Different Effects of Deglycosylation on the Lactose Sensing Ability of Mesophilic and Thermophilic Cellobiose Dehydrogenases.

Abstract

The development of an efficient lactose biosensor employing cellobiose dehydrogenases (CDHs) for monitoring and precise control of the lactose levels in dairy-based products is extremely important for the health of lactose-intolerant population. In this study, the mesophilic (Nc_CDH) and thermophilic (Ct_CDH-A, Ct_CDH-B) CDHs were successfully obtained by heterologous expression and treated with α-1,2-mannosidase and endoglycosidase H to prepare the deglycosylated forms (Nc_dCDH, Ct_dCDH-A, and Ct_dCDH-B); then, the effects of deglycosylation on the catalytic activity in solution and electrochemical performance on electrodes for lactose detection were systematically investigated. In solution, Nc_dCDH was more stable and had a higher Vmax value and lower KM value than Nc_CDH at different temperatures and pH values. In contrast, deglycosylation had adverse effects on the stability of Ct_CDH-A and Ct_CDH-B. When the CDHs mixed with multi-walled carbon nanotubes were dropped and immobilized on electrodes, with regard to Nc_CDH, in the presence of the same concentration of lactose, the detection current of the electrode modified with Nc_dCDH was higher than that of the electrode modified with Nc_CDH, and it had a lower detection limit (2.006mM) and higher sensitivity (39.37μA.mmol.L-1.cm-2). However, with respect to the thermophilic CDHs, the sensitivity was lowered and the detection limit was increased after deglycosylation. The discrepancy may result from two reasons: N-glycosylation may play a more crucial role in thermostability and structural stability of thermophilic CDHs, and the distribution sites of glycosylated residues may affect the electron transfer kinetics. This study is a step toward using CDH as an electron transfer-based lactose biosensor.