• Journal of Microbiology and Biotechnology
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• v.11 no.4
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• pp.577-584
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• 2001

An antimicrobially active bacterium which was identified as Bacillus brevis, was isolated from kimchi. The antimicrobial activity was found against various Gram-positive and Gram-negative bacteria including some pathogens food-spoilage microorganisms, and some yeast strains. The antimicrobial activity was especially strong against Bacillus anthracis and Shigella dysenteriae. The strong activity was observed during an early stationary phase in the culture when incubated at $37^{\circ}C$ with initial medium pH of 6.8. The antimicrobial activity was found to be stable at $90^{\circ}C$ for 30 min and in the pH range of 3-11, and it was insensitive to organic solvents including acetone, acetonitrile, ethanol, and methanol. Analysis of the bacterocin on tricine-sodium dodecyl sulfate-polyacrylamide gel suggested a molecular mass of approximately 4.5-6.0 kDa. The antimicrobial substance was characterized as a bacteriocin, because of its proteinaceous nature and low molecular weight. The bacteriocin could potentially be used as a food preservative, because of its thermostable property and broad antimicrobial spectrum.

• Advances in nano research
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• v.6 no.1
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• pp.81-92
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• 2018

Biosynthesis of nanoparticles has acquired particular attention due to its economic feasibility, low toxicity and simplicity of the process. Extracellular synthesis of nanoparticles by bacteria and fungi has been stated to be brought about by enzymes and other reducing agents that may be secreted in the culture medium. The present study was carried out to determine the underlying mechanisms of extracellular silver nanoparticle synthesis by Pseudomonas hibiscicola isolated from the effluent of an electroplating industry in Mumbai. Synthesized nanoparticles were characterized by spectroscopy and electron microscopic techniques. Protein profiling studies were done using Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (1D-SDS PAGE) and subjected to identification by Mass Spectrometry. Characterization studies revealed synthesis of 50 nm nanoparticles of well-defined morphology. Total protein content and SDS PAGE analysis revealed a reduction of total protein content in test (nanoparticles solution) samples when compared to controls (broth supernatant). 45.45% of the proteins involved in the process of nanoparticle synthesis were identified to be oxidoreductases and are thought to be involved in either reduction of metal ions or capping of synthesized nanoparticles.

• BMB Reports
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• v.28 no.4
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• pp.281-289
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• 1995

Homogeneous human deoxycytidine kinase was purified in one step from a variety of spontaneous human leukemic cells (T-ALL, B-ALL, B-CLL, AML, CML), and from cultured T-lymphoblast cells (MOLT-4) using the newly developed affinity medium, $dCp_4$-Sepharose. Starting with an ammonium sulfate fraction, purification was achieved in one step with the kinase being eluted from a column by the end product inhibitor, dCTP. The purified deoxycytidine kinase from T-ALL cells phosphorylated deoxyadenosine and deoxyguanosine, as well as deoxycytidine. The enzyme purified from T-ALL and B-CLL cells yielded one major band with a molecular weight of 52 kDa determined by SDS-polyacrylamide gel electrophoresis. AML and CML cells yielded one 52 kDa band and an extra band of 30 kDa molecular weight. On the other hand, B-ALL and MOLT-4 cells showed a low molecular weight band of 30 kDa only. However, the electrophoretic mobilities of enzymatic activity in 12% non-denaturing gels were identical for the dCyd kinase from all different kinds of leukemic cell lines, except that the B-ALL, B-CLL, and MOLT-4 cell preparations had an extra minor peak, all at the same position. dAdo and dCyd phosphorylating activities comigrated indicating that these activities are all associated with the same protein. Two new methods, a disk implantation method and a nitrocellulose powder method were used with a small amount of enzyme protein to raise polyclonal antibodies against dCyd kinase purified from T-ALL cells.

• BMB Reports
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• v.30 no.3
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• pp.177-181
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• 1997

We constructed a single-chain immunoglobulin in which the carboxyl end of the heavy chain variable domain is covalently joined to the amino terminus of the light chain variable domain via peptide linker and the carboxyl end of the light chain variable domain is linked to human ${\gamma}1$ Fc region through the hinge region. The molecule was expressed in Chinese hamster ovary cells, assembled into a dimeric molecule and secreted into the culture medium. The dimeric molecule (2E11) was purified from the culture supernatant by affinity chromatography on Protein G-Sepharose column. The size of the unreduced or reduced protein was the expected molecular weight of approximately 120 or 60 kDa, respectively, as assessed by SDS-polyacrylamide gel electrophoresis. The antigen-binding affinity of 2E11 was almost the same as that of a native antibody counterpart (CS131A), suggesting that the single-chain immunoglobulin may function like a native antibody.

• Microbiology and Biotechnology Letters
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• v.23 no.3
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• pp.255-262
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• 1995

Intact cells of Acinetobacter sp. T5-7 completely degraded trichloroethylene (TCE) following growth with phenol. This strain could grow on at least eleven aromatic compounds, e.g., benzaldehyde, benzene, benzoate, benzylalochol, catechol, caffeic acid, 2.4-D, p-hydroxybenzoate, phenol, protocatechuate and salicylate, and did grow on alkane, such as octane. But except phenol, other aromatic compounds did not induced TCE degradation. Phenol biotransformation products, catechol was identified in the culture media. However, catechol-induced cells did not degrade TCE. So we assumed that phenol hydroxylase was responsible for the degradation of TCE. The isolate T5-7 showed growth in MM2 medium containing sodium lactate and catechol rather than phenol, but did not display phenol hydroxyalse activity, suggesting induction of enzyme synthesis by phenol. Phenol hydroxylase activity was independent of added NADH and flavin adenine dinucleotide but was dependent on NADPH addition. Degradation of phenol produced catechols which are then cleaved by meta-fission. We identified catechol-2.3-dioxygenase by active staining of polyacrylamide gel.

• KSBB Journal
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• v.8 no.2
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• pp.126-132
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• 1993

The objective of this study was to develop an artificial dentin for easy handle and accurate observation of the mechanism on dental caries and to screen biologically active materials from the extracts of traditional plants and fruits for prevention of early dental cares. In order to produce disc PAHA (artificial dentin), the powdered hydroxylapatite was immobilized in a 20% polyacrylamide gel. The characteristics of disc PAHA was very similar to the surface, figure and lattice of human enamel. After decalcification in 0.1M citric acid based on observation with SEM. The critical point of decalcification of disc PAHA by acids was found to be pH 5.0-5.5, which was hi agreement with human enamel. The degree of decalcification from disc PAHA in 0.1M citric acid solution was sixfold higher than that of human enamel. This result suggested that disc PAHA would be useful as a substitute of human enamel for in vitro experiment. The extracts of garlic and Flower Apple A, B seemed to inhibit growth of S. mutans. Especially, when the 300$\mu\ell$ of its extracts added to the medium to incubate S. mutans, F. apple B showed strongly an inhibitory effect in both the growth of S. mutans and the synthesis of insoluble glucan.

• Applied Biological Chemistry
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• v.46 no.3
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• pp.171-175
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• 2003

The putative endochitinase gene, yheB of Escherichia coli K-12 is not expressed under lab culture conditions. The endochitinase gene was amplified by PCR and subcloned into pET28c vector and pQE9 vector, respectively. The endochitinase produced in E. coli harboring pET28c containing yheB or pQE9 vector containing yheE was partly released into the growth medium. The overproduced endochitinase was partially purified by His affinity column chromatography and DE-52 column chromatography. The apparent molecular weight of the endochitinase determined by SDS-polyacrylamide gel electrophoresis was about 97,000. The purified E. coli endochitinase showed maximal chitinolytic activity at pH 6 and $40^{\circ}C$.

• Microbiology and Biotechnology Letters
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• v.32 no.2
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• pp.117-122
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• 2004

Bacillus sp. $\beta$-mannanase was purified by DEAE-sephadex ion exchange column chromatography. The specific activity of the purified enzyme was 21.57 units/$m\ell$ protein, representing an 95.33-folds purification of the original crude extract. The final preparation thus obtained showed a single band on SDS-polyacrylamide gel electrophoresis. The molecular weight was determined to be 38.9 kDa. Guar gum galactomannan was hydrolyzed by the purified $\beta$-mannanase, and then the hydrolysates was separated by activated carbon column chromatography and Sephadex G-25 gel filtration. The main hydrolysates were composed of D.P. (Degree of Polymerization) 5 and 7 galactomannooligosaccharides. To investigate the effects of guar gum galactomannooligosaccharides on in vitro growth of Bifidobacterium longum, B. bifidum, B. infantis, B. adolescentis, B. animalis, and B. breve, Bifidobacterium spp. were cultivated individually on the modified-MRS medium containing carbon source such as D.P. 5 and D.P. 7 galactomannooligosaccharides, respectively B. longum and B. bifidum grew up l0-fold and 9.8-fold more effectively by the treatment of D.P. 5 galactomannooligosaccharides, compared to those of standard MRS medium. Especially, D.P. 5 was more effective than D.P. 7 galactomannooligosaccharide on the growth of Bifidobacterium spp.

• Journal of Microbiology and Biotechnology
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• v.22 no.7
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• pp.930-938
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• 2012

High levels of xylanase activity (143.98 IU/ml) produced by the newly isolated Paenibacillus campinasensis G1-1 were detected when it was cultivated in a synthetic medium. A thermostable xylanase, designated XynG1-1, from P. campinasensis G1-1 was purified to homogeneity by Octyl-Sepharose hydrophobic-interaction chromatography, Sephadex G75 gel-filter chromatography, and Q-Sepharose ion-exchange chromatography, consecutively. By multistep purification, the specific activity of XynG1-1 was up to 1,865.5 IU/mg with a 9.1-fold purification. The molecular mass of purified XynG1-1 was about 41.3 kDa as estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Sequence analysis revealed that XynG1-1 containing 377 amino acids encoded by 1,134 bp genomic sequences of P. campinasensis G1-1 shared 96% homology with XylX from Paenibacillus campinasensis BL11 and 77%~78% homology with xylanases from Bacillus sp. YA-335 and Bacillus sp. 41M-1, respectively. The activity of XynG1-1 was stimulated by $Ca^{2+}$, $Ba^{2+}$, DTT, and ${\beta}$-mercaptoethanol, but was inhibited by $Ni^{2+}$, $Fe^{2+}$, $Fe^{3+}$, $Zn^{2+}$, SDS, and EDTA. The purified XynG1-1 displayed a greater affinity for birchwood xylan, with an optimal temperature of $60^{\circ}C$ and an optimal pH of 7.5. The fact that XynG1-1 is cellulose-free, thermostable (stability at high temperature of $70^{\circ}C{\sim}80^{\circ}C$), and active over a wide pH range (pH 5.0~9.0) suggests that the enzyme is potentially valuable for various industrial applications, especially for pulp bleaching pretreatment.

• Applied Biological Chemistry
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• v.22 no.4
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• pp.191-197
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• 1979

${\beta}-Tyrosinase$ was purified and crystallized from cells of Escherichia intermedia A-21 grown in a medium supplemented with 0.2% L-tyrosine. Molecular weight of its subunit, Km value and absorption spectra were determined. Crystallization methods were also studied to eliminate any unnecessary procedures. The results obtained were as follows: 1. The purification procedure included ammonium sulfate fractionation, dialysis against potassium phosphate buffer, pH 6.0 and pH 7.0, and DEAE-Sephadex column chromatography. In the column chromatography, 11 mg of protein was applied per ml of DEAE-Sephadex for efficiency. 2. Steps of protamine sulfate treatment and Sephadex G-150 gel filtration could be eliminated for this enzyme from the known procedures. 3. The purified enzyme was dissolved in 0.01M potassium phosphate buffer containing 2-mercaptoethanol, with a concentration of 20mg/ml. Crystalline enzyme, which appears as hexagonal rods, was obtained by adding solid fine powdered ammonium sulfate to the enzyme solution. 4. Absorption maxima of the enzyme appeared at 340 and 430nm when associated with pyridoxal phosphate. 5. Km value of the enzyme for L-tyrosine was $2.31{\times}10^{-4}M$ and the molecular weight of its subunit was determined by SDS-polyacrylamide electrophoresis to be approximately 50,000.