• Journal of Microbiology and Biotechnology
• /
• 제31권3호
• /
• pp.483-491
• /
• 2021

Two putative genes, lip29 and est29, encoding lipolytic enzymes from the thermophilic bacterium Geobacillus thermocatenulatus KCTC 3921 were cloned and overexpressed in Escherichia coli. The recombinant Lip29 and Est29 were purified 67.3-fold to homogeneity with specific activity of 2.27 U/mg and recovery of 5.8% and 14.4-fold with specific activity of 0.92 U/mg and recovery of 1.3%, respectively. The molecular mass of each purified enzyme was estimated to be 29 kDa by SDS-PAGE. The alignment analysis of amino acid sequences revealed that both enzymes belonged to GDSL lipase/esterase family including conserved blocks with SGNH catalytic residues which was mainly identified in plants before. While Est29 showed high specificity toward short-chain fatty acids (C4-C8), Lip29 showed strong lipolytic activity to long-chain fatty acids (C12-C16). The optimal activity of Lip29 toward p-nitrophenyl palmitate as a substrate was observed at 50℃ and pH 9.5, respectively, and its activity was maintained more than 24 h at optimal temperatures, indicating that Lip29 was thermostable. Lip29 exhibited high tolerance against detergents and metal ions. The homology modeling and substrate docking revealed that the long-chain substrates showed the greatest binding affinity toward enzyme. Based on the biochemical and insilico analyses, we present for the first time a GDSL-type lipase in the thermophilic bacteria group.

• 한국생물공학회:학술대회논문집
• /
• 한국생물공학회 2001년도 추계학술발표대회
• /
• pp.625-628
• /
• 2001

토양으로부터 특이적인 광학 이성질체에 활성을 갖는 두 효소원을 선별한 후, 동정하여 Pseudomonas sp. S34와 B. stearothermophilus JY144로 명명하였고, genecloning과 sequencing을 통해 유전자의 특성 및 관련효소들과의 유연관계를 규명하였다. 규명된 정보의 분석과 비교를 통해 ketoprofen ethyl ester에 활성을 갖는 효소들의 구조적 특성을 추론할 수 있었고, 이를 바탕으로 발현시스템을 구축하였다. 대량생산된 효소를 활용한 반응의 결과 높은 수율과 순도를 갖는 각각의 광학이성질체를 경제적으로 생산할 수 있었다.

• 한국환경성돌연변이발암원학회지
• /
• 제24권2호
• /
• pp.85-90
• /
• 2004

Nickel(II) compounds are carcinogenic metals which induce genotoxicity and oxidative stress through the generation of reactive oxygen species. In search of new molecular pathways toward understanding the molecular mechanism of nickel(II)-induced carcinogensis, we performed mRNA differential display analysis using total RNA extracted from nickel(II) acetate-treated normal rat kidney cells (NRK-52E). Cells were exposed for 3 days to 160 and 240 uM nickel(II) concentrations. cDNAs corresponding to mRNAs for which expression levels were altered by nickel(II) were isolated, sequenced, and followed by a GenBank Blast homology search. Specificity of differential expression of cDNAs was determined by RT-PCR and Western blot analysis. Two of them (SH3BGRL3 and FHIT) were down-regulated and one (metallothionein) was up-regulated by nickel(II) treatment. The expression of these mRNAs were nickel(II) concentration-dependent. The levels of FHIT and metallothionein proteins were also consistent with the results for mRNAs. Overall, although the fundamental questions related to function of these genes in nickel(II)-mediated carcinogenicity are not answered, our study suggests that they can be interesting candidates for studies of molecular mechanisms of nickel(II) carcinogenesis.

• Journal of Microbiology and Biotechnology
• /
• 제24권12호
• /
• pp.1699-1706
• /
• 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.

• 한국환경독성학회:학술대회논문집
• /
• 한국환경독성학회 2002년도 추계국제학술대회
• /
• pp.167-167
• /
• 2002

Methylmercury (MeHg), one of the heavy metal compound, can cause severe damage to the central nervous system in humans. Many reports have contributed MeHg poisoning to contaminated foods and release into the environment. Despite many studies on the pathogenesis of MeHg-induced central neuropathy, no useful mechanism of toxicity has been established. To find genes differentially expressed by MeHg in neuronal cell, we peformed forward and reverse suppression subtractive hybridization (SSH) method on mRNA derived from neuroblastoma cell line, SH-SY5Y treated with solvent (DMSO) and 6.25 uM (IC$\sub$50/) MeHg. Differentially expressed CDNA clones were sequenced and the mRNAs were re-examined on Northern blots. These sequences were identified by BLAST homology search to known genes or expressed sequence tags (ESTs). Analysis of these sequences has provided an insight into the biological effects of MeHg in the pathogenesis of neurodegenerative disease and a possibility to develop more efficient and exact monitoring system of heavy metals as common environmental pollutants.

• Journal of Microbiology
• /
• 제41권4호
• /
• pp.295-299
• /
• 2003

Adenovirus (Ad) precursor to the terminal protein (pTP) plays an essential roles in the viral DNA replication. Ad pTP serves as a primer for the synthesis of a new DNA strand during the initiation step of replication. In addition, Ad pTP forms organized spherical replication foci on the nuclear matrix (NM) and anchors the viral genome to the NM. Here we identified the interferon inducible gene product 1-8D (Inid) as a pTP binding protein by using a two-hybrid screen of a HeLa cDNA library. Of the clones obtained in this assay, nine were identical to the Inid, a 13-kDa polypeptide that shares homology with genes 1-8U and Leu-13/9-27, most of which have little known functions. The entire open reading frame (ORF) of Inid was cloned into the tetracycline inducible expression vector in order to determine the biological functions related with adenoviral infection. When Inid was introduced to the cells along with adenoviruses, fifty to sixty percent of Ad-infected cells expressing Inid had rounded morphology, which was suggestive of apoptosis. Results from the terminal deoxynucleotidyl transferase (TdT) and DNA fragmentation assays confirmed that Inid induces apoptosis in Ad-infected or in uninfected cells. The Inid binding to pTP may target the cell for apoptotic destruction as a host defense mechanism against the viral infection.

• 한국식품저장유통학회지
• /
• 제17권3호
• /
• pp.410-417
• /
• 2010

우리나라 전통발효식품의 하나인 청국장으로부터 고 비활성 세포외 protease 생산능이 우수한 균주를 분리하고 그 특성을 조사하였다. 분리된 균주 중 D14 균주 배양 상징액의 protease 활성이 15.2 U/mL로서 가장 강하였으며 비활성 또한 40.0 U/mg protein으로서 가장 강하게 나타났다. 이 균주의 특성을 조사한 후 Berge's Manual of Systematic Bacteriology에 준하여 Bacillus subtilis로 동정하였다. 균주 D14의 16S rDNA 염기서열을 분석하여 B. subtilis subsp. subtilis NCIB 3610 균주와 99.9%의 상동성을 가짐을 확인 하였다. 또한 16S rDNA의 염기서열을 토대로 계통분석을 통하여 다른 Bacillus sp.의 균주들보다 NCIB 3610 균주와 유전적으로 매우 가까운 관계에 있음을 확인하였다. Soluble starch는 사용한 탄소원 중 가장 높은 수준의 protease 생산능을 보였으나 단당류, 이당류 등은 모두 효소 생산능에 대하여 저해효과를 나타내었다. 특히 fructose를 첨가한 경우에는 12.7%, glucose를 첨가한 경우에는 35.9% 수준의 효소 생산능을 나타내었다. 질소원의 경우는 soytone의 첨가구에서 대조구에 비하여 약 61.4% 수준의 효소활성을 나타내어 저해효과가 가장 높았으며 다른 질소원은 효소 생산에 크게 영향을 미치지 않았다.

• The Plant Pathology Journal
• /
• 제33권1호
• /
• pp.43-52
• /
• 2017

A survey was conducted to determine the status of Lucerne transient streak virus (LTSV) in three high-yielding alfalfa regions in central Saudi Arabia (Riyadh, Qassim, and Hail) during 2014. Three hundred and eight symptomatic alfalfa, and seven Sonchus oleraceus samples were collected. DAS-ELISA indicated that 59 of these samples were positive to LTSV. Two isolates of LTSV from each region were selected for molecular studies. RT-PCR confirmed the presence of LTSV in the selected samples using a specific primer pair. Percentage identity and homology tree comparisons revealed that all Saudi isolates were more closely related to each other but also closely related to the Canadian isolate-JQ782213 (97.1-97.6%) and the New Zealand isolate-U31286 (95.8-97.1%). Comparing Saudi isolates of LTSV with ten other sobemoviruses based on the coat protein gene sequences confirmed the distant relationship between them. Eleven out of fourteen plant species used in host range study were positive to LTSV. This is the first time to document that Trifolium alexandrinum, Nicotiana occidentalis, Chenopodium glaucum, and Lathyrus sativus are new host plant species for LTSV and that N. occidentalis being a good propagative host for it.

• 한국어병학회지
• /
• 제32권2호
• /
• pp.69-80
• /
• 2019

We performed MLSA (multilocus sequence analysis) and phenotypic characterization of Aliivibrio species isolated from walleye pollock (Gadus chalcogrammus) maintained in 3 different facilities of Gangwon Province, the east coast of Korea. Of 38 Aliivibrio species identified by 16S rDNA sequences, 12 strains were randomly selected and MLSA was conducted with 5 house-keeping genes (gapA, gyrB, pyrH, recA and rpoA) and 16S rDNA gene. Phylogenetic analysis and homology of the concatenated sequences (4,580 bp) with other Vibrionaceae genera revealed that 4 strains (GNGc16.1, YYGc16.1, YYGc16.2, GSGc18.1) were identified as Aliivibrio logei and one strain (GSGc16.1) as A. wodanis. One strain (GSGc17.1) was tentatively identified as A. logei, but needs further analysis because it did not belong to the same clade with A. logei type strain. 6 strains (GSGc17.2, GNGc16.2, GSGc16.2, GSGc17.3, GSGc18.2, GSGc17.4) need further investigation as potential novel species. Either phenotypic characterization or 16S rDNA sequence alone did not provide enough information for identification of Aliivibrio strains at the species level. A. logei and A. wodanis are generally known as non-pathogenic bacteria, but also known as opportunistic or secondary pathogens of cold water fishes. Cares should be taken to prevent potential outbreaks due to these bacteria, although there was no outbreaks during the sampling period.

• Molecules and Cells
• /
• 제27권1호
• /
• pp.47-54
• /
• 2009

A cDNA clone for a transcript preferentially expressed during an early phase of flooding was isolated from Nicotiana tabacum. Nucleotide sequencing of the cDNA clone identified an open reading frame that has high homology to the previously reported glycine-rich RNA-binding proteins. The open reading frame consists of 157 amino acids with an N-terminal RNA-recognition motif and a C-terminal glycine-rich domain, and thus the cDNA clone was designated as Nicotiana tabaccum glycine-rich RNA-binding protein-1 (NtGRP1). Expression of NtGRP1 was upregulated under flooding stress and also increased, but at much lower levels, under conditions of cold, drought, heat, high salt content, and abscisic acid treatment. RNA homopolymer-binding assay showed that NtGRP1 binds to all the RNA homopolymers tested with a higher affinity to poly r(G) and poly r(A) than to poly r(U) and poly r(C). Nucleic acid-binding assays showed that NtGRP1 binds to ssDNA, dsDNA, and mRNA. NtGRP1 suppressed expression of the fire luciferase gene in vitro, and the suppression of luciferase gene expression could be rescued by addition of oligonucleotides. Collectively, the data suggest NtGRP1 as a negative modulator of gene expression by binding to DNA or RNA in bulk that could be advantageous for plants in a stress condition like flooding.