title: Direct spectrophotometric assay of laccase using diazo derivatives of guaiacol.
abstract: Laccase (EC 1.10.3.2) is a widespread cuproenzyme able to oxidize various types of phenols and similar aromatic compounds through a one-electron transfer mechanism. The enzyme has already found its way into the market as a biocatalyst. Because of its ability to be paired by electron mediators, the expectation for employing laccases in versatile processes is very high. There are a few spectrophotometric methods for assaying the laccase activity; however, all of them are based on the formation of product(s) resulting from the enzymatic and inevitable succeeding chemical reactions. Use of diazo derivatives of guaiacol (DdG) was developed as a new spectrophotometric method based on substrate depletion allowing direct assessment of enzyme activity has been introduced. This method allows accurate comprehensive kinetic studies of laccases and provides reliable information about the quality of docking of different substrates or one substrate to the active sites of different laccases. Using this method, the kinetic parameters of various DdG carrying different electron donating and withdrawing substituents were used to assay laccase from Neurospora crassa. 2-Methoxy-4-[(4-phenyl)azo]-phenol (K(m) = 93.5 μM and V = 1.98 μM/min) was identified as an appropriate substrate for the accurate and routine spectrophotometric based assay of laccases.
authors.affiliation: Faculty of Basic Sciences, National Institute for Genetic Engineering and Biotechnology, P.O. Box 149651/161, Tehran, Iran.
authors.forename: Seyed Mohammad
authors.initials: SM
authors.lastname: Moshtaghioun
authors.forename: Kamahldin
authors.initials: K
authors.lastname: Haghbeen
authors.forename: Abbas Lotfi
authors.initials: AL
authors.lastname: Sahebghadam
authors.forename: Raymond L
authors.initials: RL
authors.lastname: Legge
authors.forename: Rabea
authors.initials: R
authors.lastname: Khoshneviszadeh
authors.forename: Sahar
authors.initials: S
authors.lastname: Farhadi
mesh_terms: D001391:Azo Compounds / Q000737:chemistry; D020134:Catalytic Domain; D057075:Enzyme Assays / Q000379:methods; D006139:Guaiacol / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D042845:Laccase / Q000378:metabolism*; D009492:Neurospora crassa / Q000201:enzymology; D010084:Oxidation-Reduction; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*; D013379:Substrate Specificity
chemical_list: D001391:Azo Compounds; D006139:Guaiacol; D042845:Laccase