• Advances in environmental research
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• v.9 no.3
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• pp.215-232
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• 2020

Crude oils are essential source of energy. It is majorly found in geographical locations beneath the earth's surface and crude oil is the main factor for the economic developments in the world. Natural crude oil contains unrefined petroleum composed of hydrocarbons of various molecular weights and it contains other organic materials like aromatic compounds, sulphur compounds, and many other organic compounds. These hydrocarbons are rapidly getting degraded by biosurfactant producing microorganisms. The present study deals with the isolation, purification, and characterization of biosurfactant producing microorganism from oil-contaminated soil. The ability of the microorganism producing biosurfactant was investigated by well diffusion method, drop collapse test, emulsification test, oil displacement activity, and blue agar plate method. The isolate obtained from the oil contaminated soil was identified as Bacillus subtilis. The identification was done by microscopic examinations and further characterization was done by Biochemical tests and 16SrRNA gene sequencing. Purification of the biosurfactant was performed by simple liquid-liquid extraction, and characterization of extracted biosurfactants was done using Fourier transform infrared spectroscopy (FTIR). The degradation of crude oil upon treatment with the partially purified biosurfactant was analyzed by FTIR spectroscopy and Gas-chromatography mass spectroscopy (GC-MS).

• Journal of Periodontal and Implant Science
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• v.29 no.3
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• pp.663-676
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• 1999

Porphyromonas gingivalis, a Gram negative, anaerobic, asaccharolytic rod, is one of the most frequently implicated pathogens in human periodontal disease and has a requirement for hemin for growth. A 30 kDa (heated 24 kDa) hemin-binding protein whose expression is both hemin and iron regulated has recently been purified and characterized in this oral pathogen. This study has identified a hemin-binding P. gingivalis protein by expression of a P. gingivalis genomic library in Escherichia coli, a bacterium which does not require or transport exogenous hemin. A library of genomic DNA fragments from P. gingivalis was constructed in plasmid pUC18, transformed into Escherichia coli strain $DH5{\alpha}$ , and screened for recombinant clones with hemin-binding activity by plating onto hemin-containing agar. Of approximately 10,000 recombinant E. coli colonies screened on LB-amp-hemin agar, 10 exhibited a clearly pigmented phenotype. Each clone contained various insert DNA. The Hind III fragment transferred to the T7 RNA polymerase/promoter expression vector system produced a sligltly smaller (21 kDa) protein, a precursor form, immunoreactive to the antibody against the 24 kDa protein, suggesting that the cloned DNA fragment probably carried an entire gene for the 24 kDa hemin-binding protein.

• Journal of Life Science
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• v.23 no.2
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• pp.259-266
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• 2013

To isolate the fibrinolytic enzyme, 268 strains from 21 samples were morphologically isolated from Cheonggukjang collected from Korea and Japan. Among the 268 strains, protease-producing bacteria were isolated in nutrient agar medium including 1% skimmed milk. As a result of this, 22 strains were isolated. Apiweb site was used to identify these strains based on their biochemical properties. In addition, 16S rRNA sequencing was performed to identify the strain. Most of the identified strains were Bacillus subtilis and B. amyloliquefaciens. Fibrinolytic enzyme activity was measured with the fibrin plate method. Five strains were finally selected: A2-14, A2-20, C1-05, C1-09, and F2-01. Of those five strains, the A2-20 strain, which is close to B. amyloliquefaciens, showed the strongest fibrinolytic activity. The fibrinolytic enzyme produced by the A2-20 strain was partially purified from culture supernatant by gel filtration and ion exchange chromatography. The optimal pH and temperature values of the partially purified enzyme were 7.0 and $35^{\circ}C$, respectively. Purified protein analysis was carried out with SDS-PAGE and zymography. A genetic analysis was also conducted by PCR based on the consensus sequence of fibrinolytic enzyme. Corresponding genes with a partial sequence of the A2-20 strain were identified.

• Journal of Microbiology and Biotechnology
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• v.16 no.2
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• pp.286-290
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• 2006

A genomic library of Streptococcus vestibularis ATCC49124 was constructed in an E. coli plasmid vector, and the urease-positive transformants harboring the urease gene cluster were isolated on Christensen-urea agar plates. The minimal DNA region required for urease activity was located in a 5.6 kb DNA fragment, and a DNA sequence analysis revealed the presence of a partial ureI gene and seven complete open reading frames, corresponding to ureA, B, C, E, F, G, and D, respectively. The nucleotide sequence over the entire ure gene cluster and 3'-end flanking region of S. vestibularis was up to 95% identical to that of S. salivarius, another closely related oral bacterium, and S. thermophilus, isolated from dairy products. The predicted amino acid sequences for the structural peptides were 98-100% identical to the corresponding peptides in S. salivarius and S. thermophilus, respectively, whereas those for the accessory proteins were 96-100% identical. The recombinant E. coli strain containing the S. vestibularis ure gene cluster expressed a high level of the functional urease holoenzyme when grown in a medium supplemented with 1 mM nickel chloride. The enzyme was purified over 49-fold by using DEAE-Sepharose FF, Superdex HR 200, and Mono-Q HR 5/5 column chromatography. The specific activity of the purified enzyme was 2,019 U/mg, and the Michaelis constant ($K_{m}$) of the enzyme was estimated to be 1.4 mM urea. A Superose 6HR gel filtration chromatography study demonstrated that the native molecular weight was about 196 kDa.

• The Korea Journal of Herbology
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• v.25 no.2
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• pp.81-88
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• 2010

Objectives : Sophorae Radix (SR), the dried root of Sophorae Flavescens Aiton, has been used to treat atherosclerosis, arrhythma and skin diseases including scabies and eczema. The present study was examined to evaluate antimicrobial activities of SR extracts against oral microorganism. Methods : Antimicrobial properties of SR extracts were determined by agar diffusion method and minimum inhibitory concentration (MIC) against Streptococcus mutans, Streptococcus sobrinus and Actinomyces viscosus. Analysis of kurarinone from SR extracts was conducted using UPLC (Ultra Performance Liquid Chromatography). Results : The ethanolic extracts of SR showed stronger antimicrobial effect than methanolic extracts, while the aqueous extracts of SR had no activity. In addition, the higher content of kurarinone was found in ethanolic extracts than methanolic extracts. The purified kurarinone from ethanolic extracts showed potent antimicrobial activity with the MIC value of $3.9{\sim}7.8{\mu}g/m{\ell}$. Conclusion : An ethanolic extract of SR showed antimicrobial properties against several oral microorganisms, and kuranrinone contributed to antimicrobial action of SR. Thus, ethanolic extracts of SR or purified kurarinone should be beneficial for the preparation of the useful agent for treating oral disease including anticaries.

• Journal of Pharmacopuncture
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• v.25 no.2
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• pp.114-120
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• 2022

Objectives: Antivenom serums have been used extensively for over a century and are the only effective treatment option for snake bites and other dangerous animal envenomations. In therapeutic serum centers, a wide range of antivenoms is made from animal serum, mainly equine and sheep, that are immunized with single or multiple venoms. This work aimed to use caprylic acid (CA) to purify therapeutic snake antivenom. Methods: Plasma was obtained from equine immunized with a mixture of venoms. Immunized plasma was obtained by precipitation of different concentrations (2-5%) of CA. This methodology was compared to that based on ammonium sulfate (AS) precipitation. Sediment plasma proteins were purified by ion-exchange chromatography. Protein assay, SDSPAGE, and agar gel diffusion were performed. Results: The total protein precipitation with AS was higher than precipitation with CA, but the best results were obtained when CA was added to the plasma until a final CA concentration of 5% was reached. Chromatography and electrophoresis indicated a stronger band for the 5% CA, and the gel diffusion assay showed antigen-antibody interaction in the purified serum. Conclusion: The use of CA compared to the routine method for purifying hyperimmune serums is a practical and cost-effective method for preparing and producing therapeutic serums. It constitutes a potentially valuable technology for alleviating the critical shortage of antivenom in Iran.

• Journal of Food Hygiene and Safety
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• v.32 no.3
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• pp.228-233
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• 2017

The aim of the current study was to examine genotoxicological safety of purified bee venom (Apis mellifera L.) The bacterial reverse mutation in Salmonella typhimurium (TA100, TA1535, TA98, and TA1537) and Escherichia coli (WP2 uvrA) were evaluated with purified bee venom at concentrations of 0, 1.5, 5, 15, 50, 150, and $500{\mu}g/plate$. Purified bee venom was negative in Ames test with both in the presence and absence of rat liver microsomal enzyme. According to these results, we concluded that purified bee venom did not cause bacterial reverse mutation. The safety of the purified bee venom at practical doses needs to be further evaluated in in vivo genotoxicity assays.

• Journal of Microbiology and Biotechnology
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• v.23 no.2
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• pp.167-176
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• 2013

A novel bioactive molecule produced by Bacillus thuringiensis subsp. kurstaki Bn1 (Bt-Bn1), isolated from a common pest of hazelnut, Balaninus nucum L. (Coleoptera: Curculionidae), was determined, purified, and characterized in this study. The Bt-Bn1 strain was investigated for antibacterial activity with an agar spot assay and well diffusion assay against B. cereus, B. weinhenstephenensis, L. monocytogenes, P. savastanoi, P. syringae, P. lemoignei, and many other B. thuringiensis strains. The production of bioactive molecule was determined at the early logarithmic phase in the growth cycle of strain Bt-Bn1 and its production continued until the beginning of the stationary phase. The mode of action of this molecule displayed bacteriocidal or bacteriolytic effect depending on the concentration. The bioactive molecule was purified 78-fold from the bacteria supernatant with ammonium sulfate precipitation, dialysis, ultrafiltration, gel filtration chromatography, and HPLC, respectively. The molecular mass of this molecule was estimated via SDS-PAGE and confirmed by the ESI-TOFMS as 3,139 Da. The bioactive molecule was also determined to be a heat-stable, pH-stable (range 6-8), and proteinase K sensitive antibacterial peptide, similar to bacteriocins. Based on all characteristics determined in this study, the purified bacteriocin was named as thuricin Bn1 because of the similarities to the previously identified thuricin-like bacteriocin produced by the various B. thuringiensis strains. Plasmid elution studies showed that gene responsible for the production of thuricin Bn1 is located on the chromosome of Bt-Bn1. Therefore, it is a novel bacteriocin and the first recorded one produced by an insect originated bacterium. It has potential usage for the control of many different pathogenic and spoilage bacteria in the food industry, agriculture, and various other areas.

• Microbiology and Biotechnology Letters
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• v.27 no.3
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• pp.208-214
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• 1999

The condition for immobilization of the partially purified agarase from Bacillus cereus ASK202 and the properties of the immobilized enzyme have been investigated. Agarase was immobilized on various supports by entrapment method. The enzyme immobilized on Na-alginate bead showed the highest activity among those studied. The optimal reaction conditions of the immobilized agarase were obtained in 3%(w/v) Na-alginate for the matrix, bead diameter of 2.5mm, 1 unit of agarase solution and 1.0%(w/v) calcium chloride solution. The optimum pH and temperature of the immobilized agarase were pH and temperature of the immobilized agarase were pH 7.0 and 4$0^{\circ}C$, respectively. Km and Vmax values were 0.5mg/ml.min, respectively. The immobilized agarase conerted agar to agarobiose, and their total conversion ratio under the optimal condition was 89%.

• The Korean Journal of Food And Nutrition
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• v.11 no.6
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• pp.629-634
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• 1998

1-Deoxynojirimycin which is a potent intestinal ${\alpha}$-glucosidase inhibitor was purified from the culture broth by ion exchange chromatography, Sephadex LH20 column chromatography, TSK gel chromatography and HPLC respectively. Acute toxicity of 1-deoxynojirimycin, which was loaded through the oral as dose of 200mg/kg, was investigated in IRC mouse. None of the tested IRC mice were not dead and increase of body weight showed also the same results in comparison with control mice. The antimicrobial susceptibility of 20 pathogenic strains against 3 antidiabetic compounds (1-deoxynojirimycin, AO-128, acarbose) were obtained by agar dilution method. All of the three antidiabetic compounds has very weak antimicrobial activity (MIC>100$\mu\textrm{g}$/ml).