Enhancing thermostability by modifying flexible surface loops in an evolved high‐redox potential laccase

Abstract

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.

Non-Hydrolyzable Plastics – An Interdisciplinary Look at Plastic Bio-Oxidation

“…Specific favorable characteristics of LMSs can be enhanced further using targeted mutations. Recent approaches have used the creation of stable and robust HRPLs mutant libraries coupled with screening assays in various directed evolution strategies, including classical adaptive evolution, consensus design, and chimeragenesis [73,74] Structure-guided evolution of HRPLs has been shown to increase thermal stability through the development of double mutants or chimeras [74,75]. The thermostability of a fungal laccase has been improved significantly, doubling its half-life at 80°C, using computer-assisted directed evolution [76,77].…”

“…The thermostability of a fungal laccase has been improved significantly, doubling its half-life at 80°C, using computer-assisted directed evolution [76,77]. In another approach, an evolved HRPL mutant from white-rot fungi subjected to structure-guided evolution displayed enhanced thermostability because of increased rigidity of flexible surface loops [74]. Computationally guided mutagenesis and directed evolution also effected a threefold increase in half-life at 70°C in another evolved basidiomycete LMS by increasing the hydrophobicity of the T1 copper environment [78].…”

Consensus Design of an Evolved High-Redox Potential Laccase

“…While we are witnessing a blossom of HRPL engineering in an attempt to cover different industrial needs, including laboratory evolution campaigns to retain activity in non-conventional media or to increase activities on different compounds, thermostability has been largely overlooked during such efforts (Hildén et al, 2009;Mate and Alcalde, 2015). However, in recent years we have paid special attention to this issue by embarking on SCHEMA recombination and structure-guided evolution experiments (Mateljak et al, 2019b;Vicente et al, 2020).…”

High production of glutathione by in vitro enzymatic cascade after thermostability enhancement

“…Fold X and Rosetta ddg_monomer, both of which had higher accuracy for prediction of thermal stability sites, were combined to predict stabilizing substitutions (folding free energy change ΔΔG < −1.0 kcal/mol or approaching) 28,29 . The amino acids, which were on or near the loop, were intentially selected due to higher flexibility 30 . Consequently, eight single variants were selected by taking folding free energy calculation and structural aspects into consideration on the basis of NSS‐L ST ‐SSC (Figure S1).…”