• Journal of Microbiology and Biotechnology
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• 제28권8호
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• pp.1346-1351
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• 2018

Oxidoreductases are effective biocatalysts, but their practical use is limited by the need for large quantities of NAD(P)H. In this study, a whole-cell biocatalyst for NAD(P)H cofactor regeneration was developed using the economical substrate glycerol. This cofactor regeneration system employs permeabilized Escherichia coli cells in which the glpD and gldA genes were deleted and the gpsA gene, which encodes $NAD(P)^+-dependent$ glycerol-3-phosphate dehydrogenase, was overexpressed. These manipulations were applied to block a side reaction (i.e., the conversion of glycerol to dihydroxyacetone) and to switch the glpD-encoding enzyme reaction to a gpsA-encoding enzyme reaction that generates both NADH and NADPH. We demonstrated the performance of the cofactor regeneration system using a lactate dehydrogenase reaction as a coupling reaction model. The developed biocatalyst involves an economical substrate, bifunctional regeneration of NAD(P)H, and simple reaction conditions as well as a stable environment for enzymes, and is thus applicable to a variety of oxidoreductase reactions requiring NAD(P)H regeneration.

• Journal of Microbiology and Biotechnology
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• 제32권8호
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• pp.1054-1063
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• 2022

Trehalose is a non-conventional sugar with potent applications in the food, healthcare and biopharma industries. In this study, trehalose was synthesized from maltose using whole-cell Pseudomonas monteilii TBRC 1196 producing trehalose synthase (TreS) as the biocatalyst. The reaction condition was optimized using 1% Triton X-100 permeabilized cells. According to our central composite design (CCD) experiment, the optimal process was achieved at 35℃ and pH 8.0 for 24 h, resulting in the maximum trehalose yield of 51.60 g/g after 12 h using an initial cell loading of 94 g/l. Scale-up production in a lab-scale bioreactor led to the final trehalose concentration of 51.91 g/l with a yield of 51.60 g/g and productivity of 4.37 g/l/h together with 8.24 g/l glucose as a byproduct. A one-pot process integrating trehalose production and byproduct bioremoval showed 53.35% trehalose yield from 107.4 g/l after 15 h by permeabilized P. moteilii cells. The residual maltose and glucose were subsequently removed by Saccharomyces cerevisiae TBRC 12153, resulting in trehalose recovery of 99.23% with 24.85 g/l ethanol obtained as a co-product. The present work provides an integrated alternative process for trehalose production from maltose syrup in bio-industry.

• Biotechnology and Bioprocess Engineering:BBE
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• 제9권3호
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• pp.147-150
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• 2004

Permeabilization is known to overcome cell membrane barriers of whole cell biocatalysts. The use of organic solvents is advantageous in terms of cost, simplicity, and efficiency. In this study, Ochrobactrum anthropi SY509 was permeabilized with various organic solvents. Treatment with organic solvents resulted in lower permeability barriers due to falling out lipids of the cell membrane. Therefore, permeabilized cells showed higher enzyme activity with no cell viability. Among various organic solvents, 0.5% (v/v) chloroform was selected as the most efficient permeabilizing reagent. Changes in the cell membrane structure were observe d and the residual amounts of phospholipids of the cell membrane were measured to investigate the mechanism of the improved permeability.

• Journal of Microbiology and Biotechnology
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• 제4권4호
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• pp.343-348
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• 1994

Enterobacter sp. producing -$\beta$-galactosidase with high transgalactosylation activity was isolated from dairy wastewater. The isolate had common biochemical features to E. aerogenes and E. cloacae. Enzyme production increased as the cell mass increased with optimum enzyme activity of 0.21 Unit/mg-protein (o-nitro-phenyl-$\beta$ -D-galactoside (ONPG) as substrate) until 8 hr of culture. Whole cells permeabilized by toluene were used to produce galacto-oligosaccharide. Optimum toluene concentration, temperature and pH for -$\beta$-galactosidase activity of permeabilized whole cells were 10% (v/v), $50^{\circ}C$ and 6.0, respectively. A maximum of 38% (w/w) of galacto-oligosaccharide was obtained with lactose concentration of 20% (w/w) at $40^\{\circ}C$ and pH 6.0.

• Journal of Microbiology and Biotechnology
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• 제18권5호
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• pp.946-951
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• 2008

The opd gene, encoding organophosphorus hydrolase (OPH) from Flavobacterium sp. capable of degrading a wide range of organophosphate pesticides, was surface- and intracellular-expressed in Synechococcus PCC7942, a prime example of photoautotrophic cyanobacteria. OPH was displayed on the cyanobacterial cell surface using the truncated ice nucleation protein as an anchoring motif. A minor fraction of OPH was displayed onto the outermost surface of cyanobacterial cells, as verified by immunostaining visualized under confocal laser scanning microscopy and OPH activity analysis; however, a substantial fraction of OPH was buried in the cell wall, as demonstrated by proteinase K and lysozyme treatments. The cyanobacterial outer membrane acts as a substrate (paraoxon) diffusion barrier affecting whole-cell biodegradation efficiency. After freeze-thaw treatment, permeabilized whole cells with intracellular-expressed OPH exhibited 14-fold higher bioconversion efficiency ($V_{max}/K_m$) than that of cells with surface-expressed OPH. As cyanobacteria have simple growth requirements and are inexpensive to maintain, expression of OPH in cyanobacteria may lead to the development of a low-cost and low-maintenance biocatalyst that is useful for detoxification of organophosphate pesticides.