• Journal of Microbiology and Biotechnology
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• 제17권11호
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• pp.1898-1903
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• 2007

We attempted to modulate the overall protein expression rate through the addition of a repressor against the araBAD promoter system of Escherichia coli, in which glucose was used as a repressor. Therefore, 0.5% L-arabinose was initially contained as an inducer in culture medium, and either 2% glucose or 2% glycerol was used as a carbon source, and it was found that the expression of recombinant interferon-${\alpha}$ could be observed at the beginning of the batch culture when glycerol was used as a carbon source. However, when glucose was used, the initiation of recombinant interferon-${\alpha}$ expression was delayed compared with that when glycerol was used. Furthermore, when the addition of 0.5% glucose was carried out once or twice after 0.5% L-arabinose induction during DO-stat fed-batch culture, the distributions of soluble and insoluble recombinant interferon-${\alpha}$ were modulated. When glucose was not added after the induction of L-arabinose, all of the expressed recombinant interferon-${\alpha}$ formed an inclusion body during the later half of culturing. However, when glucose was added after induction, the expressed recombinant interferon-${\alpha}$ did not all form an inclusion body, and about half of the total recombinant interferon-${\alpha}$ was expressed in a soluble form. It was deduced that the addition of glucose after the induction of L-arabinose might lower the cAMP level, and thus, CAP (catabolite activator protein) might not be activated. The transcription rate of recombinant interferon-${\alpha}$ in the araBAD promoter system might be delayed by the partial repression. This inhibition of the transcription rate probably resulted in more soluble interferon-${\alpha}$ expression caused by the reduction of the protein synthesis rate.

• Journal of Microbiology and Biotechnology
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• 제18권3호
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• pp.434-442
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• 2008

We observed that an inclusion body (IB) of recombinant ${\beta}$-galactosidase that was produced by the araBAD promoter system in Escherichia coli (E. coil) showed enzyme activity. In order to improve its activity, the lowering of the transcription rate of the ${\beta}$-galactosidase structural gene was attempted through competition between an inducer (L-arabinose) and an inducer analog (D-fucose). In the deep-well microtiter plate culture and lab-scale fermentor culture, it was demonstrated that the addition of D-fucose caused an improvement in specific ${\beta}$-galactosidase production, although ${\beta}$-galactosidase was produced as an IB. In particular, the addition of D-fucose after induction led to an increase in the specific activity of ${\beta}$-galactosidase IB. Finally, we confirmed that the addition of D-fucose after induction caused changes in the structure of ${\beta}$-galactosidase IB, with higher enzyme activity. Based on these results, we expect that an improved enzyme IB will be used as a biocatalyst of the enzyme bioprocess, because an enzyme IB can be purified easily and has physical durability.

• Journal of Microbiology and Biotechnology
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• 제21권11호
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• pp.1151-1158
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• 2011

In this study, a galactooligosaccharide (GOS) was synthesized using active ${\beta}$-galactosidase (${\beta}$-gal) inclusion bodies (IBs)-containing Escherichia coli (E. coli) cells. Analysis by MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) mass spectrometry revealed that a trisaccharide was the major constituent of the synthesized GOS mixture. Additionally, the optimal pH, lactose concentration, amounts of E. coli ${\beta}$-gal IBs, and temperature for GOS synthesis were 7.5, 500 g/l, 3.2 U/ml, and $37^{\circ}C$, respectively. The total GOS yield from 500 g/l of lactose under these optimal conditions was about 32%, which corresponded to 160.4 g/l of GOS. Western blot analyses revealed that ${\beta}$-gal IBs were gradually destroyed during the reaction. In addition, when both the reaction mixture and E. coli ${\beta}$-gal hydrolysate were analyzed by high-performance thin-layer chromatography (HP-TLC), the trisaccharide was determined to be galactosyl lactose, indicating that a galactose moiety was most likely transferred to a lactose molecule during GOS synthesis. This GOS synthesis system might be useful for the synthesis of galactosylated drugs, which have recently received significant attention owing to the ability of the galactose molecules to improve the drugs solubility while decreasing their toxicity. ${\beta}$-Gal IB utilization is potentially a more convenient and economic approach to enzymatic GOS synthesis, since no enzyme purification steps after the transgalactosylation reaction would be required.