• Journal of Microbiology and Biotechnology
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• v.23 no.4
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• pp.527-533
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• 2013

Polyclonal antibodies labeled with a tracer have been commonly used as secondary antibodies in immunochemical assays to quantify the concentration of antibody-antigen complexes. The majority of these antibodies conjugated with a tracer are commercially available, with the exception of few untouched targets. This study focused on the production and application of mouse anti-quail IgY as an intermediate antibody to link between quail egg yolk IgY and goat anti-mouse IgG-HRP as primary and secondary antibodies, respectively. Subsequently, the produced mouse anti-quail IgY was labeled with horseradish peroxidase (HRP) and its efficiency on enzyme linked immunosorbent assay (ELISA) was compared with that of commercial rabbit anti-chicken IgY-HRP. As an intermediate antibody, mouse anti-quail IgY was successfully produced with good affinity and sensitivity (1:10,000) to the primary and secondary antibodies. Subsequently, mouse anti-quail IgY was effectively conjugated with HRP enzyme, resulting in a secondary antibody with good sensitivity (1:10,000) to quail anti-V. parahaemolyticus and V. vulnificus IgY. The detection limit was $10^5$ CFU/ml for both V. parahaemolyticus and V. vulnificus. The efficiency of the produced conjugate to detect quail IgY on ELISA was comparable to that of the commercial rabbit anti-chicken IgY-HRP, and hence the produced and labeled mouse anti-quail IgY-HRP can be used as a secondary antibody to detect any antibody produced in quail.

• Preventive Nutrition and Food Science
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• v.13 no.3
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• pp.196-203
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• 2008

Hwangtae, dried Alaska pollack, is a major storage product in the fish processing industry. Hwangtae is prepared by removing the internal organs and drying outdoors during the cold witner months by allowing it to thaw during the daytime and re-freeze at night under sub-zero ($-10^{\circ}C$) conditions and gradually dry from December until the next April for around 5 months from Myungtae. In this study, ground Hwangtae was hydrolyzed using two proteolytic enzymes (pepsin and alcalase) which produced five soluble active peptides from Hwangtae (yellowish dried Pollack, Theragra chalcogramma) protein. Two different peptides with strong antioxidative activity were isolated from the hydrolysate using consecutive chromatographic methods of Sephadex G-25 gel, ion-exchange chromatography on a Sepharose-Sephadex C-25 gel, and high-performance liquid chromatography. The isolated peptides, APO1 and APO2, were composed of 16 and 13 amino acid residues, respectively. Both peptides contained a Gly residue at the C-terminus and the repeating motif Gly-Pro-Hyp. The peptide with a molecular weight less than 1,000 Daltons (APACE) obtained from enzymatic hydrolysates of Hwangtae exhibited the highest ACE inhibitory activity. The APACE peptides was composed of 4 amino acid residues (Gly-Leu-Leu-Pro). These results suggest that Hwangtae hydrolysates could be a good source of peptides with ACE inhibitory activity. Biochemical analysis indicated that two 70 kDa peptides (APG1 and APG2) isolated from the hydrolysate had gelatinoytic activity, which was shown to be a calcium dependent protease type as showed by gelatin SDS PAGE.

• The Journal of Natural Sciences
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• v.14 no.2
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• pp.67-82
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• 2004

A thiol-specific antioxidant protein (TSA or TPx) was purified from Halophilic archeabacteria Halococcus agglomeratus, by DEAE-Cellulose, Phnyl, sepharose, Sephadex G-75, Sephacryl S-100, Sephacryl S-200, and Q-Wepharose FF. This protein exhibited the preventeive effect against the inactivation of glutamine synthehase (GS) activity was support by a thiol-reducing equicalent such as dithiothreitol. TPx activity was maximal at NaCl concentration above 500mM. The molecular mass of the protein was determinated to be 22-kDa by SDS-PAGE. The TPx purified from Halococcus agglomeratus seems to be similar to other TPx family, except for the salt requirement for the maximal antioxidant activity.

• The Korean Journal of Mycology
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• v.31 no.3
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• pp.168-174
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• 2003

In this study, Cordyceps militaris was grown in a liquid medium containing colloidal chitin. A chitinase was purified from the supernatant or cultured medium by ammonium sulfate fractionation, DEAE-Sephadex A-25 and Sephadex G-50 column chromatography. Optimum temperature and pH of this enzyme were $35^{\circ}C$ and 5.5, respectively. The molecular weight of the chitinase was estimated to be 48.5 kDa by SDS-PAGE and its Km value was 0.57 mM. The activity of this enzyme was inhibited by $Cu^{2+},\;Mn^{2+},\;Hg^{2+},\;Zn^{2+},\;CO_{3}^{2-},\;SO_4^{2-},\;CN^-,\;ion,\;and\;OCN^-$ maleic anhydride, acetic anhydride or N-bromo succinimide, especially strongly inhibited by sodium cyanate for 84.0 percentage. But its activity wag slightly stimulated by $Mg^{2+}\;and\;K^+$ ion, respectively. The products formed during hydrolysis of the hexa-N-acetylchitohexaose with this enzyme were N,N'-diacetylchitobiose and N,N',N'-triacetylchitotriose. These results imply that this purified enzyme may be an endo-chitinase.

• Journal of Microbiology and Biotechnology
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• v.7 no.2
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• pp.107-113
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• 1997

A strain producing a high amount of chitinase was isolated from soil, identified as Pseudomonas sp., and tentatively named Pseudomonas sp. YHS-A2. An extracellular chitinase of Pseudomonas sp. YHS-A2 was purified according to the procedure of ammonium sulfate saturation, affinity adsorption, Sephadex G-100 gel filtration and Phenyl-sepharose CL-4B hydrophobic interaction column chromatography. The molecular weight of the purified enzyme was estimated to be 55 kDa on SDS-PAGE was confirmed by active staining. Optimal pH and temperature of the enzyme are pH 7.0 and $50^{\circ}C$, respectively, and the enzyme is stable between pH 5.0 and 8.0 and below $50^{\circ}C$. The main products of colloidal chitin by the chitinase were N-acetyl-D-glucosamine and N,N'-diacetylchitobiose both of which were detected by HPLC analysis. The enzyme is supposed to be a random-type endochitinase which can degrade any position of ${\beta}$-l,4-linkages of chitin and chitooligosaccharides. The chitinase inhibited the growth of some phytopathogenic fungi, Fusarium oxysporum, Botrytis cineria, and Mucor rouxii and these antifungal effects were thought to be due to the characteristics of endochitinase.

• Food Science and Biotechnology
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• v.17 no.4
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• pp.766-771
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• 2008

The proteolytic enzyme from Saccharomyces sp. B101 was purified to homogeneity by ammonium sulfate fractionation, ultrafiltration, diethyl aminoethyl (DEAE)-Sephadex A-50 ion-exchange chromatography, and Sephadex G-100 gel filtration chromatography from the culture supernatant of Saccharomyces sp. B101. The specific activity and the purification fold of the purified enzyme were 4,688.9 unit/mg and 18, respectively. The molecular weight of the purified enzyme was estimated to be 33 kDa by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature for the enzyme activity were pH 8.5 and $30^{\circ}C$, respectively. The enzyme activity was relatively stable in the pH range of 6.5-8.5 at below $35^{\circ}C$. The salt-tolerance and stability for the enzyme activity were relatively stable even at NaCl concentrations of 10 and 15%. The activity of enzyme was inhibited by $Ag^{2+}$ and $Fe^{2+}$, and activated by $Mn^{2+}$. In addition, the enzyme activity was potently inhibited by ethylenediaminetetraacetic acid (EDTA) and phenylmethyl sulfonylfluoride (PMSF). Based on these findings we concluded that the purified enzyme was a serine protease. Km and Vmax values for hammastein milk casein were 1.02 mg/mL and 278.38 unit/mL, respectively.

• Journal of Life Science
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• v.7 no.3
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• pp.198-204
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• 1997

The signal transduction through tyrosine kinases play important roles in neuronal development and synaptic regulation. We carried out immunoblot analyses to study tyrosine=phosphorylated proteins in the rat cerebellar postsynaptic density (PSD), a protein-rich cytoskeletal specialization underlying beneath the postsynaptic membrane. The overall protein composition of cerebellar PSD fractions was similar to that of the forebrain’s and only a few bands were different in Coomassie stain. Immunoblot analyses with phosphtyrosine-specific antiboy (4G10) showed that there are many more tyrosine-phosphorylated proteins in the cerebellar PSD than in the forebrain PSD. Interestiingly, a major phosphotyrosine signals in cerebellar PSD fractions was associated with a 50 kD molecular size, named as PSD-50. Migration of PSD-50 coincided with that of $\alpha$CaMKII and remained in the pellet fraction after N-octylglucoside extraction. These results indicate that tyrosine phosphorylation is important in cerebellar synaptic regulation and that the PSD-50 may be same as $\alpha$CaMKIIor a new protein which is a major substrate of tyrosine kinase.

• Preventive Nutrition and Food Science
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• v.2 no.2
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• pp.174-179
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• 1997

CMCase produced by recombinant E. coli JM109 (pCEH#4) containing CMCase gene from Cellulomonas sp. YE-5 was purified to 24.3 fold and 2.6% yield by ammoniumsulfate precipitation, DEAE-cellulose column chromatography and gel filtration on Sephadex G-100. The optimum pH and temperature for CMCase activity were pH 7.0 and 5$0^{\circ}C$. The enzyme was stable between pH 5.0 and 10.0, and up to 6$0^{\circ}C$. The molecular weight of he enzyme was estimated to be approximately 40,000 daltons by SDS-PAGE. Analysis of the amino acid composition showed that the enzyme contained many glycines and acidic amino acids. The enzyme was an endo-type CMCase and the final enzyme reaction product from hydrolysis of Cm-cellulose by the enzyme was cellobiose. {TEX}$K_{M}${/TEX} value determined with CM-cellulose was 1.28mM.

• International Journal of Industrial Entomology and Biomaterials
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• v.37 no.2
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• pp.95-99
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• 2018

In insect defense system, antimicrobial peptides (AMPs) are one of important biological molecules to survive in a variety of environments. Insect can synthesize AMPs to protect against invading pathogens in humoral immune response. Taking more advantage of biological antimicrobial molecules, we report antibacterial activity of two cecropin type peptides, cecropin and moricin, isolated from the silkworm against four salmonella species. In this work, we purified antimicrobial candidate peptides (AMCP) from the extracts of immune challenged silkworm larval hemolymph by two-step chromatographic purification procedure, cation exchange and gel permeation chromatography. The molecular weights of purified peptides were estimated to be about 4 ~ 5 kDa by Tricin SDS-PAGE analysis, and identified as silkworm cecropin and moricin by NCBI BLAST homology search with their N-terminal amino acid sequences. As antibacterial activity assay, the purified peptides showed stronger antibacterial activity against Salmonella pathogens with an MIC value of $1{\sim}4{\mu}g/mL$. Therefore two cecropin type peptides purified from the silkworm will be valuable potential materials for development of new natural antibiotics.

• Microbiology and Biotechnology Letters
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• v.47 no.3
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• pp.412-422
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• 2019

The present study reports the morphological and molecular characterization of the fungal strain, CMSS06 and evaluates its raw starch hydrolyzing ability in four different agricultural substrates (rice bran, banana peel, cassava tubers, and coconut water). The potential use of each agricultural substrate to replace the expensive fermentation media was evaluated with six different fermentation media: rice bran (RB), banana peel (BP), cassava starch (CS), cassava in coconut water (CSCW), cassava in modified coconut water (CMCW), and pure Coconut water (CW). The fungal strain CMSS06 was identified as Thielaviopsis ethacetica by the analysis of the ITS sequences. The T. ethacetica alpha amylase enzyme exhibited maximum alpha amylase activity at 72 h, pH 7.0, and $40^{\circ}C$ on soluble starch. This species resulted in the highest enzyme activity (mU/ml) of 26.06, 10.89, 58.82, 14.2, and 54.67 with the RB, BP, CS, CSCW, and CMCW fermentation media, respectively. The results indicate that CS can be used as a carbon substrate and CMCW can be used to accelerate the fermentation by T. ethacetica. The enzyme was partially purified by 40-60% ammonium sulphate fraction, and it showed total enzyme activity, total protein content, specific activity, purification fold, and a recovery of 2400 mU, 30 mg, 80 mU/mg, 2.7, and 71.1%, respectively. The molecular mass of the T. ethacetica alpha amylase was estimated on SDS-PAGE, and two bands around 50 kDa and 70 kDa were identified. The present study implies that T. ethacetica can produce alpha amylase, and it can be used to hydrolyze raw starch during the fermentation processes.