• Journal of the Korean Society of Clothing and Textiles
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• v.20 no.4
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• pp.655-666
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• 1996

The effect of protease (subtilisin Carlsberg) on the removal of hemoglobin as protein soil was studied. The relation between the renloval and the hydrolysis of hemoglobin by subtilisin Carlsberg was discussed. The soiled babric was prepared by spotting of hemoglobin solution evenly on the cotton fabric and was denatured by steaming. The soiled fabric was washed by using Terg-0-Tometer at various conditions. The removal efficiency was evaluated by analysis of protein on the fabrics before and after washing by means of copper-Folin method. 1. The removal of hemoglobin was increased in proportion to increasing of the enzyme concentration up to a certain point, but it began to decrease above the point. 2. The hemoglobin was removed effectively by adding of subtilisin Carlsberg, and more effectively removed by adding of AOS in the enzyme solution. 3. The removal of hemoglobin deviated from the first order reaction in detergency. 4. The renloval of hemoglobin was highest at $50^{\circ}C$ in detergency, Even at low temperature the removal efficiency of enzyme was relatively higher compared with the hydrolysis of hemoglobin by the enzyme. However the removal of hemoglobin was apparently decreased with the increase of temperature over $60^{\circ}C$. 5. The removal of hemoglobin was relatively high at pH 7.0~8.0 and increased continuously with the increase of pH in detergency 6. In detergency, the removal mechanism of hemoglobin by subtilisin Carlsberg could be explained as follows: Fisrt of all, the enzyme hydrolyzed hemoglobin substrates partially by forming E-S complex at the surface of hemoglobin on the cotton fiber, and decomposed cooperative binding of hemoglobin. Subsequently, the fragments of hemoglobin were easily removed by washing. According as the enzyme penetrated to inner part of hemoglobin gradually, the hemoglobin on the cotton fiber was effectively removed by the repetition of these process. The removal of hemoglobin was more effectively increased by adding both the enzyme and AOS in the washing solution. Therefore, it was regarded that AOS molecules were adsorbed at the hydrophobic surface of denatured hemoglobin, subsequently, decomposed more effectively cooperative binding of hemoglobin, and the fragments of hemoglobin were removed more efficiently by means of the interfacial reaction of AOS.

• Bulletin of the Korean Chemical Society
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• v.10 no.6
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• pp.600-604
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• 1989

In order to find the rational methods for improving the thermal stability of subtilisin Carlsberg, the mechanisms of irreversible thermoinactivation of the enzyme were studied at $90^{\circ}C.$ At pH 4, the main process was hydrolysis of peptide bond. This process followed first order kinetics, yielding a rate constant of $1.26\;{\times}\;10^{-1}h^{-1}$. Hydrolysis of peptide bond of PMS-subtilisin occurred at various sites, which produced new distinct fragments of molecular weights of 27.2 KD, 25.9 KD, 25.0 KD, 22.3 KD, 19.0 KD, 17.6 KD, 16.5 KD, 15.7 KD, 15.0 KD, 13.7 KD, and 12.7 KD. Most of the new fragments originated from the acidic hydrolysis at the C-side of aspartic acid residues. However 25.0 KD, 15.7 KD, and 13.7 KD which could not be removed in purification steps stemmed from the autolytic cleavage of subtilisin. The minor process at pH 4 was deamidation at asparagine and/or glutamine residues and some extend of aggregation was also observed. However, the aggregation was main process at pH 7 with a first order kinetic constant of $16 h^{-1}.$ At pH 9, the main process seemed to be combination of deamidation and cleavage of peptide bond.

• Journal of Korean Ophthalmic Optics Society
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• v.10 no.2
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• pp.91-97
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• 2005

We investigated the question whether protein removing activities of enzyme cleaner - protein remover for soft contact lens - are associated with the material of soft contact lens as well as action time, temperature and pH of enzyme solution. We used a subtilisin cleaner as protein remover and estimated the protein amount remained on soft contact lens after using the subtilisin cleaner under the different conditions. The remained protein in soft contact lens was greatly decreased until treatment for 60min, but no significant differences were found from 60min to 24hr. The cleaning effect of the enzymatic treatment in the range of $15{\sim}30^{\circ}C$ was constant. however, there was a significant decline of the protein removing effect at $10^{\circ}C$ and less. The pH of the solution was also important for the efficacy of the enzymatic treatment. The activity of the enzyme cleaner was highest in pH 8.0 and significantly decreased a pH below 7. The pH dependence was found to be related to the conformational change of subtilisin. Furthermore, significant differences in the protein deposit removing efficacy of the subtilisin cleaner were found among the soft contact lens materials.

• Journal of the Korean Society of Food Science and Nutrition
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• v.37 no.3
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• pp.373-378
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• 2008

The objective of this paper was to investigate optimal culture conditions for the production of an inhibitor against subtilisin-like protease from Streptomyces thermocarboxydus (S. thermocarboxydus) C12 isolated from sediments of Gwangyang coast. The optimal temperature and initial pH for the production of subtilisin-like protease inhibitor were $40^{\circ}C$ and pH 8.0, respectively. Inhibition activities were high for galactose, glucose and fructose. The best carbon source and its concentration were galactose and 1.6% (w/v), respectively. Inhibition activities were also high in medium containing polypeptone, proteose and peptone. Optimal nitrogen source and concentration were protease peptone and 0.5% (w/v), respectively. Optimal concentrations for inhibitor production were 1% (w/v) for NaCl and 1 mM LiCl for metal salts. The subtilisin-like protease inhibitor from S. thermocarboxydus C12 showed a maximum inhibitor activity after cultivation for 84 h under the optimized medium.

• Bulletin of the Korean Chemical Society
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• v.32 no.spc8
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• pp.2871-2872
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• 2011

• Proceedings of the Korean Biophysical Society Conference
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• 1997.07a
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• pp.44-44
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• 1997

Ca$^{2＋}$ is one of the most common metal ions associated with proteins, playing more or less well-defined functional roles in biological activities. In protease, the presence of $Ca^{2＋}$ slows down autolysis and enhances thermal stability. Subtilisin, one of the best studied proteases, is known to have two $Ca^{2＋}$ -binding sites.(omitted)

• Journal of Microbiology and Biotechnology
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• v.21 no.6
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• pp.617-626
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• 2011

Transglutaminase from Streptomyces mobaraensis is an enzyme of unknown function that cross-links proteins to high molecular weight aggregates. Previously, we characterized two intrinsic transglutaminase substrates with inactivating activities against subtilisin and dispase. This report now describes a novel substrate that inhibits papain, bromelain, and trypsin. Papain was the most sensitive protease; thus, the protein was designated Streptomyces papain inhibitor (SPI). To avoid transglutaminase-mediated glutamine deamidation during culture, SPI was produced by Streptomyces mobaraensis at various growth temperatures. The best results were achieved by culturing for 30-50 h at $42^{\circ}C$, which yielded high SPI concentrations and negligibly small amounts of mature transglutaminase. Transglutaminasespecific biotinylation displayed largely unmodified glutamine and lysine residues. In contrast, purified SPI from the $28^{\circ}C$ culture lost the potential to be cross-linked, but exhibited higher inhibitory activity as indicated by a significantly lower $K_i$ (60 nM vs. 140 nM). Despite similarities in molecular mass (12 kDa) and high thermostability, SPI exhibits clear differences in comparison with all members of the wellknown family of Streptomyces subtilisin inhibitors. The neutral protein (pI of 7.3) shares sequence homology with a putative protein from Streptomyces lavendulae, whose conformation is most likely stabilized by two disulfide bridges. However, cysteine residues are not localized in the typical regions of subtilisin inhibitors. SPI and the formerly characterized dispase-inactivating substrate are unique proteins of distinct Streptomycetes such as Streptomyces mobaraensis. Along with the subtilisin inhibitory protein, they could play a crucial role in the defense of vulnerable protein layers that are solidified by transglutaminase.

• Applied Biological Chemistry
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• v.31 no.3
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• pp.274-283
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• 1988

Aqualysin I is an alkaline serine protease which is secretet into the culture medium by Thermus aquaticus YT-1, an extreme thermophile. Aqualysin I was purified, and its partial amino acid sequence was determined. The gene encoding aqualysin I was cloned into E. coli using synthetic oligodeoxyribonucleotides as hybridization probes. The nucleotide sequence of the cloned DNA was determined. The primary structure of aqualysin I, deduced from the nucleotide sequenc, agreed with the determid amino acid sequences, including the $NH_2-$ and COOH terminal sequence of the tryptides derived from aqualysin I. Aqualysin I comprised 281 amino acid residues and its molecular mass was determined to be 28350. On alignment of the whole amino acid sequence, aqualysin I showed high sequence homology with the subtilisin type serine protease, and 43% identity with proteinase K, 37-30% with subtilisins and 34% with thermitase. Extremely high sequence identity was observed in the regions containing the active-site residues, corresponding to Asp32, His64 and Ser221 of subtilisin BPN'. Aqualysin I contains two disulfide bonds, Cys67-Cys99 and Cys163-Cys194, and these disulfide bonds seem to contribute to the heat stability of the enzyme. The determined positions of the twe disulfide bonds of aqualysin I agreed with those predicted previously on the basis of computer graphics of the crystallographic data for subtilisin BPN'. Therefore, these findings sugests that the three-dimensional structure of aqualysin I is similar to that of subtilisin BPN' Aqualysin I is produced as a lage precursor, which contains $NH_2-$ and COOH- terminal portions besides the mature protease sequence.

• Journal of Microbiology and Biotechnology
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• v.31 no.2
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• pp.327-337
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• 2021

Fibrinolytic enzymes with a direct mechanism of action and safer properties are currently requested for thrombolytic therapy. This paper reports on a new enzyme capable of degrading blood clots directly without impairing blood coagulation. This enzyme is also non-cytotoxic and constitutes an alternative to other thrombolytic enzymes known to cause undesired side effects. Twenty-four Bacillus isolates were screened for production of fibrinolytic enzymes using a fibrin agar plate. Based on produced activity, isolate S127e was selected and identified as B. subtilis using the 16S rDNA gene sequence. This strain is of biotechnological interest for producing high fibrinolytic yield and consequently has potential in the industrial field. The purified fibrinolytic enzyme has a molecular mass of 27.3 kDa, a predicted pI of 6.6, and a maximal affinity for Ala-Ala-Pro-Phe. This enzyme was almost completely inhibited by chymostatin with optimal activity at 48℃ and pH 7. Specific subtilisin features were found in the gene sequence, indicating that this enzyme belongs to the BPN group of the S8 subtilisin family and was assigned as AprE127. This subtilisin increased thromboplastin time by 3.7% (37.6 to 39 s) and prothrombin time by 3.2% (12.6 to 13 s), both within normal ranges. In a whole blood euglobulin assay, this enzyme did not impair coagulation but reduced lysis time significantly. Moreover, in an in vitro assay, AprE127 completely dissolved a thrombus of about 1 cc within 50 min and, in vivo, reduced a thrombus prompted in a rat tail by 11.4% in 24 h compared to non-treated animals.

• Bulletin of the Korean Chemical Society
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• v.19 no.3
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• pp.369-372
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• 1998