Laccase evolution

Liu H, Zhu L, Bocola M, Chen N, Spiess AC, Schwaneberg U (2013) Directed laccase evolution for improved ionic liquid resistance. Green Chem 15 1348-1355... 

Laccase, 36 318, 329, 40 122 see also Blue copper oxidases amino-acid sequences, 40 141 anaerobic reduction, 40 158-160 biological function, 40 124 electrochemistry, 36 360 fungal, 40 145-152 evolution, 40 153-154 inhibition, 40 162 kinetic properties, 40 157-162 molecular and spectroscopic properties, 40 125-126... 

Parallelism in the specific inhibition of electrocatalytic and enzymatic activity. The specific inhibitors of a particular enzyme are observed also to suppress its electrocatalytic activity in the adsorbed state. Experimental data demonstrate that a,a -dipyridyl completely suppresses the reaction of hydrogen evolution by immobilized hydrogenase fluorine ions inactivate laccase in the reaction of oxygen electroreduction and diphenylhydrazine has the same effect on peroxidase in the reaction of hydrogen peroxide electroreduction. A complete parallelism is also observed in the inactivating effect of hydrogen peroxide on peroxidase in the electrochemical reaction and enzymatic oxidation of o-dianisidine. 

Later we summarize the main advances made in the directed evolution of this interesting group of oxidoreductases, paying particular attention to fungal laccases. 

Several directed evolution studies of bacterial laccase CotA have successfully improved its substrate specificity and functional expression, modifying its specificities by screening mutant libraries through surface display [33-37]. The advantages of some bacterial laccases include high thermostability and activity at neu-tral/alkaline pH, although a low-redox potential at the T1 site often precludes their use in certain sectors. 

Directed Evolution of Medium-Redox Potential Laccases... 

The first successful example of the directed evolution of fungal laccase involved the laccase from the thermophile ascomycete Myceliophihora thermophila laccase (MtL). This study led to subsequent directed evolution experiments in S. cerevisiae with several high-redox potential ligninolytic oxidoreductases (see below). MtL was subjected to 10 cycles of directed evolution to enhance its functional expression in S. cerevisiae [38]. The best performing variant of this process (the T2 mutant that harbored 14 mutations) exhibited a total improvement of 170-fold in activity its expression levels were enhanced 8-fold and the around 22-fold. The... 

Directed Evolution of Ligninoiytic High-Redox Potential Laccases (HRPLs)... 

The sequence identity between PcL and PMIL is over 77%, which facilitated mutational exchange between the two parallel evolution pathways and allowed us to switch protein sequence blocks to create chimeric proteins of HRPLs with hybrid or even enhanced features. To favor mulhple crossover events between laccase scaffolds, in vitro and in vivo DNA recombination methods were combined in a single evolutionary step (see Section 1.6). Chimeras with up to six crossover events per sequence were identified, which generated active laccase hybrids with combined characteristics in terms of substrate affinity, pH activity, and thermostability [54]. Interestingly, some chimeras showed higher thermostabilities than the original... 

Expression system of CotA-laccase for directed evolution and high-throughput screenings for the oxidation of high-redox potential dyes. Biotechnol. J., 4, 558-563. 

Laboratory evolution of laccase for substrate specificity. J. Mol. Catal. B Enzym., 62, 230-234. 

Gupta, N. and Farinas, E.T. (2010) Directed evolution of CotA laccase for increased substrate specificity using Bacillus subtilis spores. Protein Eng. Des. Sel, 23, 679-682. 

Meinhold, P., Schlachtbauer, C., and Arnold, F.H. (2003) Functional expression of a fungal laccase in Saccharomyces cerevisiae by directed evolution. Appl. Environ. Microbiol., 69, 987—995. 

F.J., Ballesteros, A., and Alcalde, M. (2007) In vitro evolution of a fungal laccase in high concentrations of organic cosolvents. Chem. Biol., 14, 1052—1064. 

Mate, D., Garcfa-Ruiz, E., Camarero, S., and Alcalde, M. (2011) Directed evolution of fungal laccases. Curr. Genomics, 12, 113-122. 

Festa, G., Autore, F., Fraternali, F., Giardina, P., and Sannia, G. (2008) Development of new laccases by directed evolution functional and computational analyses. Proteins, 71, 25-34. 

Mate, D.M., Garcia-Ruiz, E., Camarero, S., Shubin, V.V., Falk, M., Shleev, S., Ballesteros, A.O., and Alcalde, M. (2013) Switching from blue to yellow altering the spectral properties of a high redox potential laccase by directed evolution. Biocatal. Biotransfor., 31, 8-21. 

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