• Journal of Microbiology
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• v.38 no.4
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• pp.218-223
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• 2000

Using two drugs, tunicamycin and brefeldin A, which affect protein processing, we investigated the intracellular processing mechanism of secreted aspartyl proteinase 1 (SAPl) of Candide albicans. Three intracellular forms of SAPI were detected by immunoblotting using menoclonal antibody (MAb) CAPl. Their molecular weights were approximately 40, 41 and 45 kDa, respectively. The 41 kDa protein is a glycoprotein and may be the same as the extracellular form judging by its molecular mass. The 40 kDa protein was the unglycosylated form and its molecular mass coincided with deglycosylated SAPl and the 45 kDa protein was also the unglycosylated form. Neither the 40 and 45 kDa proteins were detected in the culture supernatant of C. albicans. These suggested that the 40 and 45 kDa proteins might be intracellular precursor forms of SAPI. These results show that SAPI is translated as a 45 kDa precusor form in the endoplasmic reticulum and the 45 kDa precursor farm undergoes proteolytic cleavage after translocation into the Golgi apparatus, generating the 40 kDa precursor form. This 40 kDa precursor is converted into a 41 kDa mature form through glycosylation in the Golgi apparatus. The mature form of the 41 kDa protein is sorted into secretary vesicles and finally released into the extracellular space through membrane fusion. When the glycan region of SAPl was digested with N-glycosidase F, both stability and activity of the enzyme decreased. These results indicate that the glycan attached to the enzyme may, at least in parti be related to enzyme stability and activity.

• Genomics & Informatics
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• v.21 no.1
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• pp.9.1-9.13
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• 2023

Matrix metalloproteinase-9 (MMP-9) is a zinc and calcium-dependent proteolytic enzyme involved in extracellular matrix degradation. Overexpression of MMP-9 has been confirmed in several disorders, including cancers, Alzheimer's disease, autoimmune diseases, cardiovascular diseases, and dental caries. Therefore, MMP-9 inhibition is recommended as a therapeutic strategy for combating various diseases. Cinnamic acid derivatives have shown therapeutic effects in different cancers, Alzheimer's disease, cardiovascular diseases, and dental caries. A computational drug discovery approach was performed to evaluate the binding affinity of selected cinnamic acid derivatives to the MMP-9 active site. The stability of docked poses for top-ranked compounds was also examined. Twelve herbal cinnamic acid derivatives were tested for possible MMP-9 inhibition using the AutoDock 4.0 tool. The stability of the docked poses for the most potent MMP-9 inhibitors was assessed by molecular dynamics (MD) in 10 nanosecond simulations. Interactions between the best MMP-9 inhibitors in this study and residues incorporated in the MMP-9 active site were studied before and after MD simulations. Cynarin, chlorogenic acid, and rosmarinic acid revealed a considerable binding affinity to the MMP-9 catalytic domain (ΔG_binding < -10 kcal/ mol). The inhibition constant value for cynarin and chlorogenic acid were calculated at the picomolar scale and assigned as the most potent MMP-9 inhibitor from the cinnamic acid derivatives. The root-mean-square deviations for cynarin and chlorogenic acid were below 2 Å in the 10 ns simulation. Cynarin, chlorogenic acid, and rosmarinic acid might be considered drug candidates for MMP-9 inhibition.

• Journal of the Korean Society of Food Science and Nutrition
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• v.13 no.2
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• pp.193-204
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• 1984

The enzymatic properties of the alkaline AL-protease, which had been prepared from the crude zymolyase of Arthrobzoter luteus, was investigated together with its active amino acid residue. Complete inactivaton of the proteolytic activity of AL-protease by either DFP or PMSF was simultaneously accompanied by the loss of its lytic effect on the lysis of yeast cell wall. In the reaction, AL-protease showed the pattern of inactivation to decrease very slowly, as compared to that of chymotrypsin, and that enzyme and DFP were found to react with a molar ratio of 1 : 1. The preparation of AL-protease exhibited no hydrolytic activity in any substrates of polysaccharases, playing a significant role in the lysis of yeast cell wall. The optimum pH and temperature of AL-protease was pH 10.5 and $65^{\circ}C$, respectively. It also showed stability in the pH range from 5 to 11 and at the temperature below $65^{\circ}C$. Through the identification of the amino acid residue in the active site of the $^{32}P$-diisopropylph-osphorylated(DIP) AL-protease modified specifically with $^{32}P$-labeled DFP, AL-protease was found to be a DFP-sensitive which has a mole of active serine residue involved in its proteolytic activity per mole of the enzyme.

• Korean Journal of Food Science and Technology
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• v.16 no.2
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• pp.211-217
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• 1984

To study affinity of proteolytic enzymes to soy proteins, the physicochemical and functional properties of enzymatically modified protein products, kinetic parameters and degree of hydrolysis were measured using trypsin, alcalase (serine type protease) and pronase. Bacterial alcalase and pronase showed much greater affinity to soy protein than animal intestinal trypsin. This effect was very significant when unheated soy isolate was used as a substrate. Specific activities of these enzymes decreased with the increment of substrate concentration (over 2.0%, w/v) when heat denatured soy protein was used as a substrate. However, the decrease in specific activity was negligible at substrate concentrations lower than 2.0%. Polyacrylamide gel electrophoretic results showed that the pattern of 2S protein band changed distinctly in alcalase hydrolysis as compared with those of trypsin and pronase. Protein solubilities of alcalase and pronase hydrolyzates increased by 25-30%, at their pI (pH 5.0) over the control. Virtually no change was observed in solubility by trypsin hydrolysis. Heat coagulability and calcium-tolerance of the protein increased by enzymatic hydrolysis. No clear tendency, however, was observed for emulsion properties, foam expansion and the amount of free -SH groups. The enzyme treatment considerably decreased foam stability.

• 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.

• Korean Journal of Microbiology
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• v.30 no.6
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• pp.528-532
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• 1992

The ATP-dependent protease. Clp P from Esehaichia coli has been increase the stahility with or without detergent as Triton X-100 and NP-40 in the Clp P. The C]p P proteolytic activity was remained to 0.1 M salt by $Na^{-1}$, $K^{+}$, $Li^{+}$ but was inhihited by $SO_4^{2}$. An active ATPase site in Clp A is required for A TP-dependent proteolysis by Clp protease as

• Korean Journal of Food Science and Technology
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• v.27 no.5
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• pp.708-715
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• 1995

Effect of enzymatic modification with pepsin, papain and trypsin was studied on functional properties of isolated sesame meal protein hydrolysates. Solubility of protein hydrolysates distinctively increased from 2% to $53{\sim}94%$ at pH 4. Emulsifying properties showed marked increase 6 fold and 4.5 fold at degree of 10%, 20% hydrolysis by trypsin and degree of 10% hydrolysis by papain. The emulsion stability of the protein was unstable by heat treatment for 30 min. at $80^{\circ}C$. Foaming properties were also enhanced by enzymatic hydrolysis except at degree of 30% hydrolysis. Bulk density and water absorption of protein with trypsin and papain decreased about 0.1 g/ml and $0.3{\sim}0.7\;ml/g$, but oil absorption was increased about 1 ml/g.

• BMB Reports
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• v.35 no.2
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• pp.155-164
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• 2002

The structural aspects of ervatamin B have been studied in different types of alcohol. This alcohol did not affect the structure or activity of ervatamin B under neutral conditions. At a low pH (3.0), different kinds of alcohol have different effects. Interestingly, at a certain concentration of non-fluorinated, aliphatic, monohydric alcohol, a conformational switch from the predominantly $\alpha$-helical to $\beta$-sheeted state is observed with a complete loss of tertiary structure and proteolytic activity. This is contrary to the observation that alcohol induces mostly the $\alpha$helical structure in proteins. The O-state of ervatamin B in 50% methanol at pH 3.0 has enhanced the stability towards GuHCl denaturation and shows a biphasic transition. This suggests the presence of two structural parts with different stabilities that unfold in steps. The thermal unfolding of ervatamin B in the O-state is also biphasic, which confirms the presence of two domains in the enzyme structure that unfold sequentially. The differential stabilization of the structural parts may also be a reflection of the differential stabilization of local conformations in methanol. Thermal unfolding of ervatamin B in the absence of alcohol is cooperative, both at neutral and low pH, and can be fitted to a two state model. However, at pH 2.0 the calorimetric profiles show two peaks, which indicates the presence of two structural domains in the enzyme with different thermal stabilities that are denatured more or less independently. With an increase in pH to 3.0 and 4.0, the shape of the DSC profiles change, and the two peaks converge to a predominant single peak. However, the ratio of van't Hoff enthalpy to calorimetric enthalpy is approximated to 2.0, indicating non-cooperativity in thermal unfolding.

• Food Science and Biotechnology
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• v.17 no.4
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• pp.766-771
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• 2008

The proteolytic enzyme from Saccharomyces sp. B101 was purified to homogeneity by ammonium sulfate fractionation, ultrafiltration, diethyl aminoethyl (DEAE)-Sephadex A-50 ion-exchange chromatography, and Sephadex G-100 gel filtration chromatography from the culture supernatant of Saccharomyces sp. B101. The specific activity and the purification fold of the purified enzyme were 4,688.9 unit/mg and 18, respectively. The molecular weight of the purified enzyme was estimated to be 33 kDa by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimum pH and temperature for the enzyme activity were pH 8.5 and $30^{\circ}C$, respectively. The enzyme activity was relatively stable in the pH range of 6.5-8.5 at below $35^{\circ}C$. The salt-tolerance and stability for the enzyme activity were relatively stable even at NaCl concentrations of 10 and 15%. The activity of enzyme was inhibited by $Ag^{2+}$ and $Fe^{2+}$, and activated by $Mn^{2+}$. In addition, the enzyme activity was potently inhibited by ethylenediaminetetraacetic acid (EDTA) and phenylmethyl sulfonylfluoride (PMSF). Based on these findings we concluded that the purified enzyme was a serine protease. Km and Vmax values for hammastein milk casein were 1.02 mg/mL and 278.38 unit/mL, respectively.

• Korean Journal of Fisheries and Aquatic Sciences
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• v.23 no.1
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• pp.40-50
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• 1990

The purpose of this paper is to develop a colorant from krill, Euphausia superba, process wastes for use in food products. Carotenoproteins were extracted from preboiled krill processing offal(PKPO) and raw frozen krill processing offal(RKPO) with the aid of proteolytic enzymes. The long-term stability of the astaxanthin associated with the carotenoprotein by the addition of pretense inhibitor and antioxidant to the product were also investigated. Total astaxanthin contents of PKPO and RKPO were $35.1mg\%,\;22.1mg\%$ and those in carotenoproteins were $98.6mg\%,\;61.9mg\%$, respectively. The chitin contents of PKPO and RKPO were $6.9\%,\;4.5\%$, however, those of carotenoproteins were not determined. When $0.5\%$ trypsin was added to the extraction medium containing 0.5M $Na_3EDTA$ at $4^{\circ}C,\;74\%$ of astaxanthin and $83\%$ of the protein of PKPO were recovered as carotenoprotein in 24hrs. The amino acid profile in carotenoprotein was mainly composed of glutamic acid, methionine, aspartic acid and isoleurine. Their contents amounted to about 40% of the total amino acids, followed by alanine, phenylalanine, Iysine, leucine, threonine and tyrosine in that order, with a small amount of cysteine and tryptophan. The levels of essential amino acids were high as much as $38.3\%\~43.6\%$ of the total amino acids. The maximum observance of the carotenoid fraction from krill processing offal and from carotenoprotein was 469nm in petroleum ether. The separated components of carotenoprotein by TLC had Rfs $0.20\~0.23\;0.56\~0.60$ and $0.88\~0.91$. The carotenoids were comprised of astaxanthin, astaxanthin monoester and asthaxanthin diester in $25\~30\%\;,35\~40\%$and $40\~45\%$, respectively. The loss of carotenoids in the carotenoprotein can be prevented by the addition of pro-tease inhibitor(trasylol) and antioxidant(BHT) below $4^{\circ}C$.