1 research outputs found
Further Insights into the Catalytical Properties of Deglycosylated Pyranose Dehydrogenase from Agaricus meleagris Recombinantly Expressed in Pichia pastoris
The
present study focuses on fragmented deglycosylated pyranose
dehydrogenase (fdgPDH) from Agaricus meleagris recombinantly expressed in Pichia pastoris. Fragmented deglycosylated PDH is formed from the deglycosylated
enzyme (dgPDH) when it spontaneously loses a C-terminal fragment when
stored in a buffer solution at 4 °C. The remaining larger fragment
has a molecular weight of ∼46 kDa and exhibits higher volumetric
activity for glucose oxidation compared with the deglycosylated and
glycosylated (gPDH) forms of PDH. Flow injection amperometry and cyclic
voltammetry were used to assess and compare the catalytic activity
of the three investigated forms of PDH, “wired” to graphite
electrodes with two different osmium redox polymers: [Os(4,4′-dimethyl-2,2′-bipyridine)<sub>2</sub>(poly(vinylimidazole))<sub>10</sub>Cl]<sup>+</sup> [Os(dmbpy)PVI]
and [Os(4,4′-dimethoxy-2,2′-bipyridine)<sub>2</sub>(poly-(vinylimidazole))<sub>10</sub>Cl]<sup>+</sup> [Os(dmobpy)PVI]. When “wired”
with Os(dmbpy)PVI, the graphite electrodes modified with fdgPDH showed
a pronounced increase in the current density with <i>J</i><sub>max</sub> 13- and 6-fold higher than that observed for gPDH-
and dgPDH-modified electrodes, making the fragmented enzyme extraordinarily
attractive for further biotechnological applications. An easier access
of the substrate to the active site and improved communication between
the enzyme and mediator matrix are suggested as the two main reasons
for the excellent performance of the fdgPDH when compared with that
of gPDH and dgPDH. Three of the four glycosites in PDH: N<sup>75</sup>, N<sup>175</sup>, and N<sup>252</sup> were assigned using mass spectrometry
in conjunction with endoglycosidase treatment and tryptic digestion.
Determination of the asparagine residues carrying carbohydrate moieties
in PDH can serve as a solid background for production of recombinant
enzyme lacking glycosylation