• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.4
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• pp.322-328
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• 2005

Acylhomoserine lactones (AHLs) are known to be the triggering molecules in the quorum sensing mechanism of many gram-negative bacteria. In order to detect AHL inhibitors that are potential biofilm inhibitors, a convenient and sensitive bioassay was developed based on the $\beta$-galactosidase activity ($\beta$-GAL) of a recombinant Agrobacterium tumefaciens strain. A series of commercially available AHLs were tested for inducing $\beta$-GAL at varying concentrations in agar-plate and liquid cultures of the reporter strain. All AHLs tested exhibited a concentration­dependent induction, and octanoyl homoserine lactone (OHL) showed the highest sensitivity with a detection limit of 0.1 nM in the liquid culture assay. When fimbrolide, a known quorum sensing inhibitor, was added, induction of $\beta$-GAL by OHL was repressed. The repression at a constant OHL concentration was dependent on the fimbrolide concentration with the detection limit below 1 ppm, indicating that this assay is a sensitive method for screening AHL inhibitors.

• Biotechnology and Bioprocess Engineering:BBE
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• v.6 no.4
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• pp.231-236
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• 2001

How much do we know about the biology of aging from cell culture studies Most normal somatic cells have a finite potential to divide due to a process termed cellular or replicative senescence. A growing body evidence suggests that senescence evolved to protect higher eu-karyotes, particularly mammals, from developing cancer, We now know that telomere shortening due to the biochemistry of DNA replication, induces replicative senescence in human cells. How-ever in rodent cells, replicative senescence occurs despite very long telomeres. Recent findings suggest that replicative senescence is just the tip of the iceberg of a more general process termed cellular senescence. It appears that cellular senescence is a response to potentially oncogenic in-sults, including oxidative damage. In young orgainsms, growth arrest by cell senescence sup-presses tumor development, but later in life, due to the accumulation of senescent cells which se-cret factors that can disrupt tissues during aging, cellular senescence promotes tumorigenesis. Therefore, antagonistic pleiotropy may explain, if not in whole the apparently paradoxical effects of cellular senescence, though this still remains an open question.

• Biotechnology and Bioprocess Engineering:BBE
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• v.3 no.2
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• pp.71-77
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• 1998

The strain of Serratia marcescens QM B1466 produces selectively large amount of chitinolytic enzymes (about 1mg/L medium). Enzymatic hydrolysis of chitin to N-acetyl-${\beta}$-D-glucosamine (NAG) was performed with a system consisting of two hydrolases (chitinase and chitobiase) produced by optimization of a microbial host consuming chitin particles. For the development of Large-scale biological process for the production of NAG from chitinaceous waste, the selection and optimization of a microbial host, particle size of crab/shrimp chitin sources and initial induction time using chitin as a sole carbon source on chitinase/chitobiase production and NAG production were examined. Crab-shell chitin(1.5%) treated by dilute acid and , ball-milled with a normal diameter less than 250m gave the highest chitinase activity over a 7 days culture. Crude chitinase/ chitobiase solution obtained in a 10 L fed-batch fermentation showed a maximum activities of 23.6 U/mL and 5.1 U/mL, respectively with a feeding time of 3 hrs, near pH 8.5 at 30$^{\circ}C$.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.6
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• pp.556-563
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• 2005

Homogentisic acid was found to scavenge intracellular reactive oxygen species (ROS), and 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals and thus prevented lipid peroxidation in human fibroblast (Wl 38) cells. The radical scavenging activity of homogentisic acid was found to protect Wl 38 cells against hydrogen peroxide $(H_2O_2)$ induced oxidative stress, via the activation of extracellular signal regulated kinase (ERK) protein. Homogentisic acid increased the activity of catalase. Hence, from the present study, it is suggested that homogentisic acid protects Wl 38 cells against $H_2O_2$ damage by enhancing the intracellular antioxidative activity.

• Biotechnology and Bioprocess Engineering:BBE
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• v.10 no.6
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• pp.505-509
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• 2005

For the purpose of developing a direct label-free electrochemical detection system, we have systematically investigated the electrochemical signatures of each step in the preparation procedure, from a bare gold electrode to the hybridization of label-free complementary DNA, for the streptavidin-modified electrode. For the purpose of this investigation, we obtained the following pertinent data; cyclic voltammogram measurements, electrochemical impedance spectra and square wave voltammogram measurements, in $Fe(CN)_6^{3-}/Fe(CN)_6^{4-}$ solution (which was utilized as the electron transfer redox mediator). The oligonucleotide molecules on the streptavidin-modified electrodes exhibited intrinsic redox activity in the ferrocyanide-mediated electrochemical measurements. Furthermore, the investigation of electrochemical electron transfer, according to the sequence of oligonucleotide molecules, was also undertaken. This work demonstrates that direct label-free oligonucleotide electrical recognition, based on biofunctional streptavidin-modified gold electrodes, could lead to the development of a new biosensor protocol for the expansion of rapid, cost-effective detection systems.

• Biotechnology and Bioprocess Engineering:BBE
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• v.5 no.1
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• pp.61-64
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• 2000

A series of 5-oxaproline peptide derivatives was synthesized and evaluated for its ability to inhibit the prolyl 4-hydroxylase in vitro. Structure-activity studies show that the 5-oxaproline sequences, prepared by the 1,3-dipolar cycloaddition of the C-methoxycarbonyl-N-mannosyl nitrone in the presence of the ethylene, are more active than the corresponding proline derivatives. Prolyl 4-hydroxylase belongs to a family of $Fe^{2+}-dependent$ dioxygenase, which catalyzes the formation of 4-hydroxyproline in collagens by the hydroxylation of proline residues in -Gly-Xaa-Pro-Gly- of procollagen chains. In this paper we discover the more selective N-Cbz-Gly-Phe-Pro-Gly-OEt $(K_m\;=\;520\;{\mu}M)$ sequences which are showed stronger binding than others in vitro. Therefore, we set out to investigate constrained tetrapeptide that was designed to mimic the proline structure of pep tides for the development of prolyl 4-hydroxylase inhibitor. From this result, we found that the most potent inhibitor is N-Dansyl-Gly-Phe-5-oxaPro-Gly-OEt $(K_i\;=\;1.6\;{\mu}M)$. This has prompted attempts to develop drugs which inhibit collagen synthesis. Prolyl 4-hydroxylase would seem a particularly suitable target for antifibrotic therapy.

• Biotechnology and Bioprocess Engineering:BBE
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• v.5 no.5
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• pp.372-378
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• 2000

The cell culture of Angelica gigas Nakai producing decursin derivatives and immunostimulating polysaccharides was preserved in liquid nitrogen after pre-freezing in a deep freezer at -70$^{\circ}C$ for 480 min. The effects of the cryoprotectant and pretreatment before cooling were investigated to obtain the optimal procedure for cyropreservation. When compared to mannitol, sorbitol, or NaCl with a similar osmotic pressure, 0.7 M sucrose was found to be the best osmoticum for the cryopreservation of A. gigas cells. In the pre-culture medium, the cells in the exponential growth phase showed phase showed the best post-freezing survival after cryopreservation. A mixture of sucrose, glycerol, and DMSO was found to be an effective cryoprotectant and a higher concentration of the cryoprotectant provided better cell viability. When compared with the vitrification, the optimum cryopreservation method proposed in this study would seem to be more effective for the long-term storage of suspension cells. The highest relative cell viability established with the procedure was 89%.

• KSBB Journal
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• v.32 no.1
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• pp.35-45
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• 2017

In this work the optical fiber glucose and lactate biosensors were developed by using fluorescent dye and enzyme immobilized on the end tip of an optical fiber. 3-Glycidyloxypropyl)methyldiethoxysilane (GPTMS), (3-Aminopropyl) trimethoxysilane (APTMS) and Methyltrimethoxysilane (MTMS) were used to immobilize glucose oxidase (GOD), lactate oxidase (LOD) and ruthenium(II) complex (tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II), $Ru(dpp)_3^{2+}$) as oxygen sensitive fluorescent dye. MTMS sol-gel was an excellent supporting material for the immobilization of $Ru(dpp)_3^{2+}$, GOD, and LOD on the optical fiber. Storage stability of the optical fiber glucose sensor was kept constant over 20 days, while the optical fiber lactate sensor had constant storage stability over 17 days. The optical fiber glucose and lactate biosensors also maintained good operational stability for 20 hours and 14 hours, respectively. The activities of the immobilized enzymes were most excellent at pH 7 and at $25^{\circ}C$. On-line monitoring of glucose and lactate in a simulated process was performed with the optical fiber glucose and lactate biosensors. On-line monitoring results were agreed with those of off-line data measured with high performance liquid chromatography (HPLC).

• Biotechnology and Bioprocess Engineering:BBE
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• v.6 no.2
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• pp.75-88
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• 2001

In this paper, the microbial production of riboflavin is reviewed and includes descriptions of riboflavin overproducers, and the biosynthesis and details of the key-enzyme genes related to riboflavin. There kinds of riboflavin overproducers are known; Bacillus subtilis and Candida famate utilize glucose as a carbon source, but the fungus Ashbya gossypii requires plant oil as its sole carbon source. The starting material in ribofalvin biosynthesis is guanosine triphospate (GTP), which is converted to riboflavin through six enzymatic reactions. Though Bacillus subtilis, Candida famate, and Ashbya gossypii operate via different pathways until GTP, they follow the same pathway from GTP to riboflavin. From the metabolic viewpoint, with respect to improved riboflavin production, the supplementation of GTP, aprocess-limiting precursor must be considered. The GTP fluxes originate from three sources, serine, threonine and glyoxylate cycles. The development of pathways to strengthen GTP supplementation using biotechnological techniques remains an issue fro future research.

• Biotechnology and Bioprocess Engineering:BBE
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• v.9 no.3
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• pp.196-200
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• 2004

The supra molecular weight poly(〔R〕-3-hydroxybutyrate) (PH B), having a molecular weight greater than 2 million Da, has recently been found to possess improved mechanical properties compared with the normal molecular weight PHB, which has a molecular weight of less than 1 million Da. However, applications for this PHB have been hampered due to the difficulty of its production. Reported here, is the development of a new metabolically engineered Escherichia coli strain and its fermentation for high level production of supra molecular weight PHB. Recombinant E. coli strains, harboring plasm ids of different copy numbers containing the Alcaligenes latus PHB biosynthesis genes, were cultured and the molecular weights of the accumulated PHB were compared. When the recombinant E. coli XL1-Blue, harboring a medium-copy-number pJC2 containing the A. latus PHB biosynthesis genes, was cultivated by fed-batch culture at pH 6.0, supra molecular weight PHB could be produced at up to 89.8 g/L with a productivity of 2.07 g PHB/L-h. The molecular weight of PHB obtained under these conditions was as high as 22 MDa, exceeding by an order of magnitude the molecular weight of PHB typically produced in Ralstonia eutropha or recombinant E. coli.