• Journal of Nutrition and Health
• /
• v.45 no.5
• /
• pp.429-436
• /
• 2012

This study was performed to examine the characteristics of protein of red crab (Chionoecetes japonicus) shell powder hydrolyzed by commercial proteases. Red crab shell was digested by commercial proteases, such as Protamex (P), Neutrase (N), Flavourzyme (F), Alcalase (A), Protease M (PM) and Protease A (PA). Protein yield analyzed by Biuret assay, absorbance at 280 nm and brix revealed that PA was the enzyme having the highest proteolytic activity. SDS PAGE showed that molecular weight of proteins produced by protease treatments was various and below 150 kDa. Combinational treatment of proteases (PA + P, PA + PM, PA + F, PA + A) was tried whether these increase protein hydrolysis from red crab shell powder compared to a PA single treatment. Soluble protein content was similar, but amino acid concentration by combinational treatments was higher than PA single treatment [PA + P 247.4 mg/g > PA + F (206.4 mg/g) > PA + A (133.4 mg/g) > PA + PM (59.1 mg/g) > PA (54.9 mg/g)]. Amino acid composition by combinational treatments was slightly different. Most abundant essential amino acids were phenylalanine, glycine, alanine, and leucine, whereas tyrosine and cystine were not detected.

• The Journal of Korean Society of Virology
• /
• v.29 no.3
• /
• pp.175-182
• /
• 1999

An attractive target for anti-herpes chemotherapy is the herpes simplex virus 1 (HSV-1) protease encoded by the UL26 gene. HSV-1 protease is essential for DNA packaging and virus maturation. To perform high throughput for potent inhibitors, the efficient production of larger amounts of highly purified enzyme and protease activity assay method must be established. In this report, expression in E. coli and purification of the protease gene of HSV-1 strain F was investigated. The protease gene was cloned pET28, and the nucleotide sequence of protease catalytic domain of HSV-1 compared strain F with other strains (KOS and CL101). In these results the F strain was different in base sequence. However, the amino acid sequence was identifical. The HSV-1 protease was purified with His-tagged affinity column. The analysis of HSV-1 protease activity was performed by high performance liquid chromatography.

• Microbiology and Biotechnology Letters
• /
• v.31 no.3
• /
• pp.224-229
• /
• 2003

A keratinolytic protease was purified from the culture medium of Pseudomonas sp. KP-364 by use of an assay of the hydrolysis of feather keratin. Membrane ultrafiltration and DEAE-cellulose ion-exchange resin and Sephadex G-150 gel chromatographies were used to purify the enzyme. The specific activity of the purified keratinolytic protease relative to that in the original medium was approximately 72-fold high. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Sephadex G-150 chromatography indicated that the purified keratinolytic protease is monomeric and has a molecular weight of 36 kDa. The optimal pH and temperature of the keratinolytic protease activity were 6.6 and 37 C, respectively, and the keratinolytic protease was relatively stable at pH value from 3.0 to 10.0 at 37 C for 1hour. The keratinolytic protease was inhibited by EDTA and EGTA, indicating that the keratinolytic protease was a kind of metalloprotease that require Li+ for cofactor.

• Journal of Korean Ophthalmic Optics Society
• /
• v.19 no.1
• /
• pp.51-57
• /
• 2014

Purpose: The present study was conducted to establish the experimental condition for the proper evaluation of protein removal efficacy when developing protein removal agents. Its protein removal efficacy was further analyzed and compared with the result from protein removal efficacy against protein deposition on contact lens to suggest the evaluation method for efficacy of protein removal agents. Methods: Protein digestibility assay presented in the Korean pharmacopoeia was selected to establish the evaluation method for efficacy of papain, pancreatin, subtilisin A and protease itself as a ingredient and protein removal tablets or solution containing those enzymes and find a suitable test conditions. Furthermore, the cleaning efficacy of commercially available protein removal tablets and solution on balafilcon A lens deposited with protein artificially was measured and the correlation between two evaluation methods was further analyzed. Results: When pancreatin itself and the product containing pancreatin was evaluated by protein digestibility assay, both reached 28 IU/mg, the standard value of protein digestibility suggested by the Korean pharmacopoeia. In case of protease and subtilisin A tested with trichloroacetic acid B solution, both of them met the enzyme activity level proposed by the manufacturers when they were evaluated by protein digestibility assay however, papain and subtilisin A tested with trichloroacetic acid A solution were not reached the enzyme activity level. Among protein removal agents, three products except a product containing pancreatin did not meet the enzyme activity value specified by the manufacturer when they were evaluated by protein digestibility assay. However, actual protein removal efficacy of three products except a papain-containing product on the lens was greater than 90% protein removal. In the case of papain-containing protein removal product, its effect was not measured by protein digestibility assay however, its actual protein removal efficacy on the lens reached 73.72%. Conclusions: From the results, it was confirmed that the efficacy of protein removal agents for contact lens should be evaluated by different method according to the type of proteolytic enzyme contained. That is, the protein removal agents containing pancreatin, protease and subtilisin A can be evaluated by protein digestibility assay and protein removal efficiency evaluation and the products containing papain can be effectively evaluated by only the evaluation method for protein removal efficiency employing the lens.

• Journal of Life Science
• /
• v.20 no.7
• /
• pp.1005-1011
• /
• 2010

$\gamma$-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system. GABA transporters (GATs) control extracellular GABA levels by reuptake of released GABA from the synaptic cleft. However, how GATs are regulated has not yet been elucidated. Here, we used the yeast two-hybrid system to identify the specific binding protein(s) that interacts with the carboxyl (C)-terminal region of GAT1, the major isoform in the brain and find a specific interaction with the ubiquitin-specific protease 14 (Usp14), a deubiquitinating enzyme. Usp14 protein bound to the tail region of GAT1 and GAT2 but not to other GAT members in the yeast two-hybrid assay. The C-terminal region of Usp14 is essential for interaction with GAT1. In addition, these proteins showed specific interactions in the glutathione S-transferase (GST) pull-down assay. An antibody to GAT1 specifically co-immunoprecipitated Usp14 from mouse brain extracts. These results suggest that Usp14 may regulate the number of GAT1 at the cell surface.

• 한국생물공학회:학술대회논문집
• /
• 2001.11a
• /
• pp.89-92
• /
• 2001

Aspergillus niger has been used as a host system to express many heterologous proteins. It has various advantages over other expression systems in that it is a small eukaryotic GRAS (Generally Recognized aS Safe) organism with a capacity of secreting large amount of foreign proteins. However, it has been known that the presence of an abundant protease is a limiting factor to express a heterologous protein. The proteases deficient mutants of A. niger were obtained using UV -mutagenesis. A total of 1 ${\times}$ $10^5$ spores were irradiated with 10-20% survival dose of UV, 600J/M2 at 280nm, and the resulting spores were screened on the casein -gelatin plates. Ten putative protease deficient mutants were further analyzed on the starch plates to differentiate the pro from the secretory mutant. An endogenous extracellular enzyme, glucose oxidase, was also examined to confirm that the mutant phenotype was due to the proteases deficiency rather than the mutation in the secretory pathway. The reduced proteolytic activity was measured using SDS-fibrin zymography gel, casein degradation assay, and bio-activity of a supplemented hGM -CSF (human Granulocyte-Macrophage Colony Stimulating Factor). Comparing with the wild type strain, less than 30 % of proteolytic activity was observed in the culture filtrate of the protease deficient mutant (pro -20) without any notable changes in cell growth and secretion.

• The Korean Journal of Mycology
• /
• v.26 no.4 s.87
• /
• pp.583-588
• /
• 1998

A fibrinolytic enzyme has been isolated from the edible honey mushroom, Armillariella mellea and purified. The apparent molecular mass of purified enzyme was estimated to be 19800Da by SDS polyacryl amide gel electrophoresis and 19900Da by gel filtration, indicating that it was a monomer. The enzyme was optimal at pH 7, suggesting that the purified enzyme was a neutral proteinase. It shows the maximum fibrinolytic activity at $55^{\circ}C$, is completely inactivated above $65^{\circ}C$, and still indicates 40% of activity at $37^{\circ}C$. The fibrinolytic activity has been decreased by the addition of EDTA. Fifteen amino acid sequence was determined by protein sequencing techniques.

• Korean Journal of Oriental Medicine
• /
• v.10 no.1
• /
• pp.119-126
• /
• 2004

For the purpose of developing new anti-HIV agents from natural sources, the extracts of Solanum nigrum L. were tested for their inhibitory effects on HIV-1 replication and its essential enzymes as the reverse transcriptase (RT), protease and ${\alpha}$-glucosidase. In the assay of HIV-1-infected human T-cell line, water extracts inhibited the HIV- 1 -induced cytopathic effects with IC (inhibitory concentration) of 100 ug/ml. Moreover water extracts (100ug/ml) of aerial parts showed strong activity of 32.6% on anti-HIV-1 PR using the activity of the enzyme to cleave an oligopeptide. In the HIV-1 reverse transcriptase inhibition assay, aqueous extract a inhibited 17.4%, but no glucosidase inhibitory activities. We found out this result, for these samples it is possible that the inhibition of the viral replication in vitro is due to the inhibition at least one of PR and RT. It would be of great interest to identify the compounds which are responsible for this inhibition, since all therapeutically useful agent up to date are PR, RT and ${\alpha}$-glucosidase inhibitors.

• The Korean Journal of Mycology
• /
• v.39 no.2
• /
• pp.99-104
• /
• 2011

Shiitake breeding requires the procedures of mating of two different parental strains and selection of hybrid strains that have good traits for the mushroom production. In this study, we tested the possibility of the use of chromogenic plate-based assay for extracellular enzyme production in order to assess and find good biochemical properties-possessed hybrid strains that were generated from genetic cross of the monokaryotic strains derived from two different parental strains of Lentinula edodes Sanjo-101ho and Sanjo-108ho. We observed that there was difference in the ability of producing ${\beta}$-glucosidase, avicelase, CM-cellulase, amylase, pectinase, xylanase, and protease among the monokaryotic strains. We could also comparatively assess that the ability of the seven extracellular enzymes production in the hybrid strains depended on the mating combination of the monokaryotic strains. Our results demonstrate that the assessment method for extracellular enzyme production using chromogenic plate assay could be usefully applied to the assessment of the hybrid strains derived from the breeding procedure of L. edodes.

• IMMUNE NETWORK
• /
• v.5 no.4
• /
• pp.193-198
• /
• 2005

Background: Protease-activated receptor 2 (PAR2) belongs to a family of G protein coupled receptors activated by proteolytic cleavage. Trypsin-like serine proteases interact with PAR2 expressed by a variety of tissues and immune cells. The aim of our study was to investigate whether PAR2 stimulation can lead to the activation of human mac rophages. Methods: PAR2-mediated proliferation of human macrophage cell line THP-1 was measured with MTT assay. We also examined the extracellular regulated kinase (ERK) phosphorylation and cytokine production induced by trypsin and PAR2-agonist using western blot and enzyme-linked immunosorbent assay (ELISA), respectively. Results: Treatment of trypsin or PAR2-activating peptide increased cell proliferation in a dose-dependent manner, and induced the activation of ERK1/2 in THP-1 cells. In addition, trypsin-induced cell proliferation was inhibited by pretreatment of an ERK inhibitor (pD98059) or trypsin inhibitor (SBTI). Moreover, PAR2 activation by trypsin increased the secretion of TNF-${\alpha}$ in THP-1 cells. Conclusion: There results suggest that P AR2 activation by trypsin-like serine proteases can induce cell proliferation through the activation of ERK in human macrophage and that PAR2 may playa crucial role in the cell proliferation and cytokine secretion induced by trypsin-like serine proteases.