• 한국생물공학회:학술대회논문집
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• 2005.10a
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• pp.644-648
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• 2005

Dextranase (${\alpha}$-1,6-D-glucan-6-glucanogydrolase:E.C. 3.2.1.11) catalyzes the hydrolysis of ${\alpha}$-(1.6) linkages of dextran. A lsd1 gene encoding an extracellular dextranase was isolated from the genomic DNA of L. starkeyi. The lsd11 gene is a synthetic dextranase (lsd1) after codon optimization for gene expression with Pichia pastoris system. A open reading frame of lsd11 gene was 1827 bp and it was inserted into the pPIC3.5K expression vector. The plasmid linearized by Sac I was integrated into the 5'AOX region of the chromosomal DNA of P. pastoris. The lsd11 gene fragment encoding a mature protein of 608 amino acids with a predicted molecular weight of 70 kDa, was expressed in the methylotrophic yeast P. pastoris by controling the alcohol oxidase-1 (AOX1) promoter. The recombinant lds11 was optimized by using the shake-flask expression and upscaled using fermentation technology. More than 9.8 mg/L of active dextranase was obtained after induction by methanol. The optimum pH of LSD11 was found to be 5.5 and the optimum temperature $28^{\circ}C$.

• The Journal of Korean Medicine
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• v.27 no.1 s.65
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• pp.91-103
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• 2006

Objectives : Etiological studies of diabetes and its complications showed that oxidative stress might play a major role. Therefore, many effects have been fried to regulate oxygen free radicals for treating diabetes and its complications. Because Holotrichia has been known to be effective for the treatment of diabetes, the methanol extract of Holotrichia was tested for its effectiveness in reducing the oxidative stress induced by streptozotocin. Methods : Holotrichia was washed, dried in the shade and crushed. The crushed Holotrichia was extracted 3 times, each time with 3 volumes of methyl alcohol at $60^{\circ}C$ for 24h. The extract was filtered and evaporated under a reduced pressure using a rotary evaporator to yield 17 g. Holotrichia extract was oral-administed to the diabetic rats induced by streptozotocin 50 mg per 1 kg of body weight for 20 days. The efficacy of the Holotrichia extract was examined with regard to the enzymatic pathways involved in the oxygen free radical production and the glutathione balance. Results : The Effects of the methanol extract of Holotrichia in streptozotocin-induced diabolic rats with regards to body weight, blood glucose level, hepatic lipid peroxide level, hepatic superoxide anion radical content. hepatic xanthine oxidase activity and type conversion rate, hepatic glutathione level, hepatic aldose reductase activity, and hepatic sorbitol dehydrogenase activity were shown to be good enough to cure and prevent the diabetes and its complications. Conclusions : These results indicated that Holotrichia might reduce the oxidative stress in the tissues and organs by regulating the production of oxygen free radicals. Especially, Holotrichia might prevent and cure the diabetes and its complications by reducing the oxidative stress in the ${\beta}$-cells of pancreas. Some suggestions on biophoton experiments were made.

• Biomedical Science Letters
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• v.9 no.3
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• pp.151-158
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• 2003

In the biological world, there are a number of ecological fights for survival between each organism such as plants, animals and microorganism In such events, an organism can use its natural bioactive products as defence agent against other organisms. Furthermore, natural bioactive products can be utilized for medicine or functional food. Recently, we investigate the effect of Monascus pigment extracted from a fungus, Monascus anke, on the alcohol metabolism and blood lipid profile. In the present study, it is observed that Monascus pigment supplemented dietary may have a hepatoprotective effect on rat's liver damage induced with $CCl_4$ . By treatment with $CCl_4$(3 times, I.P), liver damage was reduced more in the rats fed 2% Monascus pigment extract supplemented diet than those fed standard diet, based on the serum levels of alanine aminotransferase, microsomal glucose-6-phosphatse activity and hepaic malondialdehyde content. On the other hand, oxygen free radical generating enzymes, hepatic P-450 dependent aniline hydroxylase, xanthine oxidase, and oxygen free radical scavenging enzymes, hepatic glutathione S-transferase, catalase, superoxide dismutase activities were generally higher both in $CCl_4$ treated group and control fed 2% Monascus pigment extract supplemented diet than those fed standard diet. In conclusion, the rats fed 2% Monascus pigment extract supplemented diet showed more reduced liver damage than those fed standard diet, which may be due to the acceleration of oxygen free radical metabolism.

• Herbal Formula Science
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• v.10 no.2
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• pp.127-141
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• 2002

Objectives: Haeganjeon(HGJ) has been used for the treatment of liver disease in traditional medicine. The present study was carried out to evaluate the antioxidant and protective effects of HGJ extract on oxidative damage of hepatocytes by tert-butyl hydroperoxide(t-BHP). Methods: In the linoleic acid water-alcohol system, the levels of lipid peroxide(LPO) were determined by TBA method. The scavenging effect of HGJ on ${\alpha},{\alpha}-diphenyl-{\beta}-picrylhydrazyl$(DPPH) radical was determined according to the method of Hatano. In the Fenton system(ferrous ion reaction with hydrogen peroxide), the levels of hydroxyl radical induced LPO in rat liver homogenate were determined according to the method of TBA. Inhibitory effect of HGJ on superoxide generation was measured by xanthine-xanthine oxidase system. In order to evaluate antioxidative activity of HGJ in the liver cell, cultured normal rat liver cells(Ac2F) were prepared and incubated with or without HGJ. After 18hr, cells placed in DMEM medium without serum, and then incubated with 1mM tert-butyl hydroperoxide(t-BHP) for 2hrs. Viable cells were detected by MTT assay. Conclusions: In the linoleic acid autoxidation system, HGJ extract significantly inhibited the time course of the lipid peroxidation. These effects were similar to those of BHA HGJ extracts showed about 70% scavenging effect on DPPH radical. And HGJ extract inhibited the lipid peroxide formation in rat liver homogenate induced by hydroxyl radical derived from Fenton system. In addition, HGJ extract protected the cell death induced by t-BHP and significantly increased cell viability in the normal rat liver cell. These result indicated that HGJ extract might playa protective role against oxidative hepatic cell injury by means of free radical scavenger.

• YAKHAK HOEJI
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• v.37 no.6
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• pp.647-658
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• 1993

The susceptibilities of aldehyde dehydrogenase (AldDH) and alcohol dehydrogenase (ADH) to active oxygen generated by xanthine-xanthine oxidase (XOD) system were studied. Incubation of AldDH with 2$\times$10$^{-3}$ units of XOD for 30 min at $25^{\circ}C$ resulted in the decrease of enzyme activity to 30% and it was inactivated completely when incubated with 5$\times$10$^{-3}$ units of XOD. Whereas 70% of ADH activity was retained after exposure to 5$\times$10$^{-3}$ units of XOD for 30 min, 40% of ADH activity was retained after exposure to 5$\times$10$^{-2}$ unit of XOD for 30 min. This inhibition effect by the active oxygen was preventable by catalase and glutathione, but not by SOD. The rates of the NADPH-dependent oxygen consumption by the liver S-9 mixture and microsomes were also determined in this study. Rate of oxygen consumption is increased in the liver S-9 mix and microsomes from phenobarbital-treated rat, and it was consistent with increased lipid peroxidation. In the presense of ethanol as a substrate, the oxygen consumption rates were increased. It is reported that hepatic AldDH activity is depressed in alcoholic liver diseases, however there is few report that explains the reason of depressed AldDH activity. These results are supportive of the theory that the increase in hepatic ethanol oxidation through the induced ME activity after chronic ethanol feeding generate oxygen radical at elevated rates and it leads to the depression of AldDH activity.

• Journal of Oral Medicine and Pain
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• v.22 no.1
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• pp.65-80
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• 1997

Cytochrome P450 is an oxidase involved in oxidation of alcohol and is known to be an activator of carcinogen. The present study was perfomed to analyze the effect of diabetes and cold stress on the expression of Cytochrome P450 IIE1(CYPIIE1) in the liver and salivary glands in rats by an immunoblot analysis. Sixty three divided into 4 groups; 1) 20 rats belonging to group I were allowed diabetes (40mg/kg. I.V.) 2) 20 rats of group II were bathed in cold water for 30 seconds twice a day 3) 20 rats comprising group III were received diabetes and cold stress as described above 4) 3 rats of group IV were selected as a control. The rats were sacrificed at the end of the same day 1, 2, 3, 4 weeks experiment. The liver and parotid glands were removed and stored at $-70^{\circ}C$ until use. The stored organs were homogenized for 10 seconds and the supernatants were obtained by centrifugation. The proteins of the supernatants were separated by sodium dodecyl sulfate- polyacrylamide gel electrophoresis and subjected to Western blotting. The blotted membranes were incubated with polyclonal antibodies to CYPIIE1. And sepimens were observed with light microscope also under the Hematoxillin-Eosin staining. The obtained results were as follows : 1. In diabetes group, acini had changed to degeneration severely 1 week experiment, but repaired gradually in lapse of time. 2. In diabetes group, septal connective tissue had changed to degeneration little by little from 1 week after experiment, and progressed severely in lapse of time. 3. In stress group, acini had not changed remarkably, but slightly separated each other 3 weeks after experiment. 4. In diabetes and stress group, histological feature had changed remarkably campare with in the group of diabetes only. 5. In all experimental group, CTPIIE1 had expressed remarkably in the liver tissue, but not in the parotid gland tissues. 6. In diabetes and stress group, CYPIIE1 had expressed remarkably campare with in the group of diabetes only.

• 한국균학회소식:학술대회논문집
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• 2016.05a
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• pp.27-27
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• 2016

The ascomycete fungus Fusarium graminearum is the most common pathogen of Fusarium head blight (FHB), a devastating disease for major cereal crops worldwide. FHB causes significant crop losses by reducing grain yield and quality as well as contaminating cereals with trichothecenes and zearalenone (ZEA) that pose a serious threat to animal health and food safety. ZEA is a causative agent of hyperestrogenic syndrome in mammals and can result in reproductive disorders in farm animals. In F. graminearum, the ZEA biosynthetic cluster is composed of four genes, PKS4, PKS13, ZEB1, and ZEB2, which encode a reducing polyketide synthase, a nonreducing polyketide synthase, an isoamyl alcohol oxidase, and a transcription factor, respectively. Although it is known that ZEB2 primarily acts as a regulator of ZEA biosynthetic cluster genes, the mechanism underlying this regulation remains undetermined. In this study, two isoforms (ZEB2L and ZEB2S) from the ZEB2 gene in F. graminearum were characterized. It was revealed that ZEB2L contains a basic leucine zipper (bZIP) DNA-binding domain at the N-terminus, whereas ZEB2S is an N-terminally truncated form of ZEB2L that lacks the bZIP domain. Interestingly, ZEA triggered the induction of both ZEB2L and ZEB2S transcription. In ZEA producing condition, the expression of ZEB2S transcripts via alternative promoter usage was directly or indirectly initiated by ZEA. Physical interaction between ZEB2L and ZEB2L as well as between ZEB2L and ZEB2S was observed in the nucleus. The ZEB2S-ZEB2S interaction was detected in both the cytosol and the nucleus. ZEB2L-ZEB2L oligomers activated ZEA biosynthetic cluster genes, including ZEB2L. ZEB2S inhibited ZEB2L transcription by forming ZEB2L-ZEB2S heterodimers, which reduced the DNA-binding activity of ZEB2L. This study provides insight into the autoregulation of ZEB2 expression by alternative promoter usage and a feedback loop during ZEA production.

• Journal of the East Asian Society of Dietary Life
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• v.23 no.5
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• pp.645-653
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• 2013

The results of examining total soluble solid, reducing sugar, antioxidant and sensory properties regarding LTD (Liriope Tuber Dried), LTSD (Liriope Tuber Steamed and Dried), LTASD (Liriope Tuber Alcohol-Steamed and Dried), LTDR (LTD Roasted), LTSDR (LTSD Roasted) and LTASDR (LTASD Roasted) are as follows : Total soluble solid content of the roasted samples (LTDR, LTSDR and LTASDR) was more than those of all dried samples (LTD, LTSD and LTASD). According to roasting conditions, total sugar and reducing sugar are significantly greater than the raw and dried sample (LTD) in all heat-treated samples. The browning index was significantly higher in all roasted samples. In particular, LTASDR had a high browning index. Further, the antioxidative activity of the roasted LT samples were higher than that of all dried LT samples. In particular, the LTASDR sample showed significantly high figures in DPPH scavenging activity, ABTS scavenging activity, Nitrite scavenging activity and xanthine oxidase inhibitory activity. Sensory properties showed an increased acceptance in all evaluation items among roasted samples. In this study, hot water extracts of steamed or alcohol-steamed roasted LT samples had a higher antioxidative effect than that of LTD or LTDR and attained positive results by getting high scores in the overall sensory evaluation. Therefore, when using Liriope tuber in making beverages or herbal recipes, it is appropriate to dry and roast before steaming or spreading with alcohol when treating LT.

• The Korean Journal of Mycology
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• v.32 no.2
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• pp.112-118
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• 2004

Laccase produced by Trametes hirsuta S1 isolated from Korea was partially purified and characterized using ultrafiltration, anion exchange chromatography and affinity chromatography. The laccase was produced as the predominant extracellular enzyme during primary metabolism. Neither lignin peroxidase nor veratryl alcohol oxidase (VAO) were detected in the culture fluid. Addition of 2,5-xylidine enhanced 4-fold laccase production. Purified laccase was a single polypeptide having a molecular mass of approximately 66 kDa, as determined by SDS-polyacrylamide gel electrophoresis, and carbohydrate content of 12%. $K_{m}\;and\;V_{max}$ values for laccase with ABTS [2,2-azinobis (3-ethylbenzthiazoline 6-sulfonic acid)] as a substrate (Lineweaver-Burk plot) was determined to $51.2\;{\mu}M\;and\;56.8\;{\mu}mole$, respectively. The optimal pH for laccase activity was found to be 3.0. The enzyme was very stable for 1 hour at $50^{\circ}C$. Half-life ($t_{1/2}$) of the enzyme was about 20 min at $70^{\circ}C$. Spectroscopic analysis of purified enzyme indicated that the enzyme was typical of copper-containing protein. Substrate specificity and inhibitor studies for laccase also indicated to be a typical fungal laccase. The N-terminal amino acid sequence of the T. hirsuta S1 laccase showed 100% of homology to those of laccase from C. hirsutus.

• Journal of Microbiology and Biotechnology
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• v.11 no.6
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• pp.915-921
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• 2001

Phytase improves the bioavailability of phytate phosphorus in plant foods to humans and animals, and reduces the phosphorus pollution of animal waste. In order to express a high level of fungal phytase in Saccharomyces cerevisiae, various expression vectors were constructed with different combinations of promoters, translation enhancers, signal peptides, and terminator. Three different promoters fused to the phytase gene (phyA) from Aspergillus niger were tested: a galactokinase (GAL1) promoter, glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter, and yeast hybrid ADH2-GPD promoter consisting of alcohol dehydrogenase II (ADH2) and a GPD promoter. The signal peptides of phytase, glucose oxidase (GO), and rice amylase 1A(RAmy1A) were included. Plus, the translation enhancers of the ${\Omega}$ sequence and UTR70 from the tobacco mosaic virus (TMV) and spinach, respectively, were also tested. Among the recombinant vectors, pGphyA06 containing the GPD promoter, the ${\Omega}$ sequence, RAmy1A, and GAL7 terminator expressed the highest phytase activity in a culture filtrate, which was estimated at 20 IU/ml. An intracellular localization of the expressed phytase activity in a culture filtrate, which was estimated at 20 IU/ml. An intracellular localization of the expressed phytase was also performed by inserting an endoplasmic reticulum (ER) retention signal, KDEL sequence, into the C-terminus of the phytase within the vector pHphyA-6. It appeared that the KDEL sequence directed most of the early expression of phytase into the intracellular compartment yet more than $60\%$ of the total phytase activity was still retained within the cell even after the prolonged (>3 days) incubation of the transformant. However, the intracellular enzyme activity of the transformant without a KDEL sequence was as high as that of the extracellular one, thereby strongly suggesting that the secretion of phytase in S. cerevisiae appeared to be the rate-limiting step for the expression of a large amount of extracellular recombinant phytase, when compared with other yeasts.