Evolving stability and pH-dependent activity of the high redox potential Botrytis aclada laccase for enzymatic fuel cells

Stefan Scheiblbrandner,Florian Csarman,Tamara V Tikhonova,Evgeny M Osipov,Roman Kittl,Vladimir O Popov,Dietmar Haltrich,Peter L Herzog,Roland Ludwig,Erik Breslmayr,Sergey V Shleev,Johannes Führer,Regina Paukner

doi:10.1038/s41598-017-13734-0

Abstract

Fungal high redox potential laccases are proposed as cathodic biocatalysts in implantable enzymatic fuel cells to generate high cell voltages. Their application is limited mainly through their acidic pH optimum and chloride inhibition. This work investigates evolutionary and engineering strategies to increase the pH optimum of a chloride-tolerant, high redox potential laccase from the ascomycete Botrytis aclada. The laccase was subjected to two rounds of directed evolution and the clones screened for increased stability and activity at pH 6.5. Beneficial mutation sites were investigated by semi-rational and combinatorial mutagenesis. Fourteen variants were characterised in detail to evaluate changes of the kinetic constants. Mutations increasing thermostability were distributed over the entire structure. Among them, T383I showed a 2.6-fold increased half-life by preventing the loss of the T2 copper through unfolding of a loop. Mutations affecting the pH-dependence cluster around the T1 copper and categorise in three types of altered pH profiles: pH-type I changes the monotonic decreasing pH profile into a bell-shaped profile, pH-type II describes increased specific activity below pH 6.5, and pH-type III increased specific activity above pH 6.5. Specific activities of the best variants were up to 5-fold higher (13 U mg−1) than BaL WT at pH 7.5.

Highlights

Laccases (EC 1.10.3.2, CAZy AA1), alongside ascorbate oxidase and bilirubin oxidase, belong to the group of three-domain multicopper blue oxidases[1]
The most recent work to improve a high redox potential laccases (HRPLs) in this direction by enzyme engineering was performed by Mate and co-workers[6,14,18,19,20]
To the best of the authors knowledge, none of the reported HRPLs reach chloride tolerance levels comparable to the wild-type HRPL from Botrytis aclada (BaL WT) investigated in this work[9]

Summary

Introduction

Laccases (EC 1.10.3.2, CAZy AA1), alongside ascorbate oxidase and bilirubin oxidase, belong to the group of three-domain multicopper blue oxidases[1] This group of enzymes binds four copper atoms as cofactors in two active sites. The most recent work to improve a HRPL in this direction by enzyme engineering was performed by Mate and co-workers[6,14,18,19,20] They created a variant from the unclassified basidiomycete PM1 for functional expression in S. cerevisiae by eight rounds of directed evolution[18,20], rendered it active in blood by four further evolutionary rounds[19] and functionally expressed it in P. pastoris[6,14]. To improve BaLs performance at physiological conditions (37 °C, pH 7.4) an increase of activity at neutral pH and of the longevity is essential

Methods

Results

Conclusion