• 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.

• The Korean Journal of Mycology
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• v.49 no.2
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• pp.199-209
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• 2021

The present study was performed to improve the technique used for fermenting the mushroom growth medium. Taxonomic analysis of 16S rDNA sequence from the predominant Bacillus strain CY-24 isolated during the fermentation phase of the rice straw medium identified it as Bacillus licheniformis. In addition, the growth environment of B. licheniformis was also examined in this study, which revealed the optimal growth temperature and pH to be 30 ℃ and 6.0, respectively. This study also revealed that carboxymethyl cellulase (CMCase) and polygalacturonase (PGase) enzymes isolated from B. licheniformis achieved their maximal activities at 50 ℃ and 60 ℃ respectively. Furthermore, the study confirmed that the two enzymes, i.e., CMCase and PGase in B. licheniformis are stable at temperatures above 60 ℃. The present study thus demonstrates that B. licheniformis CY-24 possesses excellent enzymatic properties. It also reveals that the action of enzymes during the production of growth mediums used for the cultivation of mushrooms is closely associated with the promotion of fermentation and softening of the rice straw. Overall, this study provides elementary information regarding the role of B. licheniformis enzymes during growth medium fermentation for Agaricus bisporus cultivation.

• Journal of Forest and Environmental Science
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• v.37 no.1
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• pp.52-61
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• 2021

Decay fungi can decompose plant debris to recycle carbon in the ecosystem. Still, they can also be fungal pathogens, which can damage living trees and/or wood material and cause a large amount of timber loss. We isolated and identified basidiomycetous fungi from the decayed bark of Mongolian oak wrapped with sticky roll traps. The degrading enzyme activities were then tested for all fungal isolates. The decay ability of selected isolates was assessed based on the weight loss of wood discs after inoculating with culture suspension of decay fungi under the different humidity levels. A total of 46 basidiomycetous fungal isolates belonged to 12 species, and 10 genera were obtained from Jong Myo (16 isolates), Chang Kyung palace (7 isolates), Cheong Gye (10 isolates), and Gun Po (13 isolates). Gymnopus luxurians was the most dominant fungus in the present study, and this species distributed in all survey sites with 9 isolates in Jong Myo, followed by 3 isolates in Chang Kyung palace, while Cheong Gye and Gun Po had only 1 isolate each. Among 46 isolates, 44 isolates secreted at least one enzyme, while 25 isolates produced both cellulase and phenol oxidase enzymes, and 2 isolates produced neither. The assessment of decay ability by artificial inoculation indicated that the weight loss of wood discs was significantly influenced by humidity conditions when inoculated with bark decay fungi. The percent weight losses by G. luxurians inoculation in RH of 90-100% and RH of 65-75% were 4.61% and 2.45%, respectively. The weight loss caused by Abortiporus biennis were 6.67% and 0.46% in RH of 90-100% and RH of 45-55%, respectively. The humidity reduction approach should be applied for further studies to control the growth and spread of bark decay fungi on the trunks wrapped with sticky roll traps.

• Journal of Marine Life Science
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• v.8 no.2
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• pp.150-159
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• 2023

To determine the toxic effects of the mirror carp, Cyprinus carpio nudus on waterborne nitric acid exposure, they were exposed to waterborne nitric acid at 0, 4, 20, 100, 500, and 2,500 mg NO₃^-/L for 96 h. The semi-lethal concentration (LC₅₀) of the scented fish by 96 hours of acute exposure to nitric acid was 1,433.54 mg NO₃^-/L. The physiological changes of waterborne nitric acid exposure on mirror carp were evaluated through hematologic properties, and RBC count showed a significantly decreased (p < 0.05). The change in the ion control ability of the mirror carp according to exposure to waterborne nitric acid was evaluated through the plasma inorganic component, and calcium and magnesium were significantly decreased (p < 0.05) in the plasma inorganic component. The health and stress status of the mirror carp due to exposure to waterborne nitric acid were evaluated through plasma organic ingredients, and the plasma organic component glucose showed a significantly increase (p < 0.05). Changes in liver damage and enzyme activity due to waterborne nitric acid exposure were evaluated through plasma enzyme components, the enzymatic activities of plasma AST, ALT, and ALP were significantly decreased (p < 0.05) in high levels of waterborne nitric acid exposure. The results of the present study suggest that waterborne nitric acid exposure to C. carpio nudus can toxicly affect survival, hematologic properties, and plasma components.

• Korean Journal of Agricultural Science
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• v.12 no.2
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• pp.324-332
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• 1985

Aspergillus niger B-15 with strong Endo-polygalacturonase (Endo-PG) activities was selected out from a total of 1,573 fungal strains isolated from various testing materials. A mutant strain, U-46, was obtained from the Aspergillus niger B-15 by repeated irradition of ultra-violet light. The objectives of the study were to investigate the fungal properties of the parental and mutant strains obtained and to study the condition of enzyme production and reaction. The results obtained are summarized as follows: 1. The size of conidial head of the U-46 mutant was smaller than that of the parental strains, B-15 and the length of the conidiophore was also shorter than that of the parental strains. 2. The optimum conditions for the Endo-PG production of the parental B-15 strain in the wheat bran Koji were obtained when 40% of water was added to the wheat bran and the temperature was 30 to $35^{\circ}C$. However, the best condition for the mutant U-46 strain was attained when 60 to 70% of water was added and the temperature was $35^{\circ}C$. The optimum growing periods were two to three days for both parental and mutant strains. 3. Under the optimum producing conditions of each strains, the enzymatic activity of the mutant U-46 was 20 times higher than the Endo-PG of the parental strain, B-15. 4. When both strains were cultured in the wheat bran Koji containing 60% of water at $35^{\circ}C$ for three days, the mutant strain. U-46, was about 46 times higher in the Endo-PG activity and about 18 times greater in Exo-PG activity than the parental strain, B-15. The activities of cellulase, $\alpha$-amylase, and glucoamylase were also highly increased in the mutant strain. 5. The mutant strain, U-46, increased its Endo-PG activity up to 20% over that of ordinary case when 1.2 to 1.5% of ammonium sulphate was added to the wheat bran. 6. The optimum condition for Endo-PG activity of crude enzyme of the mutant strain, U-46, was attained when pH of reaction solution was 4.0 to 4.5 and the temperature was $50^{\circ}C$.

• Korean Journal of Food Science and Technology
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• v.38 no.6
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• pp.793-798
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• 2006

We investigated the effects on the cytotoxicity against several cancer cells of the hydrolysis and molecular weight fractionation of crude laminaran from E. bicyclis, a brown seaweed collected from Uleung island in Korea, was extracted with boiling water and then crude laminaran was prepared by ethanol precipitation of extract obtained after elimination of calcium alginate by calcium chloride. Crude laminaran was hydrolyzed by enzyme (Econase CE), acid (0.1 N HCl) and autoclaving ($121^{\circ}C$, 180 min), and the molecular weight fractions by ultrafiltration to generate molecular weight fractions. Total sugar and sulfate contents of hydrolyzed laminaran were 72.3 and 3.5% (enzyme hydrolysate), 68.5 and 3.0% (acid hydrolysate), 70.2 and 3.2% (autoclaved), and monosaccharides of which consisted of glucose (74.7-78.5%), mannose (9.9-11.5%), galactose (8.5-9.6%) and fucose (3.1-4.5%), respectively. When the cytotoxicity of hydrolyzed laminaran on SNU-1, HeLa and SW cells was evaluated by MTT assay, growth-inhibitory activity of the enzyme hydrolysate against cancer cells was higher than that of acid hydrolysate or autoclaved laminaran. Furthermore, the fraction at a molecular weight range of 10 to 50 kDa revealed higher anti-proliferative activities. The $IC_{50}$ values of 10-50 kDa fraction at a molecular weight range of 10 to 50 kDa revealed higher anti-proliferative activities. The $IC_{50}$ values of 10-50 kDa fractions on SNU-1, HeLa and SW cells were 60.4, 58.6 and 53.9 ${\mu}g/mL$ for enzymatic hydrolysate, 75.6, 73.5 and 77.4 ${\mu}g/mL$ for acid hydrolysate, and 61.7, 68.2 and 60.8 ${\mu}g/mL$ for autoclaved, respectively.

• Korean Journal of Microbiology
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• v.51 no.2
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• pp.97-107
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• 2015

Wetlands constitute a transitional zone between terrestrial and aquatic ecosystems and have unique characteristics such as frequent inundation, inflow of nutrients from terrestrial ecosystems, presence of plants adapted to grow in water, and soil that is occasionally oxygen deficient due to saturation. These characteristics and the presence of vegetation determine physical and chemical properties that affect decomposition rates of organic matter (OM). Decomposition of OM is associated with activities of various extracellular enzymes (EE) produced by bacteria and fungi. Extracellular enzymes convert macromolecules to simple compounds such as labile organic carbon (C), nitrogen (N), phosphorus (P), and sulfur (S) that can be easily taken up by microbes and plants. Therefore, the enzymatic approach is helpful to understand the decomposition rates of OM and nutrient cycling in wetland soils. This paper reviews the physical and biogeochemical factors that regulate extracellular enzyme activities (EEa) in wetland soils, including those of ${\beta}$-glucosidase, ${\beta}$-N-acetylglucosaminidase, phosphatase, arylsulfatase, and phenol oxidase that decompose organic matter and release C, N, P, and S nutrients for microbial and plant growths. Effects of pH, water table, and particle size of OM on EEa were not significantly different among sites, whereas the influence of temperature on EEa varied depending on microbial acclimation to extreme temperatures. Addition of C, N, or P affected EEa differently depending on the nutrient state, C:N ratio, limiting factors, and types of enzymes of wetland soils. Substrate quality influenced EEa more significantly than did other factors. Also, drainage of wetland and increased temperature due to global climate change can stimulate phenol oxidase activity, and anthropogenic N deposition can enhance the hydrolytic EEa; these effects increase OM decomposition rates and emissions of $CO_2$ and $CH_4$ from wetland systems. The researches on the relationship between microbial structures and EE functions, and environmental factors controlling EEa can be helpful to manipulate wetland ecosystems for treating pollutants and to monitor wetland ecosystem services.

• Journal of fish pathology
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• v.18 no.1
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• pp.67-80
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• 2005

Phosphoinositide-specific phospholipase $C\delta$ $PLC\delta$) plays an important role in many cellular responses and is involved in the production of second messenger. The present study was conducted to obtain the biochemical characteristics of the expressed recombinant $PLC\delta$ in E. coli cloned from Misgurnus mizolepis and partially purified $PLC\delta$ enzymes from liver tissues of M. mizolepis (wild ML-$PLC\delta$). The ML $PLC\delta$ gene was cloned and expressed under the previous report (Kim et al., 2004), and purified the recombinant protein by successive chromatography using $Ni^{2+}$-NTA affinity column and gel iltration FPLC column. The wild ML-$PLC\delta$ protein was solublized with 2 M KCI and purified by successive chromatography on open heparin-Sephagel and analytical TSKgel heparin-5PW. Both the recombinant and wild ML-$PLC\delta$ form of protein showed a concentration-dependent PLC activity to phosphatidylinositol 4,5-bis-phosphate (PIP$_2$) or phosphatidylinositol (PI). Its activity was absolutely $Ca^{2+}$- dependant, which was similar to mammalian $PLC\delta$ isozymes. Maximal PI-hydrolytic activations of recombinant and wild ML- TEX>$PLC\delta$ was at pH 7.0 and pH 7.5, respectively. In addition, the enzymatic activities of recombinant and wild ML-$PLC\delta$ were increased in concentration-dependent manner by detergent, such as sodium deoxycholate SDC), phosphatidylethanolamine (PE) and phosphatidylcholine (PC). The activities decreased in contrast by a polyamine, such as spermine. Western blotting showed that several types of $PLC\delta$ isozymes exist in various organs. Taken together our results, it suggested that the biochemical characteristics of ML-$PLC\delta$ are similar with those of mammalian $PLC\delta1$ and ${\delta}3$ isozymes.

• Tuberculosis and Respiratory Diseases
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• v.48 no.6
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• pp.887-897
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• 2000

Background : Since the exact pathogenesis of sepsis-induced ARDS has not been elucidated, the mechanisms of enhanced neutrophilic respiratory burst were probed in endotoxin primed neutrophils associated with the roles of phospholipase A2(PLA2), platelet activating factor(PAF) and apoptosis. Methods : In isolated fresh human neutrophils, effects of the inhibition of PLA2 and PAF on the apoptosis were examined by the method of Annexin-FITC/dual PIflow cytometry. The roles of PLA2 and PAF on the neutrophilic respiratory burst were also examined by measuring oxidant generation in cytochrome-c reduction assay. Activities of the PLA2 and lysoPAF acetyltransferase (lysoPAF AT) of the neutrophils were determined to understand the effect of endotoxin on these enzymatic activities which may be related to the neutrophilic respiratory burst and apoptosis. In addition, the role roles of PLA2 and PAF in neutrophilic adhesion to bovine endothelial cells were examined in vitro by neutrophil adhesion assay. To investigate the effect of oxidants on pulmonary surfactant, cytochemical ultrastructural microscopy was performed. To inhibit PLA2 and PAF, non-specific PLA2 inhibitor mepacrine (100 nM) and WEB 2086 (100 nM) or ketotifen fumarate (10 ${\mu}g$/ml) were used respectively in all in vitro experimental sets. WEB 2086 is PAF receptor antagonist, and ketotifen fumarate is a lyso PAF AT inhibitor. Results: The mapacrine treatment, provided and the endotoxin (ETX) treatment, resulted in increased apoptosis of neutrophils (p<0.001) while treatments of WEB 2086 and ketotifen did not. The inhibition of PLA2 and PAF decreased (p<0.001) production of oxidants from PMA-stimulated neutrophils. While endotoxin increased the PLA2 activity of neutrophils (p<0.01), mepacrine supressed (p<0.001) the activity, provided after treatment of ETX. The lyso PAF actyltransferase activity (lyso PAF AT) increased (p<0.01) after treatment of ETX. In contrast, mepacrine, WEB 2086 and ketotifen showed a tendency of decreasing the activity after treatment of ETX. The treatment of ETX incresed (p<0.001) neutrophil adhesion to endothelial cells, which was reversed by inhibition of PLA2 and PAF (p<0.01). The binding of oxidants to pu1monary surfactant was identified histologically. Conclusions : The enhanced neutrophilic respiratory burst by ETX plays a pivotal role in the pathogenesis of ARDS in terms of oxidayive oxidative stress. Increased production of oxidants from neutrophils is mediated by the activations of PLA2 and lyso PAF AT.

• Journal of Dairy Science and Biotechnology
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• v.33 no.3
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• pp.179-196
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• 2015

Because of our aging population, there is increasing concern about the impact of dementia and age-related cognitive decline. Intense research efforts on effective dietary interventions for the prevention or amelioration of dementia and age-related cognitive decline have indicated that dairy products affect physiological health and potentially healthy brain function during aging. Milk is a rich source of proteins and peptides with nutritional and immunotropic activities. The preparation of biologically active proteins and peptides generally requires enzymatic degradation, chemical modification, or the addition of specific co-factors. Milk-derived preparations are widely available in the food industry in the form of hygiene products and infant formulas. However, milk-derived products could also be applied as preventive or therapeutic measures for a wide-range of pathological conditions not only in neonates and infants but also in adults, including the elderly. Because they have no adverse side effects, milk-derived proteins and peptides could be used as a supplementary treatment for dementia and age-related cognitive decline.