title: New colorimetric screening assays for the directed evolution of fungal laccases to improve the conversion of plant biomass.
abstract: BACKGROUND
  Fungal laccases are multicopper oxidases with huge applicability in different sectors. Here, we describe the development of a set of high-throughput colorimetric assays for screening laccase libraries in directed evolution studies.
  
  
  RESULTS
  Firstly, we designed three colorimetric assays based on the oxidation of sinapic acid, acetosyringone and syringaldehyde with λmax of 512, 520 and 370 nm, respectively. These syringyl-type phenolic compounds are released during the degradation of lignocellulose and can act as laccase redox mediators. The oxidation of the three compounds by low and high-redox potential laccases evolved in Saccharomyces cerevisiae produced quantifiable and linear responses, with detection limits around 1 mU/mL and CV values below 16%. The phenolic substrates were also suitable for pre-screening mutant libraries on solid phase format. Intense colored-halos were developed around the yeast colonies secreting laccase. Furthermore, the oxidation of violuric acid to its iminoxyl radical (λmax of 515 nm and CV below 15%) was devised as reporter assay for laccase redox potential during the screening of mutant libraries from high-redox potential laccases. Finally, we developed three dye-decolorizing assays based on the enzymatic oxidation of Methyl Orange (470 nm), Evans Blue (605 nm) and Remazol Brilliant Blue (640 nm) giving up to 40% decolorization yields and CV values below 18%. The assays were reliable for direct measurement of laccase activity or to indirectly explore the oxidation of mediators that do not render colored products (but promote dye decolorization). Every single assay reported in this work was tested by exploring mutant libraries created by error prone PCR of fungal laccases secreted by yeast.
  
  
  CONCLUSIONS
  The high-throughput screening methods reported in this work could be useful for engineering laccases for different purposes. The assays based on the oxidation of syringyl-compounds might be valuable tools for tailoring laccases precisely enhanced to aid biomass conversion processes. The violuric assay might be useful to preserve the redox potential of laccase whilst evolving towards new functions. The dye-decolorizing assays are useful for engineering ad hoc laccases for detoxification of textile wastewaters, or as indirect assays to explore laccase activity on other natural mediators.
authors.forename: Isabel
authors.initials: I
authors.lastname: Pardo
authors.forename: Xiomara
authors.initials: X
authors.lastname: Chanagá
authors.forename: Ana Isabel
authors.initials: AI
authors.lastname: Vicente
authors.forename: Miguel
authors.initials: M
authors.lastname: Alcalde
authors.affiliation: Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, 28040 Madrid, Spain. susanacam@cib.csic.es.
authors.forename: Susana
authors.initials: S
authors.lastname: Camarero
mesh_terms: D001391:Azo Compounds / Q000378:metabolism; D001463:Barbiturates / Q000378:metabolism; D018533:Biomass; D003124:Colorimetry; D003470:Culture Media / Q000737:chemistry; D019020:Directed Molecular Evolution / Q000379:methods*; D005070:Evans Blue / Q000378:metabolism; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D057166:High-Throughput Screening Assays / Q000379:methods*; D042845:Laccase / Q000235:genetics / Q000378:metabolism*; D008031:Lignin / Q000737:chemistry; D010084:Oxidation-Reduction; D019800:Phenol / Q000737:chemistry; D015202:Protein Engineering / Q000379:methods; D015203:Reproducibility of Results; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics
chemical_list: D001391:Azo Compounds; D001463:Barbiturates; D003470:Culture Media; D005656:Fungal Proteins; C094277:violuric acid; C036909:lignocellulose; D019800:Phenol; D005070:Evans Blue; C100258:methyl orange; D008031:Lignin; D042845:Laccase