Background : The purpose of the present investigation is to enhance extracellular acidic protease production by subjecting a protease producing strain Cordyceps pruinosa DK-01 to random mutagenesis by UV irradiation after ethidium bromide treatment. Methods and Results : Mutants were screened as protease producers on the basis of zone of clearance and relative proteolytic activity (RPA) on skimmed milk agar plates. In addition, mutants showed strong pink-red color intensity and different RAPD profiling compared with wild type control. Four mutants were randomly selected and their extracellular enzyme activities were investigated. In liquid culture without casein, 2.2-, 2.9-, 5.2- and 4.4-fold higher acid protease activity was achieved from mutants DK-m9, -m11 and -m12, respectively, than that of wild type strain (11.13 ± 1.60 U/ml). In liquid culture with casein, 1.1-, 1.3-, 1.3 and 1.3-fold higher acid protease activity was achieved with those mutants were found to produce, respectively, than that of wild type strain (93.95 ± 12.84 U/ml). Maximum acid protease activity was noticed from a mutant DK-m11 in liquid culture with casein (121.18 U/ml) and without casein (57.65 U/ml). The extracellular acid protease produced from DJ-m11 that was active in the pH range 4.5-6.5 and optimum temperature for the activity was 37°C. Furthermore, we found a deformed, shorten structure of setae on the elytron surface of dynastid beetles treated with culture supernatant of the DK-m11. Conclusion : These findings have more impact on enzyme economy for biotechnological and insecticidal applications of fungal proteases.

Background : With the increasing demand of the mistletoe in larger quantities for cancer therapy, it has been depleted from its natural habitat in the Far East countries including Korea because of overharvesting for high-value products (e.g., lectins and viscotoxins). The rapid multiplication of mistletoe by tissue culture can help this problem and provide the benefits in the phamaceutical industry. Methods and Results : Mistletoe plants growing on oak trees were collected and their leaf and stem segments were inoculated on MS basal medium supplemented with various concentrations of growth regulators. Calli were induced only from stem explants on MS medium containing 2,4-D and transferred onto MS medium supplemented with various concentrations of 2,4-D, NAA and BAP, respectively, for their propagation. The best callus multiplication rate of more than 15 folds (759 mg) was obtained in treatment of 2,4-D (4 mg/L) that produced yellowish-green, white and friable callus on this medium. To compare biochemical characterization, lectins were partially purified from natural mistletoe plants (nML) and in vitro cultured mistletoe calli (cML), respectively. The former was purified by lactose-agarose affinity chromatography and the latter was done by ammonium sulfate precipitation followed Sephadex G-25 chromatography. Both nML and cML showed similar molecular weight on SDS-PAGE and Western blot analyses. In addition, they showed similar carbohydrate-binding specificities and hemagglutination activities. Conclusion : From the above results, we may suggest that nML and cML showed the similarity in biochemical characters.

Background : Lectins were individually isolated from natural Korean mistletoe (nML) and its in vitro cultured calli (cML). Both of the lectins showed the difference in bioactivities such as cytotoxicity and cytokine induction. Methods and Results : Target cells (1 x 104 cells/well) were seeded independently into each well of a 96-well culture plate and incubated with different concentrations of each lectin. Survivability of target cells was determined by CCK-8 kit (Sigma, USA) according to manufacturer’s directions. The nML showed 46, 34 and 5.5 times stronger than cML in cytotoxicity (IC50) to human melanoma cell line (SK-MEL-28), human carcinoma cell line (NCI-H1650) and murine macrophage (RAW 264.7), respectively. In addition, respective lectins directly stimulated macrophage RAW 264.7 but they showed the difference in enhanced productivity of some inflammatory cytokines. Compared with cML, the nML induced both TNF-α and IL-1β at its low concentration. Administration of two kinds of lectins (10-1000 μ g/kg body weight) to ICR mouse did not show any significant changes on the level of alanine transaminase (ALT), glutamate-oxaloacetic transaminase (GOT) and blood urea nitrogen (BUN) in sera. Conclusion : From the above results, we may suggest that nML and cML showed differences in cytotoxic effects and cytokine production due to the difference in amounts from sources.