J_Environmetal Microbiology and Engineering

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Characterization of Four Soil Metagenome-Derived Lipolytic Enzymes Responsible for Hydrolysis of Autoinducer 3-Hydroxypalmitic Acid Methyl Ester of Ralstonia solanacearum. Myung Hwan LEE 1 , Weixin TAO 2 , Nam Hee KIM 1 , Eul Chul HWANG 1 , Jae Wook LEE 3 and Seon-Woo LEE* 1,2 1 Department of Applied Biology, Dong-A University, Busan 604-714, Republic of Korea. 2 Department of Medical Bioscience, Dong-A University, Busan 604-714, Republic of Korea. 3 J-52 Department of Chemistry, Dong-A University, Busan 604-714, Republic of Korea. *Corresponding author: seonlee@dau.ac.kr An autoinducer 3-hydroxypalmitic acid methyl ester (3-OH PAME) an unique quorum sensing signal molecule that regulates expression of virulence factors such as in phytopathogen Ralstonia solanacearum. Previously, we selected 4 metagenomic clones hydrolyzing 3-OH PAME among 142 142 lipolytic clones by using gas chromatography. All four clones were obtained from industrial waste contaminated soil metagenome and designated LP96, ULP104, ULP86 and UDL33. Lipolytic gene from LP96B, ULP104 and UDL33BB was over-expressed after cloning in pET-30b(+) in Escherichia coli BL21(DE3). The over-expressed, purified LP96, LP104 and Est33 exhibited hydrolysis activity toward 3-OH PAME. For determining of role of 3-OH PAME hydrolase in quorum sensing system of R. solanacearum GMI1000, pRKelp104 as pRK415 carrying lp104 gene was transformed into R. solanacearum GMI1000. Growth rate and exopolysaccharides (EPS) production of R. solanacearum GMI1000 carrying pRKelp104 was slower and reducer than R. solanacearum GMI1000. These results showed that novel beta-hydroxypalmitic acid methyl ester hydrolases were discovered from industrial waste contaminated soil metagenome. The enzymes will interfere virulence factors expression in R. solanacearum by hydrolysis of autoinducer 3-OH PAME. Keywords: Metagenome, 3-OH PAME, beta-hydroxypalmitic acid methyl ester hydrolase Acute and Chronic Toxicity Assessment for Hazardous Effect of Caffeine to Daphnia magna Nam Hui HONG 2 , Thai-Hoang LE 2 , Sung-Kyu LEE 3 and Jiho MIN* 1 1 Graduate School of Semiconductor and Chemical Engineering, Chonbuk National University, 567, Baekje-Daero, Duckjin-gu, Jeonju, Jeonbuk, 561-756, KOREA. 2 Department of Bioprocess Engineering, Chonbuk National University, 567, Baekje-Daero, Duckjin-gu, Jeonju, Jeonbuk, 561-756, KOREA. 3 J-53 Toxicology Research Center, Korea Research Institute of Chemical Technology, Daejon, 305-600, KOREA. *Corresponding author: hongnh@jbnu.co.kr Caffeine has been detected in water resulting in the chronic exposure of aquatic organisms. Because of organism exposed to hazardousness, aquatic ecosystems confusion is coming, and even it affect human. For this study, the impact was examined through the acute(48h) and chronic toxicity(21d) assay using Daphnia magna as animal model. In the acute toxicity test, 10 Daphnia neonates were exposed to several concentration of caffeine from 125㎍/l to 4000㎍/l. The lethal concentration with 50% mortability (LC50) was determined as about 2225㎍/l. In the chronic toxicity test, several small concentrations of caffeine such as 1%, 2%, 10%, 20% LC50 were used to exposed organism for 21days. We found hazardous effect of caffeine on aquatic ecosystem. Although toxicity of caffeine was known to be week, we found that survival of Daphnia neonates was significantly decreased caused by toxicity of cafferine. Based on this result, it was found that aquatic ecosystems exposed by pharmaceutics can not be ignored. Keywords: Caffeine, Daphnia magna, Acute toxicity, Chronic toxicity. 2011 International Symposium & Annual Meeting Natural Adaptation of Eubacterium limosum KIST612 for Biofuel Production Shinyoung PARK 1 , Daehee KIM 1 , Sunju CHOI 1 , In Geol CHOI 2 and In Seop CHANG* 1 1 School of Environmental Science and Engineering, Gwangju Institute of Science and Technology (GIST), 261 Cheomdan-gwagiro, Buk-gu, Gwangju 500-712, Korea. 2 J-54 College of Life Sciences and Biotechnology, Korea University, Anam-Dong, Seongbuk-Gu, Seoul,136-713, Korea. *Corresponding author: ischang@gist.ac.kr The production of chemicals by microbial reaction has become an increasingly alternative to current petroleum-based chemical synthesis. Eubacterium limosum KIST612 has been the organism of choice for the biochemicals production form hydrogen or carbon monoxide. E. limosum KIST612 produces (1) organic acids - acetic and butyric acids, (2) solventethanol and (3) gas- carbon dioxide (CO2). Among them, acetate and butyrate are major products. We hypothesized that constant transfer in certain medium condition this microbe may try to adapt to the given environmental condition. We hypothesized that if an excess of acetate or butyrate exist with CO as a main electron source, surplus electron may be utilized for production of more reduced products.Therefore, the hypothesis of this experiment is that excessive acetate and butyrate addition into the medium could change metabolic flux. We have tested the effect of acetate or butyrate addition for longer than 1 year with CO as a main energy source. We found significant increase of solvents, ethanol and butanol production after 1 year adaptation. It was also clearly observed that acetate could be converted to more reduced products in the presence of excess CO. Keywords: Acetogen, Eubacterium, Adaptation J-55 Mortality of Moth Larvae by Various Entomopathogenic Fungi Young-Hoon LIM, Eun-Soo GO and Keun KIM* Dept. of Bioscience and Biotechnology, The University of Suwon, Gyeonggi-do 445-743, Korea. *Corresponding author: limyounghoon@korea.com To determine the virulence of the various fungi, 10 8 conidia/㎖ suspension of each fungal strain was sprayed onto 2rd instar of larvae on a dampened filter paper in a petri-dish and the petri-dish was incubated at 25℃ for 15 days. The mortality by the different fungal strains varied from 20% to 100%. Among various fungi tested, 4 strains showed relativity high mortality against larvae. The mortality of the larvae increased and the 50% lethal time(LT50) decreased significantly as the applied conidial concentration was raised. LT50 values of strain No. 198, 216, 218, and 500 against larvae were 12.7, 9.5, 8.4, and 9.3 days, respectively. Mean lethal concentration(LC50) values of strain No. 198, 216, 218 and 500 against the larvae were 3.62 × 10 7 , 9.94 × 10 6 , 6.97 × 10 4 , and 3.01 × 10 5 conidia/㎖, respectively. Keywords: Entomopathogenic Fungi, Mortality, Moth Larvae The Korean Society for Microbiology and Biotechnology 409
J-56 Development of Baeyer-Villiger Monooxygenases Screening Protocols Using High-throughput screening (HTS) system Gowoun LEE, Yunjon HAN, Jae Jun SONG and Jong Hyun CHOI* Enzyme-based Fusion Technology Research Team, Microbe-based Fusion Technology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 1404 Sinjeong-dong, Jeongeup, Jeonbuk 580-185, Korea. *Corresponding author: jhchoi@kribb.re.kr Baeyer-Villiger Monooxygenases (BVMOs) are flavoenzymes that catalyze the insertion of an oxygen atom in a C-C bond using NADPH and allow the conversion of (cyclic)-ketones into lactones or esters. For the development of BVMO screening protocol, we used fluorescence based substrate and robot based HTS system. Optimized BVMO screening protocol for high throughput screening system led to 6,000 clone active verification per a day was available. As result, we screened the new type of BVMO positive clone which is named as “GomsoMF” from "Gomso" metagenomic library. This “GomsoMF” was novel BVMO like enzyme can convert cyclohexanone to ε-caprolactone. Also this enzyme was convert cyclopentanone and cycloheptanone into δ-valerolactone and 7-heptanolactone respectively. This new BVMO will be used for the synthesis of useful biochemicals or the biotechnological application. (This work was supported by a high throughput screening (HTS)-based Integrated Technology Development grant from the MEST, and a basic research grant from KRIBB.) Keywords: Baeyer-Villiger Monooxygenases (BVMOs), high throughput screening J-57 The Effect of Gamma Irradiation on Poliovirus, a Model for Norovirus inoculated in Oyster (Crassostrea Gigas) and Effect of Culture Condition Jong-Il CHOI, Eu-Ri JO, Pil-Mun JUNG, Jae-Hun KIM, Beom-Suk SONG, Jae-Kyung KIM, Jong-Heum PARK and Ju-Woon LEE* Team for Radiation Food Science and Biotechnology,Advanced Radiation Technology Institute, Korea Atomic Energy Research Institute, Jeongeup 580-185. *Corresponding author: sjwlee@kaeri.re.kr Poliovirus is a recognized surrogate for norovirus, pathogen in water and food, due to the structural and genetic similarity. Although radiation sensitivity of poliovirus in water or media had been reported, there has been no research in food model such as shellfish. In this study, oyster (Crassostrea gigas) was incubated in artificial seawater contaminated with poliovirus, and thus radiation sensitivity of poliovirus was determined in inoculated oyster. The effects of ionizing radiation on the sensitivity of poliovirus were also evaluated under different conditions such as pH (4-7) and salt concentration (1-15%) in culture broth, and temperature during irradiation. The D10 value of poliovirus in PBS buffer, virus culture broth and oyster was determined to 0.46, 2.84 and 2.94 kGy, respectively. The initial plaque forming unit (PFU) of poliovirus in culture broth was slightly decreased as the decrease of pH and the increase of salt concentration, but radiation sensitivity was not affected by pH and salt contents. However, radiation resistance of poliovirus was increased at frozen state. These results provide the basic information for the inactivation of pathogenic virus in foods by using irradiation. Keywords: Radiation sensitivity, Poliovirus, Oyster 410 www.kormb.or.kr Application of Bacillus subtilis 168 to Improve Durability of Cement Sung-jin PARK 1 , Jong-Myong PARK 1 , Wha-Jung KIM 2 and Sa-Youl GHIM* 1 J-58 1 Dep. of Microbiology, Kyungpook National University, Daegu 702-701, Korea. 2 Dept. of Architecture Engineering, Kyungpook National University, Daegu 702-701, Korea. *Corresponding author: ghimsa@knu.ac.kr Calcium carbonate precipitation, a widespread phenomenon among bacteria, has been investigated due to its wide range of scientific and technological application in concrete remediation. In this study, Bacillus subtilis 168 which could induce calcium carbonate precipitation, was used to coat on the cement surface. CaCO3 crystal aggregates and organic matrix molecules of these strains were deposited on the surface of the cement paste. To verify the effect of coating on the surface of the cement paste, water permeability of the paste was measured by water immersion test. In the results, B. subtilis 168 addition showed lower water permeability than those treated with B4 medium only, suggesting that the precipitated calcium induced by B. subtilis 168 could be used as a sealing agent to block water penetration. Also, the calcium precipitation on the surface of cement mortar could increase the compressive strength of the mortar. The compressive strength test showed that the cement mortar treated with B. subtilis 168 showed an improvement of compressive strength. Keywords: Calcium carbonate precipitation, Bacillus subtilis 168, cement J-59 Screening Enzyme from Single cell Based Polymerase Fosmid Cloning Yunjon HAN, Kyong-Cheol KO, Jong Hyun CHOI and Jae Jun SONG* Enzyme-based Fusion Technology Research Team, Microbe-based Fusion Technology Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), 1404 Sinjeong-dong, Jeongeup, Jeonbuk 580-185, Korea. *Corresponding author: jjsong@kribb.re.kr "Metagenomics," which is the genomic analysis of the microbial community attempts to overcome culturability of various microbes. In general, the diversity of metagenomic libraries was affected by abundance of bacteria in environmental sample. Therefore the research of minor microbial texa in environmental samples remains the biggest challenge. A new method was developed for enrichment minor bacteria from environmental samples. This method is based on the Fluorescence in situ hybridization(FISH), Fluorescence associated cell sorter(FACS) and Multiple displacement amplification(MDA). We demonstrated enrichment minor bacteria from artificial microbial community and single cell based MDA followed by fosmid library construction for enzyme screening. The Jeongupia naejangsanesis in which the cellulolytic activity is known was used in the minor microorganism of the artificial microbial community. As a result, we obtained MDA products from single bacterial cell. And we could generate fosmid library having an average insert size of 32 kb. Among this fosmid library we screened two new cellulose genes. (This work was supported by a high throughput screening (HTS)-based Integrated Technology Development grant from the MEST, and a basic research grant from KRIBB.) Keywords: Single cell, Multiple displacement amplification, Screening enzyme
Page 1 and 2: Isolation and Characterization of L
Page 3 and 4: Characterization of Phosphate-Solub
Page 5 and 6: Thermodynamic and Kinetic Analyses
Page 7 and 8: Identification of Novel Sugar Pathw
Page 9 and 10: J-32 Enhancement of Biohydrogen Pro
Page 11 and 12: Bioelectrochemical Fixation of CO2
Page 13: Inhibition of Quorum Sensing-Contro

J_Environmetal Microbiology and Engineering

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