• Journal of Pharmaceutical Investigation
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• v.25 no.3
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• pp.177-184
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• 1995

In order to formulate an aqueous topical preparation of epidermal growth factor(EGF) for the treatment of open wound and bum, the stability of EGF in aqueous vehicles containing various stabilizers was evaluated and the pharmacological activity of gel preparations formulated with poloxamer 407 was determined with wound model. Various additives, which are known as potent stabilizers for proteins and polypeptides so far, were used to increase the stability of EGF in aqueous vehicles. The contents of EGF in the vehicles containing stabilizers were determined with an HPLC method after the storage at $37^{\circ}C$. EGF was more stable in ultrapure water than RO water or saline. All the additives studied resulted in deleterious effects on EGF stability. Therefore, it was speculated that any additives or impurities in the vehicle made EGF unstable. However, nitrogen purge of solution increased the stability of EGF in aqueous vehicles. The aqueous topical preparations of EGF were formulated with poloxamer 407 as a gel base in saline. Gelatin or amastatin was employed as a protease inhibitor. The pharmacological effect of EGF gel was studied with open wound model in mice. EGF preparations, made of oleaginous base or poloxamer gel base, showed significant healing effect compared to the control group(p<0.05). The addition of protease inhibitor in poloxamer 407 gel resulted in significant healing effect compared to the gel without it(p<0.05). Body weights of mice treated with EGF preparation were increased at the first day after the formation of open wound, while those of the control group were decreased. The EGF gel made of poloxamer 407 containing a pretense inhibitor would be a promising aqueous topical preparation for EGF.

• Bulletin of the Korean Chemical Society
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• v.26 no.1
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• pp.161-164
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• 2005

• Proceedings of the Korean Biophysical Society Conference
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• 1997.07a
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• pp.39-39
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• 1997

Native strain of inhibitory SERPINS (Serine protease inhibitors) is thought to be used in the facile conformational switch to play biological regulation. Many heat stable variants of $\alpha$$_1$-antitrypsin, a prototype of inhibitory serpins, increased their stability by reducing the native strain.(omitted)

• Microbiology and Biotechnology Letters
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• v.17 no.4
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• pp.358-364
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• 1989

A crystalline alkaline pretense- producing Streptomyce sp. YSA-130 was isolated from soil in alkaline medium(pH 10.5). The optimum culture condition of Streptomyce sp. YSA-130 for the production of alkaline protease was as follows; 2.0% soluble starch, 1.0% soytone, 0.3% $K_2$HPO$_4$, 0.02% MgSO$_4$.7$H_2O$, 0.8% Na$_2$CO$_3$, pH 10.5, 3$0^{\circ}C$, and 12 hr. The alkaline pretense from the culture broth of Streptomyce sp. YSA-130 was purified about 24 folds by ammonium sulfate precipitation , dialysis, DEAE-cellulose ion exchange chromatography, gel filtration on Sephadex G-15 and crystallization. Optimum temperature and pH of purified enzyme were 6$0^{\circ}C$, and 11.5. Temperature and pH stability of purified enzyme were 5$0^{\circ}C$, and 5.5-12.0. Calcium ion was effective to stabilize the enzyme at higher temperature. The molecular weight of the purified enzyme was approximately 30,000. The purified enzyme was inactivated by diisopropyl flurophosphate(DFP) but not affected by metal ion, EDTA, sulfhydryl reagent and stable detergent.

• Korean journal of food and cookery science
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• v.27 no.3
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• pp.29-37
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• 2011

Protease activity of fig (Ficus carica L.), cultivated in Korea was estimated. In particular, the proteolytic effect on myofibrilar protein was studied. A crude protease extract of fig was prepared in two ways; fig was homogenized in buffer followed by centrifugation, and the supernatant was precipitated by saturated ammonium sulfate followed by dialysis. The former method resulted in 41.15 mM/g fig protease activity, whereas the latter method resulted in 17.65 mM/g fig protease activity. The crude fig protease extract showed high specificity for casein as a substrate followed by egg white, bovine serum albumin, myofibrilar protein, collagen, and elastin. The extract had stable proteolytic activity in a pH range of 6.5~9.0 (optimal at pH 7-8) but lost activity, at pH 2-3. Proteolytic activity for myofibrilar protein was sensitive to pH. The proteolytic activity of the fig extract was steady up to $60^{\circ}C$ but declined at higher temperature. It also began to lose stability in salt concentrations >0.7 M NaCl. Fig has been used as a meat tenderizer for cooking, and these results support the tenderizing effectiveness of fig, particularly for Korean style meat marinating.

• Korean Journal of Food Science and Technology
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• v.42 no.5
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• pp.554-558
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• 2010

In the study, the protease activity of kiwifruit (Actinidia deliciosa Planch) cultivated in Korea was estimated, with specific examination of proteolytic effects on myofibrilar protein. The crude protease extract of kiwifruit was prepared in two ways; one in which the kiwifruit was homogenized with buffer followed by centrifugation, and the other were the supernatant was precipitated by saturated ammonium sulfate followed by dialysis. The former had 21.23 mM/mL of protease activity, which corresponded to 112.28 mM/g kiwifruit utilized, and the latter had 11.58 mM/mL and 45.80 mM/g of kiwifruit. The crude protease extract of the kiwifruit showed high specificity for casein substrate followed by bovine serum albumin, egg white, collagen, and elastin, in order. The enzyme lost proteolytic activity in acidic conditions such as pH 2-3, and at high temperatures over $60^{\circ}C$. It showed optimal activity in both pH 3.0 and pH 7.5 as well as at $40^{\circ}C$ for casein substrate and at $50^{\circ}C$ for myofibrilar protein substrate. The proteolytic activity toward casein was high with up to 0.5M salt, followed by a sharp decrease beyond this concentration. On the other hand the proteolytic activity for myofibrilar protein decreased steadily with increasing of salt concentration. Kiwifruit has been used as a for meat tenderizer for in home cooking and these results support the its tenderizing effectiveness of kiwifruit especially for Korean style marinating of meat for cooking.

• International Journal of Industrial Entomology and Biomaterials
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• v.22 no.2
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• pp.83-93
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• 2011

Biochemical and enzymatic characterization for extracellular protease isolated from Cordyceps militaris cultivated on rice bran medium was investigated. C militaris produced proteolytic enzymes from 10 days after inoculation, maximum enzyme production was found at 25 days. The optimum temperature and pH of proteases production was at $25^{\circ}C$ and pH 7.0, respectively. The protease activity was observed in the four peaks (Pro-I, Pro-II, Pro-III, and Pro-IV) separated through Sephadex G-100 column chromatography. The separated protease was optimally active at $25^{\circ}C$. Optimum pH of the protease was between 7 and 8. Enzyme was also stable over at $30-80^{\circ}C$. The enzyme was highly stable in a pH range of 4-9. Protease activity was found to be slightly decreased by the addition of $Mg^{2+}$, $Mn^{2+}$, $Zn^{2+}$, $Fe^{2+}$ and $Cu^{2+}$, whereas inhibited by the addition of $Ca^{2+}$ and $Co^{2+}$ Protease activity was inhibited by protease inhibitor PMSF. On the other hand, the partially purified protease was investigated on proteolytic protease activity by zymogram gel electrophoresis using three substances (casein, gelatin and fibrin). Four active bands (F-I, FII, F-III, and F-IV) of fibrin degradation were revealed on fibrin zymogram gels. Both of F-II and FIII showed caseinolytic, fibrinolytic and gelatinolytic activities in three gels. Thermostability, pH stability, and pH-thermostability of the enzyme determined the residual fibrinolytic activity also displayed on fibrin zymogram gel. The only one enzyme (F-II) displayed over a broad range of temperature at $30-90^{\circ}C$. The FII displayed fibrinolytic activity in the pH range 3-5, but was inactivated in the range of pH 6-11. The F-I and F-III showed enzyme activity in the pH range of 6-11. In the pH-thermostability, the F-II only kept fibrinolytic activity after heating at $100^{\circ}C$ for 10, 20 and 30 min at pH 3 and pH 7, respectively. On the other hand, the F-II was retained activity until heating for 10 min under pH 11 condition. By using fibrin zymogram gel electrophoresis, extracellular fibrinolytic enzyme F-II from C. militaris showed unusual thermostable under acid and neutral conditions.

• Microbiology and Biotechnology Letters
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• v.26 no.5
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• pp.427-434
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• 1998

An alkaline protease was 4-fold purified, yielding 2.3% of recovery by ammonium sulfate precipitation, CM-cellulose column chromatography and Sephadex G-100 column chromatography. The purified enzyme was estimated to be monomeric with molecular weight of about 62,000 from polyacrylamide gel eletrophoresis (PAGE) and sodiumdodecylsulfate polyacrylamide gel electrophoresis (SDS-FAGE). The optimal pH and temperature of the alkaline pretense activity were 11.0 and 50$^{\circ}C$, respectively, exhibiting high stability at pH value from 6.0 to 11.0 at 50$^{\circ}C$ for 30 minute. The alkaline pretense was activated by MnSO$_4$, CaCl$_2$, and was inhibited by CuSO$_4$, ZnSO$_4$, HgCl$_2$, EDTA and EGTA. Also, the enzyme was found to be a metaloenzyme requiring Mn$\^$2+/ as cofactor. The NH$_2$-terminal amino acid of alkaline protease was alanine. The Km and Vmax values of this enzyme for casein was 4.0 mg/$m\ell$ and 5,500 unit/$m\ell$, respectively.

• Journal of Microbiology and Biotechnology
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• v.17 no.4
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• pp.624-631
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• 2007

Thermostable protease is very effective to improve the industrial processes in many fields. Two thermostable extracellular proteases from the culture supernatant of the thermophilic fungus Chaetomium thermophilum were purified to homogeneity by tractional ammonium sulfate precipitation, ion-exchange chromatography on DEAE-Sepharose, and Phenyl-Sepharose hydrophobic interaction chromatography. By SDS-PAGE, the molecular mass of the two purified enzymes was estimated to be 33 kDa and 63 kDa, respectively. The two proteases were found to be inhibited by PMSF, but not by iodoacetamide and EDTA. The 33 kDa protease (PRO33) exhibited maximal activity at pH 10.0 and the 63kDa protease (PRO63) at pH5.0. The optimum temperature for the two proteases was $65^{\circ}C$. The PRO33 had a $K_m$ value of 6.6mM and a $V_{max}$ value of $10.31{\mu}mol/l/min$, and PRO63 l7.6mM and $9.08{\mu}mol/l/min$, with casein as substrate. They were thermostable at $60^{\circ}C$. The protease activity of PRO33 and PRO63 remained at 67.2% and 17.31%, respectively, after incubation at $70^{\circ}C$ for 1h. The thermal stability of the two enzymes was significantly enhanced by $Ca^{2+}$. The residual activity of PRO33 and PRO63 at $70^{\circ}C$ after 60min was approximately 88.59% and 39.2%, respectively, when kept in the buffer containing $Ca^{2+}$. These properties make them applicable for many biotechnological purposes.

• Journal of Microbiology and Biotechnology
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• v.22 no.6
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• pp.818-825
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• 2012

Keratinases are exciting keratin-degrading enzymes; however, there have been relatively few studies on their immobilization. A keratinolytic protease from Chryseobacterium sp. kr6 was purified and its partial sequence determined using mass spectrometry. No significant homology to other microbial peptides in the NCBI database was observed. Certain parameters for immobilization of the purified keratinase on chitosan beads were investigated. The production of the chitosan beads was optimized using factorial design and surface response techniques. The optimum chitosan bead production for protease immobilization was a 20 g/l chitosan solution in acetic acid [1.5% (v/v)], glutaraldehyde ranging from 34 g to 56 g/l, and an activation time between 6 and 10 h. Under these conditions, above 80% of the enzyme was immobilized on the support. The behavior of the keratinase loading on the chitosan beads surface was well described using the Langmuir model. The maximum capacity of the support ($q_m$) and dissociation constant ($K_d$) were estimated as 58.8 U/g and 0.245 U/ml, respectively. The thermal stability of the immobilized enzyme was also improved around 2-fold, when compared with that of the free enzyme, after 30 min at $65^{\circ}C$. In addition, the activity of the immobilized enzyme remained at 63.4% after it was reused five times. Thus, the immobilized enzyme exhibited an improved thermal stability and remained active after several uses.