, in FPLC system and analyzed by SDS/PAGE. The fraction containing high autolysin activity showed a single distinct protein peak in anion column and molecular mass of about 17 kDa in SDS/PAGE. Nterminal amino acid sequencing of the protein was analyzed, of which result showed the homology with some proteases, including extracellular serine protease, Dichelobacter nodosus.

• Parasites, Hosts and Diseases
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• v.31 no.3
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• pp.259-268
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• 1993

When activity of peroxidase in auld Pnrqfonimn westermqni was monitored using o-dianisidine and $H_2O_2$ as substrates, its specific activity was 1.5 times higher In excretory-secretory product (ESP) than in crude extract. The one was purified by two purification steps of Sephacryl S-300 Superfine gel permeation and DEAE-Trisacryl M anion exchange chromatographies. Its activity increased 16.9 fold with 32.3% recovery. The enzyme was inhibited totally by 1 millimoles of dithiothreitol (DTT), 2-mercaptoethanol and azide. Molecular mass was 16 kDa in reducing SDS-polyacrylamide gel electrophoresis (PAGE) or 19 kDa in TSK-Blue gel filtration high performance liquid chromatography (HPLC). respectively. Special staining for peroxidase by diaminobenzidine on SDS-PAGE confirmed the activity. The peroxidase was less reactive to a paragonimiasis serum when observed by SDS-PAGE/immunoblot. In addition, specific activities of superoxide dismutase (SOD) and catalase were also identified in the ESP. High activities of these antioxidant enzymes in ESP indicate that they are parts of defense mechanisms against reactive oxygen intermediates from host.