Increased production of laccase by the wood-degrading basidiomycete Trametes pubescens

Abstract

The white-rot fungus Trametes pubescens MB 89 is an excellent producer of the industrially important enzyme laccase. Extracellular laccase formation can be considerably stimulated by the addition of Cu(II) in the millimolar range to a simple, glucose-based culture medium. When using glucose, a typically repressing substrate, as the main carbon source, significant laccase formation by T. pubescens only started when glucose was completely consumed from the culture medium. In addition, the nitrogen source employed had an important effect on laccase synthesis. When using an optimized medium containing glucose (40 g l⁻¹), peptone from meat (10 g l⁻¹), and MgSO₄·7H₂O and stimulating enzyme formation by the addition of 2.0 mM Cu, maximal laccase activities obtained in a batch cultivation were approximately 330 U ml⁻¹. Under these conditions it was not necessary to add aromatic compounds that are routinely used as inducers of laccase in fungi. By applying a fed-batch strategy, in which a glucose solution was fed continuously to a cultivation of T. pubescens so that the glucose concentration in the medium never exceeded a certain low, critical value, glucose repression could be avoided and production of laccase was almost doubled as compared to the batch cultivation (740 U ml⁻¹).

Introduction

Laccases (benzenediol:oxygen oxidoreductases; EC 1.10.3.2.), multicopper enzymes belonging to the blue oxidases, catalyze the one-electron oxidation of a wide variety of organic and inorganic substrates, including mono-, di- and polyphenols, aminophenols, methoxyphenols, aromatic amines and ascorbate, with the concomitant four-electron reduction of oxygen to water [1], [2], [3], [4]. Laccase was first described from the sap of the Japanese lacquer tree Rhus vernicifera[5]. Probably the first report on the presence of laccase in fungi was from Laborde in 1897 [6]. Since then, laccases have been found in the majority of white-rot fungi described to date, and they are also produced by other types of fungi, some bacteria and insects [6], [7], [8]. The biologic role for laccase has yet to be fully elucidated and appears to vary depending on the type of organism [2]. In fungi, laccases are believed to be involved in degradation of lignin and/or in the removal of potentially toxic phenols arising during this degradation [2], [9]. In addition, fungal laccases are hypothesized to take part in the synthesis of dihydroxynaphthalene melanins [10], in fungal morphogenesis [11], [12], and plant pathogenesis and fungal virulence [13], [14].

In basidiomycete fungi, extracellular laccases are constitutively produced in small amounts [15], however, their production can be considerably stimulated by the presence of a wide variety of inducing substances, mainly aromatic or phenolic compounds related to lignin or lignin derivatives, such as ferulic acid, 2,5-xylidine, p-anisidine, or veratryl alcohol [8]. In addition, laccase production can be influenced by the nitrogen concentration in the culture medium. Often, higher nitrogen levels are required to increase laccase formation [8], but with certain organisms such as Pycnoporus cinnabarinus or P. sanguineus nitrogen-limited culture conditions enhance the production of this enzyme [16], [17]. Whereas the increased production of laccase activity by fungi in response to aromatic and phenolic substances is well documented, an important effect of copper on laccase formation has only recently been reported [18], [19]. This stimulating effect was found to be especially effective in several Trametes spp., most notably in T. pubescens. For this latter organism Cu concentrations optimal for laccase production were determined to be 1.5–2.0 mM [20]. Furthermore, copper had to be supplemented during the exponential phase of growth for its maximal effect. Under these growth conditions maximum values for laccase activity obtained were approximately 65 U ml⁻¹[20].

Laccases have became important, industrially relevant enzymes because of a number of diverse applications, e.g. for biocatalytic purposes such as delignification of lignocellulosics and cross-linking of polysaccharides, for bioremediation applications such as waste detoxification and textile dye transformation, for food technological uses, for personal and medical care applications, and for biosensor and analytical applications [21]. To utilize laccases more efficiently for these biotechnological and environmental applications and to better understand the properties of these important enzymes at a molecular and kinetic level, rather large amounts of crude and purified laccases are required [8]. At present, research and application are sometimes hindered by the rather low yields of the enzyme formed by wild-type organisms but also by the difficulties to efficiently overexpress laccases heterologously in active form [22]. In the present study laccase formation by the white-rot basidiomycete Trametes pubescens, which only recently had been identified as an excellent source of this enzyme [20], was studied in more detail and was optimized pertaining to the carbon and the nitrogen source used. By employing a fed-batch strategy, in which the substrate glucose—which often represses synthesis of certain enzymes [23]—was slowly fed to an actively growing culture of the fungus, yields and productivity of laccase formation could be significantly increased.