Optimization of Agaricus blazei laccase production by submerged cultivation with sugarcane molasses

Laccases are copper polyphenol oxidases that are interesting for several applications such as in the food industry, sewage treatment and decolorization. The use of agro-industrial byproducts allows bioprocesses development for the production of large quantities at viable cost enzymes. In this study, the laccase production of Agaricus blazei was optimized in submerged cultivation (SmC). First, the following agro-industrial substrates were evaluated: sugarcane molasses, soybean molasses, coffee husks, soybean husks and pellet citrus pulp; and then these nitrogen sources: urea, ammonium sulfate, yeast extract. For the optimization of laccase production, a Plackett-Burman and 2 (4-1) incomplete factorial designs were used to evaluate the effect of KH2PO4, MgSO4, KCl, FeSO4, ZnSO4 and the ethanol on laccase activity, and for the optimization of sugarcane molasses, urea, MgSO4 and ethanol concentrations. Finally, laccase production kinetics was determined. The best substrate for laccase production by A. blazei was sugarcane molasses. After optimization, it was found that laccase activity of 9635 U/L was obtained in medium with sugarcane molasses (6 g/L), urea (1.5 g/L), MgSO4 (12 mM), ethanol (1.2 mM ) at 28°C and pH 8.0 during 10 days of cultivation. The results indicate that sugarcane molasses is a promising substrate for A. blazei laccase production.


INTRODUCTION
Laccases are copper containing polyphenol oxidases where copper atoms are distributed among three different highly-conserved binding sites, each one with an important role in the catalytic mechanism of the enzyme (Soden and Dobson, 2001;Couto and Toca-Herrera, 2006). Its presence has been described in plants, bacteria and insects (Morozova et al., 2007), but especially found in ligninolytic basidiomycete fungi (Mikolasch and Shauer, 2009). In basidiomycetes, laccases have different physiological functions such as delignification, defense against oxidative stress produced by lignin degradation and morphogenesis .
The delignification process by laccase involves the transfer of electrons from phenolic substrates to molecular oxygen, reducing it to water while oxidizing phenolic substrates to semiquinone radicals (Zhou et al., 2013). One of its most striking features is the low specificity of their substrates. The number and diversity of substrates susceptible to oxidation by laccase vary greatly from one laccase to another (Dittmer and Kanost, 2010). These peculiarities make it a very interesting enzyme for *Corresponding author. E-mail: jsvalle@unipar.br. Tel: +55-44-36212837.
A. blazei is a litter-decomposing fungus (LDF) and grows naturally on the surface layers of soil that are rich in organic matter and humus in forests and fields (Wasser et al., 2002). Due to its ecological characteristics, this fungus is able to degrade lignin and compounds that are structurally similar to lignin. However, the degradation rate is smaller as compared to white rot fungus, essentially ligninolytic ones (Durrant et al., 1991). The production of ligninolytic enzymes by LDF should not be discarded, since maximal levels of production can be improved by employing optimization of culture conditions (Elisashvili and Kachlishvili, 2009) as well as by using heterologous expression  or structural modification (Robert et al., 2011). A. blazei has been described before as a laccase producer (Soden and Dobson, 2001;D'Agostini et al., 2011) and a producer of other enzymes such as amylase, cellulase, xylanase, mannanase and pectinase (Siqueira et al., 2010;Jonathan and Adeoyo, 2011).
The growing demand for lower cost in industrial processes that are also highly specific and environmentally safe has stimulated the search for new enzymes. The use of agro-industrial residues in bioprocesses has enabled the production of enzymes employing alternative substrates at low cost as well as reducing environmental degradation caused by the disposal of these residues (Elisashvili et al., 2008;Karp et al., 2013).
The sugar and ethanol industry produces different residues and many of them have great potential for bioprocesses application (Pandey et al., 2000). Sugarcane molasses is dark syrup obtained during sugar production from sugarcane or beets, resulting from the final stage of crystallization from which further recovery of sugar is no longer economically viable (Arakaki et al., 2011). Sugarcane molasses has an average of 50% total sugars in which sucrose predominates (Villavicencio et al., 1999;Feltrin et al., 2000;Arakaki et al., 2011). The production of sugarcane in Brazil in 2012/2013 harvest reached 589 million tons, yielding from 23 to 35 tons of molasses (CONAB, 2013). Its composition and abundance makes this residue a potential substrate for the development of biotechnological processes, including the production of enzymes and other products (Miranda et al., 1999).
The objective of this study was to develop a bioprocess for A. blazei laccase production in submerged cultivation using agro-industrial byproducts.

Microorganisms and inoculum preparation
A. blazei U2-4 strain, available at the culture collection of the Molecular Biology Laboratory of Paranaense University, was selected to optimize the culture conditions for laccase production. The mycelium was kept on 1% malt extract agar medium (MEA; m/v) at 25°C and subcultured to 2% MEA (m/v) at 28°C for 7 days in order to prepare the inoculum.
For the experimental phase, the submerged cultivation (SmC) was done in a 250 mL Erlenmeyer. Each flask had 100 mL of autoclaved (121°C for 20 min) minimum medium. This medium consisted of 1.5 g/L KH2PO4, 0.5 g/L MgSO4, 0.5 g/L KCl, 0.036 g/L FeSO4 H2O and 0.035 g/L ZnSO4 H2O. All flasks were inoculated with three agar discs, 6 mm diameter each, containing mycelium. The mycelial growth was carried out at 28°C for 10 days in the dark. For all experiments, at the third day of cultivation, CuSO4 (300 g/L) was aseptically added until obtaining 150 µM in the culture medium. This elemental SmC scheme was used to evaluate the effect of the addition of substrates, nitrogen sources and salt concentrations on laccase production.

Selection of alternative substrates for laccase production
Five agro-industrial byproducts were tested as substrates: sugarcane molasses, soybean molasses, coffee husks, soybean husks and pellet citrus pulp. The molasses were added until total sugars reached 10 g/L in the minimum medium and the other particulated residues were used at 50 g/L. As nitrogen source, urea (300 g/L) was filtered (0.22 µm Millipore membrane) and added to the autoclaved medium in order to reach 100 mM.

Effect of nitrogen source on laccase production
Different nitrogen sources were added to the minimum medium in order to achieve 2.8 g/L of nitrogen. Sugarcane molasses was used as substrate until total sugars reached 10 g/L. The evaluated nitrogen sources were 13.2 g/L ammonium sulfate; 7.0 g/L yeast extract; 6.0 g/L urea; a mixture of 3.0 g/L urea and 6.6 g/L ammonium sulfate; and a mixture of 6.6 g/L ammonium sulfate and 3.5 g/L yeast extract. Water solutions of urea and ammonium sulfate were filtered (0.22 µm Millipore membrane) and aseptically added to the culture medium. The yeast extract was autoclaved with SmC media at 121°C for 20 min.

Screening of media components for laccase production
Plackett-Burman statistical experimental design is used for the screening of major constituents of the media with significant effects on laccase response (Bari et al., 2009). To achieve the best conditions for laccase production by submerged cultivation with sugarcane molasses, the concentration of salts of the minimum medium as well as the addition of urea and ethanol as inducers of laccase production were analyzed. First, a seven-factor Plackett-Burman design, resulting in eight runs, was used to determine the effect of salt concentrations of the minimum medium such as KH2PO4, MgSO4, KCl, FeSO4 H2O and ZnSO4 H2O, and the ethanol (Lomascolo et al., 2003;Meza et al., 2007) on laccase production by A. blazei. Subsequently, a 2 (4-1) incomplete factorial design was used and four components were evaluated in 11 experiments with three replications of the central point to test the concentration effect of sugarcane molasses (6; 10 or 14 g/L), urea (2; 7 or 12 g/L), MgSO4 (2; 7 and 12 g/L) and ethanol (0.2; 0.7 and 1.2 g/L) on laccase production. The significant urea variable was again studied at concentrations below 2 g/L, the lowest concentration evaluated in the 2 (4-1) incomplete factorial design. For this experiment, the composition of the culture medium was sugarcane molasses (6 g/L), magnesium sulfate (12 mM), ethanol (1.2 mM), the best conditions determined in the 2 (4-1) incomplete factorial design, and urea added at 0; 0.5; 1.0; 1.5 and 2.0 g/L.

Kinetics of laccase production by SmC with sugarcane molasses
After determining the best culturing conditions (6 g/L sugarcane molasses, 1.5 g/L urea, 12 mM MgSO4 and 1.2 mM ethanol), 10 day-kinetics was carried out by SmC. The pH of the cultivated medium, the amount of reducing sugars, the production of biomass, laccase and manganese peroxidase activities were also determined.
Manganese peroxidase (EC 1.11.1.13) was determined by the oxidation of 10 mM MnSO4 at 30°C in 50 mM sodium malonate buffer (pH 4.5) and in the presence of 0.5 mM hydrogen peroxide (Wariishi et al., 1992). The oxidation was monitored by absorbance increase at 270 nm ( = 11590 M -1 cm -1 ) caused by the complex formed by the Mn 3 + ion with malonate. The mixing of the enzyme extract and sodium malonate buffer and the mixture of MnSO4 and sodium malonate buffer were used as analytical controls. One unit (U) of enzyme activity was defined as the amount of enzyme required to oxidize 1 µmol of MnSO4 per minute.
Statistical experiments and analysis were carried out using the software package STATISTICA 10 (StatSoft, Tulsa, OK, USA). All experiments and procedures were done in triplicate and the results evaluated using ANOVA or Tukey's test (p <0.05).

Selection of alternative substrates
The selection of alternative substrates for laccase production was carried out in SmC containing various do Valle et al. 941 agro-industrial residues. The sugarcane molasses, soybean molasses and coffee husks increased laccase activity when compared with the control with glucose ( Table 1). The highest (p≤0.05) laccase activity was with sugarcane molasses and it was 1.7, 1.5 and 3.8 times higher than soybean molasses, coffee husk and glucose (control), respectively. Also, sugarcane molasses was the best for productivity, reaching 58.5 U/Lh. Thus, in our study, sugarcane molasse was chosen as an alternative substrate for the next experiments. Easily assimilated substrates seem to favor the laccase production for some fungal species, but several reports showed otherwise (Galhaup et al., 2002;Elisashvili and Kachlishvili, 2009;Majeau et al., 2010). Our results showed that the highest value for laccase activity (14052 U/L) was obtained with sugarcane molasses. This residue has 540 g/L total sugars (Rodrigues et al., 2009), 70 to 91% of which is sucrose, and 2 to 4% is glucose (Arakaki et al., 2011). For A. blazei, the easily assimilated substrates present in sugarcane molasses seem to increase the laccase production.
Our results show that when coffee husks was used as substrate, the laccase production was 9253 U/L against 3566 and 4104 U/L when citric pulp and soybean hulls were used respectively. The production of laccases in SmC using agro-industrial residues can be stimulated by the presence of aromatic and/or phenolic compounds (Elisashvili and Kachlishvili, 2009). Coffee husks, for example, are rich in caffeine, tannins and polyphenols (Pandey et al., 2000). Thus, it can be suggested that aromatic and/or phenolic compounds can improve laccase production by A. blazei. Previous data from our laboratory suggested that these compounds stimulated A. blazei laccase production (data not shown) and also stimulate laccase production of other basidiomycetes (Silva et al., 2012). Additionally, the fungal ability of producing high levels of laccase in different agroindustrial residues demonstrates the versatility of this strain as a laccase producer. Previous reports of A. blazei laccase production in SmC with agro-industrial byproducts showed lower levels of laccase activity of 5000 U/L and productivity of 12.2 U/Lh (Ullrich et al., 2005) against 14052 U/Lh for laccase activity, and 58.5 U/Lh for productivity obtained in our work with sugarcane molasses.

Selection of alternative nitrogen source
Following the selection of substrates for laccase production, aproteic (urea and ammonium sulfate), proteic (yeast extract), and combinations of aproteic and proteic nitrogen sources were evaluated. Different nitrogen sources, as well as combinations thereof, did not affect (p≤0.05) the laccase activity (Figure 1). The highest laccase activities were obtained with urea (11427 U/L) and a urea and ammonium sulfate combination   (11736 U/L). Because the nitrogen sources did not affect the laccase production, it is supposed that the nitrogen concentrations could affect it. Thus, urea was chosen for the next experiments because it is an inexpensive source of nitrogen, produced high laccase activity in our work, and is used for laccase production by A. blazei in solid state cultivation (D'Agostini et al., 2011).

Selection of minimum medium components for the production of laccase by SmC
The results of the Plackett & Burman experiments for selection of significant variables in the laccase production are shown in Table 2. The obtained value for R 2 was 0.98, indicating that 98% of the data adjusted to the Additionally, the highest laccase activity (11160 U/L) was obtained without urea addition. In this case, besides sugarcane molasses, only KCl, MgSO 4 and ethanol were added for fungus growth. Thus, laccase produced by A. blazei U2/4 can be affected by reducing the urea concentration and because of that this variable was chosen for the next experimental phase. Although other variables did not affect the laccase production, it was observed that MgSO 4 and ethanol can improve the enzyme activity of other basidiomycetes. Also, considering that the inducer concentration requires a fine adjustment, MgSO 4 and ethanol were kept as variables for the next experimental phase. In this step, the laccase production at different concentrations of urea, MgSO 4 , sugarcane molasses and ethanol was assessed.

Optimization of sugar cane molasses, urea, MgSO 4 and ethanol for the production of laccase by SmC
The highest laccase activity (14139 U/L) was with 6 g/L of total sugars (sugarcane molasses), 2 g/L of urea, 12 mM MgSO 4 and 1.2 mM ethanol ( Table 3). The average laccase production of 7 and 12 g/L of urea was of 12116 and 6970 U/L, respectively (Table 3). In addition, urea was the only variable that affected (p≤0.05) the laccase production (Table 3). However, the effect of MgSO 4 was very close to the limit of statistical significance, suggesting that an increased concentration of salt in the cultivation could be advantageous. The R 2 of this analysis was 0.81 indicating that 81% of the data fit in the statistical analysis model. These results show that laccase production by A. blazei in submerged cultivation is inversely proportional to the concentration of nitrogen, so the higher carbon/nitrogen (C/N) ratio, the better the laccase yield.
The variable and levels of sugarcane molasses, MgSO 4 and ethanol (ET) did not affect the laccase activity of A. blazei in SmC. Thus, these variables were kept at lower levels of 6 g/L sugarcane molasses, 12 mM MgSO 4 , 1.2 mM ethanol, and urea was kept as a variable.
The results of the variable selection have pointed out that the laccase production can be facilitated by reducing the urea concentration, which means an increase in C/N ratio. The effect of nitrogen concentration on laccase production appears to vary according to the species and strain of the assessed fungus . Pleurotus ostreatus cultivated in a medium containing urea (0.5 g/L) produced more laccase with lower concentration of nitrogen (Hou et al., 2004). Conversely, Stajić et al. (2006) showed that higher concentrations of nitrogen (20 g/L peptone) slightly stimulate the laccase production of this species, which is an indication that different strains of the same species may respond differently to a specific nutrient. It suggests as well that laccase production depends on C/N ratio which differs for each employed strain and should be optimized for each case.
In this study, urea concentration ranged from 2 to 12 g/L. However, the results showed that concentrations of urea lower than 2 g/L could improve laccase production. Thus, an experiment was designed to refine the best urea concentration for enzyme production. The highest level of enzyme activity (13604 U/L) was obtained with 1.5 g/L urea (Figure 2). This result did not differ significantly from  those obtained with 1 (13469 U/L) and 2 g/L urea (13590 U/L). Urea concentration of 0.5 or 0.0 g/L reduced (p≤0.05) the laccase activity to 12795 and 12996 U/L, respectively. Thus, the best conditions for laccase production of A. blazei U2/4 in SmC is 6 g/L sugarcane molasses, 1.5 g/L urea, 12 mM MgSO 4 and 1.2 mM ethanol. Smaller amounts of nitrogen favor laccase production in SmC with sugarcane molasses that reached optimum values when C/N ratio was between 1.3 (2 g/L urea) and 3.0 (1 g/L urea).

Kinetics of laccase production with the optimized conditions
After determining the best cultivation conditions, our group studied the kinetics of laccase production in SmC by analyzing the enzyme activity every 24 h for a period of 10 days. The peak of laccase production and productivity, 9635 U/L and 44.6 U/Lh, respectively, were obtained at the 9 th day of cultivation (Table 4). The pH gradually increased over time, from 5.6 to 8.0, at the peak of the enzyme activity, demonstrating that higher levels of activity occurred at higher pH and around neutrality. The microbial biomass production reached maximum value at day 8 when the enzyme production was near the maximum. This shows that laccase production in SmC is associated with A. blazei mycelium growth. The amount of reducing sugars showed no important variation up to half of the cultivation period, suffering a decrease from the increase of biomass and laccase production (Table 4). Thus, the laccase production was associated with the reduction of reducing sugars in the cultivation medium. The production of manganese peroxidase (Table 4) remained at low levels, reaching maximum activity at day 8 (1855 U/L).
There is scant information on the laccase production by A. blazei. Ullrich et al. (2005) achieved levels of enzymatic activity around 5000 U/L at the 17 th cultivation day and productivity of 12 U/Lh with tomato juice as culture medium. Our results, after optimized for laccase production of A. blazei, surmount those described in the literature demonstrating an outstanding productivity of 44.6 U/Lh and laccase activity of 9635 U/L.
The production of manganese peroxidase (MnP), which remained at relatively low levels, was also determined (Table 4) reaching maximum activity at day 8 (1855 U/L). Fenice et al. (2003) reported activity peak of 292 U/L by Panus tigrinus in SmC with waste from olive oil production, whereas Hou et al. (2004) found peak of 1500 U/L for Pleurotus ostreatus in SmC.
The pH gradually increased over time, from 5.6 to 8.0, at the peak of enzyme activity, demonstrating that higher levels of activity occurred at higher pH and around neutrality. Similarly, Ullrich et al. (2005) reported that the laccase production of A. blazei in tomato juice also showed drastic change in pH which rose from 4.5 to 7.0 during 28 cultivation days. The stability of the majority of the laccases is higher under acid pH, although this characteristic varies greatly depending on the source of laccase (Majeau et al., 2010).
The microbial biomass production reached maximum value at day 8 when the enzyme production was near the maximum. This shows that laccase production is associated with mycelial growth. The amount of reducing sugars showed no important variations up to half of the cultivation period, suffering a decrease from the increase in biomass and laccase production.

Conclusions
Sugarcane molasses is the best substrate for laccase production of A. blazei in submerged cultivation. Laccase production is negatively affected by the increase in nitrogen concentration. The highest level of laccase activity (9635 U/L) and productivity (44.6 U/hL) occurs in a cultivation medium comprising of 6 g/L sugarcane molasses, 1.5 g/L urea, 150 µM CuSO 4 , 12 mM MgSO 4 , 1.2 mM ethanol at pH of 8.0 after nine cultivation days. These results indicate the versatility of A. blazei in producing significant levels of laccase in SmC using abundant and inexpensive agro-industrial byproducts.