Cloning of a phenol oxidase gene from Acremonium murorum and its expression in Aspergillus awamori

Fungal multicopper oxidases have many potential industrial applications, si nce they perform reactions under mild conditions. We isolated a phenol oxid ase from the fungus Acremonium murorum var. murorum that was capable of dec olorizing plant chromophores (such as anthocyanins), This enzyme is of inte rest in laundry-cleaning products because of its broad specificity for chro mophores. We expressed an A, murorum cDNA library in Saccharomyces cerevisi ae and subsequently identified enzyme-producing yeast colonies based on the ir ability to decolor a plant chromophore. The cDNA sequence contained an o pen reading frame of 1,806 bp encoding an enzyme of 602 amino acids. The ph enol oxidase was overproduced by Aspergillus awamori as a fusion protein wi th glucoamylase, cleaved in vivo, and purified from the culture broth by hy drophobic-interaction chromatography, The phenol oxidase is active at alkal ine pH (the optimum for syringaldazine is pH 9) and high temperature (optim um, 60 degreesC) and is fully stable for at least I h at 60 degreesC under alkaline conditions. These characteristics and the high production level of 0.6 g of phenol oxidase per liter in shake flasks, which is equimolar with the glucoamylase protein levels, make this enzyme suitable for use in proc esses that occur under alkaline conditions, such as laundry cleaning.