In this study, we utilized a catabolite repressor to improve the enzymatic activity of recombinant beta-galactosidase inclusion bodies (IBs) produced in Escherichia coli under the araBAD promoter system. Specifically, we employed methyl alpha-D: -glucopyranoside (alpha-MG) to lower the transcription rate of the beta-galactosidase structural gene. In deepwell microtiter plate and lab-scale fermentor culture systems, we demonstrated that the addition of alpha-MG after induction improved the specific beta-galactosidase production, even though beta-galactosidase was still produced as an IB. Particularly, the addition of 0.0025% alpha-MG led to the most significant increase in the specific activity of the beta-galactosidase. Interestingly, the beta-galactosidase IBs obtained in the presence of 0.0025% alpha-MG were more loosely packed, as determined by IB solubilization in guanidine hydrochloride solution. We propose that the reduced gene transcription rate was responsible for the increased specific beta-galactosidase activity and the loose packing that characterized the IBs produced in the presence of alpha-MG. This principle could be applied throughout the enzyme bioprocessing industry in order to enhance the activity of aggregate-prone enzymes within IBs.
In this study, a galactooligosaccharide (GOS) was synthesized using active β-galactosidase (β-gal) inclusion bodies (IBs)containing Escherichia coli (E. coli) cells. Analysis by MALDI-TOF (matrix-assisted laser desorption/ionizationtime 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 β-gal IBs, and temperature for GOS synthesis were 7.5, 500 g/l, 3.2 U/ml, and 37 o 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 β-gal IBs were gradually destroyed during the reaction. In addition, when both the reaction mixture and E. coli β-gal hydrolysate were analyzed by highperformance 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. β-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.
In this study, we investigated the feasibility of producing bioethanol from the hydrolysate of rape stem. Specifically, the most ideal yeast strain was screened, and the microaeration was performed by surface aeration on a liquid medium surface. Among the yeast strains examined, Pichia stipitis CBS 7126 displayed the best performance in bioethanol production during the surface-aerated fermentor culture. Pichia stipitis CBS 7126 produced maximally 9.56 g/l of bioethanol from the initial total reducing sugars (about 28 g/l). The bioethanol yield was 0.397 (by the DNS method). Furthermore, this controlled surface aeration method holds promise for use in the bioethanol production from the xylose-containing lignocellulosic hydrolysate of biomass.
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