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Enhancing thermostability by modifying flexible surface loops in an evolved high‐redox potential laccase
Author(s) -
Vicente Ana I.,
ViñaGonzalez Javier,
Mateljak Ivan,
Monza Emanuele,
Lucas Fatima,
Guallar Victor,
Alcalde Miguel
Publication year - 2020
Publication title -
aiche journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.958
H-Index - 167
eISSN - 1547-5905
pISSN - 0001-1541
DOI - 10.1002/aic.16747
Subject(s) - thermostability , laccase , redox , morphing , directed evolution , chemistry , saccharomyces cerevisiae , mutant , materials science , enzyme , biochemistry , yeast , computer science , organic chemistry , gene , computer vision
High‐redox potential laccases (HRPLs) from white‐rot fungi are versatile biocatalysts whose practical use is highly dependent on their thermostability. In this work, an evolved HRPL variant was subjected to structure‐guided evolution to improve its thermostability. We first selected several surface flexible loops in the laccase structure by inspecting them through molecular dynamics and an analysis of B ‐factors. The resulting segments were grouped into three MORPHING (Mutagenic Organized Recombination Process by Homologous In vivo Grouping) blocks, which were constructed in Saccharomyces cerevisiae and explored at high temperatures. This evolution process gave rise to a double mutant that showed a half‐life at 70°C enhanced by 31 min with an optimum temperature for activity of 75°C and similar kinetic parameters. The Ser264Lys and Ser356Asn mutations modified the contacts established between these residues and those that surround them, altering the surface loops and thereby the enzyme properties.