• Journal of Microbiology and Biotechnology
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• v.1 no.3
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• pp.188-196
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• 1991

The ${\beta}-glucosidase$ was purified to homogeneity from the culture filtrate of P. verruculosum by column chromatography. The enzyme was a glycoprotein with a relative size of approximately 220 kDa with an isoelectric point of 4.8, which was composed of dimeric protein of 105 kDa. The enzyme was stable up to $60^{\circ}C$ and the presence of glycerol significantly increased its thermostability. The enzyme was found to hydrolyze both ${\beta}-aryl$ and ${\beta}-alkyl-glucosides$ in addition to ${\beta}-glucosyl$ glucose and catalyzed glucosyl transfer to cellobiose. The enzyme attacked laminarin in an exotype-like fashion. The apparent Km's of the enzyme toward cellobiose, laminaribiose, laminarin were 0.53 mM, 0.35 mM and 1.11 mM, respectively. Glucose and glucono-${\delta}-lactone$ were competitive inhibitors for the enzyme. Copper ($Cu^{2+}$), mercury ($Hg^{2+}$) and p-chloromercuribenzoate were strong inhibitors of the enzyme. The immunoblotting result revealed that one form of ${\beta}-glucosidase$ was biosynthesized, irrespective of carbon sources used. Polyacrylamide gel electrophoresis analysis of the in vitro translated product of total RNA from avicel grown mycelium established that the P. verruculosum ${\beta}-glucosidase$ precursor was approximately 95 kDa in size. The amino acid composition and N-terminal amino acid sequence are given.

• Journal of Microbiology and Biotechnology
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• v.25 no.12
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• pp.2090-2099
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• 2015

Recently, the cardiovascular disease has been widely problematic in humans probably due to fibrin formation via the unbalanced Western style diet. Although direct (human plasmin) and indirect methods (plasminogen activators) have been available, bacterial enzyme methods have been studied because of their cheap and mass production. To detect a novel bacterial fibrinolytic enzyme, 111 bacterial strains with fibrinolytic activity were selected from kimchi. Among them, 14 strains were selected because of their stronger activity than 0.02 U of plasmin. Their 16S rRNA sequence analysis revealed that they belong to Bacillus, Leuconostoc, Propionibacterium, Weissella, Staphylococcus, and Bifidobacterium. The strain B. subtilis ZA400, with the highest fibrinolytic activity, was selected and the gene encoding fibrinolytic enzyme (bsfA) was cloned and expressed in the E. coli overexpression system. The purified enzyme was analyzed with SDS-PAGE, western blot, and MALDI-TOF analyses, showing to be 28.4 kDa. Subsequently, the BsfA was characterized to be stable under various stress conditions such as temperature (4-40oC), metal ions (Mn²⁺, Ca²⁺, K²⁺, and Mg²⁺), and inhibitors (EDTA and SDS), suggesting that BsfA could be a good candidate for development of a novel fibrinolytic enzyme for thrombosis treatment and may even be useful as a new bacterial starter for manufacturing functional fermented foods.

• Journal of Microbiology and Biotechnology
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• v.30 no.7
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• pp.1072-1081
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• 2020

Screening suitable strains with high temperature adaptability is of great importance for reducing the cost of temperature control in microalgae cultivation, especially in summer. To obtain high temperature-tolerant diatoms, water samples were collected in summer from 7 different regions of China across the Northeast, North and East. A total of 731 water samples was collected and from them 131 diatom strains were isolated and identified based on the 18S rRNA sequences. Forty-nine strains out of the 131 diatoms could survive at 30℃, and 6 strains with relatively high biomass and lipid content at high temperature were selected and were found to be able to grow at 35℃. Cyclotella sp. HB162 had the highest dry biomass of 0.46 g/l and relatively high triacylglycerol (TAG) content of 237.4 mg/g dry biomass. The highest TAG content of 246.4 mg/g dry biomass was obtained in Fistulifera sp. HB236, while Nitzschia palea HB170 had high dry biomass (0.33 g/l) but relatively low TAG content (105.9 mg/g dry biomass). N. palea HB170 and Fistulifera sp. HB236 presented relatively stable growth rates and lipid yields under fluctuating temperatures ranging from 28 to 35℃, while Cyclotella HB162 maintained high lipid yield at temperatures below 25℃. The percentage of saturated fatty acids and monounsaturated fatty acids in all the 6 strains was 84-91% in total lipids and 90-94% in TAGs, which makes them the ideal feedstock for biodiesel.

• Journal of the Korean Magnetic Resonance Society
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• v.3 no.2
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• pp.100-108
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• 1999

Cop protein is the transcription repressor protein in rolling circle replication plasmid. With antisense RNA, Cop protein controls the copy number of plasmid. Cop family proteins have been found in various plasmids. Among Cop family proteins, Cop studied in this paper consists of 55 amino acids (Mw. 6,400), and was known to have trimer structure. Since no structural facts are elucidated, we have carried out preliminary experiments aimed at the elucidation of its three dimensional structure. The secondary structure of Cop is studied by CD and NMR. To solve the aggregation of Cop at high concentration, we tested various detergents and salts. The addition of detergents and salts could not solve the aggregation problem. However, we found that concentration is important in solving the aggregation problem. We knew that 0.18mM in 50mM potassium phosphate without any other ingredients is maximum concentration not to aggregate. Wa also investigated the pH dependence of Cop protein, and knew that Cop protein is more stable in acid state. At various temperatures, 15N-1H HSQC spectra were measured in order to find the optimal experimental condition. To enhance the peak resolution, 3D NOESY-HSQC spectrum is acquired. Since there are NOE peaks in the NH-NH region, we knew that Cop protein has $\alpha$-helical content, which was also confirmed by CD.

• Journal of Microbiology and Biotechnology
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• v.20 no.2
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• pp.254-264
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• 2010

To obtain an efficient natural lignocellulolytic complex enzyme, we screened an efficient lignocellulose-degrading composite microbial system (XDC-2) from composted agricultural and animal wastes amended soil following a long-term directed acclimation. Not only could the XDC-2 degrade natural lignocelluloses, but it could also secrete extracellular xylanase efficiently in liquid culture under static conditions at room temperature. The XDC-2 degraded rice straw by 60.3% after fermentation for 15 days. Hemicelluloses were decomposed effectively, whereas the extracellular xylanase activity was dominant with an activity of 8.357 U/ml on day 6 of the fermentation period. The extracellular crude enzyme noticeably hydrolyzed natural lignocelluloses. The optimum temperature and pH for the xylanase activity were $40^{\circ}C$ and 6.0. However, the xylanase was activated in a wide pH range of 3.0-10.0, and retained more than 80% of its activity at $25-35^{\circ}C$ and pH 5.0-8.0 after three days of incubation in liquid culture under static conditions. PCR-DGGE analysis of successive subcultures indicated that the XDC-2 was structurally stable over long-term restricted and directed cultivation. Analysis of the 168 rRNA gene clone library showed that the XDC-2 was mainly composed of mesophilic bacteria related to the genera Clostridium, Bacteroides, Alcaligenes, Pseudomonas, etc. Our results offer a new approach to exploring efficient lignocellulolytic enzymes by constructing a high-performance composite microbial system with synergistic complex enzymes.

• Journal of Veterinary Clinics
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• v.20 no.2
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• pp.150-154
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• 2003

A disease with severe diarrhea occurred in a herd of one thousand, 1-week-old piglets in Chinju, Korea, and was diagnosed as porcine epidemic diarrhea by the detection of N gene of porcine epidemic diarrhea virus (PEDV) from small intestines. A PEDV, named as Chinju99, was also isolated from the intestines after two blind-passages in Vero cells supplemented with trypsin (10 ug/ml). and the biological and physicochemical properties of the isolate were characterized. The virion was roughly spherical in shape and had spike peplomers on its outer surface. The virus exhibited cytopathic effects such as rounding degeneration at initiation of infection and syncytia formation later in Vero cells. The virus was labile to 20% ether and 5% chloroform but stable in acid with pH 4-7 at $4^{\circ}C$. The infectivity of the virus was maintained at $50^{\circ}C$ for 180 min, and the buoyant density of the virus in sucrose was 1.180 g/ml. All biological and physicochemical properties of the virus were typical features of coronaviruses.

• Biomedical Science Letters
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• v.12 no.4
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• pp.399-403
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• 2006

Cervical cancer is one of the most prevalent cancers developed in women worldwide, and human papillomavirus (HPV) type 16 is the most common agent linked to human cerivical carcinoma. Viral oncogenes E6 and E7 are selectively ratined and expressed in carcinoma cells infected with human papillomavirus type 16 and cooperated with each other in immortalization and transformation of primary keratinocytes. Because of HPV oncogenesis mechanism was not completely solved, the more studies be required thoroughly. In the present study, to investigate the telomere independent role of telomerase in HPV oncogenesis, we constructed the E6 mutant, E7, E6/E7 and hTERT over-expressed stable cells with a telomerase negative cell line, SW13. Expressions of Inserted genes were measured by RT-PCR. E6, E7 and hTERT genes were well expressed in each cell lines comparing with the control groups. By analyzing the cell morphology under the microscope, hTERT clone size was a more smaller than the mock control but oncogene expressed clones were slightly lengthened the marginal region. In addition, hTERT cells has also, a tendency of brief dividing time compared to the mock control. To determine whether telomerase activity associated with a HPV oncogenesis by oncoprotein expression, we performed the PCR based TRAP assay and Northern blot analysis. In TRAP assay data, telomerase activities in hTERT and oncogene clones were more increased than the mock control. In addition, SW13/ E6/E7 cells appeared a extremely increased activity than any other clones. Induced TERT mRNA by E6/E7 wasn't, however, detected in Northern blotting. In conclusion, these findings suggest that telomerase activity closely associated the HPV oncogenesis and E6/E7 co-expression is a most important factor of telomerase activity.

• Fisheries and Aquatic Sciences
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• v.18 no.1
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• pp.73-80
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• 2015

To examine the interrelationship between transgenic insertion patterns and transgene expression profiles in established transgenic fish lines, four stable transgenic marine medaka Oryzias dancena germlines harboring ${\beta}$-actin regulator-driven RFP reporter constructs were selected. The established transgenic strains were characterized with regard to their transgenic genotypes (insertion pattern, concatemer formation, and transgene copy number based on genomic Southern blot hybridization and qPCR assay) and expression characteristics at the mRNA (qRT-PCR), protein (western blot), and phenotypic (fluorescent appearance) levels. From comparative examinations, it was found that transgenic expression at both the transcription and translation levels could be significantly downregulated in transgenic strains, potentially through methylation-mediated transgene silencing that was particularly associated with the formation of a long tail-to-head tandem concatemer in the chromosomal integration site(s). When this occurred, an inverse relationship between the transgene copy number and fluorescence intensity was observed in the resultant transgenic fish. However, with the other transgenic genotype, transgenic individuals with an identical Southern blot hybridization pattern, containing a tandem concatemer(s), had very different expression levels (highly robust vs. low expression strengths), which was possibly related to the differential epigenetic modifications and/or degrees of methylation. The concatemer-dependent downregulation of transgene activity could be induced in transgenic fish, but the overall pattern was strain-specific. Our data suggest that neither a low (or single) transgene copy number nor tandem transgene concatemerization is indicative of strong or silenced transgene expression in transgenic fish carrying a ubiquitous transgene. Hence, a sufficient number of transgenic lineages, with different genotypes, should be considered to ensure the establishment of the best-performance transgenic line(s) for practical applications.

• Journal of Soil and Groundwater Environment
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• v.22 no.2
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• pp.41-51
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• 2017

Microbial processes that bind heavy metals and form minerals are widespread, and they represent a basic aspect of biogeochemistry. Some microorganisms can crystallize minerals by secreting a specific enzyme. In particular, calcite ($CaCO_3$) precipitation is an important part of biomineralization, and has been studied extensively because of its wide application in civil engineering technology. This process provides an effective way to stabilize heavy metals within a relatively stable crystal phase. In this study, biomineralization of calcite by three urea-hydrolyzing indigenous bacterial strains was investigated by microbiological analyses. Three bacterial strains were isolated from the sludge of B mine in Mexico and each bacterial strain was identified by the cellular fatty acid composition and 16S rRNA partial sequencing analysis. The results of the identification analysis showed that these strains were closest to Sporosarcina pasteurii, Kurthia gibsonii, and Paenibacillus polymyxa. We found that the optimum conditions for growth of these indigenous bacteria were $30-40^{\circ}C$ and pH range of 7-8. Microbiological analyses showed the possibility that the bioaccumulated heavy metals ions were deposited around the cell as crystalline carbonate minerals under the optimum conditions. The findings of our study suggest that the indigenous bacterial strains play an important role in heavy metal immobilization.

• Asian-Australasian Journal of Animal Sciences
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• v.33 no.11
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• pp.1858-1865
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• 2020

Objective: The microbiota of dairy cow milk varies with the season, and this accounts in part for the seasonal variation in mastitis-causing bacteria and milk spoilage. The microbiota of the cowshed may be the most important factor because the teats of a dairy cow contact bedding material when the cow is resting. The objectives of the present study were to determine whether the microbiota of the milk and the cowshed vary between seasons, and to elucidate the relationship between the microbiota. Methods: We used 16S rRNA gene amplicon sequencing to investigate the microbiota of milk, feces, bedding, and airborne dust collected at a dairy farm during summer and winter. Results: The seasonal differences in the milk yield and milk composition were marginal. The fecal microbiota was stable across the two seasons. Many bacterial taxa of the bedding and airborne dust microbiota exhibited distinctive seasonal variation. In the milk microbiota, the abundances of Staphylococcaceae, Bacillaceae, Streptococcaceae, Microbacteriaceae, and Micrococcaceae were affected by the seasons; however, only Micrococcaceae had the same seasonal variation pattern as the bedding and airborne dust microbiota. Nevertheless, canonical analysis of principle coordinates revealed a distinctive group comprising the milk, bedding, and airborne dust microbiota. Conclusion: Although the milk microbiota is related to the bedding and airborne dust microbiota, the relationship may not account for the seasonal variation in the milk microbiota. Some major bacterial families stably found in the bedding and airborne dust microbiota, e.g., Staphylococcaceae, Moraxellaceae, Ruminococcaceae, and Bacteroidaceae, may have greater influences than those that varied between seasons.