Wol-Suk Cha

Purification and characterization of a novel fibrinolytic enzyme from fruiting bodies of Korean Cordyceps militaris

A fibrinolytic enzyme has been purified from the fruiting bodies of Korean Cordyceps militaris. The molecular mass of the enzyme was estimated to be 34 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), fibrin-zymography, and gel filtration chromatography. The 15 amino acid residues of the N-terminal sequence of the enzyme were APVEQCDAPVGLARL, which is dissimilar to those of fibrinolytic enzymes from other mushrooms. Optimal pH and temperature values of the enzyme were 7.0 and 40°C, respectively. The enzyme activity was completely inhibited by phenylmethylsulfonyl fluoride (PMSF), TPCK, 1,10-phenanthroline, Cu(2+), and Ba(2+). It was also significantly inhibited by aprotinin, EDTA, and EGTA. The enzyme showed a higher specificity for a synthetic substrate, N-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, exhibiting that it is a chymotrypsin-like serine metalloprotease. The enzyme preferentially hydrolyzed the fibrinogen Aα-, followed by the Bβ-chains and the γ-chain. The α, β, and γ-γ chains of fibrin were also degraded by the enzyme.

Biochemical and enzymatic properties of a fibrinolytic enzyme from Pleurotus eryngii cultivated under solid-state conditions using corn cob.

Biochemical and enzymatic properties of a fibrinolytic enzyme purified from Pleurotus eryngii cultivated under solid-state conditions using corn cob as energy source were investigated. The molecular mass of the enzyme was estimated to be 14 kDa by SDS-PAGE. The enzyme exhibited the highest activity (28.96 mol/min/mg) for the substrate tosyl-Gly-Pro-Lys-p-nitroanilide. K(m) and V(max) values were 0.18 mM and 53.5 U/ml, respectively. The enzyme was completely inhibited by 1.0 mM phenylmethylsulfonyl fluoride (PMSF). The N-terminal sequence was A-M-D-S-Q-T-D-A-S-Y-G-LA-N-D. This sequence exhibited a high degree of similarity to the N-terminal sequences of the subtilisin-like serine proteases. The enzyme was very stable at pH 4.0-6.0 with an optimum pH 5.0 at 40 degrees C. The enzyme rapidly hydrolyzed the A alpha-chain of fibrinogen within 5 min of incubation, followed by the B beta-chain after 10 min. The fibrinolytic enzyme from P. eryngii cultivated under solid-state conditions using corn cob could be potentially exploited in thrombolytic therapy.

Effects ofFomitopsis pinicola extracts on antioxidant and antitumor activities

We investigated the effects ofFomitopsis pinicola extract on biological activity by examining the antioxidant and antitumor activityin vitro andin vivo. When theF. pinicola extract concentration was raised from 60 to 120 μg/mL, the DPPH scavenging rate increased from 50.3 to 88.2% and the superoxide anion radical scavenging rate increased from 45.2 to 85.3% when theF. pinicola extract concentration was raised from 500 to 700 μg/mL. After incubatingF. pinicola extract for 12 h, the linoleic acid scavenging rate increased from 35.5 to 90.5%. A similar finding was observed for butylated hydroxytoluene. The total phenolic content of theF. pinicola extracts were approximately 10- to 16-fold higher than what was observed in theP. nebrodensis andA. camphorate extracts. The glutathione production, using decoctions prepared fromF. pinicola, was 20.0 μM/g of liver, which corresponded to approximately 4.0-fold higher than the control. The glutathione peroxidase activity was 8.3 U/mg of protein, which was approximately 2.8-fold higher than the activity level observed in the control rat livers. The cell viability rates of all the human cancer cells, when 100 μg/mL of ethanol extract was used for the different types of cancer cells, decreased with increasing extract concentrations in comparison to the hot water extract. In particular, when HeLa and Hep3B cells were incubated with 1.000 μg/mL of methanol extract, the cell viability rates were 20 and 25%, respectively, which was approximately 3.0-fold higher than what was observed for the hot water extract.

Effect ofPleurotus ferulae extracts on viability of human lung cancer and cervical cancer cell lines

When SiHa cells were incubated for varying periods of time with extracts of PFF and PFM, the cytotoxicity of the ethanol extracts of PFF was higher than those of the other extracts. These results indicated that the extracts from fruiting bodies ofP. ferulae contain antitumor substances. When A549, SiHa and HeLa cells were incubated with different concentrations of PFF and PFM extracts, the ethanol extracts of PFF showed strong cytotoxicity against A549 cells at concentrations over 10 μg/mL and against SiHa and HeLa cells at concentrations over 40 μg/mL. However, the differences in the cytotoxic effects of the hot water and ethanol extracts of PFM and the hot water extracts of PFF on all 3 cancer cells were not significant. Also, the PFF ethanol extracts induced synergistic effects on the TRAIL-induced apoptosis in A549 cells, which were strongly resistant to TRAIL. These results indicated that ethanol extracts of PFF were the most prominent antitumor agents toward lung cancer cells (A549).

Analysis of Mineral, Amino Acid and Vitamin Contents of Fruiting Body of Sparassis crispa.

꽃송이 버섯을 이용한 의약품 및 건강기능성 식품 개발을 위한 기초자료의 목적으로 자실체의 미네랄, 아미노산 및 비타민 함량을 조사하였다. 꽃송이버섯 자실체의 건조중량 100g을 기준으로 조사한 미네랄성분은 K, P, Na, Mg가 주성분을 이루었으며 이 중 K가 1,299.44 mg로 가장 많이 함유되어 있었다. 유리아미노산의 ...

Studies on cultivation and biological activities ofPleurotus nebrodensis inzenga

Environmental factors affecting mycelial growth and exo-polysaccharide production fromPleurotus nebrodensis Inzenga (PN) and biological activities of PN extractsin vitro were studied. The culture conditions for effective mycelial growth and exo-polysaccharide production were found to be 25 ‡C, 5% of inoculum size, and an initial pH from 6.5 to 7.0. When 5% of glucose was used, the maximum mycelial growth and exo-polysaccharide concentrations were 8.3 and 3.07 g/L, respectively. Among the various nitrogen sources, the mycelial growth and exo-polysaccharide production were very strong when polypeptone was used. In the case of the minerals sources, K2HPO4 and MgSO4·7H2O were found to best support for mycelial growth and exo-polysaccharide production. Under optimal conditions and methods, the maximum mycelial growth and exo-polysaccharide production were obtained after 10 days of culture, 12.84 and 4.85 g/L, respectively, in a jar fermentor. The effects of the PN extracts on the viability of three human cancer cell lines and antioxidant activity were investigatedin vitro. When cancer cells of the lung (A549), cervical region (HeLa) and colon (KM12C) were incubated at 6 mg/mL of the PN ethanol extracts, the viabilities of the HeLa and KM12C cells were slightly decreased. However, the growth of the A549 cells was remarkably inhibited when the PN ethanol extract was over 4 mg/mL. The antioxidant activity showed 46.2% at 40 µL, which was about 3.2 fold higher than that of the PN methanol extract.

Optimization of culture media for solid-state culture ofPleurotus ferulae

In order to elucidate the possibility of artificial production ofP. ferulae by solid-state culture, the optimization of culture conditions was carried out. When NH4H2PO4 and CaCO3 were used in the cultures using test tube with 30 g ofPopulus sawdust at 25°C±1 in the dark, the favored mycelial growth was observed with 1% of NH4H2PO4 and the production of polysaccharide was 7.85 mg/100 mg of mycelium with 1% of CaCO3. The mixtures of 80% ofPopulus sawdust and 20% of rice bran at 60% of water content were determined to be optimal for the production of fruiting bodies in the sawdust culture. When three treatments containing various ratios of garlic powder were conducted, yields of fruiting bodies were drastically higher than those of synthetic mixture without garlic powder. The highest yield (143 g/bag) was obtained with 7% garlic powder. The yield of synthetic mixture containing 7% of garlic powder was 83% higher than that of sawdust culture. The reason why garlic powder did support growth was not clear but it is possible that garlic powder might contain effective components for the formation of fruiting body. The optimal synthetic mixture composition consisted of cotton seed 77%, lime 6.4%, K2HPO4 0.2%, KH2PO4 0.2%, CaHPO4, 0.2%, corn flour 4%, wheat flour 5%, and garlic powder 7%.

Comparative study on the antioxidant and nitrite scavenging activity of fruiting body and mycelium extract from Pleurotus ferulae

We investigated the effects of the antioxidant and the nitrite scavenging activities of the extracts from Pleurotus ferulae fruiting body grown on the solid state using corn cob and activated bleaching earth (CCABE media) and its mycelium grown in the liquid state. The total phenol and polysaccharide concentrations in hot water extract of fruiting body were approximately 3.6- and 4.3-fold higher than those of the mycelium. Using the hot water extract of fruiting body, the maximum DPPH radical scavenging activity at 9 mg/mL, hydroxyl radical scavenging activity at 12mg/mL, reducing power at 12 mg/mL, and chelating ability at 12 mg/mL were obtained, 80.5%, 72.4%, 0.99 OD (700 nm), and 77.0%, respectively. However, in the case of hydrogen peroxide scavenging activity, the ethanol extract was the highest, 78.7% at 12 mg/mL. The maximum nitrite scavenging activity was obtained, 89.7% at 6 mg/mL of hot water extract from fruiting body. Hot water extracts were more effective than ethanol extracts in scavenging activity on DPPH radicals and hydroxyl radical scavenging, reducing power, and chelating activity of ferrous, whereas ethanol extracts were more effective in hydrogen peroxide scavenging activity as evidenced by their lower EC50 values. These results indicate that the hot water extract of P. ferulae fruiting body using CCABE media has good potential to be used as a source of materials or additives for oxidation suppressant in food, cosmetics and drug compositions.

Phosphorus removal in pilot plant using biofilm filter process from farm wastewater

Various environmental conditions affecting total phosphorus removal from farm wastewater in a biofilm filter, process were investigated using loess balls andChromobacterium LEE-38 at a pilot plant. WhenChromobacterium LEE-38 was used, the removal efficiency of total phosphorous was approximately 10- or 5-fold higher than that ofAcinetobacter CHA-2-14 orAcinetobacter CHA-4-5, respectively. When a loess ball of 11–14 mm manufactured at a 960°C calcining temperature was used, the removal efficiency of total phosphorous was 90.0%. When 70% of the volume fraction was used, the maximum efficiency of total phosphorus removal was 93.1%. Notably, when the initial pH was in the range of 6.0 to 8.0, the maximum removal efficiency of total phosphorus was obtained after 30 days. When the operating temperature was in the range of 30 to 55°C, the maximum removal efficiencies of total phosphorus, 95.6 to 94.6%, were obtained. On the other hand, at operating temperatures below 20°C or above 40°C, the removal efficiency of total phosphorous decreased. Among the various processes, biofilm filter process A gave the highest removal efficiency of 96.4%. Pilot tests of total phosphorus removal using farm wastewater from the biofilm filter process A were carried out for 60 days under optimal condition. WhenAcinetobacters sp. Lee-11 was used, the average removal efficiency in thep-adsorption area was only 32.5%, and the removal efficiencies of chemical oxygen demand (COD) and biological oxygen demand (BOD) were 56.7 and 62.5%, respectively. On the other hand, whenChromobacterium LEE-38 was used, the average removal efficiency was 95.1%, and the removal efficiencies of COD and BOD were 91.3 and 93.2%, respectively.

Optimal conditions for chitinase production bySerratia marcescens

A chitinase-producing bacterium was isolated from seashore mud around Beobseongpo in Chunmam province through the use of a selective enrichment culture. The best chitinase producing strain was isolated and identified asSerratia marcescens KY from its characteristics. For effective production of chitinase, optimum pH, temperature, and agitation speed were investigated in flask cultures. The optimum pH usingSerratia marcescens KY was between pH 6 and 7 and the chitinase produced was 37.9 unit/mL. On the other hand, the optimal pH of theSerratia marcescens ATCC 27117 was 7.5, and the produced amount of chitinase was 35.2 unit/mL. The optimal temperature for chitinase production forSerratia marcescens KY andSerratia marcescens ATCC 27117 was 30°C. The cell growth pattern at different temperature was almost identical to the chitinase production. To investigate the optimal shaking speed under optimal culture, speeds were varied in the range of 0≈300 rpm. The maximum production of chitinase was carried at 200 rpm although the cell growth was the highest at 150 rpm. It indicates that oxygen adjustment is required for the maximum chitinase production. Using optimal conditions, batch cultures for comparingSerratia marcescens KY andSerratia marcescens ATCC 27117 were carried out in a 5 L fermentor. The oxygen consumption was increased with the increase of culture. Especially, at 120 h of cultureSerratia marcescens KY andSerratia marcescens ATCC 27117 produced 38.3 unit/mL, and 33.5 unit/mL, respectively.