Kraft Pulp Biobleaching and Mediated Oxidation of a Nonphenolic Substrate by Laccase from Streptomyces cyaneus CECT 3335

Abstract

A new laccase (EC 1.10.3.2) produced by Streptomyces cyaneus CECT 3335 in liquid media containing soya flour (20 g per liter) was purified to homogeneity. The physicochemical, catalytic, and spectral characteristics of this enzyme, as well as its suitability for biobleaching of eucalyptus kraft pulps, were assessed. The purified laccase had a molecular mass of 75 kDa and an isoelectric point of 5.6, and its optimal pH and temperature were 4.5 and 70°C, respectively. The activity was strongly enhanced in the presence of Cu 2؉ , Mn 2؉ , and Mg 2؉and was completely inhibited by EDTA and sodium azide. The purified laccase exhibited high levels of activity against 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) and 2,6-dimethoxyphenol and no activity against tyrosine. The UV-visible spectrum of the purified laccase was the typical spectrum of the blue laccases, with an absorption peak at 600 nm and a shoulder around 330 to 340 nm. The ability of the purified laccase to oxidize a nonphenolic compound, such as veratryl alcohol, in the presence of ABTS opens up new possibilities for the use of bacterial laccases in the pulp and paper industry. We demonstrated that application of the laccase from S. cyaneus in the presence of ABTS to biobleaching of eucalyptus kraft pulps resulted in a significant decrease in the kappa number (2.3 U) and an important increase in the brightness (2.2%, as determined by the International Standard Organization test) of pulps, showing the suitability of laccases produced by streptomycetes for industrial purposes.Because of their potential for biotechnological applications in areas such as biobleaching, increasing the strength of cellulose fibers, textile dye or stain bleaching, and bioremediation, attention is currently being paid to laccases (2,23,30,43,50). These enzymes are widely distributed in plants and fungi, but until now laccase activity has been reported in only a few bacteria, including Azospirillum lipoferum, Marinomonas mediterranea, Streptomyces griseus, and Bacillus subtilis (1,15,20,22,41,45). A number of roles for laccases in bacterial systems have been suggested and include roles in melanin production and spore coat resistance and involvement in morphogenesis (15,22). In Streptomyces cyaneus, a laccase-type phenol oxidase was found to be produced during growth under solid-substrate fermentation conditions, and it was suggested that this enzyme was involved in the solubilization and mineralization of lignin from wheat straw (5). Further studies demonstrated that this organism could be used to improve the qualities of pulp after 2 weeks of incubation under solid-substrate fermentation conditions (6). However, to date there have been no reports describing the involvement of bacterial laccases in the oxidation of nonphenolic compounds in the presence of mediators. The specific activities of these enzymes with lignin and related substrates have not been examined yet.Laccases are considered some of the most promising enzymes for future industrial applications in the pulp ...

Studies on Xylanase and Laccase Enzymatic Prebleaching to Reduce Chlorine-Based Chemicals During Ceh and Ecf Bleaching

“…The more common laccase mediators, 1-hydroxybenzotriazole (1-HBT) and ABTS, have been used by many researchers comparing effects with various laccases. Different laccases have also been shown to react differently with mediators (Arias et al 2003). 1-HBT and ABTS were better mediators for delignification than other mediators tested.…”

“…The need for an inexpensive, effective, nontoxic mediator for laccase treatment is a critical problem. Use of laccase for bleaching has been limited by the availability of the enzyme and the cost of the mediators needed for lignin destruction (Arias et al 2003). Hence, there is an immediate need for a laccase mediator that is cheap and easily available to the paper industry.…”

“…Most of the direct bleaching with enzymes is done using oxidative enzymes that directly attack the color-producing compounds in the lignin. The enzymes that attack the lignin components of the fiber are oxidative (Arias et al 2003). Laccases are considered to be some of the most promising enzymes for such applications (Reid 1998).…”

Expression of Melanocarpus albomyces laccase in Trichoderma reesei and characterization of the purified enzyme

“…Laccases are common enzymes in nature, especially in plants and fungi (Gianfreda et al, 1999). Recently, some novel bacterial laccases have also been reported (Martins et al, 2002;Arias et al, 2003). The laccases most studied hitherto are of fungal origin, especially from the class of white-rot fungi.…”

Lignin Degradation Processes and the Purification of Valuable Products

“…Promising results have been achieved by using laccase-mediator systems on eucalyptus kraft pulp, e.g. bacterial laccase produced by Streptomyces cyaneus CECT 3335 in the presence of the mediator ABTS [58,90].…”