• Title/Summary/Keyword: Metagenome

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Characterization of a Lichenase Isolated from Soil Metagenome

  • Kim, Sang-Yoon;Oh, Doo-Byoung;Kwon, Ohsuk
    • Journal of Microbiology and Biotechnology
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    • v.24 no.12
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    • pp.1699-1706
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    • 2014
  • A lichenase gene (mt-lic) was identified for the first time through function-based screening of a soil metagenomic library. Its deduced amino acid sequence exhibited a high degree of homology with endo-${\beta}$-1,3-1,4-glucanase (having both lichenase and chitosanase activities), encoded by the bgc gene of Bacillus circulans WL-12. The recombinant lichenase overexpressed and purified from Escherichia coli was able to efficiently hydrolyze both barley ${\beta}$-glucan and lichenan. The enzyme showed maximal activity at a pH of 6.0 at $50^{\circ}C$, with Azo-barley-glucan as the substrate. The metal ions $Mn^{2+}$, $Mg^{2+}$, $Ca^{2+}$, and $Fe^{2+}$ enhanced the enzymatic activity, whereas the $Cu^{2+}$ and $Zn^{2+}$ ions inhibited the enzymatic activity. The $K_m$ and $V_{max}$ values of the purified lichenase were determined to be 0.45 mg/ml and 24.83 U/min/mg of protein, respectively.

Development of an Efficient Procedure for the Construction of Metagenomic Library from Environment Samples (효율적인 Metagenomic Library의 제작 방법 탐구)

  • Lim Dongbin
    • Korean Journal of Microbiology
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    • v.40 no.4
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    • pp.359-363
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    • 2004
  • I investigated an effective way to generate a metagenomic library from DNA prepared from environental samples. The sizes of DNA extracted from environmental samples were usually in the range of 10 to 100 kbp as estimated from $0.4\%$ agarose gel electrophoresis. Because of this small size, a fosmid, rather than BAC, was chosen as a vector. It was found that, for the successful generation of metagenomic library, the selection of DNA with the sized of about 40 kbp was critical and, therefore, a simple agarose gel electrophoresis system was developed to select this size of DNA. By the procedure described in this report, I obtained metagenomic libraries containing 25,000 fosmid clones, which corresponded to 1,000 Mb of metagenomic DNA.

Cloning and Characterization of a Novel ${\alpha}$-Amylase from a Fecal Microbial Metagenome

  • Xu, Bo;Yang, Fuya;Xiong, Caiyun;Li, Junjun;Tang, Xianghua;Zhou, Junpei;Xie, Zhenrong;Ding, Junmei;Yang, Yunjuan;Huang, Zunxi
    • Journal of Microbiology and Biotechnology
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    • v.24 no.4
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    • pp.447-452
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    • 2014
  • To isolate novel and useful microbial enzymes from uncultured gastrointestinal microorganisms, a fecal microbial metagenomic library of the pygmy loris was constructed. The library was screened for amylolytic activity, and 8 of 50,000 recombinant clones showed amylolytic activity. Subcloning and sequence analysis of a positive clone led to the identification a novel gene (amyPL) coding for ${\alpha}$-amylase. AmyPL was expressed in Escherichia coli BL21 (DE3) and the purified AmyPL was enzymatically characterized. This study is the first to report the molecular and biochemical characterization of a novel ${\alpha}$-amylase from a gastrointestinal metagenomic library.

Cloning and Characterization of Cellulase Gene (cel5B) from Cow Rumen Metagenome

  • Kang, Tae-Ho;Kim, Min-Keun;Barman, Dhirendra Nath;Kim, Jung-Ho;Kim, Hoon;Yun, Han-Dae
    • Journal of agriculture & life science
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    • v.46 no.2
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    • pp.129-137
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    • 2012
  • A carboxymethyl cellulase gene, cel5B, was cloned, sequenced, and expressed in Escherichia coli. pRCS20 in E. coli was identified from metagenomic cosmid library of cow rumen for cellulase activity on a carboxymethyl cellulose agar plates. Cosmid clone (RCS20) was partially digested with Sau3AI, ligated into BamHI site of pBluescript II SK+ vector, and transformed into E. coli $DH5{\alpha}$. The insert DNA of 1.3 kb was obtained, designated cel5B, which has the activity of hydrolyzation of CMC. The cel5B gene had an open reading frame (ORF) of 1,059 bp encoding 352 amino acids with a signal peptide of 48 amino acids and the conserved region, VIYEIYNEPL, belongs to the glycosyl hydrolase family 5. The molecular mass of Cel5B protein expressed from E. coli $DH5{\alpha}$ exhibited to be about 34 kDa by CMC-SDS-PAGE. The optimal pH was 8.0, and the optimal temperature was about $50^{\circ}C$ for its enzymatic activity.

Investigation of Conserved Regions in Lipase Genes (Lipase 유전자의 보존적 영역 탐색)

  • 이동근;김철민;김상진;이상현;이재화
    • Journal of Life Science
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    • v.13 no.5
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    • pp.723-731
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    • 2003
  • For the investigation of conserved regions in lipase genes, 132 and 24 sequences were obtained from LED (Lipase Engineering Database) and COG (Clusters of Orthologous Groups of proteins), respectively. There was high diversity in lipase genes and peculiar amino acid sequences were conserved for each homologous family of LED. Similar conserved amino acid sequences were detected from COG0657 and Moraxella lipase 1 homologous group of LED. Although many studies have attempted to detect new lipase genes in procaryotes, they have been limited culturable bacteria. The importance of metagenome, including DNA from non-culturable bacteria, is known. Due to the high diversity, we assumed it might be possible to detect new lipase gene from metagenome. Due to the high diversity of nucleotide sequences in lipase genes, 10 primer sets were designed. Designed primer sets were inspected in BLAST of NCBI and they could amplify a part of the lipase gene from 222 to 713 bp. They can amplify 16.7%∼60.0% of each lipase homologous group which was 3.6 fold higher than each sets. They might offer a high probability of detecting new lipase genes, owing to high efficiency and the diversity of lipase genes.

Optimization of DNA Extraction and PCR Conditions for Fungal Metagenome Analysis of Atmospheric Particulate Matter (대기 입자상물질 시료의 곰팡이 메타게놈 분석을 위한 DNA 추출 및 PCR 조건 최적화)

  • Sookyung Kang;Kyung-Suk Cho
    • Microbiology and Biotechnology Letters
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    • v.51 no.1
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    • pp.99-108
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    • 2023
  • Several challenges arise in DNA extraction and gene amplification for airborne fungal metagenome analysis from a particulate matter (PM) samples. In this study, various conditions were tested to optimize the DNA extraction method from PM samples and polymerase chain reaction (PCR) conditions with primer set and annealing temperature. As a result of comparative evaluation of DNA extraction under various conditions, chemical cell lysis using buffer and proteinase K for 20 minutes and bead beating treatment were followed by using a commercial DNA extraction kit to efficiently extract DNA from the PM filter samples. To optimize the PCR conditions, PCR was performed using 10 primer sets for amplifying the ITS2 gene region. The concentration of the PCR amplicon was relatively high when the annealing temperature was 58℃ with the ITS3tagmix3/ITS4 primer set. Even under these conditions, when the concentration of the PCR product was low, nested PCR was performed using the primary PCR amplicon as the template DNA to amplify the ITS2 gene at a satisfactory concentration. Using the methods optimized in this study, DNA extraction and PCR were performed on 15 filter samples that collected PM2.5 in Seoul, and the ITS2 gene was successfully amplified in all samples. The optimized methods can be used for research on analyzing and interpreting the fungal metagenome of atmospheric PM samples.

Exploring the Microbial Community and Functional Characteristics of the Livestock Feces Using the Whole Metagenome Shotgun Sequencing

  • Hyeri Kim;Eun Sol Kim;Jin Ho Cho;Minho Song;Jae Hyoung Cho;Sheena Kim;Gi Beom Keum;Jinok Kwak;Hyunok Doo;Sriniwas Pandey;Seung-Hwan Park;Ju Huck Lee;Hyunjung Jung;Tai Young Hur;Jae-Kyung Kim;Kwang Kyo Oh;Hyeun Bum Kim;Ju-Hoon Lee
    • Journal of Microbiology and Biotechnology
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    • v.33 no.1
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    • pp.51-60
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    • 2023
  • The foodborne illness is the important public health concerns, and the livestock feces are known to be one of the major reservoirs of foodborne pathogens. Also, it was reported that 45.5% of foodborne illness outbreaks have been associated with the animal products contaminated with the livestock feces. In addition, it has been known that the persistence of a pathogens depends on many potential virulent factors including the various virulent genes. Therefore, the first step to understanding the public health risk of livestock feces is to identify and describe microbial communities and potential virulent genes that contribute to bacterial pathogenicity. We used the whole metagenome shotgun sequencing to evaluate the prevalence of foodborne pathogens and to characterize the virulence associated genes in pig and chicken feces. Our data showed that the relative abundance of potential foodborne pathogens, such as Bacillus cereus was higher in chickens than pigs at the species level while the relative abundance of foodborne pathogens including Campylobacter coli was only detected in pigs. Also, the microbial functional characteristics of livestock feces revealed that the gene families related to "Biofilm formation and quorum sensing" were highly enriched in pigs than chicken. Moreover, the variety of gene families associated with "Resistance to antibiotics and toxic compounds" were detected in both animals. These results will help us to prepare the scientific action plans to improve awareness and understanding of the public health risks of livestock feces.

Application of Environmental DNA (eDNA) for Marine Biodiversity Analysis (해양생물 다양성 연구를 위한 환경유전자(eDNA)의 적용)

  • Soyun Choi;Seung Jae Lee;Eunkyung Choi;Euna Jo;Jinmu Kim;Minjoo Cho;Jangyeon Kim;Sooyeon Kwon;Hyun Park
    • Journal of Marine Life Science
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    • v.8 no.2
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    • pp.93-103
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    • 2023
  • eDNA, an abbreviation for environmental DNA, means DNA derived from organisms inhabiting in a specific environment. The utilization of eDNA extracted from environmental samples allows for efficient and accurate monitoring of organisms inhabiting the respective environment. Specifically, eDNA obtained from seawater samples can be used to analyze marine biodiversity. After collecting seawater samples and extracting eDNA, metagenome analysis enables the taxonomic and diversity analysis among marine organisms inhabiting the sampled area. This review proposed an overall process of marine biodiversity analysis by utilizing eDNA from seawater. Currently, the application of eDNA for analyzing marine biodiversity in domestic setting is not yet widespread. This review can contribute to establishment of marine eDNA research methods in Korea, providing valuable assistance in standardizing the use of eDNA in marine biodiversity studies.