• Journal of Microbiology and Biotechnology
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• v.23 no.11
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• pp.1554-1559
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• 2013

A protease produced by Bacillus polyfermenticus SCD was purified and characterized as a new detergent material. The protease was purified from supernatant produced by B. polyfermenticus SCD, by ammonium sulfate precipitation, ion-exchange chromatography on a DEAE-Sephadex A-50, and finally gel filtration chromatography on Sephadex G-50. The molecular mass of this enzyme was 44 kDa based on SDS-PAGE. The optimum temperature and pH were $50^{\circ}C$ and pH 8.0. The ranges of its stability to the pH and temperature were 7.0 to 9.0 and under $40^{\circ}C$, respectively. The enzyme was highly stable in the presence of the surfactants like Triton X-100 (0.1%), showing a 2-fold increase in its proteolytic activity. However, the enzyme was slightly inhibited by the chelating agent EDTA (1 mM). The enzyme has a maximum activity at $50^{\circ}C$ and the activity can be increased by surfactants such as Triton X-100 and Tween 80.

• Animal cells and systems
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• v.16 no.6
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• pp.455-461
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• 2012

Amongst the various antimicrobial peptides, lysozyme plays a central role in initiating and maintaining the antibacterial defense response of insect. Here we propose the biosynthesis and refolding of recombinant lysozyme in Escherichia coli expressed in inclusion body form. The Agrius lysozyme gene was amplified using gene specific primers and then ligated into the pGEX-4T-1 vector, which contained the glutathione S-transferase (GST) gene as a fusion partner. A recombinant lysozyme was expressed in E. coli Rosetta cells using a pGEX-4T-1 expression vector, and the fusion protein was induced by ioporpyl-${\beta}$-D-thiogalactopyranoside (IPTG). The recombinant protein produced as an inclusion body was resolubilized in solubilization buffer, and the resultant solution was dialyzed in refolding buffer. After thrombin cleavage, the recombinant lysozyme was purified by ion exchange chromatography and reverse phase chromatography. The recombinant lysozyme was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and immunoreactivity against the anti-Agrius lysozyme was observed by western blot analysis of this protein. The recombinant lysozyme displayed antibacterial activity against Bacillus megaterium and Micrococcus luteus, which was confirmed by the inhibition zone assay.

• Journal of the Korean Society of Food Science and Nutrition
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• v.15 no.3
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• pp.276-285
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• 1986

This study was undertaken to investigate the characteristics of the proteolytic enzyme extracted from Neungee mushroom [Sarcodon aspratus (Berk.) S. Ito]. The enzyme was purified by using Tris-acryl CM-cellulose ion exchange, gel filtration on Ultrogel AcA 54, Hydroxy apatite column chromatography and preparative isoelectic focusing. The specific activity of the purified enzyme increased 8 times as compared with that of the crude enzyme. The enzyme was homogeneous on polyacrylamide gel electrophoresis (PAGE). The optimum pH was 10.1, indicating the enzyme to be alkaline protease and the optimum temperature was $57^{\circ}C$. The enzyme was stable at temperatures lower than $50^{\circ}C$and at pH values ranging from 4.0 to 10.8. However, the enzyme activity decreased by 26 and 65% at 60 and $65^{\circ}C$, respectively, when incubated for 30 minutes. The enzyme activity was activated by $Mn^{++}$ and inhibited by $Cu^{++}$ and $Hg^{++}$. The enzyme was consisted of monomer and its molecular weight estimated to be about 30,100 when determined by sodium dodecyl sulfate PAGE. Isoelectric point of the enzyme was determined to be 9.80.

• Reproductive and Developmental Biology
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• v.29 no.3
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• pp.187-191
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• 2005

This study was designed to investigate the changes of quantity and quality of proteins in medium and cytoplasm during in vitro maturation of bovine oocytes. The total quantity of proteins in medium decreased from 0 to 4.5 hr, but increased from 13.5 to 18 hr after the onset of in vitro maturation. The total quantity of protein in cytoplasm increased from 0 to 4.5 hr, decreased from 4.5 to 9 hr, and increased after 18 hr after the onset of in vitro maturation. A total of 298 protein spots was detected on a gel of 2D SDS-PAGE form maturation medium. Among 28 protein spots expressed significant differences in their quantity, 8 proteins were identified by peptide mass fingerprinting (aldose reductase, alpha enolase, apolipoprotein A-1 precursor, 43kDa collectin precursor, heat shock 27kDa protein, plasminogen activator inhibitor-1 precursor, thrombospondin 1, transitional endoplasmic reticulum ATPase). Among total of 35 protein spots detected on gel of 2D SDS-PACE from oorytes cytoplasm, $\beta$-tubulin was identified by peptide mass fingerprinting.

• BMB Reports
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• v.37 no.3
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• pp.298-303
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• 2004

In general, a SYPRO Ruby dye is well known as a sensitive fluorescence-based method for detecting proteins by one-or two-dimensional SDS-PAGE (1-DE or 2-DE). Based on the SYPRO Ruby dye system, the combined two-dimensional fibrin zymography (2-D FZ) with SYPRO Ruby staining was newly developed to identify the Bacillus sp. proteases. Namely, complex protein mixtures from Bacillus sp. DJ-4, which were screened from Doen-Jang (Korean traditional fermented food), showed activity on the zymogram gel. The gel spots on the SYPRO Ruby gel, which corresponded to the active spots showing on the 2-D FZ gel, were analyzed by a matrix-assisted laser desorption ionization time of flight (MALDI-TOF) mass spectrometric analysis. Five intracellular fibrinolytic enzymes of Bacillus sp. DJ-4 were detected through 2-D FZ. The gel spots on the SYPRO Ruby dye stained 2-D gel corresponding to 2-D FZ were then analyzed by MALID TOF MS. Three of the five gel spots proved to be quite similar to the ATP-dependent protease, extracellular neutral metalloprotease, and protease of Bacillus subtilis. Also, the extracellular proteases of Bacillus sp. DJ-4 employing this combined system were identified on three gels (e.g., casein, fibrin, and gelatin) and the proteolytic maps were established. This combined system of 2-D zymography and SYPRO Ruby dye should be useful for searching the specific protease from complex protein mixtures of many other sources (e.g., yeast and cancer cell lines).

• The Korean Journal of Zoology
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• v.32 no.4
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• pp.429-440
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• 1989

Spirometru erinocei의 유충인 sparganum에서 추출한 조항원 단백질을 항원으로 하여 sparganosis, cysticercosis, hydatidosis환자의 IgG 항체와 정상인의 IgG 항체를 혈청반응시켜 ELISA와 EITB에 의해 교차반응을 일으키는 비 특이항원 성분과 종특이항원 성분을 추구하였다. 1. sparganum에서 0.01 M PBS (PH 7.4)로 추출한 조항원 단백질을 SDS-PAGE로 전개하여 290 Kd에서 23 Kd범위의 분자량을 가진 25개의 단백질이 분획되었다. 2. sparganum의 조항원을 항원으로하여 ELISA로 sparganosis, cysticercosis, hydatidosis 환자의 190 항체와의 혈청 반응치는 sparganosis환자에게서는 0.44 $\pm$ 0.07에서 1.90 $\pm$ 0.03으로 negative control normal sera의 0.D (0.15 $\pm$ 0.03)를 기준으로 하였을 때 모두 양성이며 민감도(sensitivity)가 100 %이었으며 cysticercosis, hydatidosis환자혈청에서 양성반응이 나타났으며 교차반응도 있었다. 3. EITB에서는 spargancsis환자의 IgG항체에 의해 16개의 항원성분이 인지되었으며 이 중 6개의 항원성분이 정상인의 혈청에서도 인지되어 교차반응을 일으키는 항원성분이었으며 cysticercosis환자혈청에서 인지된 4개의 항원성분 중 2개의 항원성분이 sparganosis 환자혈청에서 인지된 것과 같았으며 hydatidosis환자의 190항체에 의해 인지된 19개의 항원성분 중 12개의 항원성분이 sparganosis환자혈청에서 인지된 항원 성분과 같았다. 4. 290 Kd, 200 Kd, 28 Kd의 항원성분은 sparganosis환자의 196항체에서만 인지되었고 228 Kd, 152 Kd, 66 Kd항원성분은 hydatidosis환자의 19G항체에서만 인지되었으며 66 Kd항원성분은 sparganosis, cysticercosis, hydatidosis, 정상인의 혈청에서 모두 인지되었다. We studied the serological reaction between the specific and nonspecific antigenic components from metacestode (plerocercoid) of spiromeko erimacei and IgG antibodies in sparganosis, cysticercosis. hydatidosis patients and normal human sera by ELISA and EITB. We prepared the crude extracts of sparganlim from snake, Matrix tigrina laterolis and used as antigenic components. By SDS-PAGE, we detected a total 25 peptide bands (fractions) with 290 Kd to 23 Kd molecular weight, and 8 bands of these detected bands developed strongly by silver stain. In serological test, ELISA, we recognized the cross-reaction of antigenic components reacting with IgG antibodies in heterogenous sera, cysticercosis and hydatidosis patients sera. The crude antigenic components of sparganum showed the high sensitivity in sparganosis, hydatidosis patients sera, but showed lower sensitivity in cysticercosis patients sera than the sparasanosis, hydatidosis patients sera. Sixteen antigenic components of these 25 separated bands were recognized by antibodies In sparsanosis patients sera,8 antigenic components in normal human sera, 4 antigenic components in cysticercosis patients sera and 19 antigenic compoenents in hydatidosis patients sera. The crude antigenic compnenets with 290 Kd, 200 Kd, 125 Kd and 28 Kd molecular weight was only recognized in sparganosis patients sera, but 64 Kd antigenic component was nonspecific antigenic components which were also cross-reacted with sparganosis, hydatidosis, cysticercosis patients sera and normal human sera.

• Journal of Microbiology and Biotechnology
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• v.34 no.2
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• pp.436-456
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• 2024

Several thermostable proteases have been identified, yet only a handful have undergone the processes of cloning, comprehensive characterization, and full exploitation in various industrial applications. Our primary aim in this study was to clone a thermostable alkaline protease from a thermophilic bacterium and assess its potential for use in various industries. The research involved the amplification of the SpSKF4 protease gene, a thermostable alkaline serine protease obtained from the Geobacillus thermoglucosidasius SKF4 bacterium through polymerase chain reaction (PCR). The purified recombinant SpSKF4 protease was characterized, followed by evaluation of its possible industrial applications. The analysis of the gene sequence revealed an open reading frame (ORF) consisting of 1,206 bp, coding for a protein containing 401 amino acids. The cloned gene was expressed in Escherichia coli. The molecular weight of the enzyme was measured at 28 kDa using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The partially purified enzyme has its highest activity at a pH of 10 and a temperature of 80℃. In addition, the enzyme showed a half-life of 15 h at 80℃, and there was a 60% increase in its activity at 10 mM Ca²⁺ concentration. The activity of the protease was completely inhibited (100%) by phenylmethylsulfonyl fluoride (PMSF); however, the addition of sodium dodecyl sulfate (SDS) resulted in a 20% increase in activity. The enzyme was also stable in various organic solvents and in certain commercial detergents. Furthermore, the enzyme exhibited strong potential for industrial use, particularly as a detergent additive and for facilitating the recovery of silver from X-ray film.

• Microbiology and Biotechnology Letters
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• v.18 no.5
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• pp.541-546
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• 1990

Bacillus sphaericus ts-Dl290 was characterized by SDS-PAGE produced by the mutant at $30^{\circ}C$ and $42^{\circ}C$. The total amount of proteins produced by the mutant at $42^{\circ}C$ decreased to one-fifth of those at $30^{\circ}C$; however, when the culture was shifted down from $42^{\circ}C$ after 4 to $30^{\circ}C$, the total amount of protein decreased to one-third and the 221 kd protein did not appear, but the 155 kd appeared remarkably. When the mutant and the wild type strain were cultured in the media containing 80$\mu g$ per ml of chloramphenicol at $42^{\circ}C$, the wild type strain synthesized half amounts of the total proteins than those at $30^{\circ}C$, and the mutant produced one-tenth of the total protein amounts. When the both strains were cultured in the media containing chloramphenicol, the 155 kd protein was produced was produced in lesser amounts than those without chloramphenicol. The 150 kd protein showed lethal activity to Culex pipiens 3rd instar larvae.

• Korean Journal of Animal Reproduction
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• v.13 no.3
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• pp.179-187
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• 1989

The experiment was carried out to study the profile of uterine specific protein during early pregnancy in sows and to test it's immunosuppressive activity. Uterine protein samples were obtained by flushing the uterine horn on Day 3, 6, 9, 12 and 15 of the estrous cycle and the pregnancy respectively and the protein concentration of each sample was determined. The change of uterine protein was examined by sodium dodecyl sulfate(SDS)-PAGE. The immunosuppressive activity of uterine secretory protein was investigated according to the lymphocyte blastogenesis response to mitogen. The results of this experiment are summarized as follows ; 1. The uterine protein during estrous cycle and early pregnancy was relatively constant up to Day 9, but increased on Day 12. Maxium total protein values were found on Day 15. The concentration of serum proteins were about 82-95 mg during estrous cycle, but decreased to about 70-82 mg during early pregnancy. 2. The proteins components similar electrophoretic patterns(PAGE) that were no differences (band ; a, b, c, d, e, f, g, I) on Days 3, 6 and 9 of the estrous cycle and pregnancy. But there were more 2 bands specifically on Day 12 of the pregnancy and on Day 15 of estrous cycle and showed more 4 bands on Day 15 of early pregnancy. They seemed to be acidoprotein and their average molecular weight were 38,000, 22,300 and 12,600. 3. When uterine protein were added 500$\mu\textrm{g}$/ml, there was no immunosuppresive activity on Day 3 of estrous cycle and lymphocyte blastogenesis was slightly suppressed on Day 3 of pregnancy. The immunosuppressive activity on Day 9 of estrous cycle and pregnancy appeared in 500$\mu\textrm{g}$/ml and 150$\mu\textrm{g}$/ml, respectively the uterine protein on Day 12 and 15 showed immunosuppresive activity, which at the level of 150$\mu\textrm{g}$/ml during non-pregnancy and at the level of 100 to 125$\mu\textrm{g}$/ml during early pregnancy, respectively.

• KOREAN JOURNAL OF CROP SCIENCE
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• v.43 no.4
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• pp.259-263
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• 1998

High molecular weight glutenin (HMW-Glu) subunit compositions of 73 Korean wheat cultivars and experimental lines were evaluated by using one dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This method is suitable for obtaining a good resolution of 1Dx2 and 1Ax2$^*$ without adverse effects on separation of other HMW-Glu subunits. Korean wheats examined in this study could be divided into 15 different groups on the basis of HMW-Glu subunit compositions. From the wheat lines tested, it was identified that there were three alleles at the Glu-Al, five at the Glu-Bl and three at the Glu-D1 loci. The null allele of the Glu-Al was occurred in high frequency (79.4%), while low frequencies for 1Ax1 (12.3%) and 1Ax2$^*$(8.2%) were found. High frequency (75.3%) of the subunit pairs of 1Bx7+1By8 at the Glu-Bl loci compared with other subunits was found. The frequencies of subunits 1Dx2. 2+1Dy12 and 1Dx2+1Dy12 from the Glu-D1 loci were 54. 8% and 37.0%, respectively. However, a few Korean wheat lines (8.2%) carried 1Dx5 + 1Dy10 subunit pair which are responsible for good breadmaking quality. The information of HMW-Glu subunit compositions provide a useful tool to characterize wheat lines, and can be directly used in selection of breeding lines of different end-use properties.