A facile and cost-effective method for simultaneous preparation of lectin and tyrosinase from edible mushroom

Agaricus bisporus is a safe and rich source of tyrosinase and lectin. To address the increasing demand for these proteins, a facile method was developed for their simultaneous preparation from A. bisporus. The method includes demelanization of the raw extract followed by stepwise precipitation of the proteins from the aqueous extract. Employing the right organic solvent in demelanization and precipitation steps reduces the intermolecular interactions between these proteins. This enhances the efficacy of gel-permeation chromatography and results in preparation of partially purified lectin and tyrosinase suitable for many applications in biotechnology and bio-research. The method can be performed without diafiltration.     

Direct spectrophotometric assay of laccase using diazo derivatives of guaiacol

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.