• Journal of Microbiology and Biotechnology
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• v.23 no.5
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• pp.689-698
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• 2013

The production and characterization of an active recombinant N-acetylgalactosamine-6-sulfate sulfatase (GALNS) in Escherichia coli BL21(DE3) has been previously reported. In this study, the effect of the signal peptide (SP), inducer concentration, process scale, and operational mode (batch and semi-continuous) on GALNS production were evaluated. When native SP was presented, higher enzyme activity levels were observed in both soluble and inclusion bodies fractions, and its removal had a significant impact on enzyme activation. At shake scale, the optimal IPTG concentrations were 0.5 and 1.5 mM for the strains with and without SP, respectively, whereas at bench scale, the highest enzyme activities were observed with 1.5 mM IPTG for both strains. Noteworthy, enzyme activity in the culture media was only detected when SP was presented and the culture was carried out under semi-continuous mode. We showed for the first time that the mechanism that in prokaryotes recognizes the SP to mediate sulfatase activation can also recognize a eukaryotic SP, favoring the activation of the enzyme, and could also favor the secretion of the recombinant protein. These results offer significant information for scaling-up the production of human sulfatases in E. coli.

• Biomedical Science Letters
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• v.9 no.3
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• pp.139-144
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• 2003

Since water borne infection causes acute diseases and results in spread of diseases by secondary infection, the prevention is very important. Therefore, it is necessary to have a method that is rapid and effective to monitor pathogenic bacteria in drinking water. In this study, we employed a systematic method, Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) analysis, to develop an effective monitoring system for possible bacterial contaminants in drinking water. For this purpose, PCR primers were derived from 992 bp region of the 16s rRNA gene that is highly conserved through the different species of prokaryotes. To test whether the PCR primers designed are indeed useful for detecting all the possible microbial contaminants in the water, the primers were used to amplify 16s rRNA regions of different microbial water-borne pathogens such as E. coli, Salmonella, Yersinia, Listeria, and Staphylococcus. As expected, all of tested microorganisms amplified expected size of PCR products indicating designed PCR primers for 16s rRNA indeed can be useful to amplify all different microbial water-borne pathogens in the water. Furthermore, to test whether these 16s rRNA based PCR primers can detect bacterial populations present in the water, water samples taken from diverse sources, such as river, tap, and sewage, were used for amplification. PCR products were for then subjected for cloning into a T-vector to generate a library containing 16s rRNA sequences from various bacteria. With cloned PCR products, RFLP analysis was done using PCR products digested with restriction enzyme such as Hae III to obtain species-specific RFLP profiles. After PCR-RFLP, the bacterial clones which showed the same RFLP profiles were regarded as the same ones, and the clones which showed distinctive RFLP profiles were subsequently subjected for sequence analysis for species identification. By this PCR-RFLP analysis, we were able to reveal diverse populations of bacteria living in water. In brief, in unsterilized natural river water, over 60 different species of bacteria were found. On the other hand, no PCR products were detected in drinking tap-water. The results from this study clearly indicate that the PCR-RFLP-sequence analysis can be a useful method for monitoring diverse, perhaps pathogenic bacteria contaminated in water in a rapid fashion.

• Journal of Microbiology and Biotechnology
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• v.27 no.11
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• pp.2044-2051
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• 2017

The main pathological hallmark of Alzheimer's disease is the deposition of amyloid-beta ($A{\beta}$) peptides in the brain. $A{\beta}$ has been widely used to mimic several aspects of Alzheimer's disease. However, several characteristics of amyloid-induced Alzheimer's disease pathology are not well established, especially in mice. The present study aimed to develop a new Alzheimer's disease model by investigating how $A{\beta}$ can be effectively aggregated using prokaryotes and eukaryotes. To express the $A{\beta}42$ complex in HEK293 cells, we cloned the $A{\beta}42$ region in a tandem repeat and incorporated the resulting construct into a eukaryotic expression vector. Following transfection into HEK293 cells via lipofection, cell viability assay and western blotting analysis revealed that exogenous $A{\beta}42$ can induce cell death and apoptosis. In addition, recombinant His-tagged $A{\beta}42$ was successfully expressed in Escherichia coli BL21 (DE3) and not only readily formed $A{\beta}$ complexes, but also inhibited the proliferation of SH-SY5Y cells and E. coli. For in vivo testing, recombinant His-tagged $A{\beta}42$ solution ($3{\mu}g/{\mu}l$ in $1{\times}PBS$ containing $1mM\;Ni^{2+}$) was injected stereotaxically into the left and right lateral ventricles of the brains of C57BL/6J mice (n = 8). Control mice were injected with $1{\times}PBS$ containing $1mM\;Ni^{2+}$ following the same procedure. Ten days after the sample injection, the Morris water maze test confirmed that exogenous $A{\beta}$ caused an increase in memory loss. These findings demonstrated that $Ni^{2+}$ is capable of complexing the 50-kDa amyloid and that intracerebroventricular injection of $A{\beta}42$ can lead to cognitive impairment, thereby providing improved Alzheimer's disease models.

• Microbiology and Biotechnology Letters
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• v.33 no.2
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• pp.84-89
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• 2005

Phosphomannomutase (PMM) is a key enzyme in prokaryotes and eukaryotes, which catalyzes the conversion of ${\alpha}$-D-mannose 6-phosphate to ${\alpha}$-D-mannose 1-phosphate. The latter is the substrate for the synthesis of GDP-mannose, which serves as the mannosyl donor for many metabolic pathways in the cells. We report here on the isolation of a gene from a genomic library of Sphingomonas chungbukensis DJ77, the pmmC gene encoding phosphomannomutase. The gene was cloned into E. coli expression vector, and the sequence was analyzed. The ribosomal binding site GGAAG lays 5 bp upstream of the ORF of 750 bp, which is initiated by ATG codon and terminated by TAG. The predicted sequence of the enzyme consists of 249 amino acids with a molecular mass of 27.4 kDa and showed $86.9\%$ similarity to that of eukaryotic phosphomannomutase after bioinformatical analyses with the conserved domain search of NCBI. The purified gene product revealed the activity of phosphomannomutase. In conclusion, we confirmed that pmmC gene encodes phosphomannomutase actually.

• Journal of Life Science
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• v.28 no.11
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• pp.1379-1385
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• 2018

Glycans are attached to proteins as in glycoproteins and proteoglycans. They are found on the exterior surface of cells. O- and N-linked glycans are very common in eukaryotic cells but may also be found in prokaryotes. The interaction of cell surface glycans with complementary glycan binding proteins located on neighboring cells, other cell types, pathogens like virus, or bacteria is crucial in biologically and biomedically important processes like pathogen recognition, cell migration, cell-cell adhesion, development, and infection. Their implication in pathological condition, suggests an important role for glycans as disease markers. In addition, a great amount of research has been shown that appropriate glycosylation of a recombinant therapeutic protein is critical for product solubility, stability, pharmacokinetics and pharmacodynamics, bioactivity, and safety. Besides, cancer-associated glycosylation changes often involve sialic acid in glycan branch which play important roles in cell-cell interaction, recognition and immunological response. This review aims at giving a comprehensive overview of the glycan's biological function and describing the relevance among the glycosylation, disease diagnosis and treatment methods. Furthermore, the high-throughput analytic methods available to measure the profile changing patterns of glycan in the blood serum as well as possible underlying biochemical mechanisms.

• Journal of Food Hygiene and Safety
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• v.35 no.6
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• pp.535-543
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• 2020

In addition to the ubiquitous roles of cellular RNA in genetic regulations, gene expression and phenotypic variations in response to environmental cues and chemotactic signals, the regulatory roles of a new type of RNA called extracellular RNAs (exRNAs) are an up-and-coming area of research interest. exRNA is transported outside the cell through membrane blebs known as membrane vesicles or extracellular vesicles (EVs). EV formation is predominant and conserved among all microbial forms, including prokaryotes, eukaryotes, and archaea. This review will focus on the three major topics concerning bacterially derived exRNAs, i.e., 1) the discovery of exRNA and influence of extraneous RNA over bacterial gene regulations, 2) the known secretion mechanism for the release of exRNA, and 3) the possible applications that can be devised with these exRNA secreted by different gram-negative and gram-positive bacteria. Further, this review will also provide an opinion on exRNA- and EV-derived applications such as the species-specific exRNA markers for diagnostics and the possible roles of exRNA in probiotics and the epigenetic regulations of the gut microbiome.

• Journal of Life Science
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• v.23 no.6
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• pp.796-803
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• 2013

Property changes and bacterial characterizations by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) were investigated during the fermentation of Makgeollies by 5 isolated yeast strains. Changes of pH were large between day 0 (pH 6) and day 2 (pH 3) and showed less variation after then. ANOVA analyses revealed that pHs were statistically different with fermentation times (p<0.001), while strains (p=0.60) did not. Acidities were changed from 0.19 to 1.04% and showed rather high increase from day 2, and fermentation times (p<0.001) and strains (p=0.006) represented statistical differences. All strains showed less than 0.150% at amino-type nitrogen contents except S strain showed 0.442% at day 8, and there were no statistical differences with fermentation times (p=0.4558) and strains (p=0.3513). Saccharinities of C strain were higher from day 4, and fermentation times (p<0.0001) and strains (p=0.007) showed statistical differences. Large variation of alcohol concentrations (%) were observed between day 0 (0%) and day 2 (10%) and showed less variation after day 2, and there was no statistical difference with strains. Dominant prokaryotes were Lactobacillus fermentum and Pediococcus pentosaceus, which producing acids and functional materials. Dominant eukaryote was Saccharomyces cerevisiae, which might be resulted from addition of yeasts.

• The Sea:JOURNAL OF THE KOREAN SOCIETY OF OCEANOGRAPHY
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• v.20 no.1
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• pp.53-62
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• 2015

To elucidate the microbial consortia responsible for the anaerobic methane oxidation in the methane hydrate bearing sediments, we compared the geochemical constituents of the sediment, the rate of sulfate reduction, and microbial biomass and diversity using an analysis of functional genes associated with the anaerobic methane oxidation and sulfate reduction between chimney site (UBGH2-3) on the continental slope and non-chimney site (UBGH2-10) on the basin of the Ulleung Basin. From the vertical profiles of geochemical constituents, sulfate and methane transition zone (SMTZ) was clearly defined between 0.5 and 1.5 mbsf (meters below seafloor) in the UBGH2-3, and between 6 and 7 mbsf at the UBGH2-10. At the UBGH2-3, the sulfate reduction rate (SRR) in the SMTZ exhibited was appeared to be $1.82nmol\;cm^{-3}d^{-1}$ at the depth of 1.15 mbsf. The SRR in the UBHG2-10 showed a highest value ($4.29nmol\;cm^{-3}d^{-1}$) at the SMTZ. The 16S rRNA gene copy numbers of total Prokaryotes, mcrA, (methyl coenzyme M reductase subunit A), and dsrA (dissimilatory sulfite reductase subunit A) showed the peaks in the SMTZ at both sites, but the maximum mcrA gene copy number of the UBGH2-10 appeared below the SMTZ (9.8 mbsf). ANME-1 was a predominant ANME (Anaerobic MEthanotroph) group in both SMTZs of the UBGH2-3 and -10. However, The sequences of ANME-2 were detected only at 2.2 mbsf of the UBGH2-3 where high methane flux was observed because of massive amount of gas hydrate at shallow depth. And Desulfosarcina-Desulfococcus (DSS) that is associated with ANME-2 was detected in 2.2 mbsf of the UBHG2-3. Overall results demonstrate that ANME-1 and ANME-2 are considered as significant archaeal groups related to methane cycle in the subsurface sediment of the East Sea, and ANME-2/DSS consortia might be more responsible for methane oxidation in the methane seeping region than in non-seeping region.

• Journal of Life Science
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• v.15 no.3 s.70
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• pp.415-422
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• 2005

library of the mutants was prepared by transposon mutagenesis of the Mycobacterium bovis BCG. We screened this library for the resistance to an anti-tuberculosis antibiotic, PA-824. Most of the mutants resistant to the PA-824 were not able to synthesize the coenzyme $F_{420}$ which is normally produced by the wild type M. bovis BCG strains. HPLC analysis of the cellular extract showed that one of those mutants which lost the ability to synthesize $F_{420}$ still produced F0. The insertion site of the transposon in this mutant was determined by an inverse PCR and the transposon was found to be inserted in the Rv2435c open reading frame (ORF). Rv2435c ORF is predicted to encode an 80.3 kDa protein. Rv2435c protein appears to be bound to the cytoplasmic membrane, its N-terminal present in the periplasm and C-terminal in the cytoplasm. The C-terminal portion of this protein is highly homologous with the adenylyl cyclases of both prokaryotes and eukaryotes. There are 15 ORFs which have homology with the class III AC proteins in the genome of the M. tuberculosis and M. bovis. Two of those, Rv1625c and Rv2435c, are highly homologous with the mammalian ACs. We cloned the cytoplasmic domain of the Rv2435c ORF and expressed it with six histidine residues attached on its C-terminal in Escherichia coli to find out if this protein is a genuine AC. Production of that protein in E. coli was proved by purifying the histidine-tagged protein by using the Ni-NTA resin. This protein, however, failed to complement the cya mutation in E. coli, indicating that this protein lacks the AC activity. All of the further attempts to convert this protein to a functional AC by a mutagenesis with UV or hydroxylamine, or construction of several different fusion proteins with Rv1625c failed. It is, therefore, possible that Rv2435c protein might affect the conversion of F0 to $F_{420}$ not by synthesizing cAMP but by some other way.

• Journal of the Korean Society of Marine Environment & Safety
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• v.27 no.7
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• pp.924-942
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• 2021

The expansion of the Changjiang Diluted Water (CDW) plume during summer is known to be a major factor influencing phytoplankton diversity, community structure, and the regional marine environment of the northern East China Sea (ECS). The discharge of the CDW plume was very high in the summer of 2020, and cruise surveys and stationary monitoring were conducted to understand the dynamics of changes in environmental characteristics and the impact on phytoplankton diversity and community structure. A cruise survey was conducted from August 16 to 17, 2020, using R/V Eardo, and a stay survey at the Ieodo Ocean Research Station (IORS) from August 15 to 21, 2020, to analyze phytoplankton diversity and community structure. The southwestern part of the survey area exhibited low salinity and high chlorophyll a fluorescence under the influence of the CDW plume, whereas the southeastern part of the survey area presented high salinity and low chlorophyll a fluorescence under the influence of the Tsushima Warm Current (TWC). The total chlorophyll a concentrations of surface water samples from 12 sampling stations indicated that nano-phytoplankton (20-3 ㎛) and micro-phytoplankton (> 20 ㎛) were the dominant groups during the survey period. Only stations strongly influenced by the TWC presented approximately 50% of the biomass contributed by pico-phytoplankton (< 3 ㎛). The size distribution of phytoplankton in the surface water samples is related to nutrient supplies, and areas where high nutrient (nitrate) supplies were provided by the CDW plume displayed higher biomass contribution by micro-phytoplankton groups. A total of 45 genera of nano- and micro-phytoplankton groups were classified using morphological analysis. Among them, the dominant taxa were the diatoms Guinardia flaccida and Nitzschia spp. and the dinoflagellates Gonyaulax monacantha, Noctiluca scintillans, Gymnodinium spirale, Heterocapsa spp., Prorocentrum micans, and Tripos furca. The sampling stations affected by the TWC and low in nitrate concentrations presented high concentrations of photosynthetic pico-eukaryotes (PPE) and photosynthetic pico-prokaryotes (PPP). Most sampling stations had phosphate-limited conditions. Higher Synechococcus concentrations were enumerated for the sampling stations influenced by low-nutrient water of the TWC using flow cytometry. The NGS analysis revealed 29 clades of Synechococcus among PPP, and 11 clades displayed a dominance rate of 1% or more at least once in one sample. Clade II was the dominant group in the surface water, whereas various clades (Clades I, IV, etc.) were found to be the next dominant groups in the SCM layers. The Prochlorococcus group, belonging to the PPP, observed in the warm water region, presented a high-light-adapted ecotype and did not appear in the northern part of the survey region. PPE analysis resulted in 163 operational taxonomic units (OTUs), indicating very high diversity. Among them, 11 major taxa showed dominant OTUs with more than 5% in at least one sample, while Amphidinium testudo was the dominant taxon in the surface water in the low-salinity region affected by the CDW plume, and the chlorophyta was dominant in the SCM layer. In the warm water region affected by the TWC, various groups of haptophytes were dominant. Observations from the IORS also presented similar results to the cruise survey results for biomass, size distribution, and diversity of phytoplankton. The results revealed the various dynamic responses of phytoplankton influenced by the CDW plume. By comparing the results from the IORS and research cruise studies, the study confirmed that the IORS is an important observational station to monitor the dynamic impact of the CDW plume. In future research, it is necessary to establish an effective use of IORS in preparation for changes in the ECS summer environment and ecosystem due to climate change.