Construction of Super-Stabilized Direct Electron Transfer Type Glucose Dehydrogenase for Long Term Continuous Glucose Sensing System (2020)

Introduction: The development of super-stabilized flavin adenine dinucleotide glucose dehydrogenase (FADGDH) was achieved based on the rational biomolecular engineering. The FADGDH from Burkholderia cepacia (BcGDH) is the ideal enzyme for continuous glucose monitoring (CGM) sensor, because of its inherent DET ability (Yamazaki 2008, Lee 2018). BcGDH is composed of three subunits, which are the catalytic subunit harboring FAD and iron-sulfur (3Fe-4S) cluster, the electron transfer subunit harboring three heme c , and the small subunit which functions as a “hitchhiker” protein. We recently reported the BcGDH catalytic subunit complexed with small subunit (Yoshida et al., 2019, PDB 6a2u), thereby elucidated the intra-molecular electron transfer pathway, and inherent disulfide bond between catalytic subunit and small subunit. BcGDH shows two distinct temperature profiles due to variations in its quaternary structure (Sode 1996, Yamazaki 1999). BcGDH composed of three subunits showed an optimum reaction temperature of approximately 45 ̊C. By increasing temperature, enzyme activity gradually decreases, due to the dissociation of electron transfer subunit. However, at temperature above 60 ̊C, enzyme activity increases again with the increase the temperature and shows second peak at around 70 ̊C, which is derived from the complex composed of catalytic subunit and small subunit. This phenomenon suggested that the dissociation of the electron transfer subunit dominates the stability of BcGDH three subunit complex, which is the essential structure to show DET ability. Namely, to stabilize BcGDH enzyme complex, the stabilization of quaternary structure is the most rational strategy for this enzyme. We recently succeeded in the elucidation of crystal structure of BcGDH complex together with the electron transfer subunit. The availability of quaternary structure of this enzyme enabled us to design inter-subunit disulfide bond in BcGDH. In this study, the designing and characterization of super-stabilized BcGDH is presented. We also characterize the DET ability of the engineered BcGDH, which is the mandatory characteristic of this enzyme for the development of the future innovative biosensing systems. Methods: Amino acid substitution to cysteine residues were carried out at the residues existing on the surface of each subunit, to form inter-subunit disulfide bonds. Recombinantly prepared BcGDH mutant complexes were purified by affinity chromatography. The catalytic activity and thermal stability were investigated. Then DET-type sensors were constructed by immobilization on the gold electrode with self-assembled monolayer (SAM) layer, and investigated their stability by continuous operation. Results and Discussion: Based on the 3D structure of BcGDH complex, disulfide bonds between electron transfer subunit and either catalytic subunit or small subunit were designed. By the screening of the cystaine residue substituted mutants, some mutants showed remarkable high thermal stability as BcGDH complex without the decrease of the catalytic activity. Furthermore, multiple inter-subunit disulfide bonds made the enzyme as super-stabilized which showed high catalytic activity even by the incubation at 70 ̊C. Thus developed engineered enzyme revealed to show DET ability with electrode, indistinguishable to the wild type one. This super-stabilized enzyme will contribute the improvement of the operational stability of enzyme sensors. References Lee et al., Bioelectrochemistry. 121 (2018) 1–6 Sode et al., Enzyme Microb. Technol. 19 (1996) 82–85 Yamazaki et al., Appl. Biochem. Biotechnol. 77 (1999) 325–336 Yamazaki et al., Anal. Lett. 41 (2008) 2363-2373. Yoshida et al., Acta Crystallogr. Sect. D Struct. Biol. 75 (2019) 841–851