• Korean Journal of Microbiology
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• v.20 no.3
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• pp.105-112
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• 1982

The strain of Aspergillus, 6368A, producing acid protease showing high activity was isolated from soil, as a result of wide research about mold group. This strain was identified as a species of Aspergillus tubingensis by the investigation of morphological characteristics. The change of the enzyme production under the various media and culture condition was also studied. The optimum pH and stability of crude acid protease are 2.5, 2.0~4.5 and the optimum temeprature and thermal inactivation waas shown $50^{\circ}C,\;55^{\circ}C$, respectively. From the result of the study on the effects of metal ions, it was found that $MnCl_2,\;CoCl_2,\;CuCl_2,\;SrCl_2,\;and\;NiCl_2$ slightly increased the enzyme activity, on the other hand $ZnCl_2,\;CaCl_2,\;MgCl_2,\;SLS,\;and\;KMnO_4$ decreased it.

• Journal of Microbiology and Biotechnology
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• v.22 no.1
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• pp.58-68
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• 2012

An investigation was conducted on the enhancement of production and purification of an oxidant and SDS-stable alkaline protease (BHAP) secreted by an alkalophilic Bacillus horikoshii, which was screened from the body fluid of a unique Korean polychaeta (Periserrula leucophryna) living in the tidal mud flats of Kwangwha Island in the Korean West Sea. A prominent effect on BHAP production was obtained by adding 2% maltose, 1% sodium citrate, 0.8% NaCl, and 0.6% sodium carbonate to the culturing medium. The optimal medium for BHAP production contained (g/l) SBM, 15; casein, 10; $K_2HPO_4$, 2; $KH_2PO_4$, 2; maltose, 20; sodium citrate, 10; $MgSO_4$, 0.06; NaCl, 8; and $Na_2CO_3$, 6. A protease yield of approximately 56,000 U/ml was achieved using the optimized medium, which is an increase of approximately 5.5-fold compared with the previous optimization (10,050 U/ml). The BHAP was homogenously purified 34-fold with an overall recovery of 34% and a specific activity of 223,090 U/mg protein using adsorption with Diaion HPA75, hydrophobic interaction chromatography (HIC) on Phenyl-Sepharose, and ion-exchange chromatography on a DEAE- and CM-Sepharose column. The purified BHAP was determined a homogeneous by SDS-PAGE, with an apparent molecular mass of 28 kDa, and it showed extreme stability towards organic solvents, SDS, and oxidizing agents. The $K_m$ and $k_{cat}$ values were 78.7 ${\mu}M$ and $217.4s^{-1}$ for N-succinyl-Ala-Ala-Pro-Phe-pNA at $37^{\circ}C$ and pH 9, respectively. The inhibition profile exhibited by PMSF suggested that the protease from B. horikoshii belongs to the family of serine proteases. The BHAP, which showed high stability against SDS and $H_2O_2$, has significance for industrial application, such as additives in detergent and feed industries.

• Proceedings of the Korean Institute of Electrical and Electronic Material Engineers Conference
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• 2001.11b
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• pp.3-6
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• 2001

In common globular proteins, the native form is in its most stable state. However, the native form of inhibitory serpins (serine protease inhibitors) and some viral membrane fusion proteins is in a metastable state. Metastability in these proteins is critical to their biological functions. Our previous studies revealed that unusual interactions, such as side-chain overpacking, buried polar groups, surface hydrophobic pockets, and internal cavities are the structural basis of the native metastability. To understand the mechanism by which these structural defects regulate protein functions, cavity-filling mutations of a 1-antitrypsin, a prototype serpin, were characterized. Increasing conformational stability is correlated with decreasing inhibitory activity. Moreover, the activity loss appears to correlate with the decrease in the rate of the conformational switch during complex formation with a target protease. We also increased the stability of a 1-antitrypsin greatly via combining various stabilizing single amino acid substitutions that were distributed throughout the molecule. The results showed that a substantial increase of stability, over 13 kcal/mol, affected the inhibitory activity with a correlation of 11% activity loss per kcal/mol. The results strongly suggest that the native metastability of proteins is indeed a structural design that regulates protein functions and that the native strain of a 1-antitrypsin distributed throughout the molecule regulates the inhibitory function in a concerted manner.

• Electrical & Electronic Materials
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• v.14 no.12
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• pp.3-6
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• 2001

In common globular proteins, the native form is n its most stable state. However, the native form of inhibitory serpins (serine protease inhibitors) and some viral membrane fusion proteins is in a metastable state. Metastability in these proteins is critical to their biological functions. Our previous studies revealed that unusual interactions, such as side-chain overpacking, buried polar groups, surface hydrophobic pockets, ad internal cavities are the structural basis of the native metastability. To understand the mechanism by which these structural defects regulate protein functions, cavity-filling mutations of $\alpha$1-antitrypsin, a prototype serpin, were characterized. Increasing conformational stability is correlated with decreasing inhibitory activity. Moreover, the activity loss appears to correlate with the decrease in the rate of the conformational switch during complex formation with a target protease. We also increased the stability of $\alpha$1-antitrypsin greatly via combining various stabilizing single amino acid substitutions that were distributed throughout the molecule. The results showed that a substantial increase of stability, over 13 kcal/mol, affected the inhibitory activity with a correlation of 11% activity loss per kcal/mol. The results strongly suggest that the native metastability of proteins is indeed a structural design that regulates protein functions and that the native strain of $\alpha$1-antitrypsin distributed throughout the molecule regulates the inhibitory function in a concerted manner.

• Proceedings of the Korean Institute of Electrical and Electronic Material Engineers Conference
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• 2001.11a
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• pp.3-6
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• 2001

In common globular proteins, the native form is in its most stable state. However, the native form of inhibitory serpins (serine protease inhibitors) and some viral membrane fusion proteins is in a metastable state. Metastability in these Proteins is critical to their biological functions. Our previous studies revealed that unusual interactions, such as side-chain overpacking, buried polar groups, surface hydrophobic pockets, and internal cavities are the structural basis of the native metastability. To understand the mechanism by which these structural defects regulate protein functions, cavity-filling mutations of ${\alpha}$1-antitrypsin, a prototype serpin, were characterized. Increasing conformational stability is correlated with decreasing inhibitory activity. Moreover, the activity loss appears to correlate with the decrease in the rate of the conformational switch during complex formation with a target protease. We also increased the stability of ${\alpha}$1-antitrypsin greatly via combining various stabilizing single amino acid substitutions that were distributed throughout the molecule. The results showed that a substantial increase of stability, over 13 kcal/mol, affected the inhibitory activity with a correlation of 11% activity loss per kcal/mol. The results strongly suggest that the native metastability of proteins is indeed a structural design that regulates protein functions and that the native strain of e 1-antitrypsin distributed throughout the molecule regulates the inhibitory function in a concerted manner.

• BMB Reports
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• v.33 no.5
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• pp.379-384
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• 2000

The native conformation of serpins (serine protease inhibitors) is strained. Upon cleavage of the reactive center loop of serpins by a protease, the amino terminal portion of the cleaved loop is inserted into the central ${\beta}-sheet$, A sheet, as the fourth strand, with the concomitant release of the native strain. We questioned the role of protease in this conformational switch from the strained native form into a stable relaxed state. Chemical cleavage of the reactive center loop of ${\alpha}_1-antitrypsin$, a prototype serpin, using hydroxylamine dramatically increased the stability of the serpin. A circular dichroism spectrum and peptide binding study suggests that the amino terminal portion of the reactive center loop is inserted into the A sheet in the chemically-cleaved ${\alpha}_1-antitrypsin$, as in the enzymatically-cleaved molecule. These results indicate that the structural transformation of a serpin molecule does not require interaction with a protease. The results suggest that the serpin conformational switch that occurred during the complex formation with a target protease is induced by the cleavage of the reactive center loop per se.

• Applied Biological Chemistry
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• v.15 no.1
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• pp.27-33
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• 1972

The alkaline protease was isolated from the culture material of monascus sp. on wheat bran culture. The crude purification of this enzyme was extracted with distilled water and precipitated with ammonium sulfate of 0.5 saturation. And, the activity of this enzyme was determind very strongly by folin's colorimetric method. The optimal pH of this enzyme was ranging from pH 10 to 13 and the optimal temperature was $50^{\circ}C$. The pH stability was ranging from pH 5 to 12 and the enzyme activity was not inactivated by heat treatment in lower temperature than $40^{\circ}C$. The enzyme was protected from heat denature by the treatment of $Pb^#$, $Ba^#$, $Co^#$, $Zn^#$, and $Cu^#$, but was inactivated with $Hg^#$, $Fe^#$ strongly. Moreover, one of these metal ions, the cupper ion, has a strong protective activity on enzyme heat denature. And, it was not effected by treatment of EDTA.

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

• Microbiology and Biotechnology Letters
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• v.26 no.4
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• pp.290-296
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• 1998

Interspecific hybrids between Aspergillus oryzae var. oryzae and Penicillium chrysogenum (Tyr$\^$-/), high alkaline protease producing fungi, were obtained by nuclear transfer technique. Nuclei isolated from the wild type Aspergillus oryzae var. oryzae strain were transferred into auxotrophic Penicillium chrysogenum mutants and selected the new strains showing an increased protein degrading capability. Maximum production of protoplasts were obtained by 1% Novozym 234 at $30^{\circ}C$ for 3 hours and the most effective osmotic stabilizers for the isolation of protoplasts were 0.6M KCl. Frequencies of hybrid formation by nuclear transfer were 1.3${\times}$10$\^$-3/∼2.8${\times}$10$\^$-3/. They could be suggested as an aneuploid by the observation of genetic stability, conidial size, DNA content, and nuclear strain. The hybrids showed 1.1～2.2 fold higher alkaline pretense activities than parental strains.

• Journal of Microbiology and Biotechnology
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• v.24 no.2
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• pp.197-208
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• 2014

A novel protease gene from Bacillus gibsonii, aprBG, was cloned, expressed in B. subtilis, and characterized. High-level expression of aprBG was achieved in the recombinant strain when a junction was present between the promoter and the target gene. The purified recombinant enzyme exhibited similar N-terminal sequences and catalytic properties to the native enzyme, including high affinity and hydrolytic efficiency toward various substrates and a superior performance when exposed to various metal ions, surfactants, oxidants, and commercial detergents. AprBG was remarkably stable in 50% organic solvents and retained 100% activity and stability in 0-4 M NaCl, which is better than the characteristics of previously reported proteases. AprBG was most closely related to the high-alkaline proteases of the subtilisin family with a 57-68% identity. The secretion and maturation mechanism of AprBG was dependent on the enzyme activity, as analyzed by site-directed mutagenesis. Thus, when taken together, the results revealed that the halo-solvent-tolerant protease AprBG displays significant activity and stability under various extreme conditions, indicating its potential for use in many biotechnology applications.