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Improving enzymatic activities and thermostability of a tri-functional enzyme with SOD, catalase and cell-permeable activities

Luangwattananun, Piriya ; Eiamphungporn, Warawan ; Songtawee, Napat ; Bülow, Leif LU ; Isarankura-Na-Ayudhya, Chartchalerm ; Prachayasittikul, Virapong and Yainoy, Sakda LU (2017) In Journal of Biotechnology 247. p.50-59
Abstract

Synergistic action of major antioxidant enzymes, e.g., superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) is known to be more effective than the action of any single enzyme. Recently, we have engineered a tri-functional enzyme, 6His-MnSOD-TAT/CAT-MnSOD (M-TAT/CM), with SOD, CAT and cell-permeable activities. The protein actively internalized into the cells and showed superior protection against oxidative stress-induced cell death over native enzymes fused with TAT. To improve its molecular size, enzymatic activity and stability, in this study, MnSOD portions of the engineered protein were replaced by CuZnSOD, which is the smallest and the most heat resistant SOD isoform. The newly engineered protein,... (More)

Synergistic action of major antioxidant enzymes, e.g., superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) is known to be more effective than the action of any single enzyme. Recently, we have engineered a tri-functional enzyme, 6His-MnSOD-TAT/CAT-MnSOD (M-TAT/CM), with SOD, CAT and cell-permeable activities. The protein actively internalized into the cells and showed superior protection against oxidative stress-induced cell death over native enzymes fused with TAT. To improve its molecular size, enzymatic activity and stability, in this study, MnSOD portions of the engineered protein were replaced by CuZnSOD, which is the smallest and the most heat resistant SOD isoform. The newly engineered protein, CAT-CuZnSOD/6His-CuZnSOD-TAT (CS/S-TAT), had a 42% reduction in molecular size and an increase in SOD and CAT activities by 22% and 99%, respectively. After incubation at 70 °C for 10 min, the CS/S-TAT retained residual SOD activity up to 54% while SOD activity of the M-TAT/CM was completely abolished. Moreover, the protein exhibited a 5-fold improvement in half-life at 70 °C. Thus, this work provides insights into the design and synthesis of a smaller but much more stable multifunctional antioxidant enzyme with ability to enter mammalian cells for further application as protective/therapeutic agent against oxidative stress-related conditions.

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author
; ; ; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Catalase, CuZnSOD, HIV-1 TAT, Protein engineering
in
Journal of Biotechnology
volume
247
pages
10 pages
publisher
Elsevier
external identifiers
  • pmid:28274879
  • wos:000400317000009
  • scopus:85014883108
ISSN
0168-1656
DOI
10.1016/j.jbiotec.2017.03.001
language
English
LU publication?
yes
id
41d7300c-601d-4c9e-96c8-52a4fc01533f
date added to LUP
2017-03-23 07:29:09
date last changed
2025-01-20 11:48:30
@article{41d7300c-601d-4c9e-96c8-52a4fc01533f,
  abstract     = {{<p>Synergistic action of major antioxidant enzymes, e.g., superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) is known to be more effective than the action of any single enzyme. Recently, we have engineered a tri-functional enzyme, 6His-MnSOD-TAT/CAT-MnSOD (M-TAT/CM), with SOD, CAT and cell-permeable activities. The protein actively internalized into the cells and showed superior protection against oxidative stress-induced cell death over native enzymes fused with TAT. To improve its molecular size, enzymatic activity and stability, in this study, MnSOD portions of the engineered protein were replaced by CuZnSOD, which is the smallest and the most heat resistant SOD isoform. The newly engineered protein, CAT-CuZnSOD/6His-CuZnSOD-TAT (CS/S-TAT), had a 42% reduction in molecular size and an increase in SOD and CAT activities by 22% and 99%, respectively. After incubation at 70 °C for 10 min, the CS/S-TAT retained residual SOD activity up to 54% while SOD activity of the M-TAT/CM was completely abolished. Moreover, the protein exhibited a 5-fold improvement in half-life at 70 °C. Thus, this work provides insights into the design and synthesis of a smaller but much more stable multifunctional antioxidant enzyme with ability to enter mammalian cells for further application as protective/therapeutic agent against oxidative stress-related conditions.</p>}},
  author       = {{Luangwattananun, Piriya and Eiamphungporn, Warawan and Songtawee, Napat and Bülow, Leif and Isarankura-Na-Ayudhya, Chartchalerm and Prachayasittikul, Virapong and Yainoy, Sakda}},
  issn         = {{0168-1656}},
  keywords     = {{Catalase; CuZnSOD; HIV-1 TAT; Protein engineering}},
  language     = {{eng}},
  month        = {{04}},
  pages        = {{50--59}},
  publisher    = {{Elsevier}},
  series       = {{Journal of Biotechnology}},
  title        = {{Improving enzymatic activities and thermostability of a tri-functional enzyme with SOD, catalase and cell-permeable activities}},
  url          = {{http://dx.doi.org/10.1016/j.jbiotec.2017.03.001}},
  doi          = {{10.1016/j.jbiotec.2017.03.001}},
  volume       = {{247}},
  year         = {{2017}},
}