Laccase (benzenediol: oxygen oxidoreductases; EC 1.10.3.2; AA1) is a blue multi-copper oxidoreductase enzyme produced in nature by different species of organisms, i.e. fungi, plant and bacteria.  Laccases are able to catalyze the oxidation of phenolic compounds using oxygen as final electron acceptor. Due to this capability laccases are currently receiving high attention because of its potential role in lignin degradation.  <br/>Enzymatic laccase activity has traditionally been determined spectrophotometrically by measuring the oxidation of artificial chemical compounds such as syringaldazine and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid), also known as ABTS. However, these artificial structures can poorly be related to lignin and such assays provide little information about the true laccase oxidative mechanism. We have developed a highly sensitive, accurate methodology based on UPLC-MS to assess laccase activity on more “lignin-like” compounds. In contrast to spectrophotometric assays, this method also provides information about product formation and time-dependent reaction development. We provide proof-of-concept for methodology to study the ability of different laccases to oxidize three different hydroxycinnamic acids (sinapic acid, ferulic acid and p-coumaric acid) and a dimeric lignin model compound, determination of kinetic parameters and product profile evaluation. The results show that laccases exhibit highest activity on substrates containing methoxylated phenolic moieties, and that the product profile during the initial part of the reaction consists of a limited number of dimeric species derived from the substrate. The method is independent of laccase protein purity and can therefore also be used to assess activity of unpurified laccases.<br/>

Design of a new, accurate laccase activity assay method based on “lignin-like” compounds

255th National Meeting (2018)

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