Many redox enzymes are covered with an insulating carbohydrate core and have their active centers deeply buried inside of the protein structure. Several studies on glycon depletion showed that the carbohydrate shell hinders the electron communication between the active center and the electrode surface. Therefore, accessibility of the prosthetic group can be achieved by removal of the carbohydrate shell, thus giving a possibility to develop mediator-less biosensors.

Present methods for production of deglycosylated enzymes are based on either in-solution digestion of native enzymes, or on production of recombinant enzymes, which lack the carbohydrate shell. However, these methods are time-consuming and expensive. More efficient and cheap way to produce deglycosylated enzymes is desirable.   

In attempt to develop a technique for continuous, fast and inexpensive production of deglycosylated enzymes, we have prepared immobilised enzyme reactors (IMERs) with immobilised PNGase F. PNGase F belongs to the class of endoglycosidases, which cleave glycons from Asp residues in N-linked proteins. It was covalently attached to the surface of controlled pore glass (CPG) beads and packed into the IMER. PNGase F was used to deglycosylate glucose oxidase (GOx) containing 6 GlcNAc residues and 3 Man residues. Two IMERs containing 1500 U and 15000 U of the immobilised enzyme were prepared. The efficiency of the two IMERs at different temperatures, as well as time required for complete deglycosylation of GOx, was studied. The degree of deglycosylation was monitored using SDS-PAGE with silver staining. The deglycosylation of GOx was performed both in stop-flow and flow formats with continuous supply of the GOx.

Partial deglycosylation of GOx, subjected to a PNGaseF treatment, resulted in a sharper band on the gel for the native enzyme compared to the non-treated GOx. In a flow experiments band broadening was reduced with decrease of the flow rate from 0.5 mL/min to 0.05 mL/min. For complete deglycosylated enzyme no band for the native GOx was observed.

Complete deglycosylation of GOx was twices faster (1 hour) when using higher amount of PNGase F for preparation of the IMER in a stop-flow experiment. For the same incubation time and the same concentration of GOx, the degree of deglycosylation was higher when performing the reaction at higher temperature (37 °C).

No band for the deglycosylated form of GOx was observed on the gel in any experiments. This is probably due to change in solubility of the deglycosylated form of the enzyme compared to the native form. Since complete removal of the carbohydrate shell from the enzyme results in increased hydrophobicity, it was suggested that the deglycosylated enzyme precipitates on the surface of CPG beads inside the IMER. Addition of up to 20% of organic solvent (methanol) and non-ionic detergent (1% of Triton X-100) did not improve solubility of the deglycosylated GOx.

The advantage of the proposed IMER is that it utilises immobilised enzyme, thus the robustness of the system is greatly improved in terms of operational stability and chemical resistance. The IMER can be reused several times without loss of activity of the immobilised PNGase F, which open an interesting possibility for broad production of deglycosylated enzymes. Therefore, further studies are aiming at fabricating an IMER containing exoglycosidases, which will only perform partial deglycosylation of GOx and other redox enzymes, leaving its solubility properties unaffected.         

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