High-throughput activity screening and sorting of single catalyst particles with a droplet microreactor using dielectrophoresis

Biofilm-forming bacteria are sources of infections because they are often resistant to antibiotics and chemical removal. Recombinant biofilm-degrading enzymes have the potential to remove biofilms gently, but they can be toxic toward microbial hosts and are therefore difficult to produce in bacteria. Here, we investigated Nicotiana species for the production of such enzymes using the dispersin B-like enzyme Lysobacter gummosus glyco 2 (Lg2) as a model. We first optimized transient Lg2 expression in plant cell packs using different subcellular targeting methods. We found that expression levels were transferable to differentiated plants, facilitating the scale-up of production. Our process yielded 20 mg kg−1 Lg2 in extracts but 0.3 mg kg−1 after purification, limited by losses during depth filtration. Next, we established an experimental biofilm assay to screen enzymes for degrading activity using different Bacillus subtilis strains. We then tested complex and chemically defined growth media for reproducible biofilm formation before converting the assay to an automated high-throughput screening format. Finally, we quantified the biofilm-degrading activity of Lg2 in comparison with commercial enzymes against our experimental biofilms, indicating that crude extracts can be screened directly. This ability will allow us to combine high-throughput expression in plant cell packs with automated activity screening.

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A Cell-Based Adrenaline Assay for Automated High-Throughput Activity Screening of Epoxide Hydrolases

Chemistry - An Asian Journal ◽

10.1002/asia.200700325 ◽

2008 ◽

Vol 3 (2) ◽

pp. 233-238 ◽

Cited By ~ 10

Author(s):

Daniel Kahakeaw ◽

Manfred T. Reetz

Keyword(s):

High Throughput ◽

Epoxide Hydrolases ◽

A Cell ◽

Activity Screening

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A High-Throughput Cell-Based Assay for Inhibitors of ABCG2 Activity

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057105284576 ◽

2005 ◽

Vol 11 (2) ◽

pp. 176-183 ◽

Cited By ~ 78

Author(s):

Curtis J. Henrich ◽

Heidi R. Bokesch ◽

Michael Dean ◽

Susan E. Bates ◽

Robert W. Robey ◽

...

Keyword(s):

Natural Products ◽

High Throughput ◽

High Throughput Screening ◽

Biochemical Characterization ◽

Cytotoxic Agents ◽

Specific Substrate ◽

Pheophorbide A ◽

Transport Studies ◽

Cell Accumulation ◽

Activity Screening

ABCG2 is a member of the adenosine triphosphate (ATP)-binding cassette family of multidrug transporters associated with resistance of tumor cells to many cytotoxic agents. Evaluation of modulators of ABCG2 activity has relied on methods such as drug sensitization, biochemical characterization, and transport studies. To search for novel inhibitors of ABCG2, a fluorescent cell-based assay was developed for application in high-throughput screening. Accumulation of pheophorbide a (PhA), an ABCG2-specific substrate, forms the basis for the assay in NCI-H460/MX20 cells overexpressing wild-type ABCG2. Treatment of these cells with 10 μM fumitremorgin C (FTC), a specific ABCG2 inhibitor, increased cell accumulation of PhA to 5.6 times control (Z′ 0.5). Validation included confirmation with known ABCG2 inhibitors: FTC, novobiocin, tariquidar, and quercetin. Verapamil, reported to inhibit P-glycoprotein but not ABCG2, had insignificant activity. Screening of a library of 3523 natural products identified 11 compounds with high activity (≥ 50% of FTC, confirmed by reassay), including 3 flavonoids, members of a family of compounds that include ABCG2 inhibitors. One of the inhibitors detected, eupatin, was moderately potent (IC50 of 2.2 μM) and, like FTC, restored sensitivity of resistant cells to mitoxantrone. Application of this assay to other libraries of synthetic compounds and natural products is expected to identify novel inhibitors of ABCG2 activity.

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A low cost reactor for high-throughput activity screening of heterogeneous catalysts by mass spectrometry

Solid State Sciences ◽

10.1016/s1293-2558(03)00115-8 ◽

2003 ◽

Vol 5 (6) ◽

pp. 909-916 ◽

Cited By ~ 13

Author(s):

Jörg Urschey ◽

Pierre-Alain W. Weiss ◽

Jens Scheidtmann ◽

Rudolf Richter ◽

Wilhelm F. Maier

Keyword(s):

Mass Spectrometry ◽

High Throughput ◽

Heterogeneous Catalysts ◽

Low Cost ◽

Activity Screening

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Identification of a Novel Laser Dye Substrate of Mammalian Cytochromes P450: Application in Rapid Kinetic Analysis, Inhibitor Screening, and Directed Evolution

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057107301496 ◽

2007 ◽

Vol 12 (5) ◽

pp. 677-682 ◽

Cited By ~ 3

Author(s):

Santosh Kumar

Keyword(s):

Kinetic Analysis ◽

Directed Evolution ◽

High Throughput ◽

Cumene Hydroperoxide ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Significant Activity ◽

Laser Dye ◽

Screening Assay ◽

Inhibitor Screening ◽

Activity Screening

The author sought to develop a high-throughput activity screening assay to carry out rapid kinetic analysis, inhibitor screening, and directed evolution of cytochrome P450 2C enzymes. Initially, of the 9 fluorescent substrates and 10 P450 2C enzymes tested, several P450 2C enzymes showed > 1 nmol/min/nmol P450 activity in cumene hydroperoxide (CuOOH)—supported reaction with a laser dye, 7-dimethylamino-4-trifluoromethylcoumarin (C152). A high-throughput steady-state kinetic analysis of the human P450 2C8, 2C9, and 2C19 showed 1) kcat = 3 to 6 min—1, 2) Km, CuOOH = 100 to 200 µM, and 3) S50, C152 = 10 to 20 µM in the CuOOH system. In addition, P450 2C9 and 2C19 showed a very high kcat (27 and 38 min—1, respectively) in the nicotinamide adenine dinucleotide phosphate (NADPH)—supported reaction. Subsequently, when mammalian P450s from the other subfamilies were tested, P450 2B1dH, 2B4dH, 2B5dH, 3A4, and 3A5 exhibited a significant activity in both CuOOH and NADPH systems. Furthermore, a high-throughput activity screening assay using whole-cell suspensions of the human P450 2C8, 2C9, and 2C19 was optimized. Overall, the data suggested that C152 can be used as a model substrate for mammalian P450s in CuOOH-supported reaction to perform rapid kinetic analysis, inhibitor screening, and directed evolution. ( Journal of Biomolecular Screening 2007:677-682)

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