• Journal of Microbiology
• /
• v.41 no.2
• /
• pp.73-77
• /
• 2003

Four proteolytic bacteria were isolated and identified from a rotating biological contactor based on Bacillus. The four isolates, Ni 26, 36, 39 and 49 were identified as B. vallismortis, B. subtilis, Aeromonas hydrophila and B. amyioliquefaciens, respectively, based on their biochemical properties and 16S rDNA sequence analyses. The optimal proteolytic activity was observed in the temperature and pH ranges of 40-70$^{\circ}C$ and 8.0-8.5, respectively. The proteolytic activities of all the isolates were partially inhibited by phenylmethylsulfonylfluoride (PMSF), and the isolates Ni 26, Ni 39 and Ni 49 were inhibited by the metalloprotease inhibitor, 1,10-phenanthroline. Zymographic analyses of the culture supernatants revealed the presence of at least two pretenses in all isolates.

• KSBB Journal
• /
• v.10 no.4
• /
• pp.374-385
• /
• 1995

Nutritional requirements and cultural conditions for the production of extracellular gelatin-degrading proteolytic enzyme by Bacillus subtilis B0021 were investigated. Optimum carbon source for proteolytic enzyme production was salicin, but it was substituted by glucose for economical reason. The fermentation medium giving a maximum proteolytic enzyme activity was consisted of 1.5%(w/v) glucose, 2.5%(w/v) yeast extract, and 0.001%(w/v) manganese sulfate and 0.002%(w/v) ferrous sulfate. Proteolytic enzyme activity of B. subtilis B0021 was completely inhibited by 0.5%(w/v) tannic acid. Initial pH was optimal at 7.0 and the enzyme activity in the flask culture usually reached a maximal level after 36 hours of fermentation at $30^{\circ}C$. In the $5\ell$ fermentor fermentation at $30^{\circ}C$, enzyme activity was maximum at 36 hour of cultivation but after this enzyme activity was decreased rapidly. Initial viscosity of 45%(w/v) gelatin(2,800mPas) was decreased rapidly to 96%(mPas) after hydrolysis for 4hr at $40^{\circ}C$ by crude enzyme of B. subtilis B0021.

• BMB Reports
• /
• v.32 no.6
• /
• pp.579-584
• /
• 1999

Mantis egg fibrolase (MEF-3) was purified from the egg cases of Tenodera sinensis using ammonium sulfate fractionation, gel filtration on Bio-Gel P-60, DEAE Affi-Gel blue gel affinity chromatogragphy, and MONO-Q anion-exchange chromatography. This protease had a molecular weight of 35,600 Da as determined by SDS-polyacrylamide gel electrophoresis under reducing conditions and its isoelectric point was 6.0. The N-terminal amino acids sequence was Ala-Thr-Gln-Asp-Asp-Ala-Pro-Pro-Gly-Leu-Ala-Arg-Arg. This sequence was 80% homologous to the serine protease from Tritirachium album. MEF-3 readily digested the ${\alpha}$-and ${\beta}$-chains of fibrinogen and more slowly the ${\gamma}$-chains. It showed strong proteolytic and fibrinolytic activities. Phenylmethanesulfonyl fluoride and chymostatin inhibited its proteolytic activity, while EDTA, EGTA, cysteine, ${\beta}$-mercaptoethanol, elastinal, tosyl-lysine chloromethylketone, and tosyl-amido-2-phenylethyl chloromethyl ketone did not affect its proteolytic activity. Among the chromogenic protease substrates, the most sensitive one to the hydrolysis of MEF-3 was benzoyl-Phe-Val-Arg-p-nitroanilide. Based on these experimental results, we speculated that MEF-3 is a serine protease with a strong fibrin(ogen)olytic activity.

• Korean Journal of Food Science and Technology
• /
• v.34 no.2
• /
• pp.283-289
• /
• 2002

This study was performed to search for potential microorganism that has rapid fermenting and physiological function from anchovy sauce. We isolated three bacterial strains, JM-1, JM-2, and JM-3 with proteolytic and fibrinolytic activity from anchovy sauce. Among the 3 bacterial strains, JM-3 showed the strongest proteolytic and fibrinolytic activity. Bacterial strain JM-3 was gram-positive rod, motile and formed endospore. The 16S rRNA of bacterial strain JM-3 was amplified by PCR and then its sequence was determined by ABI 310 genetic analyzer. The 16S rRNA sequence of bacterial strain JM-3 was compared to BLAST DNA database and identified to Bacillus subtilis with 99% of homology. The optimum temperature, pH and NaCl concentration for growth of B. subtilis JM-3 were $40^{\circ}C$, 5.0 and 0%, respectively. The optimum temperature, pH and NaCl concentration for proteolytic and fibrinolytic enzyme production of B. subtilis JM-3 were same as optimum conditions for growth. At 20% of NaCl concentration which is common NaCl concentration of fish sauce, B. subtilis JM-3 showed about 60% of proteolytic and fibrinolytic activity of 0% NaCl concentration. From above results, we found that B. subtilis JM-3 will be able to used for starter of functional fish sauce.

• The Korean Journal of Food And Nutrition
• /
• v.7 no.1
• /
• pp.51-57
• /
• 1994

A nuluk, a Korean traditional koji for brewing, was made with wheat flour and Aspergillus usamii mot. shirousamii S1 which had strong abilities in producing amylase and protease. The cultural conditions for the production of saccharogenic and proteolytic enzymes were tested. The productivities of saccharogenic and dextrogenic enzymes were improved when nuluk was made with unsteamed wheat flour as compared with steamed one, but those of proteolytic enzyme and organic acid were reduced. The addition of water containing 0.5% of hydrochloric acid was unfavorable for the production of saccharogenic, dextrogenic and proteolytic enzymes. The optimum ratios of water added to wheat flour for the production of saccharogenic enzyme and proteolytic enzyme were 32% and 28%, respectively on the basis of wheat flour. The optimum temperatures for the production of saccharogenic enzyme and proteolytic enzyme were 36$^{\circ}C$ and 28$^{\circ}C$, respectively. The activity of saccharogenic enzyme reached its maximum after 120 hours of cultivation at 36$^{\circ}C$, but that of proteolytic enzyme 96 hours. The productivity of saccharogenic enzyme was enhanced when the nuluk was molded after 24 hours of precultivation but that of proteolytic enzyme was reduced as compared with no molding.

• Journal of Pharmaceutical Investigation
• /
• v.19 no.2
• /
• pp.81-86
• /
• 1989

The proteolytic enzyme extracted from Neungee [Sarcodon aspratus (Berk.) S. Ito] was purified by using Tris-acryl CM-cellulose column chromatography and chromatofocusing. The specific activity of the purified enzyme increased 15.8 times as compared with that of the crude enzyme. The enzyme was homogeneous on polyacrylamide gel electrophoresis and stable at pH values ranging from 4.0 to 10.8. The enzyme activity remained unchanged when the mushroom and the purified enzyme were stored for 3 years and 6 months at 4°C, respectively. The enzyme was found to be an endogeneous protease.

• Applied Biological Chemistry
• /
• v.7
• /
• pp.67-77
• /
• 1966

For the production of proteolytic enzyme wilth Aspergillus, the examination is made on the culture-time and koji extracting conditions, during producing koji. 1. The highest activity showed up when the culture-time took 50 hours for Aspergillus sojae and 60 hours for Aspergillus flavus. 2. When the cultured koji was extracted by a buffer solution and water, the former gave the product of higher activity until pH 7 through pH 12, and water until pH 3 through pH 7. 3. In the method of crushing and granule extractions, crushing extraction produced the one of higher activity than granule. 4. The highest activity showed up when Aspergillus sojae took 5 hours (Aspergillus flavus 4 hours) in the time of extracting enzyme solution. 5. The highest activity showed up when both Aspergillu sojae and Aspergillus flavus reacted and indicated $37.60^{\circ}C$ in the reaction temperature and activity.

• Korean Journal of Fisheries and Aquatic Sciences
• /
• v.32 no.4
• /
• pp.458-465
• /
• 1999

Distribution of the proteolytic activities of crude pretense extracted from the viscera of ten kinds of fish was examined. Their proteolytic activities on proteinous substrates (azocasein, hemoglobin, and casein) from the viscera of anchovy, bastard flatfish, mackerel and red sea bream were higher than those of other fishes, and the crude pretenses were further fractoinated with acetone or ammonium sulfate. Optimum concentrations for pretenses fractionation were $0\~55\%$ for acetone and $30\~70\%$ for ammonium sulfate. The fractionated viscera pretense of mackerel showed the highest proteolytic activity among four kinds of fishes. Activities of cathepsin D- and pepsin-like enzymes at pH 3.0, cathepsin L-, B-, H- and G-like enzyme at pH 6,0, and Hypsin- and chymotrypsin- like enzymes (pH 8.0) were detected in the fractionated viscera pretense, whereas activities of cathepsin L- and chymoeypsin-like enzyme were observed in commercial pretenses. Proteolytic activities of Alcalase, Protamex, and Aroase AP-10 for azocasein were slightly higher than the fractionated viscera pretenses, but their amidolytic activities at pH 6.0 and 8.0 toward synthetic substrates were lower than counterpart. The fractionated pretenses from fish viscera would be utilized as commercial pretenses.

• Korean Journal of Fisheries and Aquatic Sciences
• /
• v.29 no.3
• /
• pp.296-308
• /
• 1996

Proteolytic enzymes responsible for post-mortem degradation of the fish tissues have been studied in regard with screening the proteases distributed in the fish body by reacting with the specific synthesized substrates. Activities of cathepsin L, B, H, G, and D like enzymes were detected in the muscle crude protease from the both kind of fish, dark fleshed fish (anchovy, Engraulis japonica, and gizzard-shad, Clupanodo punctatus) and white fleshed fish (seabass, Lateolabrax japonicus, and sole, Pleuronichthys cornutus), however, those of chymotrypsin, trypsin, pepsin, and peptidase like enzymes were observed 3n the viscera crude pretense from the fish. Proteolytic activities of the muscle crude protease at pH 6.0 were similar to those of the viscera crude protease at pH 8.0, but, those of the viscera crude protease at pH 8.0 were about 2 times higher than those at pH 6.0. The muscle and viscera crude protease from anchovy showed the strongest proteolytic activity among the four fish crude proteases and the proteolytic activity of the viscera crude protease was approximately 100 times higher than that of the muscle crude protease, which suggest that viscera proteases were more contributed on the development of post-mortem changes than muscle proteases. With the degradation patterns on SDS-polyacrylamide gel electrophoresis against yellowtail myofibrillar proteins, the muscle and viscera crude protease of the four fishes were primary responsible for the degradation of myosin heavy chain, and myosin light chain and actin, respectively.

• Ocean and Polar Research
• /
• v.30 no.4
• /
• pp.467-472
• /
• 2008

Antarctic krill has a strong proteolytic enzyme system, which comes from a combination of several proteases. This powerful activity can be easily detected by krill's superior post mortem autolysis. Mammalian skin consists of epidermis and dermal connective tissue, and functions as a barrier against threatening environments. A clot in a wound site of the skin should be removed for successful skin regeneration. Epithelial cells secrete proteases to dissolve the clot. In previous studies Antarctic krill proteases were purified and characterized. The proteolytic enzymes from Antarctic krill showed higher activity than mammalian enzymes. It has been suggested that these krill clean up the necrotic skin wound to induce a natural healing ability. The enzymes exhibited additional possibilities for several other biomedical applications, including dental plaque controlling agent and healing agent for corneal alkali burn. Considering that these versatile activities come from a mixture of several enzymes, discovering other proteolytic enzymes could be another feasible way to enhance the activity if they can be used together with krill enzymes. Molecular cloning of the krill proteases should be carried out to study and develop the applications. This review introduces possible roles of the unique Antarctic krill proteases, with basic information and suggestion for the development of an application to skin regeneration.