• Korean Journal of Fisheries and Aquatic Sciences
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• v.44 no.1
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• pp.8-17
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• 2011

To identify and examine the distribution of proteolytic inhibitory activity in crude extracts from fish eggs, and to determine the applicability of these protease inhibitors as anti-degradation agents in surimi-based products and fish meat, we compared the inhibitory activities of various extracts from fish eggs to those of commercial proteases, such as trypsin and papain. We used the optimal conditions for the screening of trypsin activity: 30 ug/uL of 0.1% trypsin and 0.6 mM Na-benzoyl-L-arginine-p-nitroanilide (BAPNA) with a pH of 8.0 at $40^{\circ}C$ for 60 min. The activities of papain and four commercial proteases were investigated after mixing with 100 ug/uL enzymes and 0.3% casein with a pH of 8.0 at $40^{\circ}C$ for 60 min. We performed a screening assay to detect the inhibitory activity (%) of crude extracts from eight species of fish eggs against the target proteases trypsin and papain. The assay revealed a wide distribution of trypsin and papain inhibitors in fish eggs. The specific inhibitory activities (11.6.28.6 U/mg) of crude extracts from fish eggs against trypsin and BAPNA substrate were higher than that (0.64 U/mg) of egg whites, used as a commercial inhibitor. The inhibitory activities of crude extracts from fish eggs against trypsin, and of egg whites against casein substrate (1.94.4.51 U/mg), were higher than those of papain (0.24.1.57 U/mg) and commercial protease (0.04.0.32 U/mg). The extracts from fish eggs were rich in protease inhibitors that exhibited strong inhibitory activity against trypsin, a serine protease, and papain, a cysteine protease.

• Biomolecules & Therapeutics
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• v.5 no.3
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• pp.223-227
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• 1997

The protective effect of human urinary trypsin inhibitor(UTI) on acute hemorrhagic shock in beagle dog was studied. Hemorrhagic shock was experimentally induced in thoracotomized beagle dogs by removing blood and maintaining low arterial blood pressure for 30 min, and then blood removed was entirely transfused back into the dogs within one hour. When the blood was transfused, UTI was administered together to check the potential protective effect of UTI on hemorrhagic shock. The arterial blood pressure recovery was accelerated slightly by UTI treatment. Blood pH and $P_{a co2}$ returned to normal level in shorter time in the UTI treatment group. These data suggest that UTI may have protective effects on experimentally induced hemorrhagic shock.

• Fisheries and Aquatic Sciences
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• v.4 no.2
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• pp.84-87
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• 2001

Multimeric angiotensin-converting-enzyme-inhibitor (ACE}) containing a trypsin cleavable linker peptide between ACEI was constructed. We made synthetic DNA coding for the ACEI peptide with asymmetric and complementary cohesive ends of linker nucleotides. A tandemly repeated DNA cassette for the expression of concatameric short peptide multimers was constructed by ligating the basic units. The resultant multimeric peptide expressed as soluble and trypsin treated peptide was shown at the same retention time with chemically synthetic ACEI by HPLC. The present results showed that the technique developed for the production of the ACEI multimers with trypsin cleavable linker peptides can be generally applicable to the production of short peptide.

• Journal of Microbiology and Biotechnology
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• v.10 no.2
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• pp.181-186
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• 2000

MAPI (microbial alkaline protease inhibitor) was isolated from cultrue broth of Streptomyces chromofuscus SMF28. The Ki values of MAPI for the representative serine proteases such as chymotrypsin and proteinase K were 0.28 and $0.63{\;}\mu\textrm{M}$, respectively, and for the cysteine proteases cathepsin B and papain were 0.66 and $0.28{\;}\mu\textrm{M}$, respectively. These data indicate that MAPI is not a potent selective inhibitor of serine or cysteine proteases. Progress curves for the inhibition of three proteases by MAPI exhibithe characteristic patterns; MAPI exhibited slow-binding inhibition of cathepsin B. It was rapidly associated with chymotrypsin before the addition of substrate and then reactivation of MAPI-inhibited enzyme was investigated in the presence of substrate. On the other hand, MAPI-proteinase K interaction was typical for those classical inhibitors. When MAPI was incubated with trypsin, there was an extensive reduction in the ingibitory activities of MAPI corresponding to 66.5% inactivation of MAPI, indicating that trypsin-like protease may play a role in the decrease of the inhibitory activity during cultivation.

• Korean Journal of Food Science and Technology
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• v.26 no.1
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• pp.81-87
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• 1994

Two different trypsin inhibitors, TRTI-1 and TRTI-2, were purified to near homogenity from Trichosanthes kirilowii root, by $0{\sim}90%$ saturated ammonium sulfate salting out, DEAE-Sephacel ion exchange chromatography, Sephadex G-50 gel filtration chromatography and trypsin-affinity chromatography. The molecular weight of TRTI-1 and TRTI-2 were estimated to be about 5,000 Da and 24,000 Da, respectively, by gel filtration and must be monomer and homodimer since they contain 4,000 Da and 10,000 Da each on SDS-polyacrylamide gel electrophoresis. TRTI-1 was stable after heating for at least 2 hr at $100^{\circ}C$ but TRTI-2 was completely inactivated after heating for 10 min at $90^{\circ}C$. When Bz-dl-Arg-pNA was used as a substrate of TPCK-treated trypsin, half-maximal inhibitions of TRTI-1 and TRTI-2 were observed at $0.8\;{\mu}M$ and 6\;${\mu}M$, repectively. Both TRTI-1 and TRTI-2 inhibited the hydrolysis of trypsin competitively and Km values were $0.97\;{\mu}M$ and $0.63\;{\mu}M$, respectively. Both TRTI-1 and TRTI-2 specifically inhibited trypsin but they did not inhibit other proteases tested, chymotrypsin, papain, elastase, collagenase, thermolysin, Nagarase, pepsin, and thrombin.

• 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.

• Applied Biological Chemistry
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• v.33 no.2
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• pp.109-115
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• 1990

This study was carried out to investigate the effect of L-cysteine and sodium sulfite on heat inactivation of soybean trypsin inhibitor(STI) and to determine cultivar difference in the inhibitory activity of STI. Effect of L-cysteine and sodium sulfite at different concentrations, pH's, and lengths of treatment on inactivation of STI were studied. The inactivation of STI was spectrophotometrically determined by measuring the rate of production of p-nitroaniline from synthetic substrate, N-benzoyl-DL-arginine-p-nitroanilide. Addition of L-cysteine and sodium sulfite increased magnitude of heat inactivation and greatly inhibited the re-activation of STI. There was no difference STI inactivation in among soybean cultivars employed. The trypsin inhibitory activity of STI of the soybean cultivars ranged from 64.7 to 86.4 TIU(trypsin inhibitor unit) per gram soyflour and the decreasing order of the TIU was Jangback>Hill>Jangyeab, Kwangkyo> Danyeab>Dangkyung>Paldal, Saeal, Duckyu>Hwangkeum. Inhibitory activity of STI was correlated with cysteine $content(r=0.6568^*)$ and with $digestivility(r=-0.7695^{**})$, but there was no correlation between the protein content and the inhibitory activity of STI.

• Natural Product Sciences
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• v.26 no.3
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• pp.207-213
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• 2020

Characterization and in vitro inhibition studies of protease inhibitor from the mushroom Pleurotus floridanus (PfTI) towards the pest Papilio demoleus is studied. The addition of 1 mM Mn²⁺, Na²⁺, Ba²⁺ and Ni ²⁺ enhanced the PfTI activity. The ICP-atomic emission spectrum showed the presence of Ca²⁺, Mg²⁺ and Zn²⁺ in the PfTI. Surfactants SDS and CTAB at a concentration of 1% reduced the PfTI activity whereas, the nonionic detergents Triton X and Tween 80 increased the activity. The inhibitory activity gradually decreased with increase in concentration of DMSO and H₂O₂. The activity was increased by dithiothreitol up to a concentration of 80 μM and inactivated at 140 μM. The activity of PMSF modified PfTI was drastically reduced to 0.234 U/mL at 4 mM concentration and similar results were obtained for modification of cysteine by N-Ethylmaleimide at slightly higher concentrations. The complex of trypsin and PfTI showed complete loss in fluorescence intensity at 343 nm compared with control. In vitro inhibition studies of PfTI with midgut proteases isolated from citrus pest P. demoleus with protease activity of 1.236 U was decreased to 0.613 U by 50 μL (0.1 mg/mL) of the inhibitor. Inhibitor was stable up to 0.04 M concentration of HCl.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.15 no.4
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• pp.263-270
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• 1982

In an attempt to evaluate the nutritional quality of seaweed protein, the effects of heat treatment on the in vitro digestibility and trypsin inhibitor content in seaweed were determined. In this study, the nitrogen-to-protein conversion factors were also calculated on the basis of quantitative amino acid data. The results are as follows : 1. The in vitro protein digestbilty of red seaweeds (P. teoera anc P. suborbiculata) were ranged from 78.5 to 82.2, and green seawerd (E. linza) and brown seaweeds showed value under 80 in vitro digestibility. In general, trypsin inhibitor contents in brown seaweed were higher (0.33-0.54 mg/g) than those of red seaweeds (0.26-0.39 mg/g). And it is noted that the lowest trypsin inhibitor content was shown in green seaweed (E. linza) in spite of lowest in spite digestibility (78.5). 2. The in vitro protein digestibility of sun dried laver (P. tenera) was increased with cooling time (microwave heating), but it was not significant. Hot plate cooking raised the in vitro digestibility from 81. 1 to 84.5. The influence pot cooking time on trypsin inhibitor content was inversely proportional to in vitro digestibility. 3. Computed nitrogen factor, based on amino acid content (Factor method) and Kjeldahl nitrogen content (Kjeldahl mettled), were 5.83 (H. fusiforme)- 6.52 (P. tencra) as Factor method and 5.40 (U. pinnatifida)-6.29 (P. tenera) as Kjeldahl method. Individual value for each nitrogen conversion factor differed by species, especially in brown seaweeds. The best estimate of the protein content of seaweed can be calculated, from multiplying the summed amino acid content by conversion factor (Factor method).

• Journal of the Korean Society of Food Science and Nutrition
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• v.37 no.1
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• pp.117-123
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• 2008

The protein profiles of domestic potato cultivars were evaluated for total protein determination, amino acid composition, SDS-PAGE analysis and scanning densitometry. There were statistically significant differences in the levels of amino acids among potato cultivars. Total nitrogen amount was also significantly different among cultivars, ranging from 1.27 to 1.64%. SDS-PAGE analysis showed that there were significant differences in the content of major potato proteins such as papatin (40 kDa), trypsin inhibitor (20 kDa) and protease inhibitor (15 kDa) among cultivars (p<0.05). The amount of papatin among cultivars with a range of 22.16 to 25.81 mg/g d.w. was higher in Jopung, Shepody and Superior, whereas the amount of protease inhibitors including 15 kDa and 20 kDa was the highest in Jopung (37.0%). The Shepody contains the highest amount of papatin (25.8%) and the lowest of trypsin inhibitor (5.22%). Thus, it is suggested that Shepody is the most desirable cultivar for better nutrition based on the protein profile.