• Microbiology and Biotechnology Letters
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• v.47 no.3
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• pp.412-422
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• 2019

The present study reports the morphological and molecular characterization of the fungal strain, CMSS06 and evaluates its raw starch hydrolyzing ability in four different agricultural substrates (rice bran, banana peel, cassava tubers, and coconut water). The potential use of each agricultural substrate to replace the expensive fermentation media was evaluated with six different fermentation media: rice bran (RB), banana peel (BP), cassava starch (CS), cassava in coconut water (CSCW), cassava in modified coconut water (CMCW), and pure Coconut water (CW). The fungal strain CMSS06 was identified as Thielaviopsis ethacetica by the analysis of the ITS sequences. The T. ethacetica alpha amylase enzyme exhibited maximum alpha amylase activity at 72 h, pH 7.0, and $40^{\circ}C$ on soluble starch. This species resulted in the highest enzyme activity (mU/ml) of 26.06, 10.89, 58.82, 14.2, and 54.67 with the RB, BP, CS, CSCW, and CMCW fermentation media, respectively. The results indicate that CS can be used as a carbon substrate and CMCW can be used to accelerate the fermentation by T. ethacetica. The enzyme was partially purified by 40-60% ammonium sulphate fraction, and it showed total enzyme activity, total protein content, specific activity, purification fold, and a recovery of 2400 mU, 30 mg, 80 mU/mg, 2.7, and 71.1%, respectively. The molecular mass of the T. ethacetica alpha amylase was estimated on SDS-PAGE, and two bands around 50 kDa and 70 kDa were identified. The present study implies that T. ethacetica can produce alpha amylase, and it can be used to hydrolyze raw starch during the fermentation processes.

• Korean Journal of Veterinary Research
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• v.37 no.3
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• pp.541-546
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• 1997

There are two conserved regions with a significantly high amino acid sequence homology among the A subunits of STX, SLTs and ricin. To produce an inactive Verotoxin-2 (VT-2), two different mutants, pE167D and pDE5A, were constructed by site-directed mutagenesis, respectively, on the basis of the previous reports that two regions lie within the active-site clefts of the A subunits of ricin and STX family. The cytotoxicity ($10^3$ $CD_{50}/ml$) of VT-2 holotoxin with E167D mutation was reduced by $10^3$-fold compared with wild-type level. In addition, VT-2 with DE5A ($Trp_{202}GlyArgIleSer_{206}$) deletion mutation showed a significantly low cytotoxicity ($10^1$ $CD_{50}/ml$), resulting in $10^5$- and $10^2$-fold reductions, respectively, compared with the wild-type and E167D mutatant. SDS-PAGE for protein samples showed a 33-kDa band corresponding to the A subunit of VT-2. These results indicate that reduction in cytotoxic activity was affected not by amount of VT-2 protein produced but by mutation.

• The Journal of the Korean Society for Microbiology
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• v.35 no.2
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• pp.97-108
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• 2000

Two-dimensional gel electrophoresis (2-DE) is an essential tool of proteomics to analyse the entire set of proteins of an organism and its variation between organisms. Helicobacter pylori was tried to identify differences between strains. As the first step, whole H. pylori was lysed using high concentration urea contained lysis buffer [9.5 M Urea, 4% CHAPS, 35 mM Tris, 65 mM DTT, 0.01% SDS and 0.5% Ampholite (Bio-Rad, pH 3-10)]. The extract ($10\;{\mu}g$) was rehydrated to commercially available immobilised pH gradient (IPG) strips, then the proteins were separated according to their charges as the first dimensional separation. The IPG strips were placed on Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) to separate according to molecular mass of the proteins as the second dimension. The separated protein spots were visualised by silver staining in order to compare different expression of proteins between strains. Approximately 120 spots were identified in each mini-protein electrophoresised gel, furthermore about 65 to 75 spots were regarded as identical proteins in terms of pI value and molecular weight between strains used. In addition, distinct differences were found between strains, such as 219-1, Y7 and Y14, CH150. Two representative strains were examined using strips which had pH range from 4 to 7. This strips showed a number of isoforms which were considered large spots on pH range 3-10. Furthermore, the rest of spots on pH 4-7 IPG strips appeared very distinctive compared to broad range IPG strips. 2-DE seems to be an excellent tool for analysing and identifying variations between H. pylori strains.

• Applied Biological Chemistry
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• v.34 no.3
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• pp.249-257
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• 1991

To elucidate enzymatic properties of ${\alpha}-galactosidase$ (EC 3, 2, 1, 22) from germinated soybean, changes in the enzyme activities and oligosaccharide contents during germination of soybean were determined. ${\alpha}-Galactosidase$ from germinated soybean was purified by ammonium sulfate fractionation, ion exchange chromatography and gel filtration. Their chemical and enzymatic properties was investigated. ${\alpha}-galactosidase$ activity of sobeam was maximized when it was germinated at $25^{\circ}C$ for 120 hour. Raffinose and stachyose in soybean were decomposed completely after 96 hours and 120 hours of germination, respectively. Soybean ${\alpha}-galactosidase$ was purified by 6.6 fold by ammonium sulfate fractionation, ion exchange chromatography on DEAE-Cellulose and Sephadex A-50, and gel filtration on Sephadex G-150. Its specific activity was 825 Units/mg protein and the yield was 2.5% of the total activity of crude extracts. The purified ${\alpha}-galactosidase$ of soybean was found to be homogeneous by polyacrylamide gel electrophoresis and by HPLC. Isoelectric point of soybean ${\alpha}-galactosidase$ was determined analytical isoelectric focusing to be pH 4.8. The soybean ${\alpha}-galactosidase$ was monomeric and its molecular weight was estimated to be 30,000 by SDS-PAGE. The optimal temperature and pH for the soybeam ${\alpha}-galactosidase$ activity were $40^{\circ}C$ and pH 6.0 and 75% of its activity was lost by heating at $60^{\circ}C$ for 10 min. The enzyme was appeared to have higher affinity to raffinose than to stachyose. The Km value of soybean enzyme was 5.3 mM for ${\rho}-nitrophenyl-{\alpha}-D-galactopyranoside$ and the activation energy on PNPG was calculated to be 13.02 Kcal per mole.

• Korean Journal of Weed Science
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• v.9 no.3
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• pp.245-249
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• 1989

The effects of varying concentrations and durations of butachlor [N-(bytoxymety 1,)-2-chlor -2, 6-diethy lacetanilide treatment on oat(Avena sativa L.) root cell division and protein synthesis were studied. The highest concentration ($1{\times}10^{-3}M$) of butachlor caused the significant inhibition of cell division after 18hrs treatment. After 18hrs treatment, 59% and 82% inhibition of cell division occurred at $1{\times}10^{-4}M$ and $1{\times}10^{-3}M$, respectively, while 9% inhibition of cell division did at $1{\times}10^{-6}M$ concentration at the same exposure period. To investigate protein synthesis, the oats were treated for 18 and 24hrs with concentrations ranging from $1{\times}10^{-6}M$ to $1{\times}10^{-3}M$ butachlor. After 18hrs, butachlor treatment of oat with $1{\times}10^{-4}M$ inhibitited 23% protein synthesis, and butachlor treatment with $1{\times}10^{-4}M$ caused 34% inhibition after 24hrs. With SDS-PAGE of proteins extracted from oat root tips, butachlor usually inhibited the 16, 18, 30, 43 and 43.5 kD polypeptide, and proteins of root tips are made up of subunits below 100 kD polypeptide.

• Plant Resources
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• v.5 no.1
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• pp.29-44
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• 2002

In this study, plant regeneration through in vitro culture from plantlet stems of Yooja (C. junos Sieb.) and trifoliate orange (P. trifoliata Rafin.) was attempted to make mass-production system of virus-free plants having the same genotype with mother plant. In order to investigate physiological change depending on the developmental stage of plant regeneration, the changes of total protein, peroxidase and esterase activity and their isozyme patterns as well were examined in 1/2 MS medium. The results are as follows : 1. The MS medium for the optimal callus induction and shoot formation was utilized. The medium was supplemented either with 2,4-D and Kinetin or with BA and NAA. The optimal concentrations were the combination of 1.0mg/ 2,4-D +0.3mg/ Kinetin and 1.0mg BA +0.3mg NAA in callus induction and shoot formation, respectively. 2. For the plant regeneration from somatic embryos, 1/2 MS medium was used with supplements of growth regulators (free, 1.0mg/ IBA +1.0mg/ BA ,0.5mg/ IBA +0.5mg/ BA). Shooting and rooting were the best in the treatment of 0.5mg/ IBA and 0.5mg/ BA combination. 3. The total protein content has a tendency of increase with the developmental stage of embryo, but it was decreased at the plantlet. Also it was the highest at 8 and 6 weeks stage in C. junos Sieb. and P. trioliata Rafin, respectively. In the SDS-PAGE pattern of protein, C. junos Sieb. showed bands of 29.0 and 40kDa at 10 weeks. The 45,66 and 97.4 kDa bands at 10 weeks of culture were shown in P. trifoliata Rafin. 4. The highest esterase activity was shown at the 6 and 8 weeks of culture in C.junos Sieb. and P. trifoliata Rafin.., respectively. 5. Esterase isozyme patterns were shown difference according to the developmental stage. In C. junos Sieb. a new band was observed at pl 7.7 following 4 weeks culture. On the other hand, new bands in P. trifoliata Rafin. were observed at pl 7.5~6.5 following 4 and 6 weeks culture, respectively.

• Clinical and Experimental Reproductive Medicine
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• v.22 no.2
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• pp.191-201
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• 1995

Transcriptional activation of the embryonic genome initiates at 2-cell stage in mouse embryo and is characterized by the synthesis of TRC which is restricted to 2-cell stage. To investigate the roles of various protein kinases on the embryonic gene activation, the effects of protein kinase inhibitors on in vitro development and protein synthetic profiles of the early mouse embryos were examinded. None of ${\alpna}-amanitin$ which is a mRNA synthetic inhibitor, H8 which is a PKA inhibitor, and H7 which is a PKC inhibitor, affected on first cleavage of mouse 1-cell embryos in vitro. However, all of these drugs inhibited the second cleavage. When the drugs were removed following treatment for 6 hours, H8 or H7 treatment showed little inhibition on subsequent development of 1-cell embryos to 2-cell stage or further. In contrast, ${\alpna}-amanitin$ irreversibly inhibited the development of 1-cell embryos to 2-cell stage following removal of the drug. Genistein, a TPK inhibitor, inhibited both the first cleavage of 1-cell embryos and the second cleavage of 2-cell embryos, suggesting that TPK activity may be important during the early cleavages. All of the above four drugs inhibited TRC synthesis as shown by the fluorographic analysis of $[^{35}S]-Met$ labeled protein profiles. When late 1-cell embryos were treated with H7 and analyzed synthetic patterns of $[^{35}S]-Met$ labeled protein, the quantitative differences of protein synthesis on SDS-PAGE appeared on 77 kD and 33 kD region at $32{\sim}38$ hours post hCG. From these studies, transcriptional activation of embryonic genome is not essenting to the mouse 1-cell embryos to develop to 2-cell stage. Hawever, TPK activity is reguisite for both the first cleavage and second cleavage. Similarly, both PKC and PKA activities are required for the second cleavage of mouse embryos, but not for the first cleavage.

• Journal of Life Science
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• v.28 no.1
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• pp.99-104
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• 2018

Streptococcus pneumoniae is one of the major pathogens in community-acquired diseases, and it contains several factors that promote its pathogenesis, including pneumolysin (PLY). PLY is a member of the cholesterol-dependent cytolysin family, which attacks cholesterol-containing membranes, thereby forming ring-shaped pores. Thus, it is a major key target for vaccines against pneumococcal disease. We cloned the PLY gene from S. pneumoniae D39 and inserted it into the pQE-30 vector. Recombinant PLY (rPLY) was overexpressed in Escherichia coli M15 and purified by $Ni^{2+}$ affinity chromatography. Similarly, a PLY-EGFP fusion gene was produced by inserting the EGFP gene at the 3' end of the PLY gene in the same vector, and the recombinant protein was purified. Sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) showed that both recombinant proteins were purified. rPLY exhibited significant hemolytic activity against 1% human red blood cells (RBCs). Complete hemolysis was obtained at 500 ng/ml, and 50% hemolysis was found with a 240 ng/ml concentration. In contrast, rPLY-EGFP did not show hemolytic activity. However, rPLY-EGFP did bind the RBC membrane, indicating that rPLY-EGFP lost hemolytic activity via EGFP fusion, while retaining its membrane-binding ability. These data suggest that PLY's C terminus is important for its hemolytic activity. Therefore, these two recombinant proteins can be extremely useful for investigating the toxin mechanism of PLY and cell damage during pneumonia.

• Journal of Life Science
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• v.21 no.12
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• pp.1726-1731
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• 2011

Human Stem Cell Factor (hSCF) is a cytokine that binds to the c-Kit receptor and plays an important role in hematopoiesis, spermatogenesis, and melanogenesis. To produce the human Stem Cell Factor (hSCF) recombinant protein, we constructed a germline transgenic silkworm using the piggyback vector. The expression of the hSCF gene was driven by the Drosophila heat shock protein 70 (dHsp70) promoter. 3XP3 promotor-driven EGFP was used as a marker which allowed us to rapidly distinguish the transgenic silkworm. A mixture of the donor and helper vector was micro-injected into 1,020 eggs of bivoltin silkworms, Keomokjam. We obtained approximately 22 G1 broods that were EGFP-positive. The expression of the hSCF gene in the transgenic silkworm was analyzed by SDS-PAGE and immunoblotting. Also, analysis of insertion sites into the silkworm genome using inverse PCR showed that exogenous DNA was inserted into the transgenic silkworm genome. These results show that successfully constructed transgenic silkworm expresses the hSCF recombinant protein.

• Korean Journal of Weed Science
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• v.12 no.4
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• pp.368-373
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• 1992

The effect of alachlor treatment on protein synthesis was studied. Protein synthesis was inhibited by $1{\times}10^{-4}$ M and $1{\times}10^{-3}$M of alachlor 5.8% and 86.5%, respectively, while did not occur blow $1{\times}10^{-5}$M alachlor. Soluble protein of alachlor treated oat root tips was examined by polyacrylamide gel electrophoresis. The proteins extracted from oat root tips showed that they were made up of subunits blow 100 kd polypeptides by SDS-PAGE. As compared to control, high molecular proteins(above 47 kd) were inhibited of oat root treated with alachlor, while low molecular proteins(below 23 kd) were increased. Two-D gels showed that alachlor caused decrease(1-6 spots) or increase(7-10 spots) in number of polypeptides on silver staining. The intensity of some polypeptides of soluble proteins (molecular mass of 83 kd : 1, 2 spots, 70 kd : 3, 4 spots, and 47.5 kd : 5, 6 spots) decreased in alachlor treatment, whereas the intensity of other peptide bands (20 kd : 7 spot and 16 kd : 8, 9, 10 spots) increased. Oat root tip proteins present in the neutral zone are masked by diffusing of major proteins, but proteins in acid zone are resolved minor proteins.