Optimization of laccase production from Marasmiellus palmivorus LA1 by Taguchi method of Design of experiments
Laccases (EC 1.10.3.2; benzenediol: oxygen oxidoreductases) are a major group of ligninolytic enzymes which are present in all the eukaryotic kingdoms described in the five kingdom classification by R.H Whittaker in 1969 [1–5]. Laccases non-specifically catalyse one-electron oxidation of four equivalent substrates concomitant with the four-electron reduction of molecular oxygen to water with the help of a copper containing catalytic apparatus [6, 7]. Physiologically, laccase fulfil diverse roles from plant lignin polymerisation [8] to fungal morphogenesis [9]. Being less substrate specific, energy-saving, and biodegradable, laccases were suitable in the development of highly effective, sustainable, and eco-friendly enterprises [10] in the areas of biofuel production [11], chemical transformation of xenobiotics [12], dye decolourisation [13], as biofuel cells [14], effluent treatment [15], pulp bleaching [16], as biosensors [17] and in general food quality improvement [18, 19]. Any application of laccase requires large scale production of the enzyme preferably in a cost effective manner.
Even though other enzyme production systems prefer submerged fermentation, enzyme production from fungi, especially filamentous fungi is better adapted to Solid state fermentation (SSF) as only SSF offers an adherence surface to filamentous fungi [20]. In SSF, growth and enzyme production occur in inert or natural solid material under near or complete absence of free flowing liquid. SSF have advantages like high volumetric productivity [21], effective utilization of agro industrial wastes as substrates that even mimic the natural living surface of fungi and economy [22] due to its static nature. SSF utilizes materials like orange peel [23], banana waste [24], barley bran [25] and pine apple leaves [26] for useful enzyme production, which otherwise pose solid waste disposal problems. This reutilization is appreciated in the context of sustainable development. However, robust control of parameters (both media composition and cultural conditions) in SSF is difficult particularly on an industrial scale, which explains the failure of adapting successful lab scale production systems to an industrial level in the past [27]. This can be overcome by the thorough optimization of the different factors that influence production. Classical single factor method of optimization is an inadequate choice as it is time consuming [28] and will not yield any outcome regarding the relative influence of any of the involving factors. Statistical methods which also accounts for variations in the production process would be appropriate for optimization. Taguchi method of design of experiment is an approach for optimization of parameters, where the production quality stands intact even in an altered environment [29].
The Taguchi method of Design of Experiments (DOE) was developed by Genechi Taguchi who was involved in modifying the Japanese telephone system [30]. The main aim of this method is to determine the optimal process characteristic that is weakly sensitive to noise factors [31]. The taguchi method operates systematically with fewer trials, thus reducing the time, cost and effort, but offer more quantitative information [32]. The method can work even if the parameters are discrete and qualitative. It functions by reducing the sensitivity of the system [33] through thorough parameter designing. For the purpose, taguchi employs a fractional factorial design in the form of an orthogonal array. This array includes representatives from all possible combinations of selected experimental parameters, which are apt to increase the efficiency and precision and simultaneously reducing any experimental errors [34]. Analysis of individual factor contribution along with their interactive effects eventually leads to the identification of finest factors which was further optimized through Analysis of variance (ANOVA). All these advantages contribute to its greater application in other fields of science especially biotechnology.
A newly isolated strain LA1 from rarely explored species palmivorus, is the laccase producing fungus that is selected in the present study. The strain was found to be utilizing pineapple leaves, an inexpensive, unused agro-residue, as substrate for laccase production. The initial laccase activity expressed by Marasmiellus palmivorus LA1 was as good as or even higher than that of the initial activities of some of the other reported fungi [35–38]. The present study applies taguchi method for the optimization of extracellular laccase enzyme production in SSF from the fungi Marasmiellus palmivorus LA1. The experimental design comprises of seven different factors that proceeds at two levels with L8 (27) array layout for laccase production. This is the first attempt reported for the optimization of laccase production from any Marasmiellus palmivorus, which is generally viewed only in the context of palm pathogens.