Tài liệu Báo cáo Y học: Oxidation of phenols by laccase and laccase-mediator systems doc

Oxidation of phenols by laccase and laccase-mediator systems

Solubility and steric issues

Francesca d’Acunzo, Carlo Galli and Bernardo Masci

Dipartimento di Chimica and Centro CNR Meccanismi di Reazione, Universita`
La Sapienza, 00185 Roma, Italy

To investigate how solubility and steric issues affect the

laccase-catalysed oxidation of phenols, a series of oligomeric

polyphenol compounds, having increasing size and

decreasing solubility in water, was incubated with laccase.

The extent of substrate conversion, and the nature of the

products formed in buffered aqueous solutions, were com￾pared to those obtained in the presence of an organic

cosolvent, and also in the presence of two mediating species,

i.e. N-hydroxyphthalimide (HPI) and 2,2,6,6-tetramethyl￾piperidin-1-yloxy (TEMPO). This approach showed not

only an obvious role of solubility, but also a significant role

of the dimension of the substrate upon the enzymatic reac￾tivity. In fact, reactivity decreases as substrate size increases

even when solubility is enhanced by a cosolvent. This effect

may be ascribed to limited accessibility of encumbered sub￾strates to the enzyme active site, and can be compensated

through the use of the appropriate mediator.While TEMPO

was highly efficient at enhancing the reactivity of large, less

soluble substrates, HPI proved less effective. In addition,

whereas the laccase/HPI system afforded the same products

as laccase alone, the use of TEMPO provided a different

product with high specificity. These results offer the first

evidence of the role of
oxidation shuttles that the media￾tors of laccase may have, but also suggest two promising

routes towards an environmentally friendly process for

kraft pulp bleaching: (a) the identification of mediators

which, once oxidized by laccase, are able to target strategic

functional groups present in lignin, and (b) the introduction

of those strategic functional groups in an appropriate

pretreatment.

Keywords: laccase; phenols; lignin degradation; HPI;

TEMPO.

Lignin is a three-dimensional, insoluble aromatic polymer

that constitutes 15–33% of biomass. Its structure encom￾passes a number of different types of links between its

constituents, namely ether and C-C diaryl linkages [1].

White-rot fungi achieve the oxidative depolymerization of

lignin by secreting several enzymes, such as lignin peroxidase

[2], manganese peroxidase [3], and laccase (EC 1.10.3.2) [4].

In contrast with lignin peroxidase and manganese peroxi￾dase, laccase can only oxidize the phenolic constituents of

lignin, due to its lower oxidation potential. On the other

hand, it is more readily available and easier to manipulate

than the other two enzymes, and its substrate specificity is

low, as long as a good match of oxidation potentials is

provided [5–8]. In addition, the use of appropriate low

molecular-mass compounds (viz., mediators), in combina￾tion with laccase, makes this enzyme competent for the

oxidation of
non-natural nonphenolic substrates [9–12]. In

fact, the oxidized mediator (Fig. 1) can rely on an oxidation

mechanism that is not available to the enzyme [13].

Laccase can therefore be turned into a much more

versatile enzyme, and this opens up various possible

applications, as in the textile dye bleaching [14], or for

environmentally respectful kraft pulp delignification [10,15],

or also in selective organic transformations [16–19]. The

study presented here is part of our efforts to elucidate the

mechanisms of action of the laccase/mediator systems [20] in

the oxidation of lignin model compounds, as well as non￾lignin-related structures (Fig. 1).

A conceivable role of the mediator could be that of a sort

of
electron shuttle between the enzyme and the substrate

[21]. Once the mediator is oxidized by the enzyme, it diffuses

away from the enzymatic pocket and in turn oxidizes

substrates that, due to their size, could not directly enter the

enzymatic pocket. Within this framework, we wished to

investigate the influence of substrate size and solubility on

the effectiveness of laccase oxidation, and also the effect of

mediators endowed with possibly different mechanisms of

action. To this aim, we needed to start from a simple

phenolic structure, which laccase could recognize as a

natural substrate, and modify it into bigger and more

insoluble derivatives. The oligomeric series shown in Fig. 2

served our purposes for the following reasons: (a) each

repeat unit is a phenol, and therefore subject to oxidation

by laccase, at least in terms of redox potential; (b) the

number of repeat units in each oligomer, and therefore its

size, is exactly determined, because directed synthesis and

Fig. 1. Catalytic cycle of a laccase-mediator oxidation system.

Correspondence to C. Galli, Dipartimento di Chimica and Centro

CNR Meccanismi di Reazione, Universita`
La Sapienza,

00185 Roma, Italy. Fax: + 39 06 490421,

E-mail: [email protected]

Abbreviations: HP I, N-hydroxyphthalimide; TEMPO, 2,2,6,6-

tetramethylpiperidin-1-yloxy; ABTS, 2,2¢-azinobis-(3-ethylbenzothi￾azoline-6-sulfonate).

(Received 18 June 2002, revised 9 September 2002,

accepted 12 September 2002)

Eur. J. Biochem. 269, 5330–5335 (2002) FEBS 2002 doi:10.1046/j.1432-1033.2002.03256.x