• Asian-Australasian Journal of Animal Sciences
• /
• v.17 no.12
• /
• pp.1751-1757
• /
• 2004

Recombinant heterologous proteins can be produced as insoluble aggregates partially or perfectly inactive in Escherichia coli. One of the strateges to improve the solubility of recombinant proteins is fusion with a partner that is excellent in producing soluble fusion proteins. To improve the production of soluble $\beta$-galactosidase, the gene of Thermus thermophilus KNOUC112 $\beta$-galactosidase (KNOUC112 $\beta$-gal) was fused with thioredoxin gene, and optimization of its expression in E. coli TOP10 was performed. KNOUC112 $\beta$-gal in pET-5b was isolated out, fused with thioredoxin gene in pThioHis C, and transformed to E. coli TOP10. The $\beta$-galactosidase fused with thioredoxin was produced in E. coli TOP10 as dimer and trimer. The productivity of fusion $\beta$ -galactosidase expressed via pThioHis C at 37$^{\circ}C$ was about 5 times higher than that of unfused $\beta$-galactosidase expressed via pET-5b at 37$^{\circ}C$. Inclusion body of $\beta$-galactosidase was formed highly, regardless of the induction by IPTG when KNOUC112 $\beta$ -gal was expressed via pET-5b at 37$^{\circ}C$. Fusion $\beta$ -galactosidase expressed at 37$^{\circ}C$ via pThioHis C without the induction by IPTG was soluble, but the induction by IPTG promoted the formation of inclusion body. Lowering the incubation temperature for the expression of fusion gene under 25$^{\circ}C$ prevented the formation of inclusion body, optimally at 25$^{\circ}C$. 0.07 mM of IPTG was sufficient for the soluble expression of fusion gene at 25$^{\circ}C$. The soluble production of Thermus thermophilus KNOUC112 $\beta$-galactosidase could be increased about 10 times by fusion with thioredoxin, and optimization of incubation temperature and IPTG concentration for induction.

• Journal of Microbiology and Biotechnology
• /
• v.18 no.3
• /
• pp.434-442
• /
• 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
• /
• v.21 no.9
• /
• pp.972-978
• /
• 2011

In this study, we investigated the performance of an immobilized ${\beta}$-galactosidase inclusion bodies-containing Escherichia coli cell reactor, where the cells were immobilized in alginate beads, which were then used in repeated-batch operations for the hydrolysis of o-nitrophenyl-${\beta}$-D-galactoside or lactose over the long-term. In particular, in the Tris buffer system, disintegration of the alginate beads was not observed during the operation, which was observed for the phosphate buffer system. The o-nitrophenyl-${\beta}$-D-galactoside hydrolysis was operated successfully up to about 80 h, and the runs were successfully repeated at least eight times. In addition, hydrolysis of lactose was successfully carried out up to 240 h. Using Western blotting analyses, it was verified that the ${\beta}$-galactosidase inclusion bodies were sustained in the alginate beads during the repeated-batch operations. Consequently, we experimentally verified that ${\beta}$-galactosidase inclusion bodies-containing Escherichia coli cells could be used in a repeated-batch reactor as a biocatalyst for the hydrolysis of o-nitrophenyl-${\beta}$-D-galactoside or lactose. It is probable that this approach can be applied to enzymatic synthesis reactions for other biotechnology applications, particularly reactions that require long-term and stable operation.

• Journal of Microbiology and Biotechnology
• /
• v.21 no.11
• /
• pp.1151-1158
• /
• 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.

• Journal of Microbiology and Biotechnology
• /
• v.22 no.11
• /
• pp.1486-1493
• /
• 2012

In this study, we investigated whether galactooligosaccharide (GOS) can be stably and steadily synthesized using immobilized ${\beta}$-galactosidase (${\beta}$-gal) inclusion body (IB)-containing E. coli cells during long-term repeated-batch operation. To improve the operational stability of this enzyme reactor system, immobilized E. coli cells were crosslinked with glutaraldehyde (GA) after immobilization of the E. coli. When we treated with 2% GA for E. coli crosslinking, GOS production continued to an elapsed time of 576 h, in which seven batch runs were operated consecutively. GOS production ranged from 51.6 to 78.5 g/l ($71.2{\pm}10.5$ g/l, n = 7) during those batch operations. In contrast, when we crosslinked E. coli with 4% GA, GOS production ranged from 31.5 to 64.0 g/l ($52.3{\pm}10.8$, n = 4), and only four consecutive batch runs were operated. Although we did not use an industrial ${\beta}$-gal for GOS production, in which a thermophile is used routinely, this represents the longest operation time for GOS production using E. coli ${\beta}$-gal. Improved stability and durability of the cell immobilization system were achieved using the crosslinking protocol. This strategy could be directly applied to other microbial enzyme reactor systems using cell immobilization to extend the operation time and/or improve the reactor system stability.