• 생명과학회지
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• 제15권6호
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• pp.1013-1021
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• 2005

미생물 메타게놈은 특이한 생체촉매의 다양한 원료로 제공된다. 한우의 반추위에서 게놈 DNA를 분리한 후 메타게놈 은행을 구축하고 $\alpha$-amlylase를 암호화하는 유전자를 클로닝하여 DNA 및 아미노산 서열을 밝히고 생화적 특징을 조사하였다. amyA유전자는 1,893 bp로 631개의 아미노산 잔기를 가진 단백질을 암호화였으며 효소의 분자량은 단백질 전기영동 결과 약 71,000 Da으로 확인되었다. 이 효소를 다른 아밀라제와 비교한 결과 $21-59\%$의 상동성을 보였다. AnyA는 pH $6.40\%$에서 최적 활성을 나타내었고, pl값은 5.87이었다. E. coliDH5$\alpha$에서 발현된 AmyA의 활성은 $\Mg^{2+}$(20mM), $Ca^{2+}$ (30 mM) 존재 시 그 활성이 증가하였고, $Fe^{2+}$, $Cu^{2+}$ 존재 시 저해되었다. amyA 유전자의 internal primer를 사용하여 인공적으로 배양할 수 있는 49종의 반추세균에서 분리한 게놈 DNA을 주형으로 PCR분석한 결과 해당하는 벤드를 확인할 수 없었다. AmyA는 현재로 배양할 수 없는 반추 미생물에서 온 것으로 추정된다.

• Genomics & Informatics
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• 제20권4호
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• pp.44.1-44.13
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• 2022

Brugada syndrome (BS) is an autosomal dominant inheritance cardiac arrhythmia disorder associated with sudden death in young adults. Thailand has the highest prevalence of BS worldwide, and over 60% of patients with BS still have unclear disease etiology. Here, we performed a new viral metagenome analysis pipeline called VIRIN and validated it with whole genome sequencing (WGS) data of HeLa cell lines and hepatocellular carcinoma. Then the VIRIN pipeline was applied to identify viral integration positions from unmapped WGS data of Thai males, including 100 BS patients (case) and 100 controls. Even though the sample preparation had no viral enrichment step, we can identify several virus genes from our analysis pipeline. The predominance of human endogenous retrovirus K (HERV-K) viruses was found in both cases and controls by blastn and blastx analysis. This study is the first report on the full-length HERV-K assembled genomes in the Thai population. Furthermore, the HERV-K integration breakpoint positions were validated and compared between the case and control datasets. Interestingly, Brugada cases contained HERV-K integration breakpoints at promoters five times more often than controls. Overall, the highlight of this study is the BS-specific HERV-K breakpoint positions that were found at the gene coding region "NBPF11" (n = 9), "NBPF12" (n = 8) and long non-coding RNA (lncRNA) "PCAT14" (n = 4) region. The genes and the lncRNA have been reported to be associated with congenital heart and arterial diseases. These findings provide another aspect of the BS etiology associated with viral genome integrations within the human genome.

• 한국자원식물학회:학술대회논문집
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• 한국자원식물학회 2023년도 임시총회 및 춘계학술대회
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• pp.36-36
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• 2023

천마(天麻, Gastrodia elata Blume)는 난초과(蘭草科, Orchidaceae)에 속하는 식물로 잎과 뿌리가 없어 탄소동화능력이 없으며, 뽕나무버섯균과 공생하는 기생식물이다. 천마는 노지 재배에 따른 안정생산 문제가 지속적으로 발생하고 있다. 혹한, 폭우 등 기상환경에 따른 연차간 수량성 차가 673~1,175kg/10a로 크고, 연작에 따른 수량성이 연작 1회 시 29%, 연작 2회 시 68%가 감소하는 경향을 보이고 있다. 따라서, 본 연구는 천마의 연작에 따른 토양내 미생물상의 변화를 분석하기 위해 수행하였다. 본 실험은 초작지, 연작 1회지, 연작 2회지 및 자생지의 각각 3곳에 대한 토양을 Metagenome 분석법을 활용하여 미생물상을 분석하였다. metagenome 분석 결과, 초작지 3곳의 시료를 이용하여 확보한 총 read는 699,221개였으며, 이 중에서 Eukaryota로 분류되지 않은 read는 1,377개(0.2%), no hit, not assign된 read는 10,510개 (1.5%), Bacteria로 분류된 read는 342,916개(49.0%), Eukaryota로 분류된 read는 총 344,418개(49.3%)였다. 그리고 천마의 생육에 영향을 주는 Fusarium 속과 종은 3곳 포장에서 고르게 분포하고 있으며, 총 11,242 read로 전체의 3.3%를 차지하였다. 연작 1회지 3곳의 시료를 이용하여 확보한 총 read는 655,097개였으며, 이 중에서 Eukaryota로 분류되지 않은 read는 1,694개(0.3%), no hit, not assign된 read는 18,985개(2.9%), Bacteria로 분류된 read는 312,201개(47.7%), Eukaryota로 분류된 read는 총 322,217개 (49.2%)였다. 그리고 천마의 생육에 영향을 주는 Fusarium 속과 종은 3곳 포장에서 고르게 분포하고 있으며, 총 11,597 read로 전체의 3.6%를 차지하였다. 연작 2회지 3곳의 시료를 이용하여 확보한 총 read는 651,624개였으며, 이 중에서 Eukaryota로 분류되지 않은 read는 1,753개(0.3%), no hit, not assign된 read는 7,995개(1.2%), Bacteria로 분류된 read는 307,178개(47.1%), Eukaryota로 분류된 read는 총 334,698개(51.4%)였다. 그리고 천마의 생육에 영향을 주는 Fusarium 속과 종은 총 43,877 read로 전체의 13.1%를 차지하였다. 자생지 3곳의 시료를 이용하여 확보한 총 read는 731,719개였으며, 이 중에서 Eukaryota로 분류되지 않은 read는 2,828개(0.4%), no hit, not assign된 read는 585개(0.1%), Bacteria로 분류된 read는 356,690개(48.7%), Eukaryota로 분류된 read는 총 371,616개(50.8%)였다. 그리고 천마의 생육에 영향을 주는 Fusarium 속과 종은 MJH01 자생지에서만 전체의 0.2%(총 660 read)로 극히 일부가 분리되었다. 따라서, 천마의 생육에 영향을 미치는 Fusarium 속과 종은 초작지 3.3%, 연작 1회지 3.6%, 연작 2회지 13.1%, 자생지 0.2%로 나타났으며, 연작 2회 시 급격히 증가하는 것으로 판단되었다.

• Animal Bioscience
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• 제37권4호
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• pp.709-717
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• 2024

Objective: Abnormally increased somatic cell counts (SCCs) in milk is usually a sign of bovine subclinical mastitis. Mutual interaction between the host and its associated microbiota plays an important role in developing such diseases. The main objective of this study was to explore the difference between cows with elevated SCCs and healthy cattle from the perspective of host-microbe interplay. Methods: A total of 31 milk samples and 23 bovine peripheral blood samples were collected from Holstein dairy cattle to conduct an integrated analysis of transcriptomic and metagenomics. Results: The results showed that Ralstonia and Sphingomonas were enriched in cows with subclinical mastitis. The relative abundance of the two bacteria was positively correlated with the expression level of bovine transcobalamin 1 and uridine phosphorylase 1 encoding gene. Moreover, functional analysis revealed a distinct alternation in some important microbial biological processes. Conclusion: These results reveal the relative abundance of Ralstonia and Sphingomonas other than common mastitis-causing pathogens varied from healthy cows to those with subclinical mastitis and might be associated with elevated SCCs. Potential association was observed between bovine milk microbiota composition and the transcriptional pattern of some genes, thus providing new insights to understand homeostasis of bovine udder.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2003년도 생물공학의 동향(XIII)
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• pp.815-820
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• 2003

A huge database resulted from whole genome sequencing has provided a possibility of new information that is likely to extent the scope and thus changes the way of approach for the functional assigning of putative open reading frames annotated by whole genome sequence analyses. These are mainly realized by ease, one-step identification of putative genes using genomics or proteomics tools. A major challenge remained in biotechnology may translate these informations into better ways to screen or select a gene as a representative sequence. Further attempts to mine the related whole genes or partial DNA fragment from whole genome treasure, and then the incorporation of these sequences into a representative template, will result in the use of putative genes that can be translated into functional proteins or allowed the generation of new lineages as a valuable pool. Such screens enable rapid biochemical analysis and easy isolation of the target activity, thereby accelerating the screening of novel enzymes from the expanded library with related sequences. Information-based PCR amplification of whole genes and reconstitution of functional DNA fragments will provide a platform for expanding the functional spaces of potential enzymes, especially when used mixed- or metagenome as gene resources.

• 한국생물정보학회:학술대회논문집
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• 한국생물정보시스템생물학회 2003년도 제2차 연례학술대회 발표논문집
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• pp.245-250
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• 2003

There are currently about 6000 bacterial species with validly published names, but scientists assume that these may be less than 1% of bacterial species present on the earth. Microbial resource is one of the most important bioresources in bioinderstry and provides us with high economic values. To find missing ones, the studies of metagenome, metabolome, and proteome about microbes have started recently in developed countries. We construct the information system that integrates information on microbial genome resources and manages the information to support efficient research of microbial genome application, and name this system 'Bio-Meta Information System (Bio-MIS)'. Bio-MIS consists of integrated microbial genome resources database, microbial genome resources input system, integrated microbial genome resources search engine, microbial resources on-line distribution system, portal service and management via internet. In the future, we will include public database connection and implement useful bioinformatics software for analyzing microbial genome resources. The web-site is accessible at http://biomis.probionic.com

• Journal of Microbiology and Biotechnology
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• 제26권10호
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• pp.1717-1722
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• 2016

A novel phytase of Acidobacteria was identified from a soil metagenome, cloned, overexpressed, and purified. It has low sequence similarity (<44%) to all the known phytases. At the optimum pH (2.5), the phytase shows an activity level of 1,792 μmol/min/mg at physiological temperature (37℃) and could retain 92% residual activity after 30 min, indicating the phytase is acidophilic and acidostable. However the phytase shows poor stability at high temperatures. To improve its thermal resistance, the enzyme was redesigned using Disulfide by Design 2.0, introducing four additional disulfide bridges. The half-life time of the engineered phytase at 60℃ and 80℃, respectively, is 3.0× and 2.8× longer than the wild-type, and its activity and acidostability are not significantly affected.

• Journal of Microbiology and Biotechnology
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• 제15권5호
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• pp.1067-1072
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• 2005

A metagenomic library was constructed using a fosmid vector, and total genomic DNA was extracted directly from soil at Cisolok (hot spring area, Indonesia). This library was composed of 10,214 clones and screened for lipolytic enzyme on tributyrin agar plates. An esterase gene (estMa) was subcloned and sequenced from a positive lipolytic active clone. Esterase EstMa was encoded by a 954-bp open reading frame and showed low ($11-33\%$) amino acid similarity to known esterases. The amino acid sequence analysis demonstrated that the enzyme is a new member of lipolytic enzyme family VI. The estMa gene encodes a preprotein of 317 amino acids with a predicted molecular mass of 34,799 Da. The purified enzyme exhibited optimal activity at $50^{\circ}C$ and pH 6.5. The $K_m,\;and\;V_{max}$ values of EstMa for the hydrolysis of p-nitrophenyl valerate were $45.3\;{\mu}M$ and 4.45 U/mg, respectively.

• Journal of Microbiology and Biotechnology
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• 제19권2호
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• pp.187-193
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• 2009

A metagenomic library of surface-water microbes from the Yangtze River in China was constructed, and a novel esterase, designated as EstY, was isolated and characterized. EstY had 423 amino acids with an estimated molecular mass of 44 kDa and pI of 7.28. It hydrolyzed various p-nitrophenyl esters(acetate, butyrate, caprate, caprylate, laurate, myristate, and palmitate) and its best substrate was p-nitrophenyl caprate(C8). The optimum pH for EstY activity was 9.0 and the optimum temperature was $50^{\circ}C$. Metal ions, such as $Mn^{2+},\;Co^{2+},\;Hg^{2+},\;Zn^{2+},\;and\;Fe^{3+}$, strongly inhibited the activity of EstY, whereas $Mg^{2+}$ was required for maximal activity. Activity remained in the presence of 10% alcohol, acetone, isopropanol, and dimethyl sulfoxide, respectively. An analysis of the amino acid sequence deduced from estY revealed that it had 7 closely related lipolytic enzymes. Moreover, a sequence analysis showed that EstY, like its 7 relatives, did not belong to any known lipolytic enzyme family.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVII)
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• pp.243-246
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• 2005

A novel esterase showing high enantioselectivity to (S)-ketoprofen ethyl ester was selected from fosmid environmental DNA library which is provided by Microbial Genomic & Applications Center. As a result of Blast search, the gene wasn't registerated in Gene Bank yet. And as we know, conserved domain region of esterase , G-X-S-X-G, wasn't discovered.$^{4)}$ And it is similar to Beta-lactamase. The DNA sequence of cloned esterase include an open reading frame consisting of 1170 bp, designated as EST-Y29, encoding a protein of 389 amino acids with a molecular mass of about 42.8 kDa. And amino acid sequence analysis revealed only a few identity (28%) to tile known esterases/lipases in the databases containing the conserved sequence motifs of esterases/lipases. when being comparison to other esterase revealed , this enzyme seems to be classified as a new member of esterase family. EST-Y29 was functionally overexpressed in a soluble form in E. coli with maximum conversion yield of (S)-ketoprofen at $65^{\circ}C$. This study demonstrates that functional screening combined with the sequential uses of restriction enzymes to exclude already known enzymes is a useful approach for isolating novel enzyme from a metagenome.