• Journal of the Korean Society of Food Science and Nutrition
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• v.19 no.5
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• pp.434-442
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• 1990

Aspergillus sp. CC-29 ws selected for its strong protease activity among various stains of molds found in soil. It was found that the production of alkaline protease reached to maximum when the wheat bran medium containing glucose as carbon source had been cultured for 4 days. Alkaline proteased was purified 36.10 fold from Aspergillus sp. CC-29 The purification procedures included ammonium sulfate fractunation gel filteration on Sepha-dex G-75 G-150 and DEAE-cellulose ion-exchange chromatography, The yield of the purified enzyme was 22.40% The purified enzyme was confirmed as a single band by the polyacryla-mide. When the purified enzyme was applied to SDS-PAGE the molecular weight was estima-ted 24000. The optimum pH for the enzyme activity was 9.0 and the optimum temperature was 4$0^{\circ}C$ The reaction of this enzyme followed typical Michaelis-Menten kinetics with the Km value of 2.10$\times$10-4M with the Vmax of 29.41 $\mu$g/min. The enzyme was reactively stable in alkalic condition and unstable by heat treatment. The activity of alkaline protease was increased by the addition of Ca2+ whereas it was inhibited by Hg2+ Zn2+ at concentration of 1$\times$10-3M.

• The Plant Pathology Journal
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• v.28 no.2
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• pp.197-201
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• 2012

Potyviruses express their RNA genomes through the production of polyproteins that are processed in host cells by three virus-encoded proteases. Soybean plants produce large amounts of protease inhibitors during seed development and in response to wounding that could affect the activities of these proteases. The in vitro activities of two of the proteases of Soybean mosaic virus (SMV) and Tobacco vein mottling virus (TVMV) were compared in the rabbit reticulocyte lysate in vitro translation system using synthetic RNA transcripts. Transcripts produced from SMV and TVMV cDNAs that included the P1 and helper component-protease (HC-Pro) coding regions directed synthesis of protein products that were only partially processed. Unprocessed poly-proteins were not detected from transcripts that included all of the P1, HC-Pro, P3 and portions of the cylindrical inclusion protein coding regions of either virus. Addition of soybean trypsin inhibitor to in vitro translation reactions increased the accumulation of the unprocessed polyprotein from TVMV transcripts, but did not alter the patterns of proteins produced from SMV. These experiments suggest that SMV-and TVMV-encoded proteases are differentially sensitive to protease inhibitors.

• Journal of Microbiology and Biotechnology
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• v.28 no.12
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• pp.2079-2094
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• 2018

Sunflower trypsin inhibitor (SFTI) is a 14-amino-acid bicyclic peptide that contains a single internal disulfide bond. We initially constructed chimeras of SFTI with N-terminal secretion signals from the Escherichia coli OmpA and Pseudomonas aeruginosa ToxA, but only detected small amounts of protease inhibition resulting from these constructs. A substantially higher degree of protease inhibition was detected from a C-terminal SFTI fusion with E. coli YebF, which radiated more than a centimeter from an individual colony of E. coli using a culture-based inhibitor assay. Inhibitory activity was further improved in YebF-SFTI fusions by the addition of a trypsin cleavage signal immediately upstream of SFTI, and resulted in production of a 14-amino-acid, disulfide-bonded SFTI free in the culture supernatant. To assess the potential of the secreted SFTI to protect the ability of a cytotoxic protein to kill tumor cells, we utilized a tumor-selective form of the Pseudomonas ToxA (OTG-PE38K) alone and expressed as a polycistronic construct with YebF-SFTI in the tumor-targeted Salmonella VNP20009. When we assessed the ability of toxin-containing culture supernatants to kill MDA-MB-468 breast cancer cells, the untreated OTG-PE38K was able to eliminate all detectable tumor cells, while pretreatment with trypsin resulted in the complete loss of anticancer cytotoxicity. However, when OTG-PE38K was co-expressed with YebF-SFTI, cytotoxicity was completely retained in the presence of trypsin. These data demonstrate SFTI chimeras are secreted in a functional form and that co-expression of protease inhibitors with therapeutic proteins by tumor-targeted bacteria has the potential to enhance the activity of therapeutic proteins by suppressing their degradation within a proteolytic environment.

• Korean Journal of Food Science and Technology
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• v.36 no.1
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• pp.152-157
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• 2004

Gluten was extracted from domestic wheat flour using UTH buffer (4 M urea in 0.1 M Tris-HCl, pH 8.6) and validated by SDS-PAGE analysis for production of wheat flour products with reduced gluten content.. Anti-gluten polyclonal antibody was made by administering extracted gluten fraction on animal model. Anti-gluten serum titer of extracted gluten fraction was evaluated by ELISA, and that of antibody titer according to administration period. Anti-gluten sera were used for ELISA and immunoblot analysis before and after hydrolysis of gluten fraction at optimal pH and temperature condition for each protease. Gluten fraction separated by SDS-PAGE showed several bands covering 75 to 10 kDa, in which anti-gluten sera were 25, 34, and 45 kDa. Enzyme hydrolysis of gluten fraction revealed protein band sizes to be lower than 15 kDa. Content of pretense from bovine pancreas (b.p. protease) for gluten hydrolysis was estimated as 1 mg in 10 mL gluten fraction extracted for 4 hr.

• Korean Journal of Agricultural Science
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• v.29 no.2
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• pp.78-90
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• 2002

This study was carried out to investigate whether peptide produced from wheat protein by enzyme hydrolysis can be used as a natural antimicrobial agent. Antimicrobial peptide was obtained from wheat protein by protease of 7 species. The produced antimicrobial peptide was purified through ultrafiltration, membrane filtration and HPLC, and molecular weight and amino acid sequence of the purified antimicrobial peptide were determined. Among hydrolysate produced from wheat protein by protease of 7 species, antimicrobial activity was observed for the peptide obtained from Asp. saito protease. The Asp. saito protease did production antimicrobial hydrolysate showing the highest antimicrobial activity at reaction condition of $37^{\circ}C$ and pH 6.0, but not at reaction condition above $50^{\circ}C$. Wheat protein hydrolysate was fractionated by membrane filtration and showed antimicrobial activity between molecular weight 1,000 - 3,000. The antimicrobial activity fraction obtained by membrane filtration was separated through HPLC and showed antimicrobial activity in the peak of retention time 31.1 - 31.8 min. Since after wheat protein protease hydrolysate was heated during 15 min at $121^{\circ}C$, antimicrobial activity was maintained, we could be conviction as heat-stable peptide. Molecular weight of antimicrobial peptide identified by MALDI-mass was 1,633. Amino acid sequence of antimicrobial peptide was cysteine, glycine, prolin, prolin, prolin, valine, valine, alanine, alanine and arginine.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.12
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• pp.1761-1767
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• 2016

Two experiments were conducted to evaluate effects of coated compound proteases (CC protease) on apparent total tract digestibility (ATTD) of nitrogen (N) and energy, and apparent ileal digestibility (AID) of amino acids (AA) and nutrients in diets for pigs. In Exp. 1, 12 crossbred barrows (initial body weight: $20.79{\pm}1.94kg$) were housed in individual metabolism crates and allotted into 2 treatments with 6 piglets per treatment according to weight in a randomized complete block design. The 2 diets were corn-soybean meal basal diets with (0.2 g/kg) or without CC protease supplementation. The CC protease supplementation increased (p<0.05) the digestible and metabolizable N and energy values and the digestibility and retention rate of N in the diet. The ATTD of energy and nutrients had been improved (p<0.05) in the diet supplemented with CC protease. In Exp. 2, 12 crossbred barrows (initial body weight: $20.79{\pm}1.94kg$), fitted with T-cannulas at the distal ileum, were blocked by body weight into 2 groups with 6 pigs each. The diets were the same as those in Exp. 1. The CC protease increased (p<0.05) the AID of crude protein and some essential AA including arginine, isoleucine and leucine. The AID and ATTD of energy and nutrients had been improved (p<0.05) by supplemental CC protease, but the hindgut digestibility of nutrients was unaffected. Overall, the CC protease improved the ATTD of N and energy and AID of some indispensible AA and nutrients in the corn-soybean meal diet for pigs. Therefore, the CC protease supplement could improve the utilization of protein in the corn-soybean meal diet and thus contribute to lower N excretion to the environment.

• Parasites, Hosts and Diseases
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• v.46 no.2
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• pp.95-99
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• 2008

Eosinophil degranulation plays a crucial role in tissue inflammatory reactions associated with helminth parasitic infections and allergic diseases. Paragonimus westermani, a lung fluke causing human paragonimiasis, secretes a large amount of cysteine proteases, which are involved in nutrient uptake, tissue invasion, and modulation of hos's immune responses. There is, however, limited information about the response of eosinophils to direct stimulation by cysteine proteases (CP) secreted by P. westermani. In the present study, we tested whether degranulation and superoxide production from human eosinophils can be induced by stimulation of the 2 CP (27 kDa and 28 kDa) purified from excretory-secretory products (ESP) of P. westermani newly excysted metacercariae (PwNEM). A large quantity of eosinophil-derived neurotoxin (EDN) was detected in the culture supernatant when human eosinophils isolated from the peripheral blood were incubated with the purified 27 kDa CP. Furthermore, the 27 kDa CP induced superoxide anion production by eosinophils in time- and dose-dependent manners. In contrast, the purified 28 kDa CP did not induce superoxide production and degranulation. These findings suggest that the 27 kDa CP secreted by PwNEM induces superoxide production and degranulation of human eosinophils, which may be involved in eosinophil-mediated tissue inflammatory responses during the larval migration in human paragonimiasis.

• Asian-Australasian Journal of Animal Sciences
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• v.28 no.9
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• pp.1317-1326
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• 2015

This study investigated the metabolizable energy (ME) intake, net energy of production (NEp), heat production (HP), efficiencies of ME use for energy, lipid and protein retention as well as the performance of broiler chickens fed diets based on cassava chips or pellets with or without supplementation with an enzyme product containing xylanase, amylase, protease and phytase. The two products, cassava chips and pellets, were analysed for nutrient composition prior to feed formulation. The cassava chips and pellets contained 2.2% and 2.1% crude protein; 1.2% and 1.5% crude fat; and 75.1% and 67.8% starch, respectively. Lysine and methionine were 0.077%, 0.075%, and 0.017%, 0.020% protein material, respectively, while calculated ME was 12.6 and 11.7 MJ/kg, respectively. Feed intake to day 21 was lower (p<0.01) on the diet containing cassava chips compared to diets with cassava pellets. Enzyme supplementation increased (p<0.01) feed intake on all diets. Live weight at day 21 was significantly (p<0.01) reduced on the diet based on cassava chips compared to pellets, but an improvement (p<0.01) was noticed with the enzyme supplementation. Metabolizable energy intake was reduced (p<0.01) by both cassava chips and pellets, but was increased (p<0.01) on all diets by enzyme supplementation. The NEp was higher (p<0.01) in the maize-based diets than the diets containing cassava. Enzyme supplementation improved (p<0.01) NEp in all the diets. Heat production was highest (p<0.01) on diets containing cassava pellets than on cassava chips. It is possible to use cassava pellets in diets for broiler chickens at a level close to 50% of the diet to reduce cost of production, and the nutritive value of such diets can be improved through supplementation of enzyme products containing carbohydrases, protease, and phytase.

• Microbiology and Biotechnology Letters
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• v.30 no.4
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• pp.325-331
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• 2002

investigated to maximize the production of fibrinolytic enzyme from Fomitella fraxinea mycelia. Among the tested media, Coriolus versicolor medium (CVM) showed the highest production for the enzyme. 2% galactose, 0.6% yeast extract and 0.1% $NaNO_3$, 0.1% $K_2HPO_4$, and 0.05% $MgSO_4$.$7H_2O$ as carbon, nitrogen, phosphorus, and inorganic salt sources resulted in the maximum level of the enzyme activity, respectively. The enzyme production from F. fraxinea was reached to highest level after the cultivation for 10 days at $25^{\circ}C$ and pH 9. The enzyme activity of culture supernatant was most active at $40^{\circ}C$ and pH 10. The activity of the enzyme was inhibited by phenylmethylsulfonylfluoride and aprotinin, suggesting that it is a serine protease.

• KSBB Journal
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• v.20 no.3
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• pp.215-219
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• 2005

Production of alkaline pretense by Bacillus clausii I-52 was optimized by experimental design methods. Among 7 medium components, three (wheat flour, sodium citrate, sodium carbonate) were selected as components affecting the pretense activity significantly by Plackett-Burman methods. Furthermore the ranges of effective concentrations were determined by Box-Behnken methods. The objective function describing the alkaline pretense production was obtained and optimum concentration of 3 components was determined by using response-surface methods (RSM). Theoretical maximum production was 74000 U/mL (Wheat flour: 0 g/L, Sodium citrate: 5 g/L, Sodium carbonate: 10 g/L). With the optimized medium composition, 92000 U/mL alkaline protease was produced experimentally, resulting in $90\%$ increase compared to before-optimization production (49000 U/mL).