Further Insights into the Catalytical Properties of Deglycosylated Pyranose Dehydrogenase from Agaricus meleagris Recombinantly Expressed in Pichia pastoris
(2013) In Analytical Chemistry 85(20). p.9852-9858- Abstract
- 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 degrees C. The remaining larger fragment has a molecular weight of similar to 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... (More)
- 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 degrees C. The remaining larger fragment has a molecular weight of similar to 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)(2)(poly(vinylimidazole))(10)Cl](+) [Os(dmbpy)PVI] and [Os(4,4'-dimethoxy-2,2'-bipyridine)(2)(poly-(vinylimidazole))(10)Cl](+) [Os(dmobpy)PVI]. When "wired" with Os(dmbpy)PVI, the graphite electrodes modified with fdgPDH showed a pronounced increase in the current density with J(max). 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-75, N-175, and N-252 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. (Less)
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https://lup.lub.lu.se/record/4212829
- author
- organization
- publishing date
- 2013
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Analytical Chemistry
- volume
- 85
- issue
- 20
- pages
- 9852 - 9858
- publisher
- The American Chemical Society (ACS)
- external identifiers
-
- wos:000326126600061
- scopus:84886885900
- pmid:24016351
- ISSN
- 1520-6882
- DOI
- 10.1021/ac4023988
- language
- English
- LU publication?
- yes
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- 13ad2deb-aaa5-4628-8cb2-7b4ff8342488 (old id 4212829)
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- 2016-04-01 10:03:45
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- 2023-09-13 16:55:04
@article{13ad2deb-aaa5-4628-8cb2-7b4ff8342488,
abstract = {{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 degrees C. The remaining larger fragment has a molecular weight of similar to 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)(2)(poly(vinylimidazole))(10)Cl](+) [Os(dmbpy)PVI] and [Os(4,4'-dimethoxy-2,2'-bipyridine)(2)(poly-(vinylimidazole))(10)Cl](+) [Os(dmobpy)PVI]. When "wired" with Os(dmbpy)PVI, the graphite electrodes modified with fdgPDH showed a pronounced increase in the current density with J(max). 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-75, N-175, and N-252 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.}},
author = {{Yakovleva, Maria and Killyeni, Aniko and Seubert, Oliver and Conghaile, Peter O. and MacAodha, Domhnall and Leech, Donal and Gonaus, Christoph and Popescu, Ionel Catalin and Peterbauer, Clemens K. and Kjellström, Sven and Gorton, Lo}},
issn = {{1520-6882}},
language = {{eng}},
number = {{20}},
pages = {{9852--9858}},
publisher = {{The American Chemical Society (ACS)}},
series = {{Analytical Chemistry}},
title = {{Further Insights into the Catalytical Properties of Deglycosylated Pyranose Dehydrogenase from Agaricus meleagris Recombinantly Expressed in Pichia pastoris}},
url = {{http://dx.doi.org/10.1021/ac4023988}},
doi = {{10.1021/ac4023988}},
volume = {{85}},
year = {{2013}},
}