• 한국생물공학회:학술대회논문집
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• 2002.04a
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• pp.457-460
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• 2002

In an effort to isolate novel strains expressing a thermostable esterase that hydrolyzed the rac-ketoprofen ethyl ester to ketoprofen in the stereospecific manner, we screened various soils and composts from broad ecological niches in which the activity was expected to be found. Three hundreds of microbial strains were tested to determine their ester-hydrolyzing activity by using an agar plate containing insoluble tributyrin as an indicative substrate, and then further screened by activity on the (R,S)-ketoprofen ethyl ester. Twenty-six strains were screened primarily at high growth and incubation temperature and further compared the ability to ethyl ester-hydrolyzing activity in terms of conversion yield and chiral specificity. Consequently, a strain JYl44 was isolated as a novel strain that produced a (R)-stereospecific esterase with high stability and systematically identified as a Bacillus stearothermophilus JY144. The enzyme indeed stables at a broad range of temperature, upto 65 $^{\circ}C$, and pH ranging from 6.0 to 10.0. The optimal temperature and pH for enzymatic conversion were 50 $^{\circ}C$ and 9.0, respectively. Based on the observations that resulted a poor cell growth, and enzyme expression in wild type strain, we further attempted the gene cloning into a general host Escherichia coli and determined its primary structure, concomitantly resulting a high level expression of the enzyme. The cloned gene had an open reading frame (250 amino acids) with a calculated molecular mass of 27.4 kDa, and its primary structure showed a relative high homology (45-52 %) to the esterases from Streptomyces and Bacillus strains. The recombinant whole cell enzyme could efficiently convert the rac-ketoprofen ethyl ester to (R)-ketoprofen, with optical purity of 99 % and yield of 49 %.

• Korean Journal of Microbiology
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• v.44 no.2
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• pp.116-121
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• 2008

In my search of microbial source of novel enzymes, a marine actinomycetes, A1460, producing haloperoxidase was isolated from macroalgae from south sea, Korea and studied for physiological and biochemical properties. The haloperoxidation reaction was followed by the bromination of phenol red in the presence of hydrogen peroxide and potassium bromide. The haloperoxidase was partially purified from the cell extract with $35\sim75%$ ammonium sulfate precipitation, High-Q anion exchange chromatography, gel filtration chromatography, hydroxyapetite chromatography and hydrophobic interaction chromatography to a yield of 42% and purification fold of 70. This enzyme showed relatively high heat stability without losing 50% of activity after 1 hr incubation at $60^{\circ}C$. The highest activity was found at $45^{\circ}C$, and the optimal pH was about pH 7, but higher stability was observed at pH 8. Azide and cyanide ion showed strong inhibition at less than 1 $\mu M$ level suggesting that the enzyme was Fe ion dependent haloperoxidase.

• Korean Journal of Environmental Agriculture
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• v.31 no.1
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• pp.68-74
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• 2012

BACKGROUND: Some microorganisms extant in nature have ability to suppress various plant pathogens, and also can promote plant growth. Thus microorganisms are such great source of antimicrobial agents to develop antagonistic microorganism production and eco-friendly crop management. We isolated the microorganisms in various eco-friendly formulations. The suppressive abilities against plant pathogens have been characterized in vitro level. METHODS AND RESULTS: The indigenous microorganisms have been isolated from Cooked rice, Black sugar, Rice Bran, and Red clay using dilution plating method. Population of bacteria and fungi were above 107 in the all formulations. We isolated and pure cultured the microorganisms based on morphological characteristics. Three major plant pathogens (Fusarium oxysporum, Rhizoctonia solani, Phytophthora capsici) have been used to select antagonistic microorganisms. Total 20 bacteria and 9 fungi showed the pathogen growth suppression ability in vitro condition. The selected microorganisms were identified by ITS sequence similarity. CONCLUSION: All tested eco-friendly formulations contained high-density of the microorganisms. Among the isolated microorganisms, Bacillus spp. and Streptomyces spp. showed the most effective antifungal activity against the plant pathogens such as F. oxysporum, R. solani, and P. capsici. Among the selected fungi Trichoderma sp. demonstrated antifungal activity. Our results suggest that the currently adapted eco-friendly formulations might useful for sustain agricultural system.

• Food Science of Animal Resources
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• v.28 no.5
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• pp.675-680
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• 2008

The physical quality and microbial safety of Korean beef jerky was evaluated at various steps during its preparation. Microbial counts in raw beef demonstrated mesophillic bacteria at 4.20 Log CFU/g, psychrotrophic bacteria at 3.85 Log CFU/g, anaerobic bacteria at 4.90 Log CFU/g, and yeast and molds at 1.92 Log CFU/g. Spore-forming bacteria and coliforms were not detected in raw beef samples. Spices and spiced meats showed similar trends in microbial counts, demonstrating minimal microbial contamination during these stages of preparation. The final beef jerky product exhibited counts of mesophillic bacteria at 1.15-1.66 Log CFU/g, psychrotrophic bacteria at 1.15-1.66 Log CFU/g, and anaerobic bacteria at 0.81-1.72 Log CFU/g. Spore-forming bacteria, yeast and molds, and coliforms were not detected in beef jerky. Significant differences from added ingredients occurred for instron textural profile analysis traits for hardness. In general, Korean beef jerky with humectant and tenderizer had lower hardness than control (without humectant and tenderizer). Also, the sample added with 0.01% protease from Streptomyces griseus had lower hardness than all samples. All samples had 0.7l to 0.72 water activities, and the color and pH were not shown in significant changes of all samples.

• KSBB Journal
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• v.22 no.4
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• pp.218-221
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• 2007

The polyene antifungal antibiotics, mostly produced by Gram-positive soil actinomycetes, are a family of type I polyketide macrolide ring compounds with 20$\sim$40 carbon backbone contain 3$\sim$8 conjugated double bonds. Using polyene-specific genomic screening strategy, we previously isolated three novel polyene-producing actinomycetes strains from soil, implying the potential application of these strains' spores as microbial pesticides. Here, we report that the sonication is a very efficient method for actinomycetes spore generation with a sonicator power-dependent manner. In addition, these sonication-driven actinomycetes spores retained significant portion of their cell viabilities as well as antifungal activities after freeze-drying procedure, implying the potential application of these strains' spores as microbial pesticides.

• Journal of Microbiology and Biotechnology
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• v.16 no.9
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• pp.1384-1391
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• 2006

The signal transduction system is one of the most important devices involved in maintaining life, and many protein kinases are included in the cellular signal transduction system. Finding a protein kinase inhibitor is very valuable, as it can be used to study cell biology and applied to pharmaceuticals. For the efficient and rapid screening of protein kinase inhibitors, two assay systems were combined; the nonradioactive protein kinase assay system that uses an FITC-labeled IRS-2 peptide and the cell-based paper disc assay system that uses Streptomyces griseus as the indicator strain. Among 330 kinds of herb extracts tested, the extract of Dalbergia odorifera exhibited the strongest inhibitory activity in the two assay systems and was selected for further isolation. Based on solvent extraction and many steps of chromatography, seven compounds were finally separated to homogeneity and their structures determined by $^{1}H$ and $^{13}C$ NMR spectroscopies. Four were to be flavonoids and identified as butin ($C_{15}H_{12}O_5$, Mw=272.07), 3'-hydroxymelanetin ($C_{16}H_{12}O_6$, Mw=300.06), liquiritigenin ($C_{15}H_{12}O_4$, Mw=256.07), and 2'-hydroxyformononetin ($C_{16}H_{12}O_{5}$, Mw=284.07). 3'-Hydroxymelanetin inhibited the phosphorylation of the GSK3 protein by Akt to 37% at a concentration of $10{\mu}g/ml$ and showed the strongest cytotoxicity ($ED_{50}<50{\mu}g/ml$) against the human cancer cell line HCT116. Under the same conditions, liquiritigenin also inhibited the phosphorylation of GSK3 by Akt to 26%, and its cytotoxicity against the HCT116 cell line was lower than $100{\mu}g/ml$.

• Journal of Microbiology and Biotechnology
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• v.21 no.9
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• pp.979-987
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• 2011

Culture pH change has some important roles in signal transduction and secondary metabolism. We have already reported that acidic pH shock enhanced actinorhodin production in Streptomyces coelicolor. Among many potential governing factors on pH variation, the putative $Na^+/H^+$ antiporter (sha) genes in S. coelicolor have been investigated in this study to elucidate the association of the sha on pH variation and secondary metabolism. Through the transcriptional analysis and overexpression experiments on 8 sha genes, we observed that most of the sha expressions were promoted by pH shock, and in the opposite way the pH changes and actinorhodin production were enhanced by the overexpression of each sha. We also confirmed that sha8 especially has a main role in maintaining cell viability and pH homeostasis through $Na^+$ extrusion, in salt effect experiment under the alkaline medium condition by deleting sha8. Moreover, this gene was observed to have a function of pH recovery after pH variation such as the pH shock, being able to cause the sporulation. However, actinorhodin production was not induced by the only pH recovery. The sha8 gene could confer on the host cell the ability to recover pH to the neutral level after pH variation like a pH drop. Sporulation was closely associated with this pH recovery caused by the action of sha8, whereas actinorhodin production was not due to such pH variation patterns alone.

• Journal of Microbiology and Biotechnology
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• v.22 no.3
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• pp.316-325
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• 2012

Xylanase has been used extensively in the industrial and agricultural fields. However, the low-yield production of xylanase from native species cannot meet the increasing demand of the market. Therefore, improving the heterologous expression of xylanase through basic gene optimization may help to overcome the shortage. In this study, we synthesized a high-GC-content native sequence of the thermostable xylanase gene xynB from Streptomyces olivaceoviridis A1 and, also designed a slightly AT-biased sequence with codons completely optimized to be favorable to Pichia pastoris. The comparison of the sequences' expression efficiencies in P. pastoris X33 was determined through the detection of single-copy-number integrants, which were quantified using qPCR. Surprisingly, the high GC content did not appear to be detrimental to the heterologous expression of xynB in yeast, whereas the optimized sequence, with its extremely skewed codon usage, exhibited more abundant accumulation of synthesized recombinant proteins in the yeast cell, but an approximately 30% reduction of the secretion level, deduced from the enzymatic activity assay. In this study, we developed a more accurate method for comparing the expression levels of individual yeast transformants. Moreover, our results provide a practical example for further investigation of what constitutes a rational design strategy for a heterologously expressed and secreted protein.

• Korean Journal of Food Science and Technology
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• v.11 no.3
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• pp.192-199
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• 1979

With cells of Streptomyces spp K-45 isolated from soil, the immobilization of glucose isomerase by a series of treatments ; heat, carefully manipulated drying, extrusion with a thickening agent, and glutaraldehyde-induced crosslinking, was presented. This was aimed to obtain a mechanically stable form of whole cell containing glucose isomerase. The resulted pellet form had a good mechanical strength, compared with a commercial product, and showed 26 % of the activity recovery. The specific activity was 48.1 units per g of the dry material. The immobilized glucose isomerase generally showed properties similar to those of the soluble enzyme ; optimal pH at $7.5{\sim}9.0$, optimal temperature at $80{\sim}85^{\circ}C$, activation energy of 10.9 kcal/mole, and $K_m$ for glucose of 10.9M. The immobilized enzyme was very thermostable and pH stable.

• Applied Microscopy
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• v.28 no.2
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• pp.225-236
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• 1998

Adriamycin is a one of anthracyclin antibiotics isolated from the culture media of Streptomyces peucetius var casius. The formation of reactive oxygen metabolite by redox cycling during the metabolism and the inhibition of DNA synthesis results in antineoplastic effects of adriamycin. The authors have demonstrated the effects of SOD(superoxide dismutase) or DMTU (dimethyl thiourea), which are used as an antioxidant, on the ultrastructural changes of the gastric chief cells after the administration of adriamycin in the rat. Adriamycin (30 mg/kg) was administered intraperitoneally to the Sprague-Dawley rats weighing about 220 gm and SOD (15000 unit/kg) or DMTU (500 mg/kg) were administered intraperitoneally to the rats 30 minutes after the administration of adriamycin. The gastric chief cells 24, 48 and 72 hours after the administration of adriamycin were observed with Hitachi-600 electron microscope. The results were as follows. 1. SOD or DMTU alone did not affect the ultra structures of the gastric chief cells in the rat. 2. Dilation, sacculation and segmentation of the cisternae of rough endoplasmic reticulum, dilation of the saccules of Golgi complex and dilated mitochondria with electron lucent matrix were seen in the adriamycin treated rats. In the course of time, the ultrastructures of the chief cell changed markedly. 72 hours after drug administration, severely dilated cisternae of rough endoplasmic reticulum, with clumping of chromatin around the nuclear envelope and mitochondria with electron lucent matrix and dilated cristae were seen in the chief cell. 3. The treatment of SOD is more effective than DMTU to attenuated the ultrastructural changes of the chief cells in the adriamycin administered rat. Consequently it is suggested that adriamycin would induce the degenerative changes of the organelles of the chief cell. The treatment of SOD is more effective than DMTU to attenuate the adriamycin induced damage.