• Korean Journal of Food Science and Technology
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• v.46 no.1
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• pp.115-120
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• 2014

The purpose of this study was to develop a rice straw-derived Bacillus cereus (B. cereus)-free starter culture for traditional soybean fermented products using a B. cereus-specific bacteriophage, BCP8-2. To determine the optimal medium that supports the growth of rice straw-derived microorganisms and BCP8-2 activity, 5 different culture media were tested. The 5% ground bean (GB) medium was selected for further study. No B. cereus was detected in the BCP8-2-treated rice straw in GB medium, whereas B. cereus at a level of $10^7$ CFU/mL was recovered in the no-phage control. The total bacterial count reached approximately $10^9$ CFU/mL regardless of phage addition. When the 16S rRNA sequence-based microbial community was monitored using denaturing gradient gel electrophoresis (DGGE) and pyrosequencing, a similar microbial community was observed in the phage-treated and control samples. In conclusion, we demonstrate that phage can be used to prepare a rice straw-derived B. cereus-free starter culture with minimal effect on natural microflora.

• Journal of Korean Ophthalmic Optics Society
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• v.12 no.1
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• pp.9-15
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• 2007

The aim of this study was to find the proper UV-A blocking percentage that could protect the denaturation of ribonuclease A (RNase A), one of protein enzymes in eye, induced by UV-A. RNase A was irradiated at 365 nm for 1, 3, 6, 24, 48, 72, 96 hr and the extent of denaturation was monitored by polyacrylamide gel electrophoresis. Furthermore, it was investigated whether blocking of UV-A by 20, 50, 80 and 99% eyeglass lens could protect the denaturation of RNase A or not. The denaturation of RNase A was induced by only 1 hr UV-A irradiation and the extent of denaturation became severe depending on UV-A irradiation time. The mild denaturation of RNase A induced by irradiation for 1 hr could be sufficiently protected by 20% UV-A blocking lens. When RNase A was irradiated for 3 hr, more that 50% blocking of UV-A needed to prevent the denaturation. Even though 99% UV-A blocking lens was used, the denaturation of RNase A induced by 6 hr irradiation could not be prevented perfectly. However, 99% UV-A blocking lens could dramatically decrease the severe denaturation of RNase A induced by irradiation for 96 hr.

• Microbiology and Biotechnology Letters
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• v.34 no.1
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• pp.40-46
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• 2006

A bacterial strain producing high level of a phytase was isolated from cattle feces and identified as Bacillus subtilis, and designated as Bacillus sp. CF 5-26. The production of the phytase from Bacillus sp. CF 5-26 reached the highest level after 72 hours at $37^{\circ}C$. The optimum condition of the media for the production of phytase was 10% rice bran extract, 0.1% whey protein powder, $0.01%\;CaCl_{2},\;0.01%\;KH_{2}PO_4$. The phytase was purified 20.3 folds with ethanol precipitation, Sephadex G-100, CM Sepharose CL-6B and Sephacryl S-100-HR column chromatography. The molecular weight of the purified enzyme was estimated to be 66 kDa on SDS-polyacrylamide gel electrophoresis. The purified phytase activity was stable up pH 5.0, 7.0, 11.0 and the remaining activity was 50% when it was treated at $100^{\circ}C$ for 1 hour. The substrate specificity of phytase was most active against sodium phytate and inositol polyphosphate compound. And the phytase hydrolysed tripolyphosphate and pyrophosphate a little. The Km value for the sodium phytate was 0.64 mM and the Vmax value was $4.41\;{\mu}mol/min$.

• Journal of Microbiology and Biotechnology
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• v.19 no.12
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• pp.1656-1664
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• 2009

Bioaugmentation of bioreactors focuses on the removal of numerous organics, with little attention typically paid to the maintenance of high and stable nitrite accumulation in partial nitrification. In this study, a bioaugmented membrane bioreactor (MBR) inoculated with enriched ammonia-oxidizing bacteria (AOB) was developed, and the effects of dissolved oxygen (DO) and temperature on the stability of partial nitrification and microbial community structure, in particular on the nitrifying community, were evaluated. The results showed that DO and temperature played the most important roles in the stability of partial nitrification in the bioaugmented MBR. The optimal operation conditions were found at 2-3 mgDO/l and $30^{\circ}C$, achieving 95% ammonia oxidization efficiency and nitrite ratio ($NO_2^-/{NO_x}^-$) of 0.95. High DO (5-6 mg/l) and low temperature ($20^{\circ}C$) had negative impacts on nitrite accumulation, leading to nitrite ratio drop to 0.6. However, the nitrite ratio achieved in the bioaugmented MBR was higher than that in most previous literatures. Denaturing gradient gel electrophoresis (DGGE) and fluorescence in situ hybridization (FISH) were used to provide an insight into the microbial community. It showed that Nitrosomonas-like species as the only detected AOB remained predominant in the bioaugmented MBR all the time, and coexisted with numerous heterotrophic bacteria. The heterotrophic bacteria responsible for mineralizing soluble microbial products (SMP) produced by nitrifiers belonged to the Cytophaga-Flavobacterium-Bacteroides (CFB) group, and $\alpha$-, $\beta$-, and $\gamma$- Proteobacteria. The fraction of AOB ranging from 77% to 54% was much higher than that of nitrite-oxidizing bacteria (0.4-0.9%), which might be the primary cause for the high and stable nitrite accumulation in the bioaugmented MBR.

• Microbiology and Biotechnology Letters
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• v.41 no.2
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• pp.145-152
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• 2013

The culture conditions to maximize the production of endoglucanase (EC 3.2.1.4) from the brown rot fungus Fomitopsis pinicola MKACC 54347 mycelia were investigated. Among the tested media for endoglucanase production, Mandel's mineral salts medium (MSM; 1% cellulose, 0.1% peptone, 0.14% $(NH_4)_2SO_4$, 0.03% urea, 0.2% $KH_2PO_4$, 0.03% $MgSO_4{\cdot}7H_2O$, 0.03% $CaCl_2$, and 0.1% trace metal solution (19.8 mM $FeSO_4$, 13.0 mM $MnSO_4$, 12.2 mM $ZnSO_4$, and 15.4 mM $CoCl_2$)) produced the highest activity of the enzyme. To optimize the medium composition for enzyme activity, the effects of various carbon, nitrogen, phosphorus, and inorganic sources were investigated in MSM. Maximal enzyme production was accomplished using a medium containing 2% carboxymethyl cellulose (CMC), 2% yeast extract, 0.2% $KH_2PO_4$, 0.03% $MnSO_4$, and 0.3% trace metal solution. Different physiological conditions, like incubation period and temperature, were also examined to assess their influence on enzyme production. Enzyme production from F. pinicola reached its highest level after cultivation for 8 days at $25^{\circ}C$. Nondenaturing polyacrylamide gel electrophoresis (PAGE), followed by the endoglucanase activity staining using CMC as the substrate, was performed to identify the endoglucanase under the culture conditions studied. Zymogram analysis of the culture supernatant revealed an endoglucanase band with a molecular mass of 52 kDa. The optimum pH and temperature for enzyme activity were $55^{\circ}C$ and pH 5.0, respectively.

• Journal of Microbiology and Biotechnology
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• v.28 no.9
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• pp.1547-1553
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• 2018

Hyaluronidases are a family of enzymes that catalyse the breakdown of hyaluronic acid, which is abundant in the extracellular matrix and cumulus oocyte complex. To investigate the activity of recombinant bovine sperm hyaluronidase 1 (SPAM1) and determine the effect of the Asn-X-Ser/Thr motif on its activity, the bovine SPAM1 open reading frame was cloned into the mammalian expression vector pCXN2 and then transfected to the HEK293 cell line. Expression of recombinant bovine hyaluronidase was estimated using a hyaluronidase activity assay with gel electrophoresis. Recombinant hyaluronidase could resolve highly polymeric hyaluronic acid and also caused dispersal of the cumulus cell layer. Comparative analysis with respect to enzyme activity was carried out for the glycosylated and deglycosylated bovine sperm hyaluronidase by N-glycosidase F treatment. Finally, mutagenesis analysis revealed that among the five potential N-linked glycosylation sites, only three contributed to significant inhibition of hyaluronic activity. Recombinant bovine SPAM1 has hyaluronan degradation and cumulus oocyte complex dispersion ability, and the N-linked oligosaccharides are important for enzyme activity, providing a foundation for the commercialization of hyaluronidase.

• Journal of Korean Society of Environmental Engineers
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• v.30 no.11
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• pp.1161-1169
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• 2008

The removals of TCE and PCE vapor with or without a supply of toluene as a primary substrate were compared in a biofiltration process, and the variations of microbial communities associated with the removal were also investigated. As a result of investigations on the removals of TCE/PCE in a biofilter B within which TCE/PCE-acclimated sludge was attached on the surface of media without a supply of primary substrate, and those in another biofilter A where toluene-acclimated sludge was attached with a supply of toluene as a primary substrate, followings were found: (i) parts of microbes responsible to the decomposition of toluene vapor participate in the removal of chlorinated VOCs such as TCE and PCE, and (ii) effective biological removals of TCE and PCE vapor do not necessarily need cometabolism. Sequencing of 16S rDNA obtained from the band profile of DGGE (Denaturating Gradient Gel Electrophoresis), it was confirmed that: (i) uncultured alpha proteobacterium, uncultured Desulfitobacterium, uncultured Rhodobacteraceae bacterium, Cupriavidus necator, and Pseudomonas putida were found to be toluene-decomposing microbes, (ii) alpha proteobacterium HTCC396 is a TCE-removing microbe, (iii) Desulfitobacterium sp. is a PCE-decomposing microbe, and (iv) particularly, uncultured Desulfitobacterium sp. is probably a microbe decomposable not only toluene but also various chlorinated VOC vapor including TCE and PCE.

• Applied Biological Chemistry
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• v.39 no.1
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• pp.1-8
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• 1996

A one-stage, continuous-flow bioreactor with both immobilized and suspended cells was used to investigate the roles of lipid supplements in ethanol production by Saccharomyces cerevisiae. The reactor performance and the level of alcohol dehydrogenase(ADH) activities of the suspended cells, grown under various conditions, were measured. When ergosterol and/or oleic acid were added with surfactants to the yeast culture grown under non-aerated conditions, remarkable increases in ethanol production and cell growth was achieved, but specific ADH activities were not affected. Especially, no difference of specific ADH activities of the suspended cells grown under aerated and non-aerated condition was observed. The addition of the surfactant as a supplement also resulted in significant increases in ethanol production, cell growth, and specific ADH activity. When ergosterol and oleic acid were added to the yeast culture exposed to higher ethanol concentration($>40\;g/{\ell}$) level, ethanol production, cell growth, and specific ADH activity were increased, but the addition of surfactant was as effective as at lower ethanol concentration level. The results indicated that lipid supplements were more effective at higher ethanol concentration level than at lower ethanol concentration level during ethanol production. ADH isozyme patterns of the yeast cultures grown under various conditions on starch gel electrophoresis showed only one major band, probably ADH I. The migrating distance of the major isozyme, however, varied slightly according to the culture conditions of the cells. No apparent correlation was found between specific ADH activity and amount of ethanol produced. Cell mass was more important factor for ethanol production than specific ADH activity of the cells.

• Reproductive and Developmental Biology
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• v.29 no.2
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• pp.83-91
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• 2005

Somatic cell nuclear transfer in cattle has limited efficiency in terms of production of live offspring due to high incidence of fetal failure after embryo transfer to recipients. Such low efficiency of cloning could possibly arise from abnormal and poorly developed placenta. In the present study the placental proteome in late pregnancy established from in vitro fertilization (IVF) and nuclear transfer (NT) was analysed. Proteome alternation was tested using two-dimensional polyacrylamide gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI- TOF). Comparing placenta from NT embryos to those from IVF counterparts, significant changes in expression level were found in 18 proteins. Of these proteins 12 were not expressed in NT placenta but expressed in IVF counterpart, whereas the expression of the other 6 proteins was limited only in NT placenta. Among these proteins, cytokeratin 8 and vimentin are considered to be involved in regulation of post-implantation development. In particular, cytokeratin 8 and vimentin may be used as makers for placental development during pregnancy because their expression levels changed considerably in NT placental tissue compared with its IVF counterpart. Data from 2-DE suggest that protein expression was disorientated in late pregnancy from NT, but this distortion was eliminated with progression of pregnancy. These findings demonstrate abnormal placental development during late pregnancy from NT and suggest that alterations of specific placental protein expression may be involved in abnormal function of placenta.

• Journal of Applied Biological Chemistry
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• v.54 no.2
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• pp.94-100
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• 2011

A gene encoding a putative phospholipase D was isolated from Bacillus licheniformis and cloned into pGEM-T easy vector. The gene was expressed in E. coli BL21 (DE3) using a pET-21(a) vector containing His6 tag. Affinity purification of the recombinant phospholipase D with nickel-nitrilotriacetic acid (Ni-NTA) resin resulted major one-band by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The purified enzyme showed a molecular weight of 44 kDa. The optimum activity of enzyme was around pH 7.0 and the enzyme was also the most stable around this condition. The optimum temperature was about $40-45^{\circ}C$ and the enzyme still showed considerable activities at wide range of temperature. Among various detergents, Triton X-100 significantly increased the enzyme activity, resulting in 181% activity of control at 0.6 mM of the detergent. Calcium ion did not significantly affect the enzyme activity, suggesting that the enzyme might be classified into $Ca^{2+}$-independent PLD.