Application to Photocatalytic H2Production of a Whole-Cell Reaction by RecombinantEscherichia coliCells Expressing [FeFe]-Hydrogenase and Maturases Genes

2016 ◽  
Vol 128 (28) ◽  
pp. 8177-8180 ◽  
Author(s):  
Yuki Honda ◽  
Hidehisa Hagiwara ◽  
Shintaro Ida ◽  
Tatsumi Ishihara
Keyword(s):  
Cell Reaction ◽  
Whole Cell

scholarly journals P212A Mutant of Dihydrodaidzein Reductase Enhances (S)-Equol Production and Enantioselectivity in a Recombinant Escherichia coli Whole-Cell Reaction System

2016 ◽  
Vol 82 (7) ◽  
pp. 1992-2002 ◽  
Author(s):  
Pyung-Gang Lee ◽  
Joonwon Kim ◽  
Eun-Jung Kim ◽  
EunOk Jung ◽  
Bishnu Prasad Pandey ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Reaction System ◽  
Gut Bacteria ◽  
Site Directed Mutagenesis ◽  
Anaerobic Growth ◽  
Efficient Production ◽  
Cell Reaction ◽  
Whole Cell ◽  
Content Type ◽  
Postmenopausal Symptoms

ABSTRACT(S)-Equol, a gut bacterial isoflavone derivative, has drawn great attention because of its potent use for relieving female postmenopausal symptoms and preventing prostate cancer. Previous studies have reported on the dietary isoflavone metabolism of several human gut bacteria and the involved enzymes for conversion of daidzein to (S)-equol. However, the anaerobic growth conditions required by the gut bacteria and the low productivity and yield of (S)-equol limit its efficient production using only natural gut bacteria. In this study, the low (S)-equol biosynthesis of gut microorganisms was overcome by cloning the four enzymes involved in the biosynthesis fromSlackia isoflavoniconvertensintoEscherichia coliBL21(DE3). The reaction conditions were optimized for (S)-equol production from the recombinant strain, and this recombinant system enabled the efficient conversion of 200 μM and 1 mM daidzein to (S)-equol under aerobic conditions, achieving yields of 95% and 85%, respectively. Since the biosynthesis oftrans-tetrahydrodaidzein was found to be a rate-determining step for (S)-equol production, dihydrodaidzein reductase (DHDR) was subjected to rational site-directed mutagenesis. The introduction of the DHDR P212A mutation increased the (S)-equol productivity from 59.0 mg/liter/h to 69.8 mg/liter/h in the whole-cell reaction. The P212A mutation caused an increase in the (S)-dihydrodaidzein enantioselectivity by decreasing the overall activity of DHDR, resulting in undetectable activity for (R)-dihydrodaidzein, such that a combination of the DHDR P212A mutant with dihydrodaidzein racemase enabled the production of (3S,4R)-tetrahydrodaidzein with an enantioselectivity of >99%.

scholarly journals Integration of whole-cell reaction and product isolation: Highly hydrophobic solvents promote in situ substrate supply and simplify extractive product isolation

2017 ◽  
Vol 257 ◽  
pp. 110-117 ◽  
Author(s):  
Dorothea Leis ◽  
Bernhard Lauß ◽  
Robert Macher-Ambrosch ◽  
Andreas Pfennig ◽  
Bernd Nidetzky ◽  
...  
Keyword(s):  
Cell Reaction ◽  
Whole Cell ◽  
Substrate Supply ◽  
Highly Hydrophobic

HVEM Analysis of Microfilament Patterns in The Margins of Tissue Cells

1980 ◽  
Vol 38 ◽  
pp. 808-811
Author(s):  
Carol Allen
Keyword(s):  
Cell Movement ◽  
Cell Spreading ◽  
Living Cells ◽  
Tissue Cell ◽  
Large Sample Size ◽  
Cell Periphery ◽  
Whole Cell ◽  
Critical Point Drying ◽  
Lamellar Region ◽  
Tissue Cells

When provided with a suitable solid substrate, tissue cells undergo a rapid conversion from the spherical form expressed in suspension culture to a characteristic flattened morphology. As a result of this conversion, called cell spreading, the cell nucleus and organelles come to occupy a central region of “deep cytoplasm” which slopes steeply into a peripheral “lamellar” region less than 1 pm thick at its outer edge and generally free of cell organelles. Cell spreading is accomplished by a continuous outward repositioning of the lamellar margins. Cell translocation on the substrate results when the activity of the lamellae on one side of the cell become dominant. When this occurs, the cell is “polarized” and moves in the direction of the “leading lamellae”. Careful analysis of tissue cell locomotion by time-lapse microphotography (1) has shown that the deformational movements of the leading lamellae occur in a repeating cycle of advance and retreat in the direction of cell movement and that the rate of such deformations are positively correlated with the speed of cell movement. In the present study, the physical basis for these movements of the cell margin has been examined by comparative light microscopy of living cells with whole-mount electron microscopy of fixed cells. Ultrastructural observations were made on tissue cells grown on Formvar-coated grids, fixed with glutaraldehyde, further processed by critical-point drying, and then photographed in the High Voltage Electron Microscope. This processing and imaging system maintains the 3-dimensional organization of the whole cell, the relationship of the cell to the substrate, and affords a large sample size which facilitates quantitative analysis. Comparative analysis of film records of living cells with the whole-cell micrographs revealed that specific patterns of microfilament organization consistently accompany recognizable stages of lamellar formation and movement. The margins of spreading cells and the leading lamellae of locomoting cells showed a similar pattern of MF repositionings (Figs. 1-4). These results will be discussed in terms of a working model for the mechanics of lamellar motility which includes the following major features: (a) lamellar protrusion results when an intracellular force is exerted at a locally weak area of the cell periphery; (b) the association of cortical MFs with one another determines the local resistance to this force; (c) where MF-to-MF association is weak, the cell periphery expands and some cortical MFs are dragged passively forward; (d) contact of the expanded area with the substrate then triggers the lateral association and reorientation of these cortical MFs into MF bundles parallel to the direction of the expansion; and (e) an active interaction between these MF bundles associated with the cortex of the expanded lamellae and the cortical MFs which remained in the sub-lamellar region then pulls the latter MFs forward toward the expanded area. Thus, the advance of the cell periphery on the substrate occurs in two stages: a passive phase in which some cortical MFs are dragged outward by the force acting to expand the cell periphery, and an active phase in which additional cortical MFs are pulled forward by interaction with the first set. Subsequent interactions between peripheral microfilament bundles and filaments in the deeper cytoplasm could then transmit the advance gained by lamellar expansion to the bulk of the cytoplasm.

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