Laccases directed evolution

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)... 

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. 

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. 

Zumarraga, M., Plou, F.J., Garcia, H., Ballesteros, A., and Alcalde, M. (2007) Bioremediation of Polycyclic Aromatic Hydrocarbons by a fungal Laccases engineered by directed evolution. Biocatal. Biotransfor., 25, 219-228. 

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. 

In order to screen mutants with improved direct electron transfer, it is necessary to use an electrochemical screening system. Currently, only a few electrochemical screening methods were described in literature such as the system developed by the Bartlett group used to screen NADH electro-oxidation. This system uses a multichannel potentiostat with sixty electrodes to screen zinc(n) or ruthenium(ii) complexes bearing the redox phenidione as a mediator for NADH oxidation. It allows the complete evaluation of the electrochemical kinetic constants of the mediators and the immobilization procedure. Unfortunately, this system could only be used with a single electrolyte solution for all the electrodes (e.g., when a single reaction condition or enzyme is assayed), and it requires mL-scale reaction volumes. Recently, another system was described which makes it possible to screen bioelectrocatalytic reactions on 96 independent electrodes screen-printed onto a printed-circuit-board. It showed the possibility to screen direct or mediated electron transfer between oxidoreductases and electrode by intermittent pulse amperometry at the pL-scale (Fig. 6). The direct electron transfer assay was validated with laccase and unmodified electrodes.As an example of the mediated electron transfer assay, the 96 carbon electrodes were modified by phenazines to sereen libraries of a formate dehydrogenase obtained by directed evolution. ... 

Similar to laccase, a number of methods have been developed to engineer GOx for increased activity and specificity. An enzymatic assay coupled with site-directed mutagenesis was used to alter the substrate specificity of GOx to increase the dynamic range of glucose biosensors [34], and a directed evolution screen was developed to improve GOx activity in BFCs [35]. 

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... 

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