Faculty of Basic Sciences, National Institute for Genetic Engineering and Biotechnology, P.O. Box 149651/161, Tehran, Iran.
Anal Chem. 2011 Jun 1;83(11):4200-5. doi: 10.1021/ac200501w. Epub 2011 May 11.
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.
漆酶（EC 1.10.3.2）是一种广泛存在的铜酶，能够通过单电子转移机制氧化各种类型的酚类和类似的芳香族化合物。该酶已作为生物催化剂进入市场。由于其能够与电子介体配对，因此人们对在多功能过程中使用漆酶的期望非常高。有几种分光光度法可用于测定漆酶活性；然而，所有这些方法都是基于酶促和不可避免的后续化学反应形成的产物。开发了愈创木酚重氮衍生物（DdG）作为一种新的分光光度法，基于底物消耗，允许直接评估酶活性。该方法允许对漆酶进行准确全面的动力学研究，并提供有关不同底物或一个底物与不同漆酶的活性位点对接质量的可靠信息。使用该方法，对具有不同给电子和吸电子取代基的各种携带 DdG 的动力学参数进行了测定，以测定粗糙脉孢菌中的漆酶。2-甲氧基-4-[(4-苯基)偶氮]-苯酚（K(m) = 93.5 μM 和 V = 1.98 μM/min）被鉴定为用于准确和常规分光光度法测定漆酶的合适底物。
