• Applied Biological Chemistry
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• v.40 no.3
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• pp.178-183
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• 1997

A protease was purified from Bacillus subtilis CCKS-111 by ammonium sulfate treatment, DEAE-cellulose ion-exchange chromatography, Sephadex G-100 gel filtration and high performance liquid chromatography (HPLC). The specific activity of the purified enzyme was 24.3 unit/mg protein and the purification fold of enzyme was 50.6. Molecular weight of the purified enzyme estimated about 28,000 by HPLC gel filtration. The amino acid residues of this enzyme were 251.3 except threonine, serine and glycine. This result was similar to Bacillus subtilis subtilisin DY. From the first N-terminal amino acid to the 32th amino acid, the amino acid sequence was estimated after RP-HPLC elution. N-terminal and the 32th amino acids were alanine and aspartic acid. Alanine, serine, glycine and arginine were four major acids in the enzyme.

• Journal of Life Science
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• v.23 no.2
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• pp.259-266
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• 2013

To isolate the fibrinolytic enzyme, 268 strains from 21 samples were morphologically isolated from Cheonggukjang collected from Korea and Japan. Among the 268 strains, protease-producing bacteria were isolated in nutrient agar medium including 1% skimmed milk. As a result of this, 22 strains were isolated. Apiweb site was used to identify these strains based on their biochemical properties. In addition, 16S rRNA sequencing was performed to identify the strain. Most of the identified strains were Bacillus subtilis and B. amyloliquefaciens. Fibrinolytic enzyme activity was measured with the fibrin plate method. Five strains were finally selected: A2-14, A2-20, C1-05, C1-09, and F2-01. Of those five strains, the A2-20 strain, which is close to B. amyloliquefaciens, showed the strongest fibrinolytic activity. The fibrinolytic enzyme produced by the A2-20 strain was partially purified from culture supernatant by gel filtration and ion exchange chromatography. The optimal pH and temperature values of the partially purified enzyme were 7.0 and $35^{\circ}C$, respectively. Purified protein analysis was carried out with SDS-PAGE and zymography. A genetic analysis was also conducted by PCR based on the consensus sequence of fibrinolytic enzyme. Corresponding genes with a partial sequence of the A2-20 strain were identified.

• Applied Biological Chemistry
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• v.46 no.3
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• pp.176-182
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• 2003

An alkaline fibrinolytic protease-producing bacteria was isolated front Korean traditional soy sauce and identified as Bacillus subtilis K7 from the results of analyses of its morphological and physiological properties, $API^{\circledR}$, and Biolog system. The enzyme was purified by 75% ammonium sulfate fractionation, QAE-Sephadex anion and SP-Sephadex cation exchange column chromatography and Sephadex G-100 gel filtration. The specific activity of the purified enByme was 233.9 unit/mg protein and the yield of enzyme was 3.8%. The homogeneity of the purified enzyme was confirmed by polyacrylamide gel electrophoresis. Molecular mass of the enzyme was estimated about 21,500 Da by SDS-polyacrylamide get electrophoresis and gel chromatography. The optimum temperature and pH for the enzyme activity were $40^{\circ}C$ and 9.0, respectively. The enzyme was stable in a pH range of 5.0 to 12.0, and 60% of its activity was lost on heat treatment at $50^{\circ}C$ for 20 min. The activity of the purified enzyme was inhibited by the presence of $Fe^{2+},\;Ag^{2+},\;Cu6{2+}$, iodoacetate, ethylene diamine tetraacetic acid (EDTA), and trans-1,2-diaminocycloheane-N,N,N',N'-tetraacetic acid (CDTA). The results indicates that the enzyme requires a metal ion for its enzymatic activity.

• 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.

• Journal of the Korean Society of Food Science and Nutrition
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• v.12 no.4
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• pp.387-394
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• 1983

The factor, which was capable of accelerating the yeast cell wall lysis of the zymolyase(${\beta}-1$, 3-glucanase), was purified to a homogeneous state from the protease fraction of the crude zymolyase by Sephadex G-75 gel filtration and preparative polyacrylamide gel disc electrophoresis. The molecular weight of the purified factor was estimated to be 40,500 by SDS-polylacrylamide gel disc electrophoresis and it's iso-electric point was pH 9.6. The factor was found to be a basic protease consisted of single polypeptide chain with 395 amino acid residues and it showed the $E_{280,cm}^{1%}$ of 11.9 and the molecular extinction coefficient of $4.83{\times}10^4$, respectively.

• Korean Journal of Food Science and Technology
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• v.34 no.2
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• pp.141-150
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• 2002

Owing to a need for simple extraction and purification for analysis of water soluble vitamins in food samples by RP-HPLC with UV-detector, the methods of bromelain and protease hydrolysis and $C_{18}$ Sep-Pak solid phase extraction were employed. The recoveries of standard water soluble vitamins by the bromelain and protease hydrolysis and $C_{18}$ Sep-Pak solid phase extraction were significantly high compared to AOAC methods in most of vitamins. The contents of pyridoxal determined with protest in the pork was similar, but in the bromelain hydrolysis and AOAC method, was high compared to the results of reference. The niacinamide, thiamin and riboflavin determined with bromelain and protease hydrolysis showed similar values to the results of references. In the potato, pyridoxamine was detected in the AOAC method, which was not detected in the bromelain and protease hydrolysis methods. Pyridoxal contents in the protease hydrolysis and AOAC methods were very similar to the results of references. The recoveries of fortified standard vitamins in food samples were significantly high and accurate compared to those of AOAC methods. The extraction and purification with $C_{18}$ Sep-Pak solid extractor might be considered superior method for the determination of water soluble vitamins in food samples.

• Journal of Nutrition and Health
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• v.19 no.6
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• pp.363-373
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• 1986

This study was devised to elucidate whether soused shrimp exhibits a digestive action on boiled pork meats. and the mechanism by which sousing with a high concentration of sodium chloride preserves nutrients in foods for a prolonged pe\ulcornerriod. Protease was isolated from soused shrimp using a combination of ammonium sulfate fractionation. DEAE - cellulose ion exchange chromatography and gel filtra\ulcornertion. The isolated protease had specific activity of 1.560 units. 210 purification fo\ulcornerld with an yield of 38%. Its optimum pH and temperature were 8.0 and $43^{\circ}C$ respectively. The molecular weight of the enzyme was 35.000. The Km value of the enzyme for casein was 1.6 x $10^{-6}$ M The e=yme required the presence of cu\ulcornerpric ion to exhibit its full activity. Eighty eight percent of the enzyme activity was in\ulcornerhibited by 3.5M NaCI showing a reversibly linear decrease of the enzyme activity as NaCI concentration increased. The nature of the inhibition by NaCl was rever\ulcornersible and noncompetitive. The protease activity in soused shrimp was well preser\ulcornerved with the elapse of time at least in part due to NaCI induced suppression of autodigestion. The enzyme was denatured by acid easily. i.e. 1% of the original activity remained after staying at pH 2 for 10 minutes. which is within the norm\ulcorneral range of pH of the human stomach. Soused shrimp was observed to be one of those containing the highest protease activity compared with the other soused foo\ulcornerds such as soused oyster. squid. clam. and Pollack intestine with respect to spec\ulcornerific activities of dialized 1:4 whole homogenates(w/v) in 5 mM sodium phospha\ulcornerte - 2.4 mM j3 - mercaptoethanol buffer. pH 8.0. Casein and boiled meats including pork, beef, and chicken appeared to be the good substrates for the protease. Casein was the best. Therefore. the ingestion of boiled meats including pork together with soused sh\ulcornerrimp would help digestion of boiled pork in human not only by increasing appe\ulcornertite also by the direct proteolytic digestion of boiled meats by soused shrimp to\ulcorner some extent. And a high concentration of sodium chloride inhibited the protease activity reversibly in a remarkable degree, which ensued in a significant retardat\ulcornerion of autodigestion of protein in foods by proteases, and hereby contributed to the preservation of foods for an extended period.

• Journal of Life Science
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• v.6 no.4
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• pp.241-249
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• 1996

A collagenase was isolated from the culture filtrate of Vibrio mimicus (ATCC 33658). The enzyme was purified to homogeneity by ammonium sulfate precipitation and DEAE-Sephadex A-50 chromatography, which an activity recovery of 22%. The molecular weight of the purified enzyme was estimated to be 42 kDa by SDS-polyacrylamide gel electrophoresis and gel filtration, indication a monomer structure. The optimum pH and temperature od the enzyme for insoluble collagen (Type I) were around 7.75 and 28$\circ$C, respectively. Some chelating agents and serine protease inhibitor inactivated the enzyme, but L-cysteine and histidine did not affect the activity. The amino acid composition indicated that the collagenase contained high amounts of amino acid residues of glycine and alanine. The K$_{m}$ and R$_{cat}$/K$_{m}$ values for the collagenase, using insoluble collagen (type I) as substrate, were 2.86 mg/ml and 972.28 U/mg-protein, 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.

• Journal of Microbiology and Biotechnology
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• v.2 no.1
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• pp.14-20
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• 1992

Some microorganisms isolated from soil, including some bacteria and fungi, were found to secrete an extracellular soymilk-clotting enzyme. Among them, an isolated fungus showed the highest soymilk-clotting activity and the strain was assigned to genus Penicillium based on its cultural and morphological characteristics, and designated as Penicillium sp. L-151K. Soymilk-clotting enzymes A and B produced by Penicillium sp. L-151K were purified by ammonium sulfate precipitation and chromatographies on Sephadex G-25, CM-Sephadex, Sephadex G-100 and phenyl-Toyopearl gel. The two purified enzymes A and B were found to be homogeneous by polyacrylamide gel electrophoresis at pH 9.5. The molecular weights of enzyme A and B were 24, 000 and 40, 000, respectively, by gel filtration on Sephadex G-100. Enzymes A and B coagulated soymilk optimally at $60^\circ{C}$ and were stable up to $50^\circ{C}$. Both enzymes were most active at pH 5.8 for soymilk coagulation, and were stable with approximately 80% of original activity from pH 3.0 to 5.0. Each enzyme was an acidic protease with an optimum pH of 3.0 for casein digestion. The soymilk-clotting efficiency of these enzymes was improved with $CaCl_2\;or\;MgCl_2$ when making soymilk-curd.