• KSBB Journal
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• v.10 no.3
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• pp.271-282
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• 1995

The authentic hGH converted from met-hGH by immobilized ApM was purified by successive chromatographic processes based on the differences in isoelectric points, hydrophobicities and charges. The final recovery yield was about 14.1% and the specific activity of the purified hGH was 2.75IU per mg when assayed by enzyme immunoassay. The purified hGH was verified to be authentic hGH through the analysis of amino acid composition, amino-terminal amino acid sequence, carboxy-terminal amino acid and tryptic peptide map. The purity of purified hGH was higher than that of commercial hGH when assessed by SDS-PAGE, PAGE, IEF and HSGF. In weight-gain assay and tibia test with hypophysectomized rats, the hGH produced in this study showed the same growth effect as the commercial hGH.

• Journal of Microbiology and Biotechnology
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• v.25 no.8
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• pp.1281-1290
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• 2015

Thermolysin and its homologs are a group of metalloproteases that have been widely used in both therapeutic and biotechnological applications. We here report the identification and characterization of a novel thermolysin-like protease, BtsTLP1, from insect pathogen Bacillus thuringiensis serovar Sichuansis strain MC28. BtsTLP1 is extracellularly produced in Bacillus subtilis, and the active protein was purified via successive chromatographic steps. The mature form of BtsTLP1 has a molecule mass of 35.6 kDa as determined by mass spectrometry analyses. The biochemical characterization indicates that BtsTLP1 has an apparent K_m value of 1.57 mg/ml for azocasein and is active between 20℃ and 80℃. Unlike other reported neutral gram-positive thermolysin homologs with optimal pH around 7, BtsTLP1 exhibits an alkaline pH optimum around 10. The activity of BtsTLP1 is strongly inhibited by EDTA and a group of specific divalent ions, with Zn²⁺ and Cu²⁺ showing particular effects in promoting the enzyme autolysis. Furthermore, our data also indicate that BtsTLP1 has potential in cleaning applications.

• Journal of Microbiology and Biotechnology
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• v.22 no.7
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• pp.947-959
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• 2012

This study was aimed at an evaluation of the potential inheritance of electroporation effects on Lactobacillus fermentum BT 8219 through to three subsequent subcultures, based on their growth, isoflavone bioconversion activities, and probiotic properties, in biotin-supplemented soymilk. Electroporation was seen to cause cell death immediately after treatment, followed by higher growth than the control during fermentation in biotin-soymilk (P<0.05). This was associated with enhanced intracellular and extracellular ${\beta}$-glucosidase specific activity, leading to increased bioconversion of isoflavone glucosides to aglycones (P<0.05). The growing characteristics, enzyme, and isoflavone bioconversion activities of the first, second, and third subcultures of treated cells in biotin-soymilk were similar to the control (P>0.05). Electroporation affected the probiotic properties of parent L. fermentum BT 8219, by reducing its tolerance towards acid (pH 2) and bile, lowering its inhibitory activities against selected pathogens, and reducing its ability for adhesion, when compared with the control (P<0.05). The first, second, and third subcultures of the treated cells showed comparable traits with that of the control (P>0.05), with the exception of their bile tolerance ability, which was inherited to the treated cells of the first and second subcultures (P<0.05). Our results suggest that electroporation could be used to increase the bioactivity of biotin-soymilk via fermentation with probiotic L. fermentum BT 8219, with a view towards the development of functional foods.

• Mycobiology
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• v.44 no.4
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• pp.260-268
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• 2016

Regulation of alkaline-resistant laccase from Perenniporia tephropora KU-Alk4 was proved to be controlled by several factors. One important factor was the initial pH, which drove the fungus to produce different kinds of ligninolytic enzymes. P. tephropora KU-Alk4 could grow at pH 4.5, 7.0, and 8.0. The fungus produced laccase and MnP at pH 7.0, but only laccase at pH 8.0. The specific activity of laccase in the pH 8.0 culture was higher than that in the pH 7.0 culture. At pH 8.0, glucose was the best carbon source for laccase production but growth was better with lactose. Low concentrations of glucose at 0.1% to 1.0% enhanced laccase production, while concentrations over 1% gave contradictory results. Veratryl alcohol induced the production of laccase. A trace concentration of copper ions was required for laccase production. Biomass increased with an increasing rate of aeration of shaking flasks from 100 to 140 rpm; however, shaking at over 120 rpm decreased laccase quantity. Highest amount of laccase produced by KU-Alk4, 360 U/mL, was at pH 8.0 with 1% glucose and 0.2 mM copper sulfate, unshaken for the first 3 days, followed by addition of 0.85 mM veratryl alcohol and shaking at 120 rpm. The crude enzyme was significantly stable in alkaline pH 8.0~10.0 for 24 hr. After treating the pulp mill effluent with the KU-Alk4 system for 3 days, pH decreased from 9.6 to 6.8, with reduction of color and chemical oxygen demand at 83.2% and 81%, respectively. Laccase was detectable during the biotreatment process.

• Mycobiology
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• v.48 no.5
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• pp.383-391
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• 2020

Use of nanoparticles (NPs) in several commercial products has led to emergence of novel contaminants of air, soil and water bodies. The NPs may exhibit greater ecotoxicity due to nano-scale dependent properties over their bulk counterparts. The present investigation explores the effect of in vitro supplementation of TiO₂, silica and silver NPs on radial growth and ultrastructural changes in the hyphae and spores of two mushroom genera, Ganoderma lucidum and Volvariella volvaceae. A concentration dependent decrease in radial growth on NP amended potato dextrose agar medium was recorded. However, in comparison to control, there was decrease in radial diameter on supplementation with TiO₂ NPs while an increase was recorded for silica and silver NPs amendments as compared to their bulk salts at same concentrations after 48 h of incubation. Optical microscopy studies showed decrease in the number of spores while increase in spore diameter and thinning of hyphal diameter on NPs supplementation. Scanning electron microscopy analysis of fungal growth showed presence of deflated and oblong spores in two fruiting strains of Ganoderma while Volvariella exhibited decreased sporulation. Further, hyphal thinning and branching was recorded in response to NP amendments in both the test mushrooms. Enhancement of protein content was observed on NP compared to bulk supplementation for all cultures, concentrations and hours of incubation except for TiO₂ NPs. Likewise, bulk and NP supplementations (at 100 mg L^-1) resulted in enhanced laccase activity with occurrence of laccase specific protein bands on SDS-PAGE analysis.

• Journal of Ginseng Research
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• v.39 no.1
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• pp.14-21
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• 2015

Background: Colorectal cancer is a leading cause of cancer-related death, and inflammatory bowel disease is a risk factor for this malignancy. We previously reported colon cancer chemoprevention potential using American ginseng (AG) in a xenograft mice model. However, the nude mouse model is not a gut-specific colon carcinogenesis animal model. Methods: In this study, an experimental colitis and colitis-associated colorectal carcinogenesis mouse model, chemically induced by azoxymethane/dextran sodium sulfate (DSS) was established and the effects of oral AG were evaluated. The contents of representative ginseng saponins in the extract were determined. Results: AG significantly reduced experimental colitis measured by the disease activity index scores. This suppression of the experimental colitis was not only evident during DSS treatment, but also very obvious after the cessation of DSS, suggesting that the ginseng significantly promoted recovery from the colitis. Consistent with the anti-inflammation data, we showed that ginseng very significantly attenuated azoxymethane/DSS-induced colon carcinogenesis by reducing the colon tumor number and tumor load. The ginseng also effectively suppressed DSS-induced proinflammatory cytokines activation using an enzyme-linked immunosorbent assay array, in which 12 proinflammatory cytokine levels were assessed, and this effect was supported subsequently by real-time polymerase chain reaction data. Conclusion: AG, as a candidate of botanical-based colon cancer chemoprevention, should be further investigated for its potential clinical utility.

• Molecules and Cells
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• v.42 no.11
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• pp.783-793
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• 2019

When endoplasmic reticulum (ER) functions are perturbed, the ER induces several signaling pathways called unfolded protein response to reestablish ER homeostasis through three ER transmembrane proteins: inositol-requiring enzyme 1 (IRE1), PKR-like ER kinase (PERK), and activating transcription factor 6 (ATF6). Although it is important to measure the activity of ATF6 that can indicate the status of the ER, no specific cell-based reporter assay is currently available. Here, we report a new cell-based method for monitoring ER stress based on the cleavage of $ATF6{\alpha}$ by sequential actions of proteases at the Golgi apparatus during ER stress. A new expressing vector was constructed by using fusion gene of GAL4 DNA binding domain (GAL4DBD) and activation domain derived from herpes simplex virus VP16 protein (VP16AD) followed by a human $ATF6{\alpha}$ N-terminal deletion variant. During ER stress, the GAL4DBD-VP16AD(GV)-$hATF6{\alpha}$ deletion variant was cleaved to liberate active transcription activator encompassing GV-$hATF6{\alpha}$ fragment which could translocate into the nucleus. The translocated GV-$hATF6{\alpha}$ fragment strongly induced the expression of firefly luciferase in HeLa Luciferase Reporter cell line containing a stably integrated 5X GAL4 site-luciferase gene. The established double stable reporter cell line HLR-GV-$hATF6{\alpha}$(333) represents an innovative tool to investigate regulated intramembrane proteolysis of $ATF6{\alpha}$. It can substitute active pATF6(N) binding motif-based reporter cell lines.

• Journal of Life Science
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• v.23 no.2
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• pp.167-174
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• 2013

Peroxiredoxin 6 (Prx 6) is a member of the thiol-specific antioxidant protein family, which may play a role in protection against oxidative stress and in regulating phospholipid turnover. The aim of this study was to determine whether a human Prx 6/Luc vector was stably expressed and responded to antioxidants in a lung cell line (NCI-H460). To achieve this, the luciferase signal, hPrx 6 mRNA expression, and superoxide dismutase (SOD) activity were measured in transfectants with a hPrx 6/Luc plasmid after treatment with four antioxidant extracts, including Korea white ginseng (KWG), Korea red ginseng (KRG), Liriope platyphylla (LP), and red Liriope platyphylla (RLP). First, the hPrx 6/Luc plasmid was successfully constructed with DNA fragments of human Prx 6 promoter, amplified by PCR using genomic DNA isolated from NCI-H460 cells, and cloned into the pTransLucent reporter vector. The orientation and sequencing of the hPrx 6/Luc plasmid were identified with restriction enzyme and automatic sequencing. A luciferase assay revealed significant enhancement of luciferase activity in the four treatment groups compared with a vehicle-treated group, although the ratio of the increase was different within each group. The KRG- and LP-treated groups showed higher activity than the KWG- and RLP-treated groups. Furthermore, the luciferase activity against RLP occurred roughly in a dose-dependent manner. However, the level of endogenous hPrx 6 mRNA did not change in any group treated with the four extracts. The SOD activity was in agreement with the luciferase activity. Therefore, these results indicate that the hPrx 6/Luc vector system may successfully express and respond to antioxidant compounds in NCI-H460 cells. The data also suggest that the Prx 6/Luc vector system may be effectively applied in screening the response of hPrx 6 to antioxidant compounds in transgenic mice.

• Asian Journal of Turfgrass Science
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• v.25 no.1
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• pp.17-21
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• 2011

Korean bennudagrass collections showed diverse genetic variations in their morphology, growth habit, and cytological aspects. Chromosome number and nuclear DNA content of the bennudagrasses indicated a ploidy level ranging from triploid (2n=3x) to hexaploid (2n=6x). In this study, we investigated the different responses of antioxidant enzymes (superoxide dismutase, catalase, peroxidase, ascorbate peroxidase) and cell membrane stability of those bennudagrass cytotypes to lower temperature and shorter day length, which meets a dormant induction in Korea. All the antioxidant enzymes were found to be higher during dormant stage, while the heme-containing catalase which converts hydrogen peroxide ($H_2O_2$) to water and oxygen molecules was activated before dormant initiation in the three cytotypes except for hexaploid bennudagrass. The triploid and tetraploid which exhibited relatively finer leaves and a rapid establishment speed were found to show increased activities of superoxide dismutase and peroxidase enzyme. The malondialdehyde(MDA) which is a product of lipid peroxidation in the cell membrane damaged by the hydroxyl radical was increased in all cytotypes as temperature declined, and tri- and tetraploids which had more protective antioxidant enzymes demonstrated a significantly lower MDA production. Similarly electrolyte leakage was higher in penta- and hexaploidy, seemingly more damage to cell membrane when low temperature was implemented. Results indicated that antioxidant responses of different cytotypes were genetically specific, which needs to be investigated the relevance with the low temperature tolerance in the bermudagrass further at the molecular level.

• The Journal of the Korean bone and joint tumor society
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• v.7 no.2
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• pp.41-50
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• 2001

Purpose : To investigate biochemical markers for osteosarcoma, activities of deoxyribocuclease(DNase), ribonuclease(RNase), 5'-nucleotidase, alkaline phosphatase and amylase were determined in the osteosarcoma tissue and serum of patients with osteosarcoma. Also studied were DNase, RNase in osteosarcoma tissue, isolating the enzymes from the sarcoma tissue and investigating the sarcoma specific enzymes. Materials and Methods : The experimental tissue and serum were obtained from twelve patients with osteosarcoma. The control group were obtained from the normal healthy tissue of the same patients. The tissue were centrifugalized to obtain extracts. The extracts were analized for the estimation of nucleic acid, protein contents and enzyme activities. And then each enzymes were isolated and analized by DEAE-cellulose chromatography and estimated for activities. Result : Activities of acid DNase, RNase, 5'-nucleotidase and alkaline phosphatase were significantly increased in osteosarcoma tissue. Neutral RNase in osteosarcoma tissue was shown to bo highly active, exhibiting secretory form of RNase inhibitor associated with the RNase was also increased. In the serum of patients with osteosarcoma, RNase activity was significantly increased. DEAE-cellulose column chromatographical analysis revealed that acid DNase was isolated as a single enzyme and neutral RNase as five isozymes in osteosarcoma tissue. Conclusion : The results indicated that combination of these enzymes could be used as markers for osteosarcoma. The results indicated that acid DNase and neutral RNase might play a role in genesis of sarcoma and suppression of sarcoma.