ChitO-based assay and Schales’ method for chitinase detection
The chitinase ChitO-based assay is based on the oxidation of the chito-oligosaccharides by ChitO, which are formed by the action of the chitinases on the chitin. Upon oxidation of these substrates, a stoichiometric amount of H2O2 is produced by reduction of molecular oxygen. The hydrogen peroxide is used by HRP to convert 4-aminoantipyrine (AAP) and 3,5-dichloro-2-hydroxybenzenesulfonic acid (DCHBS) into a pink and stable compound [12]. As a result, the intensity of the pink color is proportional to the concentration of the available ChitO substrates. To test our assay for the detection of chitinase activity, a chitinase from Streptomyces griseus and colloid chitin as a substrate were used. Colloidal chitin is a natural unmodified substrate, easy to prepare and convenient for pipetting compared to chitin flakes. Varying amounts of chitinase were incubated with colloid chitin for 60 minutes to allow degradation of the chitin. Subsequently, the ChitO assay components (ChitO, AAP, DCHBS, and HRP) were added to the incubations in the 96-well microtiter plate. Development of a clear pink color is indicative of chitinase activity. By measuring the absorbance at 515 nm, the activity of ChitO, and hence the activity of chitinase, could be determined. A clear relationship was observed between the measured absorbance and increasing units of chitinase (Figure 1). In fact, the data shows a saturation curve which can be fitted with a simple hyperbolic formula:
Interestingly, the assay could detect as low as 10 μU of chitinase with an assay time of only 15 minutes and using 0.12 U ChitO (P <1%). The blank reaction (colloidal chitin incubated without chitinase) revealed that colloidal chitin itself is a very poor substrate for ChitO. For such incubation a very weak signal (A515 = 0.12) was recorded and used as a blank. The reproducibility of the ChitO-based assay was assessed by comparing the corrected absorbance values on nine replicates of colloidal chitin treated with 50 μU of chitinase, to nine replicates of untreated colloidal chitin (Figure 2). The assay showed high reproducibility with a low standard deviation (<0.3%) for both samples.
For benchmarking, we compared the ChitO-based assay to the Schales’ procedure since it is one of the most common methods for the detection of chitinase activity [7, 13]. The Schales’ reagent is yellow in color and reaction with reducing sugars results in color fading, which can be measured at 420 nm. Figure 3 shows the absorbance signal obtained in relation to the concentration of chitinase. A chitinase activity of 600 μU was found to be the lowest detection limit (P <3%). This value is 60 times higher than the detection limit of the ChitO-based assay (10 μU) indicating a higher sensitivity in favor of the ChitO assay. In addition, the recorded signal intensity of the ChitO assay was higher, by approximately two-fold, than Schales’ procedure. This can be concluded from comparing the signal responses in Figures 1 and 3, particularly when considering the range of 600 μU to 3,000 μU chitinase. It is important to note that the boiling step, an essential step in the Schales’ procedure, is omitted from the ChitO assay, which represents one of the main advantages (Figure 4).
ChitO-based assay for cellulase detection
To adapt the ChitO-based assay for monitoring activity of cellulolytic enzymes, a ChitO mutant (ChitO-Q268R) was used instead of wild-type ChitO. ChitO-Q268R has a higher enzymatic efficiency towards glucose, cellobiose, cellotriose and cellotetraose compared to wild-type ChitO. We applied the assay using the same conditions as for detection of chitinase activity. As a model cellulase, an endocellulase from Aspergillus niger was used with a filter paper as a substrate. Endoglucanases typically hydrolyze accessible parts of the cellulose polymer and generate new chain ends. The generated cellotetraose and lower fragments will be substrates for ChitO-Q268R and consequently will allow H2O2 generation and development of the pink-colored product. The signal intensity, which is based on endocellulase activity, depends on the fraction of accessible β-glycosidic bonds in the substrate.
It was gratifying to see that using ChitO-Q268R in combination with HRP resulted in a clear and immediate color development. As was found for the ChitO-based chitinase assay, a directly proportional relationship of the absorbance to the amount of cellulase units was observed (Figure 5). The response curve started to level off when using >100 mU of the hydrolase. The lowest tested amount of endocellulase was 6 mU which could be detected with an assay time of 15 minutes using 0.13 U of ChitO-Q268R (P <0.5%). The commonly used colorimetric reagent to measure the cellulose saccharification is DNS [14]. There are many drawbacks of this method, such as non-reproducibility, complexity of reagents preparations and time-consuming. It also requires a strict control of temperature for proper color development and stability [15]. Moreover, the use of toxic reagents and phenolic compounds in large amounts makes it not a very environmentally-friendly method. Trials have been made to improve the DNS assay, such as reducing the amount of reagents used and adapting it to a microtiter plate assay. However, heating or boiling is still required in all of these approaches [16]. Both the DNS assay and the ChitO-based cellulase assay cannot distinguish between the contributions given by the different sugars present in the reaction mixture. However, the ChitO-based assay does not require alkaline medium and harsh treatment as in DNS, which results in degradation of the sugar content and decreased sensitivity [15, 16]. On the whole, the assay represents a faster, high-throughput and ‘green’ method for cellulase detection when compared with the established DNS assay.
ChitO-based assays: detecting defined substrates
The color that develops in the aforementioned assay experiments is a sum of the ChitO activity on a mixture of different hydrolytic products produced by chitinase or cellulase activity. In order to identify the sensitivity of the assay for individual hydrolysis products, response curves were determined. Two sets of compounds were tested: chito-oligosaccharides and cello-oligosaccharides. The experiments were performed at pH 6 and 5, respectively, similar to the ChitO-chitinase and ChitO-cellulase detection experiments. Figure 6A shows a direct response of the signal when testing varying concentrations of N-acetyl-D-glucosamine, chitobiose and chitotetraose, representatives of the chitin degradation products. The limit of detection for N-acetyl-D-glucosamine, chitobiose and chitotetraose was 5 μM (P <5%). Based on the observed slopes, N-acetyl-D-glucosamine showed the highest signal response followed by chitobiose and chitotetraose. A similar trend has also been found with the Schales’ procedure and described in literature by Horn and Eijsink [9]. The second set of compounds tested represented cellulose degradation products: glucose, cellobiose and cellotetraose. Figure 6B shows a direct response of the ChitO assay signal to the increasing concentration of the compounds. The limit of detection was 25 μM for glucose and 10 μM for cellobiose and cellotetraose (P <5%). No specific trend of signal response to the compound’s length was observed. Cellobiose showed the highest signal response followed by cellotetraose and glucose.
ChitO-based assays: monitoring hydrolysis of complex natural substrates
The ChitO assay showed applicability for detection of chitinase and cellulase activity on processed substrates such as colloid chitin and filter paper. We have tested the applicability of the assay on unprocessed and complex materials, that is, ground shrimp shell and wheat straw. In both cases a strong signal was observed (Figure 7). The assay was found to be very specific as the blank reactions did not yield any significant signal. The measured absorbance values for the triplicate samples showed only marginal differences, which confirms the above results of the assay reproducibility.
In the context of comparing the ChitO-based assay to the Schales’ procedure, the availability of reagents should also be addressed. The oxidases used in the ChitO-based assay are expressed in a standard expression system using Escherichia coli as host. The enzymes are stable at room temperature and active under the assay pH condition. A His-tag has been fused to the recombinant enzymes to facilitate the purification process. Expression in E. coli and subsequent purification can yield 40 mg (170 U) of purified protein per liter of culture [11]. Considering the low amount of ChitO used in the present experiments (0.12 U per sample), a 1 L culture provides sufficient ChitO for assaying over 1,400 samples.
Several strategies can be foreseen for further development of the ChitO-based assay. The formation of H2O2, that is a reactive oxidative species, can be used for detection by highly sensitive techniques. For example, an amperometric redox polymer-based biosensor replacing the colorimetric reagents can be utilized as has been done for cellobiose dehydrogenase [17]. Alternatively, the use of a fluorescent dye such as Amplex Red for H2O2 detections will enable the ChitO-based assay to work in real-time analyses and turbid materials such as soil samples. The present study has shown the applicability of ChitO-based assay for cellulose or chitinase activity detection. However, it can also find a potential application in the food industry, for example, for the detection of chitin and chitosan content in edible mushrooms [18].