A pyranose dehydrogenase-based biosensor for kinetic analysis of enzymatic hydrolysis of cellulose by cellulases

https://doi.org/10.1016/j.enzmictec.2014.03.002Get rights and content

Highlights

  • An electrochemical biosensor based on pyranose dehydrogenase was developed.

  • The enzyme biosensor is not anomer specific.

  • The enzyme biosensor showed high sensitivity and stability.

  • The method can be used for real-time monitoring of cellulases activity on cellulose.

Abstract

A novel electrochemical enzyme biosensor was developed for real-time detection of cellulase activity when acting on their natural insoluble substrate, cellulose. The enzyme biosensor was constructed with pyranose dehydrongease (PDH) from Agaricus meleagris that was immobilized on the surface of a carbon paste electrode, which contained the mediator 2,6-dichlorophenolindophenol (DCIP). An oxidation current of the reduced form of DCIP, DCIPH2, produced by the PDH-catalyzed reaction with either glucose or cellobiose, was recorded under constant-potential amperometry at +0.25 V (vs. Ag/AgCl). The PDH-biosensor was shown to be anomer unspecific and it can therefore be used in kinetic studies over broad time-scales of both retaining- and inverting cellulases (in addition to enzyme cocktails). The biosensor was used for real-time measurements of the activity of the inverting cellobiohydrolase Cel6A from Hypocrea jecorina (HjCel6A) on cellulosic substrates with different morphology (bacterial microcrystalline cellulose (BMCC) and Avicel). The steady-state rate of hydrolysis increased towards a saturation plateau with increasing loads of substrate. The experimental results were rationalized using a steady-state rate equation for processive cellulases, and it was found that the turnover for HjCel6A at saturating substrate concentration (i.e. maximal apparent specific activity) was similar (0.39–0.40 s−1) for the two substrates. Conversely, the substrate load at half-saturation was much lower for BMCC compared to Avicel. Biosensors covered with a polycarbonate membrane showed high operational stability of several weeks with daily use.

Introduction

A common observation in the kinetics of enzymatic cellulose degradation is a declining hydrolysis rate with both time and conversion [1], [2]. The origin of the slowdown remains unclear and both enzyme- and substrate related properties have been proposed [1], [3] and further progress in this area seems to require better descriptions of structural and kinetic aspects. Fundamental insights into the complex enzymatic hydrolysis process can be obtained from kinetic studies, but such work is challenged by the insoluble and heterogeneous nature of cellulose. Some progress has been obtained using novel real-time experimental approaches such as quartz crystal microbalance (QCM) measurements [4], [5], [6], [7], [8], electrochemical sensors [9] and isothermal titration calorimetry (ITC) [10], [11], [12], [13]. Enzyme biosensors constitute another real-time approach, which in some cases provides advantageous sensitivity, specificity, and response time. This was utilized in the development of amperometric enzyme biosensors for cellulase activity based on immobilized enzymes including glucose oxidase (GOx), pyrroloquinoline quinine-dependent glucose dehydrogenase (GDH) or cellobiose dehydrogenase (CDH) [14], [15], [16]. The CDH biosensor was found to have a particularly high resolution in time and analyte concentration, and this allowed elucidation of the pre-steady state kinetics of the cellobiohydrolase Cel7A [17], [18] and endoglucanase Cel7B [16] from Hypocrea jecorina (anamorph: Trichoderma reesei) on their insoluble substrates. Sensors based on either GOx, GDH or CDH share the feature of specifically detecting the β-anomer of their analytes. In some special cases this specificity provides analytical advantages, but in general activity measurements, it sets up a number of severe limitations. Hence, these sensors cannot be directly used when the product of the enzymatic reaction is an α-anomer (e.g. for inverting cellulases such as Cel6A). More importantly, anomeric specificity limits the time-scales over which a biosensor can be used for any hydrolytic enzyme in real-time measurements. This is because mutarotation (i.e. equilibration of the α-β distribution) will occur in parallel with the enzymatic hydrolysis, and hence impede quantification of the product. In practice this means that the anomer-specific biosensors can be used over time-scales that are either much faster or much slower than the mutarotation because under these conditions mutarotation will either be negligible or fully equilibrated and hence easy to account for. Between these extremes there will be a broad interval where biosensor measurements will be either unfeasible or dependent on extensive and error-prone corrections [14]. In the light of this, a biosensor without anomeric specificity appears useful in attempts to elucidate cellulolytic enzymes and their ubiquitous activity loss.

Pyranose dehydrogenase (PDH, pyranose:acceptor oxidoreductase, EC 1.1.99.29) (PDH) is a glycosylated, extracellular, monomeric flavin-dependent sugar oxidoreductase secreted by several wood degrading fungi and a member of the glucose–methanol–choline oxidoreductase family [19], [20]. PDH from Agaricus meleagris (AmPDH) appears promising for biosensor-based cellulase activity studies because it shows a broad electron-donor substrate specificity which includes both mono-, di- and oligosaccharides, is inert towards oxygen, shows broad optimal pH range (pH 4–10) and is stable for months when stored at 4 °C [19], [21]. AmPDH can perform both single oxidizations on the C-1, C-2 or C-3 position or double oxidation (C-1,2 or C-3,4 positions) depending on the substrate and it is not specific to one of the anomeric forms [21]. AmPDH has been successfully “wired” with Osmium redox polymers and immobilized on electrodes both for detection of sugars [22], [23] and as anode in enzymatic biofuel cells [24], [25], [26].

In the present work a mediated amperometric biosensor based on immobilized AmPDH was developed and the biosensor was applied for real-time activity measurements of a cellulase hydrolyzing insoluble cellulose. The experimental results were analyzed with respect to a recent published steady-state rate equation for processive cellulases [27].

Section snippets

Chemicals

Unless otherwise stated, all chemicals were of HPLC grade (>99% purity) and supplied by Sigma–Aldrich (St. Louis, USA). α-d-(+)-Glucose (>99.0%) was supplied by Acros Organics (NJ, USA), β-d-(+)-Glucose (>99.0%) was from ChromaDex™ (Irvine, USA) and Cellotriose (Fine grade, >95%) was purchased from Seikagaku Biobusiness Corporation (Tokyo, Japan). All solutions were prepared with 50 mM sodium acetate and 2 mM CaCl2 buffer adjusted to pH 5.0. Stock solutions of sugars were prepared at least 24 h

Mediator selection for PDH-biosensor

From the steady-state kinetic parameters of AmPDH with various electron acceptors reported by Sygmund et al. [19] Fc+, BQ and DCIP were tested for the development of a AmPDH biosensor. BQ and ferrocene have been used as electron mediators in second-generation enzyme biosensors [14], [15], [16], [17], [33], [34], [35], [36], [37], [38]. DCIP is frequently used in enzyme assays in solution and as a pH/redox indicator. It is electrochemically active and has been used as a redox coupling agent in

Conclusion

In conclusion, it has been shown that a mediated amperometric biosensor using immobilized pyranose dehydrogenase from A. meleagris on a carbon paste electrode and DCIP as mediator provides an advantageous approach to kinetic studies of cellulases. The PDH-biosensor is anomer unspecific and can therefore be used in continuous studies of both retaining- and inverting cellulases over different time-scales. The PDH-DCIP-biosensor showed high sensitivity and when covered with a polycarbonate

Conflict of interest statement

Novozymes is a enzyme producing company.

Acknowledgments

This work was supported by the Danish Agency for Science, Technology and Innovation, Programme Commission on Sustainable Energy and Environment (Grant # 2104-07-0028 to P.W.) and the Ministry of Education, Culture, Sports, Science, and Technology in Japan (grant-in-aid for young scientists # 23760746 and special coordination funds for promoting science and technology to H.T.).

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