Comparative Studies on the Blue and Yellow Laccases

Pankaj Kumar Chaurasia, Shashi Lata Bharati, Sunil Kumar Singh

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Comparative Studies on the Blue and Yellow Laccases

Pankaj Kumar Chaurasia1, Shashi Lata Bharati1, Sunil Kumar Singh1,

1Department of Chemistry, Gorakhpur University, Gorakhpur (U. P.), India

Abstract

In the modern time enzymatic works have achieved a sound attention due to their successful involvements in several types of tedious syntheses without generating any type of environmentally harmful pollutants. It has also been proved that laccases have such types of capabilities to perform several environmentally safe roles in food, paper-pulp, textile, cosmetics, nanotechnology, sugar industries, synthetic organic and drugs chemistry. In most cases, laccase mediator systems play very important roles and in such cases, reactions were difficult or impossible without mediators but the studies of yellow laccases and their capabilities to oxidize phenolic lignin model compounds without help of any mediator systems have good future with several possibilities in industrial and medicinal areas. In the above respects, this review represents the concise and compact comparative studies on blue and yellow laccases with their different important properties.

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Cite this article:

  • Chaurasia, Pankaj Kumar, Shashi Lata Bharati, and Sunil Kumar Singh. "Comparative Studies on the Blue and Yellow Laccases." Research in Plant Sciences 1.2 (2013): 32-37.
  • Chaurasia, P. K. , Bharati, S. L. , & Singh, S. K. (2013). Comparative Studies on the Blue and Yellow Laccases. Research in Plant Sciences, 1(2), 32-37.
  • Chaurasia, Pankaj Kumar, Shashi Lata Bharati, and Sunil Kumar Singh. "Comparative Studies on the Blue and Yellow Laccases." Research in Plant Sciences 1, no. 2 (2013): 32-37.

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1. Introduction

Laccase (benzenediol: oxygen oxidoreductase; EC 1.10.3.2) is a polyphenol oxidase, which belongs to the superfamily of multicopper oxidases [1, 2, 3, 4]. It catalyzes the four electron reduction of molecular oxygen to water according to the equation

Four molecules of the substrate are oxidized to radicals which react non-enzymatically. Laccases are widely distributed in higher plants [6, 7], fungi [8-18][8], bacteria [19, 20, 21], insects [22] and wasp venom [23].

Laccases are the lygnolytic enzymes and abundantly occur in the fungal systems [8] mainly in Ascomycetes, Deuteromycetes and Basidiomycetes and its production in lower fungi has never been demonstrated. They occur in fungal causative agents of the soft rot, in most, white rot causing fungi, soil saprophytes and edible fungi. These laccases producing fungi are generally called wood degrading fungi. White rot fungi are the highest producers of laccases but litter decomposing and ectomicorrhizal fungi also secrete laccases. Almost all white rot fungi are laccase producers [8, 9] except for Phanerochaete chrysosporium. Laccases are also reported in bacteria as Azospirrullum lipoferum [20] which was the first laccase reporting bacteria. Except fungi, plants and bacteria, the presence of laccases have also been reported in wasp venom [23] as well as insects [22].

Figure 1. Three different redox sites of four copper atoms present in laccases

2. Properties

Since laccase recycles on molecular oxygen, it is the most promising enzyme of oxidoreductases group [5, 24, 25, 26]. Ortho and para diphenols, amino phenols, polyphenols, polyamines, lignins and arylmines and some of the inorganic ions are the substrates for laccases. The biotechnological importance of laccases have increased after the discovery that oxidizable reaction substrate range could be further extended in the presence of small readily oxidizable molecules called mediators [27, 28]. During the last two decades, laccases have turned out to be the most promising enzymes for industrial uses [5, 25, 26] to name a few in foods, paper-pulp, textile, cosmetics and synthetic organic chemistry. The laccase contains four copper atoms per monomer distributed in three redox sites [29]. Type I Cu (T1), is characterized by an intense charge transfer band at about 600 nm which impart blue colour to the enzyme. Moreover, its EPR spectrum exhibits narrow hyperfine splitting [A׀׀= (40-90) × 10-4 cm-1]. Type 2 copper (T2) or normal copper site, is characterized by lack of strong absorption in the visible range and its EPR spectrum exhibits normal large hyperfine splitting [A׀׀ = (140-200) × 10-4 cm-1]. Type 3 site consists of two copper atoms antiferromagnetically coupled and EPR silent. This site is characterized by an intense charge transfer band at about 330 nm. Type 2 and type 3 coppers form a trinuclear cluster as shown in Figure 1.

The organic substrate is oxidized at type I Cu by one electron generating a radical and Cu (I). The electron received at Cu type I is shuttled to the trinuclear cluster where oxygen is reduced to water as shown in Figure 2.

Figure 2. Diagrammatical representation of reduction of molecular oxygen to water

Most of the kinetic and spectroscopic studies on laccases are summarized in the book by Messerschmidt [3] and a number of recent reviews [4, 5].There is a recent review by Dwivedi et al [5] which includes the research work done on laccases up to Oct. 2010. It has been reported in the literature [30, 31] that the fungal strains, which secrete blue laccases in submerged culture, secrete yellow laccases when grown on solid lignin containing substrates. Most of the studies reported so far are on blue laccases. Yellow/white laccases have rarely been studied [30, 31]. Yellow/white laccases differs from blue laccases in two respects. Yellow/white laccases lack absorption band around 610 nm always found in blue laccases [30] and yellow/white laccases oxidize non-phenolic substrates in absence of mediator molecules [31] which are required in cases of blue laccases. Since yellow/white laccases oxidize non-phenolic substrates in absence of mediator molecules, they are better biocatalysts than blue laccases.

Figure 3. UV-Visible spectrum of a purified blue laccase of Phellinus linteus MTCC-1175
Figure 4. UV-Visible spectra of a purified laccase from Coriolopsis floccosa MTCC-1177. (a) Spectrum lacking the peak around 610 nm for yellow laccase. (b) Spectrum of oxidation of veratryl alcohol to veratraldehyde by yellow laccase

A characteristic spectrum for blue laccase obtained from the laccase of Phellinus linteuis MTCC-1175 studied by Pankaj et. al. [32] is given Figure 3, in which the band appears around 610 nm while a characteristic spectrum for yellow laccase is given in Figure 4. Figure 4(a) is the spectrum of purified laccase of Coriolopsis floccosa MTCC-1177 [33] and Figure 4(b) represents the oxidation of veratryl alcohol into veratraldehyde by this yellow laccase [33].

3. Reaction Mechanism of Yellow and Blue Laccases

It is well known that blue laccases generally reacts with organic substrates in the presence of mediators while yellow laccases perform the same act without help of any mediator molecules. Here it is necessary to know what mediators are.

Bourbonnais and Paice [34] showed that in the presence of substrates such as Remazol Blue and 2,2’-Azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS), Coriolus versicolour laccase can oxidize non-phenolic lignin model compounds like veratryl alcohol which can not be oxidized by the laccase alone. Thus, the oxidation of lignin model compounds was dependent on the presence of primary laccase substrates. Such primary substrates were termed as mediator molecules.

After the discovery of mediators, laccases become much more significant in the area of several biotechnological applications. Laccase mediator systems and their applications have been reviewed by Morozova et al. [31]. The well-known mediators are ABTS (2,2 [Azino-bis-(3-ethylbonzthiazoline-6-sulphonic acid) diammonium salt]) and HOBT (1-hydroxybenzotriazole). An ideal mediator is that which can perform many cycles without degrading itself and without any side reactions. The oxidized mediator formed during the course of laccase catalyzed reaction can oxidize non-phenolic substrate, non-enzymatically. The difference between the redox potentials of the substrate to be oxidized and T1 copper ions is the driving force of the reaction. Mediators are a group of low molecular weight compounds with high redox potential (above 900 mV) which have abilities to enhance the range of substrates of laccases and those act as a sort of ‘electron shuttle’: once it is oxidized by the enzyme generating a strongly oxidizing intermediate that is known as co-mediator or oxidized mediator, it diffuses away from the enzymatic pocket and in turn oxidizes any substrate that, due to its size could not directly enter into the active site. Due to its large size and steric hindrance, enzyme and polymer do not have to interact in a direct manner, then, the use of mediators allows the oxidation of polymers by side-stepping the inherent steric hindrance problems [35]. Alternatively, the oxidized mediator could rely on an oxidation mechanism not available to the enzyme, thereby extending the range of substrates accessible to it [36]. Other organic redox-mediators are N-hydroxyphthalamide (NHPI), and inorganic redox-mediators are iron complexes with o-phenanthroline and 4,4-dimethylbipyridine.

The different steps involve in the general oxidation reaction done by laccase with the help of mediator molecules and without the help of mediator molecules are shown in Figure 5 (a-b) [37]. Figure 5(a) shows the oxidation of non-phenolic structures in which laccase firstly, oxidizes the mediator molecule and then this oxidized mediator oxidizes the non-phenolic structures (by blue laccase), while Figure 5(b) represents the direct oxidation of substrate (phenolic) without the help of any type of mediators (by yellow laccase). A typical example of oxidation of phenolic as well as non-phenolic lignin model compounds by laccase and laccase mediator systems are given in Fgure 6(a) and Fgure 6(b) [38]. Selective biotransformations of aromatic methyl group of toluene, 3-nitrotoluene, 4-chlorotoluene etc. to its aldehyde group have been done successfully by laccase-mediator systems [39, 40] and also by laccase without using any mediators [33]. The laccase catalyzed reactions which do not need any type of mediators are more efficient and valuable than those of laccase catalyzed reactions which need of mediators to accomplish their reactions.

Figure 5. A schematic representation of reaction mechanism of blue as well as yellow laccases for different substrates (a) Oxidation of non-phenolic substrates with mediators generally done by blue laccases (b) Oxidation of phenolic substrates without mediators generally done by yellow laccases
Figure 6. A typical example of oxidation of phenolic (a) as well as non-phenolic lignin model compounds (b) by laccase and laccase mediator systems, respectively

4. Conclusions

This review shows the clear explanations about blue and yellow laccases and their style to play significant roles in several functions. There is a need of intelligent and more research in the area of yellow laccases because they have strong possibilities to perform several tedious applications without the involvement of any types of mediator molecules which are necessary for blue laccases as in organic syntheses of several important pharmaceutical drugs, hormones, polymers and other industrial applications.

Acknowledgement

The authors acknowledge the financial support of CSIR-HRDG, New Delhi for the award of JRF (NET) and SRF (NET), award no. 09/057(0201)/2010-EMR-I to Mr. Pankaj Kumar Chaurasia.

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