High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

The high-throughput and multiplexing capacity of this assay is based on insoluble chromogenic polymer (or protein) hydrogel (CPH) substrates arranged in 96-well filter plates. Enzymes as well as negative controls are added to the assay kit plate (Figure 1A) and the enzymes degrade the corresponding substrate producing a colored supernatant (Figure 1B). After the reaction is finished, the supernatant is transferred into a clear-well product plate and the absorbance can be measured directly using a spectrophotometer suitable for 96-well plates (Figure 1C).

An example of a dose response of CPH-arabinoxylan to xylanase at different concentrations of the enzyme (0.00 – 0.75 U/ml) is shown in Figure 1D where the decreasing enzyme concentration can be observed visually. A more detailed spectrophotometric quantification can be used to plot the absorbance versus enzyme concentration (Figure 1E). The signal intensity corresponds to the enzyme activity. The reproducibility of the assay is shown by the error bars (standard error of mean, SEM, of three replicas). More detailed experiments on the reproducibility of this assay are published elsewhere⁸.

Figure 1. Xylanase treatment of CPH-arabinoxylan. A) A scheme of the assay kit plate with the CPH substrate (e.g., CPH-arabinoxylan) loaded into 96-well filter plate wells just before the addition of enzymes 1 and 2 and the buffer-only control (enzyme 1 had endo-xylanase activity); B) Degradation of CPH-arabinoxylan by enzyme 1 produced a colored supernatant; C) Upon vacuum-assisted filtration of the supernatants in to the product plate, the absorbance is measured spectrophotometrically; D) product plate containing the reaction products after treatment of CPH-arabinoxylan in 4 different colors with different concentrations of endo-β-1,4-xylanase in 100 mM sodium acetate buffer, pH 4.5 for 60 min at room temperature; E) Quantification of the reaction products from D using spectrophotometry. Please click here to view a larger version of this figure.

There are different options for using this assay in enzyme screening. One option is to use a 96-well plate containing different polysaccharides for screening, e.g., a small number of (purified) endo-enzymes with unknown activity. In this case the result will show which polysaccharides are degradable by the target enzyme. To show this principle, an endo-cellulase was tested against different CPH substrates at 25 °C. Three different enzyme concentrations (0.5 U/ml, 1.0 U/ml and 5 U/ml) were incubated for 30 min. The result is clearly visible in the product plate (Figure 2A). The product sheet for this endo-cellulase provided by the supplier specifies side-activity for xyloglucan (tamarind), barley β-glucan, glucomannan, birchwood xylan and low side-activity for galactomannan. Consistent with this, activity additional to cellulase was found against CPH-β-glucan (barley), CPH-xyloglucan (tamarind), CPH-xylan (beechwood) and low activity against CPH-galactomannan (Figure 2B). Glucomannan was not tested. The same CPH substrates were digested with commercial available enzymes (three different enzyme concentrations: 0.1 U/ml, 0.5 U/ml and 1.0 U/ml) used as positive controls under the same conditions than the previous experiment. All substrates were degraded by the positive control enzyme and the signal intensity increased corresponding to higher enzyme concentration (Figure 2C).

Figure 2. Eight different CPH substrates were incubated under agitation at 25 °C for 30 min. A) The product plate of different CPH substrates digested with an endo-cellulase, at different concentrations. B) Quantification of the activity and various side activity of the endo-cellulase. The error bars represent the standard error of mean of three replicas. C) Activity of different commercial enzymes to the corresponding CPH substrate (endo-cellulase and 2-HE-cellulose; E-LAMSE and CPH-pachyman, CPH-curdlan, CPH-β-glucan (barley); E-XYAN4 and CPH-xylan, E-XEGP and CPH-xyloglucan, E-BLAAM and CPH-amylose, E-BMACJ and CPH-galactomannan; all enzymes from Megazyme). The error bars represent the standard error of mean of two replicas. Please click here to view a larger version of this figure.

The insoluble chromogenic biomass (ICB) substrates are a useful addition to the chromogenic substrate repertoire because they retain in part the natural arrangement of polysaccharides in plant cell walls which are the major constituent of biomass. CPH and ICB substrates are used in our example to analyze the secreted enzymes of Phanerochaete chrysosporium when cultivated in liquid medium. The plate setup of the assay kit plate is shown in Figure 3A, with 19 CPH substrates and 5 ICB substrates (4 wells for each substrate, Figure 3B). P. chrysosporium was cultivated for three days and then the culture supernatant analyzed. Therefore, 125 µl 200 mM buffer was transferred to each well and 25 µl culture supernatant added. Three different pH conditions have been tested using sodium acetate buffer pH 4.0, sodium phosphate buffer pH 6.0 or pH 8.0 (Figure 3C). The plate was incubated shaking (150 rpm) at 25 °C for 2 hr.

The reaction products were transferred to the product plate (Figure 3D) and analyzed. P. chrysosporium produced enzymes for the degradation of various glucans, starch and xylans (Figure 3E). Lower signals could be detected for the hemicelluloses arabinan (sugar beet) and pectic galactan as well as for RGI (soybean). The enzymes produced were more active in acidic conditions (pH 4.0) than in neutral or slightly basic conditions (pH 8.0). Lower activity towards ICB substrates (Figure 3F) demonstrates that when the polysaccharides are in a more natural context, the efficiency of the enzyme is not the same as with a pure polysaccharide and that is why ICB substrates demonstrate a more realistic view on enzyme efficiency, if it was applied to raw or pre-treated plant material.

Figure 3. Screening of a culture supernatant from a 3-day old liquid culture of Phanerochaete chrysosporium using a multi substrate plate containing 19 CPH and 5 ICB substrates. A) A scheme of the plate setup with 4 wells for each individual substrate (grey background = CPH substrates, orange background = ICB substrates). B) Picture of the assay plate containing the substrate. C) Scheme showing the buffer conditions used in that experiment (200 mM sodium acetate pH 4.0, sodium phosphate pH 6.0 and sodium phosphate pH 8.0). D) picture of the product plate after 2 hr at 25 °C. E) Absorbances were detected at 517 nm and plotted for each individual CPH substrate and F) ICB substrate results for the respective enzymes. Please click here to view a larger version of this figure.

The chromogenic substrates can be also used to study synergistic effects by using a mixture of different colored CPH substrates in one well and analyzing the reaction supernatant after treatment using single enzymes or enzyme cocktails.

In the following example shown in Figure 4, red CPH-cellulose and yellow CPH-xylan substrates were mixed together at an approximately equal ratio in 96-well filter plate wells. Figure 4A shows the colored reaction products after 1 hr treatment at room temperature with no enzyme (control), cellulose cel (2 U/ml), xylanase xyl (1 U/ml) and a mixture of both enzymes (3 replicates for each approach) in 100 mM sodium acetate buffer pH 4.5. For analysis, the reaction product was quantified spectrophotometrically by scanning the absorbance spectrum from 350 nm to 700 nm (Figure 4B). Often visual inspection alone can give an indication of whether the enzyme is acting on one or multiple substrates, however recorded absorbance spectra stemming from different dyes can also be resolved using simple linear regression⁸ to give a more accurate indication of the extent of degradation of each substrate from the mixture.

Using CPH substrates as mixtures substantially adds to the throughput of the assay, enabling screening against up to 4 different substrates in one experiment (one well). In the example shown are four different substrates used: blue CPH-β-glucan (barley), yellow CPH-xylan (beechwood), green CPH-amylose and red CPH-pectic galactan (lupin). The layout of the substrate plate is shown in Figure 4C and a picture of the assay plate in Figure 4D. The reaction was performed in 100 mM sodium acetate buffer pH 4.5 for 30 min at 25 °C and 150 rpm. First single enzymes with increasing enzyme concentration were tested with the corresponding CPH substrate (Figure 4E) and the colored supernatant was received as expected in the product plate (Figure 4F, row 1A – 12D). Row E contained the two different CPH substrates yellow CPH-xylan and blue CPH-β-glucan, which were degraded with different ratios of the corresponding enzymes endo-xylanase and endo-glucanase. After the reaction, the result is visible in the product plate: the color of the reaction product was darker green-blue, when more endo-glucanase was present (Figure 4F, 4E-6E) and turned into a lighter yellow-green, when the endo-xylanase concentration increased (Figure 4F, 10-12E). The same is seen in row F, where the two substrates red CPH-pectic galactan and yellow CPH-xylan were degraded with endo-galactanase and endo-xylanase. All four different colored CPH substrate were present (Figure 4F, 1G-12F) and single enzymes degraded the appropriate CPH substrate and by adding additional enzymes a combination of the colored reaction products was received.

Figure 4. A combination of two different CPH substrates, red CPH-cellulose and yellow CPH-xylan, treated with different enzymes. A) Reaction supernatants after treatment of two substrates with endo-cellulase (cel) or endo-xylanase (xyl) or both enzymes. B) Absorbance spectra of the reaction supernatants. A combination of four different CPH substrates treated with different enzymes. C) Scheme of the substrate plate containing the CPH substrates: blue CPH-β-glucan (barley), yellow CPH-xylan (beechwood), green CPH-amylose and red CPH-pectic galactan (lupin). D) Picture of the assay plate containing the different CPH substrates. E) Scheme of the product plate showing the ratio of the added enzymes (nominal concentrations (NC): glu = 1 U/ml endo-glucanase, xyl = 1 U/ml endo-xylanase, amy = 5 U/ml endo-amylase and gal = 0,5 U/ml endo-galactanase). F) Picture of the product plate after 30 min incubation at 25 °C. Please click here to view a larger version of this figure.

Table 1. List of available Chromogenic Polymer Hydrogel (CPH) and Insoluble Chromogenic Biomass (ICB) substrates.