The effect of carbon source succession on laccase activity in the co-culture process of Ganoderma lucidum and a yeast
Introduction
Laccases (benzenediol: oxygen oxidoreductase, EC1.10.3.2) are a group of blue multicopper oxidases which has been extensively studied [1], [2]. They can catalyze the oxidation of many substances coupled to the reduction of molecular oxygen to water [3], [4]. Because of their remarkable non-specificity regarding the reducing substrate, they are receiving increasing attention in various applications such as delignification and bleaching of pulp, dye decolourization, wastewater treatment, enzymatic removal of phenolic compounds in beverages, food processing, construction of biosensors and bioremediation [5], [6]. All these applications require a large quantity of enzyme, which makes the production of laccase an important issue.
The optimization of medium to reduce the cost of laccase is the basic research in the industrial applications [7], [8]. Another approach is to overproduce laccase in a suitable host. Thus, screening new microorganisms producing laccase is a focus of much attention [9], [10]. Furthermore, Hong et al. [11], [12] cloned the heterologous laccase genes and made them express in new hosts, which proved that gene engineering technology was also an efficient approach to increase laccase yield.
Recently, many researchers have shown great interest in overproduction of laccase by microbial associations [13], [14], [15] such as Ferreira-Gregorio et al. found that a rapid increase of laccase activity was detected in the culture supernatant when the liquid cultures of Marasmius pallescens and Marasmiellus troyanus were mixed. Moreover, Savoie et al. proved that cell-free culture fluids of Trichoderma sp. were effective to increase laccase yield of Lentinus edodes because they induced the changes of laccases in the laccase isoenzyme pattern. As for the interaction mechanism between the co-cultured microbes, although it was discussed in some literature [15], [16], there is still a lot of crucial information not being revealed [17], [18].
In the present paper, a phenomenon was reported: in the culture process of G. lucidum, laccase yield was improved remarkably when a strain of yeast, Candida sp. HSD07A, was seeded to the medium. This phenomenon is worthy of exploring because it presents a specific method to overproduce G. lucidum laccase. Thus, the mechanism of overproduction of laccase was investigated. The result shows that the mechanism which leads to the overproduction of laccase is interesting and unrevealed in the previous literature [16], [18]. Therefore, this work may be helpful for us to understand the interaction between microbes and also be beneficial to the development of laccase fermentation industry.
Section snippets
Chemicals and microorganisms
All chemicals are of spectral or analytical grade unless otherwise stated. 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS), α-amylase from Aspergillus oryzae and cellulase from Aspergillus niger are all obtained from Sigma. Both G. lucidum and Candida sp. HSD07A are obtained from Laboratory of Applied and Environmental Microbiology, Henan Normal University.
Effects of nutrition sources on laccase activity
G. lucidum and strain HSD07A were incubated at 28 °C on PDA plates, respectively. After 1 week, mycelial
Effects of carbon, nitrogen and sulfur source on laccase activity
In G1, the secretion of laccase mainly started from day 4 when reducing sugar was 2.89 g/l (Fig. 1), indicating that the cells of G. lucidum were in a state of glucose limit. However, when sterile glucose was added to G2, the synthesis of laccase was inhibited and enzyme activity decreased markedly (Fig. 1a). These facts prove that the synthesis of laccase is inhibited by higher concentration of glucose (≥3.50 g/l). Thus, it is glucose limit in the medium that induces the production of laccase.
Conclusions
In view of the results obtained, it can be concluded that:
Nitrogen source, sulfur source, hydrolytic enzymes and inducers from the yeast do not have the significant influence on laccase activity. Glucose deprivation is also not the crucial reason why G. lucidum overproduces laccase although it can improve laccase yield at a certain extent. Glucose deprivation state in the medium is made by strain HSD07A, and NMR and GC data reveal that the yeast can convert glucose into glycerol and ethanol. In
Acknowledgements
This work is supported by the natural science foundation of Education Department of Henan Province (no. 2008B610004) and the technology development program of Xinxiang Science and Technology Bureau (no. 09G038).
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