• Journal of Veterinary Clinics
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• v.25 no.5
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• pp.317-323
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• 2008

Canine trypsin-like immunoreactivity (cTLI), which is a mirror of the concentration of trypsin and trypsinogen, is a pancreas-specific enzyme and a suitable marker for canine pancreatitis and especially exocrine pancreatic insufficiency (EPI). To develop the immunochromatographic test kit, monoclonal antibodies that recognize cTLI were prepared. Anionic trypsin, cationic trypsin, and chymotrypsin from canine pancreas were successfully purified to homogeneity, using ammonium sulfate fractionation and benzamidine-affinity chromatography. The purification fold for anionic trypsin was 108 times when compared with that of the homogenation of pancreas. The molecular weights by SDS-PAGE analysis were approximately 23 kDa for chymotrypsin and approximately 20 kDa for cationic trypsin and anionic trypsin, respectively. Using the purified trypsin-like proteins, ten hybridomas which secret canine trypsin-specific monoclonal antibody were prepared. Klotz plot indicated that hybridomas, 5G2H10G4 and 2F4A11, have high affinity constant (Ka) of $4.1\;{\times}\;10^{9}$ and $1.8\;{\times}\;10^{9}$, respectively. Especially, 5F9H3 showed the cationic typsin-specific binding pattern and its Ka was determined to $4.5\;{\times}\;10^{9}$. The development of immunochromatographic test kit using these monoclonal antibodies against cTLI will be very useful in the diagnosis of canine EPI or canine pancreatitis.

• Journal of Dairy Science and Biotechnology
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• v.23 no.1
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• pp.1-8
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• 2005

The purpose of this study was to demonstrate biochemical properties and antibacterial activity of lactoferrin(Lf) obtained from the colostrum of Korean native cow. Lactoferrin was isolated from the colostrum of Korean native cow by purification steps using batch extraction, ion exchange chromatography, gel filtration chromatography, affinity chromatography. Other whey protein components that is similar molecular weight and affinity to lactoferrin were gradually removed from crude Korean Native cow's lactoferrin during the purification steps. The molecular weight of the purified Korean native cow's Lf(K-Lf) was 81 kDa, the isoelectric point was 9, and the content of iron was 0.56mg/g, which is indicated that iron saturation of the K-Lf was 40.6%. Amino acid composition and a-helix content were different K-Lf from bovine Lf(B-Lf). Antibacterial activity of E. coli O111 by K-Lf was lower than that of B-Lf. A minimal inhibitory concentration(MIC) of K-Lf and B-Lf was 2.75mg/ml and 1.5mg/ml respectively.

• Journal of Microbiology and Biotechnology
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• v.24 no.4
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• pp.545-555
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• 2014

It has been previously demonstrated that laccases exhibit great potential for use in several industrial and environmental applications. In this paper, two laccase isoenzyme genes, lccB and lccC, were cloned and expressed in Pichia pastoris GS115. The sequence analysis indicated that the lccB and lccC genes consisted of 1,563 and 1,584 bp, and their open reading frames encoded 520 and 527 amino acids, respectively. They had 72.7% degree of identity in nucleotides and 86.7% in amino acids. The expression levels of LccB and LccC were up to 32,479 and 34,231 U/l, respectively. The recombinant laccases were purified by ultrafiltration and $(NH_4)_2SO_4$ precipitation, showing a single band on SDS-PAGE, which had a molecular mass of 58 kDa. The optimal pH and temperature for LccB were 2.0 and $55^{\circ}C$ with 2,2'-azinobis-[ 3-ethylbenzthiazolinesulfonic acid (ABTS) as a substrate, whereas LccC exhibited optimal pH and temperature at 3.0 and $60^{\circ}C$. The apparent kinetic parameters of LccB were 0.43 mM for ABTS with a $V_{max}$ value of 51.28 U/mg, and the Km and $V_{max}$ values for LccC were 0.29 mM and 62.89 U/mg. The recombinant laccases were able to decolorize five types of dyes. Acid Violet 43 (100 g/ml) was completely decolorized by LccB or LccC (2 U/ml), and the decolorization of Reactive Blue KN-R (100 g/ml) was 91.6% by LccC (2 U/ml). Thus, the study characterizes useful laccase isoenzymes from T. versicolor that have the capability of being incorporated into the treatment of similar azo and anthraquinone dyes from dyeing industries.

• Journal of the Korean Chemical Society
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• v.29 no.3
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• pp.295-303
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• 1985

Carboxypeptidase B from Salmo Salar eggs was purified by CM-cellulose, 0.5 ammonium sulfate saturation, DEAE-cellulose, and Sephadex G-75 gel filtration and its enzymatic properties were investigated. Optimum temperature was 55$^{\circ}C$, pH optima were 4.0 and 7.0 at 37$^{\circ}C$, and the enzyme was stable at pH 2.0∼3.0 and 5.5∼7.0 for 1.5h. This enzyme showed substrate specificity hydrolyzing the peptide bond of glycyl-L-arginine. Its K$_m$ values was 0.21mM, and the enzyme activity was stimulated by Cu$^{2+}$ and Fe$^{3+}$, while inhibited by Zn$^{2+}$. The lysine was found to be competitive inhibitor and its K$_i$ value was determined to be 4.3mM. Molecular weight of this enzyme was determined to be 36,400 daltons by SDS-PAGE and the enzyme was monomeric protein composed of 19 kinds of amino acid residues.

• Food Science of Animal Resources
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• v.39 no.3
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• pp.459-473
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• 2019

Lactobacillus rhamnosus GG (LGG) has low resistance to low pH and bile salt in the gastrointestinal juice. In this study, the gel made from whey protein concentrate (WPC) and pullulan (PUL) was used as the wall material to prepare the microencapsulation for LGG protection. The gelation process was optimized and the properties of gel were also determined. The results showed the optimal gel was made from 10% WPC and 8.0% PUL at pH 7.5, which could get the best protective effect; the viable counts of LGG were 6.61 Log CFU/g after exposure to simulated gastric juice (SGJ) and 9.40 Log CFU/g to simulated intestinal juice (SIJ) for 4 h. Sodium dodecyl sulphite polyacrylamide gel electrophoresis (SDS-PAGE) confirmed that the WPC-PUL gel had low solubility in SGJ, but dissolved well in SIJ, which suggested that the gel can protect LGG under SGJ condition and release probiotics in the SIJ. Moreover, when the gel has highest hardness and water-holding capacity, the viable counts of LGG were not the best, suggesting the relationship between the protection and the properties of the gel was non-linear.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.55 no.6
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• pp.791-801
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• 2022

Boil-dried concentrates (BDC) and steam-dried concentrates (SDC) were prepared from highly nutritious olive flounder Paralichthys olivaceus roes (OFR) as seafood processing by-products and their nutritional characteristics were investigated. Although SDS-PAGE profiles of the BDC and SDC proteins were similar to each other, it was observed that three of the five OFR protein bands in the 50-100 kDa range had disappeared. We also detected significant differences in the Hunter's color of the two concentrates in terms of color difference (𝚫E) and whiteness. The recovery amounts of BDC and SDC prepared from 100 g of OFR were 18.6 and 21.4 g, respectively, with respective protein contents of 67.7% and 68.9%. The main amino acids of OFR and concentrate proteins were valine, leucine, lysine, arginine, aspartic acid, glutamic acid and alanine, whereas major minerals were sulfur, potassium, sodium and phosphorus, the amounts of which in concentrates had been significantly reduced. We established that by sterilizing, inactivating endogenous enzymes, and inhibiting microbial growth, the cook-dried process contributes to enhancing the concentration and storage stability of nutrients by reducing water activity, volume, and weight. Accordingly, we suggest that concentrates (BDC and SDC) prepared from OFR have considerable potential as nutritionally fortified materials.

• Journal of Nutrition and Health
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• v.45 no.5
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• pp.429-436
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• 2012

This study was performed to examine the characteristics of protein of red crab (Chionoecetes japonicus) shell powder hydrolyzed by commercial proteases. Red crab shell was digested by commercial proteases, such as Protamex (P), Neutrase (N), Flavourzyme (F), Alcalase (A), Protease M (PM) and Protease A (PA). Protein yield analyzed by Biuret assay, absorbance at 280 nm and brix revealed that PA was the enzyme having the highest proteolytic activity. SDS PAGE showed that molecular weight of proteins produced by protease treatments was various and below 150 kDa. Combinational treatment of proteases (PA + P, PA + PM, PA + F, PA + A) was tried whether these increase protein hydrolysis from red crab shell powder compared to a PA single treatment. Soluble protein content was similar, but amino acid concentration by combinational treatments was higher than PA single treatment [PA + P 247.4 mg/g > PA + F (206.4 mg/g) > PA + A (133.4 mg/g) > PA + PM (59.1 mg/g) > PA (54.9 mg/g)]. Amino acid composition by combinational treatments was slightly different. Most abundant essential amino acids were phenylalanine, glycine, alanine, and leucine, whereas tyrosine and cystine were not detected.

• Microbiology and Biotechnology Letters
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• v.34 no.1
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• pp.40-46
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• 2006

A bacterial strain producing high level of a phytase was isolated from cattle feces and identified as Bacillus subtilis, and designated as Bacillus sp. CF 5-26. The production of the phytase from Bacillus sp. CF 5-26 reached the highest level after 72 hours at $37^{\circ}C$. The optimum condition of the media for the production of phytase was 10% rice bran extract, 0.1% whey protein powder, $0.01%\;CaCl_{2},\;0.01%\;KH_{2}PO_4$. The phytase was purified 20.3 folds with ethanol precipitation, Sephadex G-100, CM Sepharose CL-6B and Sephacryl S-100-HR column chromatography. The molecular weight of the purified enzyme was estimated to be 66 kDa on SDS-polyacrylamide gel electrophoresis. The purified phytase activity was stable up pH 5.0, 7.0, 11.0 and the remaining activity was 50% when it was treated at $100^{\circ}C$ for 1 hour. The substrate specificity of phytase was most active against sodium phytate and inositol polyphosphate compound. And the phytase hydrolysed tripolyphosphate and pyrophosphate a little. The Km value for the sodium phytate was 0.64 mM and the Vmax value was $4.41\;{\mu}mol/min$.

• Journal of Life Science
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• v.20 no.6
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• pp.838-844
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• 2010

A fibrinolytic enzyme of Streptomyces corcohrussi from soil sediment was purified by chromatography using DEAE-Sephadex A-50 and Sephadex G-50. The analysis of SDS-polyacrylamide gel suggested that the purified enzyme is a homogeneous protein and the molecular mass is approximately 34 kDa. The purified enzyme showed activity of 0.8 U/ml in a plasminogen-rich fibrin plate, while its activity in a plasminogen-free fibrin plate was only 0.36 U/ml. These results suggested that the purified enzyme acts as a plasminogen activator. The fibrinolytic activity of the enzyme under the supplementation of protease inhibitors, $\varepsilon$-ACA, t-AMCHA and mercuric chloride in the enzyme reaction was less than 24%, indicating that it could be modulated by the plasmin and/or fibrinogen inhibitors involved in the fibrinogen-to-fibrin converting process. As time passed, $Zn^{2+}$, a heavy metal ion, inhibited the activity to 34.1%. The optimum temperature of the purified enzyme was approximately $50^{\circ}C$ and over 92% of the enzyme activity was maintained between pH 5.0 and 8.0. Therefore, our results provide a potential fibrinolytic enzyme as a noble thrombolytic agent from S. corcohrussi.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.31 no.5
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• pp.637-644
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• 1998

[ $\beta$ ]-Galactosidase was extracted from the internal organ of sea urchin, Hemicentrotus pulcherrimus The enzyme was purified 384.6-fold over the crude extract by the sequential chromatographic methods including DEAE-Sephadex A-25, CM-Cellulose, and Con A-Sepharose 4B affinity chromatography with a recovery $1.26\%$. The molecular weight of the purified enzyme was estimated approximately 94 kDa as monomeric term by SDS-PAGE and Sephadex G-150 gel chromatography. The maximum enzymatic activity was observed at pH 3.0 and $50^{\circ}C$ but the one was stable over the ph range or 3.0$\~$5.0 and below $37^{\circ}C$. The $K_m$ and $V_{max}$ values against PNPG (P-nitrophenyl $\beta$-D-galactopyranoside) were 15.0 mM and 214 $\mu$mole/min per mg protein, respectively. The enzymatic activity was activated by $Ba^{2+}$, but significantly inhibited by $DEP,\;Hg^{2+},\;Sn^{2+}$ and galactose.