• 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.

• The Journal of the Korean Society for Microbiology
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• v.7 no.1
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• pp.21-27
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• 1972

A simple and effective procedure is described for semi-purification of viable Myco. leprae from biopsied lepromatous nodules by trypsinization and high-speed centrifugation. An unique characteristic of this method is a complete omission of conventional grinding or homogenization of minced lepromatous tissues prior to purification. Inoculation of trypsin-purified preparation of Myco. leprae into foot pads and ear lobes of Korean chipmunk(Tamias sibiricus asiaticus, Gmelin) resulted in apparent increases in total number of acid-fast bacilli per inoculated tissue 8 and 12 months after inoculation.

• Journal of Life Science
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• v.12 no.5
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• pp.528-534
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• 2002

A kypsin inhibitor was isolated and purified from Mung bean (Vigna radiata L. wilczek) which has been used as a galenic and traditional food. In addition, we evaluated the pharmacological effect of the mung bean trypsin inhibitor (MBTI) using septic shock induced guinea pig model. Purification was carried out by Sephadex G-50 gel filtration, DEAE-cellulose ion exchange chromatography, and trypsin affinity column. The molecular weight of MBTI was estimated to be about 8,000 Da by 20% SDS-PACE under reducing condition. The chemically determined partial amino acid sequences of the purified MBTI perfectly coincide with those of previously reported MBTI which is BBI type trypsin inhibito. (Bowman-birk inhibitor type). These results suggest that the purified MBTI is authentic. Hypotension shock was prevented by the pretreatment of the MBTI (10 mg/kg of the body weight) on the septic shock guinea pig model caused by psedomonal elastase.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.24 no.5
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• pp.273-288
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• 1991

To elucidate the physiological and biochemical differences between chondrichthyes and osteichthyes, the properties of the specific digestive enzymes in cat-shark, Cephaloscyllium umbratile, and mackerel, Scomber japonicus, were studied. Homogenous trypsin proved through the disc-electrophoresis, SDS-PAG electrophoresis and gel filtration was obtained from the pancreas of cat-shark by $50-70\%$ saturated ammonium sulphate fractionation, DEAE-Sephadex A-50 column chromatography, benzamidine-Sepharose 6B affinity chromatography and Sephadex G-75-120 gel filtration. Two types of trypsins were also obtained from the pyloric caeca of mackerel by $30-70\%$ saturated ammonium sulphate fractionation and the slightly modified procedure from the method adopted in the purification of cat-shark trypsin. The two trypsins, designated trypsin A and B, were proved their homogeneity by disc- and SDS-PAG electrophoresis and gel filtration. The molecular weights of the trypsins were estimated to be 31,700 for cat-shark trypsin, 30,000 for mackerel trypsin A and 29,000 for mackerel trypsin B by SDS-PAG electrophoresis, but those were estimated to be 21,500 for cat-shark trypsin, 23,700 for mackerel trypsin A and 21,500 for mackerel trypsin B by gel filtration. The trypsins exhibited their optimum conditions at pH 9.0 and on temperature ranged from $45^{\circ}C\;to\;50^{\circ}C$ for cat-shark, and at pH 8.0 and a temperature of $50^{\circ}C$ for mackerel trypsin A and B, respectively. The cat-shark trypsin was stable at pH 10.0 and the temperature below $10^{\circ}C$, whereas the mackerel trypsin A and B, were stable in the range over pH 7.0 to pH 9.0 below $10^{\circ}C$ and at pH 8.0 below $35^{\circ}C$, respectively. The mackerel trypsins were severely inhibited by some heavy metal ions such as $Ag^{2+},\;Cu^{2+}\;and\;Hg^{2+}$ compared to cat-shark trypsin. All of the enzymes were also inhibited by antipain, leupeptin, TLCK(tosyllysine chloromethyl ketone) and SBTI(soybean trypsin inhibitor) remarkably. The inhibitory effects of PMSF(phenylmethane sulphonylfluoride), DFP(diisopropyl fluorophosphate) and benzamidine were indicated that these enzymes belong to serine-proteases.

• Fisheries and Aquatic Sciences
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• v.2 no.2
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• pp.218-225
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• 1999

A protease without tryptic and chymotryptic activities was purified from the hepatopancreas of shrimp, Penaeus orientalis, using Q-Sepharose ionic exchange, benzamidine Sepharose-6B affinity, Mono-Q, and gel chromatography. Molecular weight (M.W.) of the protease was estimated to be 27kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS­PAGE). The amino acid composition of the protease was different from that of protease from P. japonicus or trypsin from P. orientalis. The protease was completely inhibited by benzamidine, $N\alpha-p-tosyl-L-lysine$ chloromethyl ketone (TLCK), and phenylmethylsulfonyl fluoride (PMSF) and was not affected by leupeptin, pepstatin, N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), iodoacetate, and ethylenediamine tetra acetate (EDTA). The enzyme did not have any activity against Na-benzoyl-DL-arginine p-nitroanilide (BAPNA) or N-benzoyl-L-tyrosine ethyl ester (BTEE) which are specific substrates of trypsin and chymotrypsin, respectively. However, the protease showed hydrolytic activity for a carboxyl terminal of Tyr, Trp, Phe, Glu, and Cys.

• Applied Chemistry for Engineering
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• v.5 no.2
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• pp.305-312
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• 1994

The fed-batch process which combinded high selectivity of affinity chromatography and membrane process was developed. The mixture of trypsin and chymotrypsin, having almost the same molecular weight and the chemical structure, were used as model enzymes. The water soluble polymer having more affinity for trypsin and celluose acetate membrane gelated in 50vol.% ethanol for removing free enzymes and retentating trypsin-affinity polymer complex simutaneously were used in this system. The membrane pore size was controlled by ethanol concentration in the gellation bath, and the affinity polymer was prepared by polymerization of acrylamide with N-acryloyl-m-aminobenzamidine at $4^{\circ}C$. The trypsin could be effectively concentrated by utilizing an affinity polymer and a prepared UF-50 ultrafiltration membrane. As a result, 86% purity trypsin was recovered by the current purification process.

• Microbiology and Biotechnology Letters
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• v.10 no.4
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• pp.275-281
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• 1982

One strain of Streptomyces sp. (AS-707) isolated from soil was found to produce a biologically active substance that showed a strong inhibitory activity against proteolytic enzymes viz. trypsin, papain, $\alpha$-chymotrypsin, Azotobacter protease, and Bacillus pretense. The substance was separated from culture filtrate by ion exchange column chromatography using Amberlite IRC-50 and CM-cellulose column chromatography. It was found that the recovery yield was 26% as activity basis. The substance was stable in wide pH range from 2.0 to 12.0 at 37$^{\circ}C$, but it was unstable in alkaline pH values at 6$0^{\circ}C$. The activity was thermostable to give 90% activity compared to the intact sample when it was treated at pH5.6 at 10$0^{\circ}C$ for 2 hours.

• Korean Journal of Food Science and Technology
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• v.30 no.1
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• pp.82-89
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• 1998

This study was performed to elucidate the purification and characterization of pretease from saewoo-jeot, a Korean traditional salt-fermented shrimp product. The protease in saewoo-jeot (Acetes japonicus) were extracted, desalted through electrodialysis and purified by ammonium sulfate fractionation, Sephadex G-100 gel filtration and DEAE-cellulose column chromatography. Purified enzyme had specific activity of 8.4 unit/mg, yield of 14% and purification fold of 9.8. Purified enzyme was confirmed as single band protein by polyacrylamide gel electrophresis and the molecular weight was estimated to be about 24 kDa. The optimal pH and temperature for the enzyme activity were 8.0 and $40^{\circ}C$, respectively. The range of its stability to the pH and temperature were 7.0 to 10.0 and $30^{\circ}C\;to\;60^{\circ}C$, respectively. The activity of enzyme to synthetic substrate showed BAPNA and TAME. The enzyme was activated significantly by manganese ions, while inhibited by STI, TLCK. metals $(K^+,\;Li^+,\;Na^+,\;Ca^{++},\;Co^{++},\;Cu^{++},\;Mg^{++},\;Ba^{++},\;Hg^{++},\;Zn^{++},\;Fe^{+++})$. The Km value of the enzyme was $5.1{\times}10^{-7}\;M$ to hammersten casein. It's suggested that purified protease from saewoo-jeot seemed to be trypsin-like enzyme.

• Microbiology and Biotechnology Letters
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• v.36 no.1
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• pp.28-33
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• 2008

The expression vector (pWHM3-TR1R2) for sprT gene encoding Streptomyces griseus trypsin (SGT) followed by two regulatory genes, sgtR1 and sgtR2, was introduced into Streptomyces lividans TK24 and Streptomyces griseus IFO 13350. Various media with different compositions were used to maximize the productivity of SGT in the recombinant trains. he SGT productivity was best when the transformant of S. lividans TK24 was cultivated in R2YE medium (0.74 unit/mL) at 5 days of cultivation. C5/L (0.66 unit/mL) medium also gave a good productivity, but Livid (0.08 unit/mL) and NDSK (0.06 unit/mL) yielded poor productivities. S. griseus IFO 13350/pWHM3-TR1R2 produced SGT by 1.518 unit/mL (C5/L), 1.284unit/mL (R2YE),0.932 unit/mL (NDSK), and 0.295 unit/mL (Livid) at 7 days of cultivation, which was much higher than those from S. lividans TK24/TR1R2. The SGT protein was purified from the culture broth of S. griseus IFO 13350/pWHM3-TR1R2 in C5/L to homogeneity via ammonium sulfate fractionation, and CM-sepharose and SP-sepharose column chromatographies. The specific activity of purified SGT was 69,252 unit/mg, and the final purification fold and recovery yield were 6.5 and 1.4%, respectively.

• The Korean Journal of Zoology
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• v.33 no.1
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• pp.119-125
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• 1990

A Potent inhibitor of trypsin and other various serine proteases including chymotrypsin, elastase, kallikrein, plasmin and subtilisin, has been purified to homogeneity from chick skeletal muscle by convendonal chromatographic procedures. The Inhibitor has an apparent molecular weight of 66, 000 dalton as determined by gel filtration. When the purified inhibitor was electrophoresed in the presence of sodium dodecyl sulfate, there appeared rwo protein bands having molecular weights of 66, 000 and 64, 000 dalton. The 64, 000 dalton protein seems to be the product of 66, 000 dalton protein by a lin'ited proteolysis during the purification procedure or in viuo. Thus, it seems to consist of a single polypeptide. The inhibitor appeared to be glycoprotein and have an isoelectric point of 7.4. It contains relatively large amount (8.33 mole%) of cysteine residues.