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Tytuł pozycji:

Computational design of noncanonical amino acid-based thioether staples at N/C-terminal domains of multi-modular pullulanase for thermostabilization in enzyme catalysis

Tytuł:
Computational design of noncanonical amino acid-based thioether staples at N/C-terminal domains of multi-modular pullulanase for thermostabilization in enzyme catalysis
Autorzy:
Jiahua Bi
Xiaoran Jing
Lunjie Wu
Xia Zhou
Jie Gu
Yao Nie
Yan Xu
Temat:
Computational design
Thermostabilization
Noncanonical amino acids
Thioether staple
Multi-modular enzyme
Biotechnology
TP248.13-248.65
Źródło:
Computational and Structural Biotechnology Journal, Vol 19, Iss , Pp 577-585 (2021)
Wydawca:
Elsevier, 2021.
Rok publikacji:
2021
Kolekcja:
LCC:Biotechnology
Typ dokumentu:
article
Opis pliku:
electronic resource
Język:
English
ISSN:
2001-0370
Relacje:
http://www.sciencedirect.com/science/article/pii/S2001037021000015; https://doaj.org/toc/2001-0370
DOI:
10.1016/j.csbj.2020.12.043
Dostęp URL:
https://doaj.org/article/a65c9eff9ed04ad8ba940cb93b7d7ef9  Link otwiera się w nowym oknie
Numer akcesji:
edsdoj.65c9eff9ed04ad8ba940cb93b7d7ef9
Czasopismo naukowe
Enzyme thermostabilization is considered a critical and often obligatory step in biosynthesis, because thermostability is a significant property of enzymes that can be used to evaluate their feasibility for industrial applications. However, conventional strategies for thermostabilizing enzymes generally introduce non-covalent interactions and/or natural covalent bonds caused by natural amino acid substitutions, and the trade-off between the activity and stability of enzymes remains a challenge. Here, we developed a computationally guided strategy for constructing thioether staples by incorporating noncanonical amino acid (ncAA) into the more flexible N/C-terminal domains of the multi-modular pullulanase from Bacillus thermoleovorans (BtPul) to enhance its thermostability. First, potential thioether staples located in the N/C-terminal domains of BtPul were predicted using RosettaMatch. Next, eight variants involving stable thioether staples were precisely predicted using FoldX and Rosetta ddg_monomer. Six positive variants were obtained, of which T73(O2beY)-171C had a 157% longer half-life at 70 °C and an increase of 7.0 °C in Tm, when compared with the wild-type (WT). T73(O2beY)-171C/T126F/A72R exhibited an even more improved thermostability, with a 211% increase in half-life at 70 °C and a 44% enhancement in enzyme activity compared with the WT, which was attributed to further optimization of the local interaction network. This work introduces and validates an efficient strategy for enhancing the thermostability and activity of multi-modular enzymes.

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