Description

• Title:
  Recombinant C-Terminal Catalytic Domain of Rat L-Gulono Lactone Oxidase Produced in Bacterial Cells Is Enzymatically Active
• Creator:
  Gad, Abdul Aziz M.;
  Gora-Sochacka, Anna;
  Sirko, Agnieszka
• Description:
  The L-gulonolactone oxidase enzyme (GULO) catalyzes the last step of L-ascorbic acid (vitamin C) biosynthesis. This enzymatic activity is lost in primates. The full-length rat GULO has been previously produced in plants and demonstrated to be active. In this study, we compared the activity of two variants of GULO produced in Escheriachia coli cells, full-length rat GULO (fGULO) and its C-terminal catalytic domain (cGULO). The expression and purification of the recombinant proteins were optimized, and their biological activity was confirmed by two methods, the GULO activity assay in the protein extracts and the ‘in-gel’ staining for GULO activity. Both variants of recombinant GULO were biologically active in both assays. However, cGULO is more promising than fGULO for ascorbic acid production because it is more efficiently produced by bacteria. Furthermore, the optimal activities of the fGULO and cGULO recombinant proteins were observed at pH 7 and 6.5, and at temperatures of 40 and 30 °C, respectively. Kinetic studies revealed that at low substrate concentrations, Km values for fGULO and cGULO were 53.5 ± 5 and 42 ± 6.3 µM, respectively.
• Object availability:
  free access
• Date:
  2024
• Type:
  Article;
  PeerReviewed
• Language:
  English
• Format:
  application/pdf
• Relation: http://eprints.ibb.waw.pl/2451 ;http://doi.org/10.3390/cimb46080529 ;doi:10.3390/cimb46080529
• Subject:
  Q Science (General)
• Identifier: http://eprints.ibb.waw.pl/2451/1/cimb-46-00529-v3.pdf ;
  Gad, Abdul Aziz M. and Gora-Sochacka, Anna and Sirko, Agnieszka (2024) Recombinant C-Terminal Catalytic Domain of Rat L-Gulono Lactone Oxidase Produced in Bacterial Cells Is Enzymatically Active. Current Issues in Molecular Biology, 46 (8). pp. 8958-8968. ISSN 1467-3045
• Data provider:
  Repozytorium Instytutu Biochemii i Biofizyki Polskiej Akademii Nauk
• Can I use it?:
  seek permission
• Type:
  other