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Enhanced trypsin thermostability in Pichia pastoris through truncating the flexible region

Liu, L; Yu, H; Du, K; Wang, Z; Gan, Y; Huang, H; (2018) Enhanced trypsin thermostability in Pichia pastoris through truncating the flexible region. Microbial Cell Factories , 17 (1) , Article 165. 10.1186/s12934-018-1012-x. Green open access

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Abstract

BACKGROUND: High thermostability is required for trypsin to have wider industrial applications. Target mutagenesis at flexible regions has been proved to be an efficient protein engineering method to enhance the protein thermostability. RESULTS: The flexible regions in porcine trypsin were predicted using the methods including molecular dynamic simulation, FlexPred, and FoldUnfold. The amino acids 78-90 was predicted to be the highly flexible region simultaneously by the three methods and hence selected to be the mutation target. We constructed five variants (D3, D5, D7, D9, and D11) by truncating the region. And the variant D9 showed higher thermostability, with a 5 °C increase in Topt, 5.8 °C rise in [Formula: see text], and a 4.5 °C rise in Tm, compared to the wild-type. Moreover, the half-life value of the variant D9 was also found to be dramatically improved by 46 min. Circular dichroism and intrinsic fluorescence indicated that the structures had no significant change between the variant D9 and the wild-type. The surface hydrophobicity of D9 was measured to be lower than that of wild-type, indicating the increased hydrophobic interaction, which could have contributed to the improved thermostability of D9. CONCLUSIONS: These results showed that the thermostability of variant D9 was increased. The variant D9 could be expected to be a promising tool enzyme for its wider industrial applications. The method of truncating the flexible region used in our study has the potential to be used for enhancing the thermostability of other proteins.

Type: Article
Title: Enhanced trypsin thermostability in Pichia pastoris through truncating the flexible region
Location: England
Open access status: An open access version is available from UCL Discovery
DOI: 10.1186/s12934-018-1012-x
Publisher version: https://doi.org/10.1186/s12934-018-1012-x
Language: English
Additional information: This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
Keywords: Flexible region, Thermostability, Truncation, Trypsin, Ω-loop
UCL classification: UCL > Provost and Vice Provost Offices
UCL > Provost and Vice Provost Offices > UCL BEAMS
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences
UCL > Provost and Vice Provost Offices > UCL BEAMS > Faculty of Maths and Physical Sciences > Dept of Chemistry
URI: http://discovery.ucl.ac.uk/id/eprint/10061145
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