Chromogenic Polymer Hydrogel-Based Enzyme Screening Assay: A High Throughput Method to Screen the Carbohydrate-Active Enzymes Using Synthetic Substrates

Protocolo

1. Activation of the assay kit plate

1. Activate the 96-well filter (containing blue CPH-xylan) assay kit plate (List of Materials) by adding 200 µl activation solution (obtained with the kit) into each well, followed by 10 min incubation at room temperature without agitation.
2. Apply vacuum using a vacuum manifold (with the spacer block inside and any standard, transparent 96-well plate as a collection plate) to remove the extant activation solution. It is also possible to use a centrifuge at 2,700 x g for 10 min for this step instead of the vacuum manifold.
3. Wash the CPH substrates by adding 100 µl sterile water and apply a vacuum (or centrifugal force) to remove the stabilizer. Repeat this step two more times and the plates are now ready to use.

2. Enzyme reaction

NOTE: Always include buffer alone as a negative control and if possible previously characterized enzymes as positive controls. Use a statistically appropriate number of replicates. The CPH substrates are stable between pH 3.0 to 10.0 and the total volume of buffer end enzyme solution in each well should not exceed 180 µl. Plant extract or culture broth can also be used instead of purified enzyme solution.

1. Add 150 µl 100 mM sodium acetate buffer, pH 4.5, and 5 µl endo-cellulase solution (to a final concentration of 1 U/ml) to each well of the assay kit plate.
2. Put the product plate (a clear-well plate compatible with the microtiter-plate reader) underneath the assay kit plate to collect any potential leakage from the reaction plate during shaking.
3. Incubate the assay kit plate at 25 °C for 30 min in a horizontal shaker at 150 rpm.
   NOTE: Mixing the reaction in the assay kit plate during the incubation is crucial for achieving a consistent and reproducible result. CPH substrates are stable up to 90 °C. The incubation time should be increased up to 24 hr when testing culture broths containing unknown enzyme concentrations with CPH substrates. Note that appropriate incubation times depend on the activity of the enzyme(s), but in general, if there is no detectable activity within 24 hr, it is likely that the enzyme will not degrade the tested substrate. An active enzyme is degrading the insoluble chromogenic polysaccharides of the CPH substrate into soluble chromogenic oligosaccharides, which are visible as colored supernatant.
4. Place the clean product plate inside the vacuum manifold with the spacer block inside.
5. Place the assay kit plate on top and apply a vacuum (maximum negative pressure of -60 kPa). It is also possible to use a centrifuge at 2,700 x g for 10 min.
   NOTE: The filtrate containing the colored oligosaccharides as the reaction product is now in the product plate for further analysis.

3. Detection and Quantification

1. Check that the volume of liquid in each well of the product plate is approximately the same by visual inspection.
2. Read the absorbance of the collection plate at 595 nm for blue CPH-xylan using a plate reader.
3. When doing data analysis, subtract the buffer-only negative control values from the values from the wells where an enzyme was added. Calculate the mean value and the standard error of means (SEM) from the replicate wells.

Materiales

Name	Company	Catalog Number	Comments
Assay kit plates	Glycospot		customized assay kit plates
Activation solution	Glycospot		for activating CPH substrates
350 ml receiver plate spacer block for vacuum manifold	Pall Corporation	5015	spacer block
96-well MultiScreen HV filter plate,  0.45 µm, clear, non-sterile	Millipore	MSHVN4510	assay plate
96-Well Microplates, Polypropylene	Greiner Bio-One	651201	collection plate after washing the substrates
Nunc MicroWell 96-Well Microplates	Thermo Scientific	269620	product plate
Diaphragm pump MZ 2 NT	Vacuubrand	732000	vacuum pump used with the vacuum manifold
Infors HT Ecotron	Infors HT	4950132 (Buch & Holm)	horizontal shaker
SpectraMax M5	Molecular Devices	10067-750 (VWR)	96-well plate absorbance reader
Vacuum manifold	Pall Corporation	5017	vacuum manifold
Endo-cellulase (EGII) (Trichoderma longibrachiatum)	Megazyme	E-CELTR	cellulase [cel]
α-amylase (Bacillus licheniformis)	Megazyme	E-BLAAM	amylase [amy]