• Journal of Microbiology and Biotechnology
• /
• v.25 no.12
• /
• pp.2034-2042
• /
• 2015

A one-pot process of enzymatic synthesis of deoxythymidine-5'-triphosphate (5'-dTTP) employing whole cells of recombinant Escherichia coli coexpressing thymidylate kinase (TMKase) and acetate kinase (ACKase) was developed. Genes tmk and ack from E. coli were cloned and inserted into pET28a(+), and then transduced into E. coli BL21 (DE3) to form recombinant strain pTA in which TMKase and ACKase were simultaneously overexpressed. It was found that the relative residual specific activities of TMKase and ACKase, in pTA pretreated with 20 mM ethylene diamine tetraacetic acid (EDTA) at 25℃ for 30 min, were 94% and 96%, respectively. The yield of 5'-dTTP reached above 94% from 5 mM deoxythymidine 5'-monophosphate (5'-dTMP) and 15 mM acetyl phosphate catalyzed with intact cells of pTA pretreated with EDTA. The process was so effective that only 0.125 mM adenosine-5'-triphosphate was sufficient to deliver the phosphate group from acetyl phosphate to dTMP and dTDP.

• Microbiology and Biotechnology Letters
• /
• v.22 no.1
• /
• pp.85-91
• /
• 1994

The characteristcs of monascus fermentation using a hyperpigment-producing mutant, Monascus sp. J101, were analyzed, and the extractive fermentations employing permeabilizing agents and resin were carried out to increase the productivity of red pigment. And the kinetic analysis was also carried out in case of the monascus fermentation using Amberlite XAD-7. The extracellular content of the red pigment produced by Monascus sp. J101 was about 17% of the total, and the production of pigment was regulated by its own product. The cell growth reached a stationary phase at 48 hours ofter inoculation, whereas the pigment production continued up to 100 hours, which showed the pattern of a mixed growth-associated type. During the fermentation, various permeabilizing agents were added to the culture medium and their effects on pigment production were examined. By adding 0.05% Triton X-100 at 48 hours of cultivation, about an 18% increase in pigment production was accomplished as compared to the control, 12% ethyle acetate and 15% for 0.05% deoxycholate, respectively. When a nonionic adsorbent, Amberlite XAD-7 was added to the culture medium at a concentration of 12.0% at 48 hours of cultivation, the pigment production was enhanced by about 48.9% as compared to the control.

• Biotechnology and Bioprocess Engineering:BBE
• /
• v.2 no.2
• /
• pp.77-81
• /
• 1997

Removal of nitrogen compound from waste water is essential and often accomplished by biological process. To prevent washout and to develop an efficient bioreactor, immobilization of sutibal microorganisms could be sensible approach. Strains and permeabilized cell encapsulated in cellulose nitrate microcapsules and immobilized on polystyrene films were prepared by the method described in the previous study. In the wastewater treatment system, nitrification of ammonia component is generally known as rate controlling step. To enhance the rate of nitrification, firstly nitrifying strains Nitrosomonas europaea(IFO14298), are permeabilized chemically, and immobilized on polystyrene films and secondly oxidation rates of strain system and permeabilized strain system are compared in the same condition. with 30 minute permeabilized cells, it took about 25 hours to oxidize 70% of ammonia in the solution, while it took about 40 hours to treat same amount of ammonia with untreated cells. All the immobilization procedures did not harm to the enzyme activity and no mass transfer resistance through the capsule well was shown. In the durability test of immobilized system, the system showed considerable activity for the repeated operation for 90 days. With these results, the system developed in this study showed the possibility to be used in the actual waste water treatment system.

• Microbiology and Biotechnology Letters
• /
• v.38 no.1
• /
• pp.19-23
• /
• 2010

Recently, marine organisms are emerging as a leading group for identifying and extracting novel bioactive substances. These substances are known to possess a potential regarding not only as a source of pharmaceutical products but also their beneficial effects on humans. Among the substances, antimicrobial peptides (AMPs) specifically have attracted considerable interest for possible use in the development of new antibiotics. AMPs are characterized by relatively short cationic peptides containing the ability to adopt a structure in which cationic or hydrophobic amino acids are spatially scattered. Although a few reports address novel marine organisms-derived AMPs, their antimicrobial mechanism(s) are still remain unknown. In this review, we summarized the peptides previously investigated, such as Pleurocidin, Urechistachykinins, Piscidins and Arenicin-1. These peptides exhibited significant antimicrobial activities against human microbial pathogens without remarkable hemolytic effects against human erythrocytes, and their mode of actions are based on permeabilization of the plasma membrane of the pathogen. Therefore, the study of antimicrobial peptides derived from marine organisms may prove to be useful in the design of future therapeutic antimicrobial drugs.

• Molecules and Cells
• /
• v.21 no.1
• /
• pp.7-20
• /
• 2006

The major event in human immunodeficiency virus type 1 (HIV-1) infection is the death of many cells related to host immune response. The demise of these cells is normally explained by cell suicide mechanism, apoptosis. Interestingly, the decrease in the number of immune cells, such as non-CD4+ cells as well as CD4+ T cells, in HIV infection usually occurs in uninfected bystander cells, not in directly infected cells. It has, therefore, been suggested that several soluble factors, including viral protein R (Vpr), are released from the infected cells and induce the death of bystander cells. Some studies show that Vpr interacts directly with adenine nucleotide translocator (ANT) to induce mitochondrial membrane permeabilization (MMP). The MMP results in release of some apoptogenic factors such as cytochrome-c (cyt-c) and apoptosis-inducing factor (AIF). Vpr also has indirect effect on mitochondria through enhancing the level of caspase-9 transcription and suppressing nuclear factor-kappa B (NF-${\kappa}B$). The involvement of p53 in Vpr-induced apoptosis remains to be studied. On the other hand, low level of Vpr expression has anti-apoptotic effect, whereas it's high level of expression induces apoptosis. Extracellular Vpr also exhibits cytotoxicity to uninfected bystander cells through apoptotic or necrotic mechanism. The facts that Vpr has cytotoxic effect on both infected cells and bystander cells, and that it exhibits both proand anti-apoptotic activity may explain its role in viral survival and disease progression.

• Bulletin of the Korean Chemical Society
• /
• v.35 no.9
• /
• pp.2809-2812
• /
• 2014

The 43-residue defensin-like peptide coprisin, which is isolated from dung bettle, Copris tripartitus, is a potent antimicrobial peptide. In our previous work, we determined the tertiary structure of coprisin and found that alpha helical region of coprisin from residue 19 to residue 30 is important for its antimicrobial activities. Here, we designed cop12mer and cop9mer analogs of coprisin based on the tertiary structure of coprisin. To investigate the relationship between hydrophobicity and antimicrobial activities and develop the potent peptide antibiotics, we designed cop9mer-1 with substitution of $His^2$ with Trp in cop9mer. The results showed that cop9mer-1 has higher toxicities as well as improved antimicrobial activities compared to cop9mer. In order to reduce the toxicity of cop9mer-1, we designed cop9mer-2 and cop9mer-3 with substitution of $Cys^3$ with Lys or Ser. Substitution of $Cys^3$ with these hydrophilic amino acids results in lower cytotoxicities compared to cop9mer-1. Cop9mer-2 with substitution of $Cys^3$ with Lys in Cop9mer-1 showed high antibacterial activities against drug resistant bacteria without cytotoxicity. Antibiotic action of cop9mer-1 analog appears to involve permeabilization of the bacterial cell membrane while cop9mer-2 and cop9mer-3 may have different mechanism of action. These results imply that that optimum balance in hydrophobicity and hydrophilicity in these 9-meric peptides plays key roles in their antimicrobial activities as well as cytotoxicities.

• Journal of Microbiology and Biotechnology
• /
• v.28 no.3
• /
• pp.482-490
• /
• 2018

Candida albicans infections are often problematic to treat owing to antifungal resistance, as such infections are mostly associated with biofilms. The ability of C. albicans to switch from a budding yeast to filamentous hyphae and to adhere to host cells or various surfaces supports biofilm formation. Previously, the ethanol extract from Paeonia lactiflora was reported to inhibit cell wall synthesis and cause depolarization and permeabilization of the cell membrane in C. albicans. In this study, the P. lactiflora extract was found to significantly reduce the initial stage of C. albicans biofilms from 12 clinical isolates by 38.4%. Thus, to assess the action mechanism, the effect of the P. lactiflora extract on the adhesion of C. albicans cells to polystyrene and germ tube formation was investigated using a microscopic analysis. The density of the adherent cells was diminished following incubation with the P. lactiflora extract in an acidic medium. Additionally, the P. lactiflora-treated C. albicans cells were mostly composed of less virulent pseudohyphae, and ruptured debris was found in the serum-containing medium. A quantitative real-time PCR analysis indicated that P. lactiflora downregulated the expression of C. albicans hypha-specific genes: ALS3 by 65% (p = 0.004), ECE1 by 34.9% (p = 0.001), HWP1 by 29.2% (p = 0.002), and SAP1 by 37.5% (p = 0.001), matching the microscopic analysis of the P. lactiflora action on biofilm formation. Therefore, the current findings demonstrate that the P. lactiflora ethanol extract is effective in inhibiting C. albicans biofilms in vitro, suggesting its therapeutic potential for the treatment of biofilm-associated infections.

• Journal of Microbiology and Biotechnology
• /
• v.19 no.12
• /
• pp.1635-1643
• /
• 2009

The aim of this study was to provide new insight into the mechanism whereby the housekeeping enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) locates to cell walls of Lactobacillus plantarum 299v. After purification, cytosolic and cell wall GAPDH (cw-GAPDH) forms were characterized and shown to be identical homotetrameric active enzymes. GAPDH concentration on cell walls was growth-time dependent. Free GAPDH was not observed on the culture supernatant at any time during growth, and provoked cell lysis was not concomitant with any reassociation of GAPDH onto the cell surface. Hence, with the possibility of cw-GAPDH resulting from autolysis being unlikely, entrapment of intracellular GAPDH on the cell wall after a passive efflux through altered plasma membrane was investigated. Flow cytometry was used to assess L. plantarum 299v membrane permeabilization after labeling with propidium iodide (PI). By combining PI uptake and cw-GAPDH activity measurements, we demonstrate here that the increase in cw-GAPDH concentration from the early exponential phase to the late stationary phase is closely related to an increase in plasma membrane permeability during growth. Moreover, we observed that increases in both plasma membrane permeability and cw-GAPDH activity were delayed when glucose was added during L. plantarum 299v growth. Using a double labeling of L. plantarum 299v cells with anti-GAPDH antibodies and propidium iodide, we established unambiguously that cells with impaired membrane manifest five times more cw-GAPDH than unaltered cells. Our results show that plasma membrane permeability appears to be closely related to the efflux of GAPDH on the bacterial cell surface, offering new insight into the understanding of the cell wall location of this enzyme.

• Korean Journal of Plant Tissue Culture
• /
• v.21 no.6
• /
• pp.363-367
• /
• 1994

In an effort to investigate the role of calmodulin (CaM) as a modulating molecule in the signal transduction system in plant cells, we established methods for introduction of purified CaM into cultured soybean cells. CaM was purified from bovine testis, and was labelled with fluorescein isothiocyanate (FITC). Suspension -cultured cells were healed with saponin (0.1 mg/mL) to permeabilize the plasma membrane and coincubated with FITC-CaM complex. Saponin pretreatment was found to increase the fluorescence in the suspension cultured cells, indicating that the FITC-CaM complex could be incorporated into the cytoplasm. Optimal conditions for introducing FITC-CaM complex into protoplasts by electroporation were established with various electric pulses. With increasing field strength, the fluorescence in the protoplase was increased, while the viability of the protoplase decreased. FITC-CaM complex was successfully introduced into the protoplasts by electroporation and the amount of FITC-CaM complex in the protoplase was estimated.

• Korean Journal of Food Science and Technology
• /
• v.22 no.7
• /
• pp.812-816
• /
• 1990

This study describes the sorbitol production with permeabilized cells of Zymomonas mobilis immobilized in Ca-alginate. Toluene treated cells lose activity of glucose-fructose oxidoreductase due to the leaking of enzyme from the cells. To prevent this leakage, the permeabilized cells were treated with 0.25% glutaraldehyde by stirring for 1 h at room temperature. A continuous process with glutaraldehyde treated cells was developed and no significant reduction in the degree of conversion occurred during 210 h operation. The productivities were estimated to be about $7.2{\sim}7.5\;g/l-h$ for sorbitol at dilution rate $0.18\;h^{-1}$.