Abstract

The aim of this work was to find a new stable laccase against inhibitors and study the decolorization ability of free and immobilized laccase on different classes of dyes. Spores from a halotolerant bacterium, Bacillus safensis sp. strain S31, isolated from soil samples from a chromite mine in Iran showed laccase activity with maximum activity at 30 °C and pH 5.0 using 2, 2-azino-bis (3-ethylbenzothiazoline-6-sulfonate) (ABTS) as the substrate. The enzyme retained about 60% of its initial activity in the presence of 10% (v v−1) methanol, ethanol, and acetone. In contrast to many other laccases, NaN3, at 0.1 and 1 mM concentrations, showed a slight inhibitory effect on the enzyme activity. Also, the spore laccase (8 U l−1) decolorized malachite green, toluidine blue, and reactive black 5 at acidic pH values; the highest decolorization percent was 75% against reactive black 5. It was observed that addition of ABTS as a redox mediator enhanced the decolorization activity. Furthermore, immobilized spore laccase encased in calcium alginate beads decolorized 95% of reactive black 5 in the absence of mediators. Overall, this isolated spore laccase might be a potent enzyme to decolorize dyes in polluted wastewaters, especially those containing metals, salts, solvents, and sodium azide.

INTRODUCTION

Microbial enzymes have been applied in various industrial and biotechnological fields, and research studies to find new enzymes tolerating harsh industrial conditions have increased in recent years (Delgado-García et al. 2012). Enzymes from extremophiles (extremozymes) which are adapted to extreme environments have provided opportunities to develop new applications and replace their mesophilic counterparts used in extreme industrial conditions. Halophilic microorganisms are extremophiles with the ability to live in hypersaline environments, and their enzymes (haloenzymes) have unique features such as high resistance against denaturizing agents and activity in low water activity or non-aqueous media (Madern et al. 2000; Enache & Kamekura 2010). Along with discovering new sources of enzymes for applied purposes, immobilization of biocatalysts is a promising approach that protects them against harsh industrial conditions and prepares them for usage several times, because of the feasibility of separating immobilized enzymes from reaction mixtures to save them for other rounds of the process (Fernández-Fernández et al. 2013; Rodrigues et al. 2013).

Laccase (EC 1.10.3.2, p-diphenol: dioxygen oxidoreductase) is a multicopper oxidase enzyme considered as a copper protein due to the presence of copper atoms in its catalytic site. This oxidoreductase enzyme transfers four electrons from the substrates (such as diphenols, methoxy-substituted monophenols, aromatic, and aliphatic amines) through four copper atoms in the catalytic site to molecular oxygen as the final electron acceptor to oxidize substrates and reduce oxygen to water (Mayer 1986; Kudanga & Le Roes-Hill 2014). Applications of laccases in food, textile, pharmaceutical, paper, pulp, and biofuel industries are increasing, mainly because of their wide substrate range. Moreover, laccases can be applied in other fields such as bioremediation, organic synthesis, and biosensor technologies (Kudanga & Le Roes-Hill 2014). The great ability of laccases to oxidize phenolic substrates makes them appropriate biocatalysts to degrade industrial dyes and other xenobiotic phenolic pollutants (Fernández-Fernández et al. 2013).

Triphenylmethane, azo, and anthraquinone dyes are the most common and stable colorants, with different colors that could be easily used for dyeing different types of textiles. Among them, azo dyes are the most commonly used dyes, with resistance to breakdown by temperature, light, or acids and bases that makes their remediation very tough (Alexandre & Zhulin 2000). It has been estimated that 10–15% of the used colorants in textile industries end up in textile effluents and, because of their toxicity for living organisms, these dyes should be detoxified or eliminated (generally decolorized) before releasing the effluents to the environment. Physicochemical approaches for decolorization of dye-containing wastewaters are expensive, inefficient, and in some cases may cause secondary wastewater sludge that needs further treatments. On the other hand, biological decolorization of these effluents is an alternative green and cost-effective approach that does not produce secondary wastewaters. Solvents, salts, surfactants, metals, and some other chemicals may be present in the final wastewater of textile factories, and cause difficulties for using biological treatments that raise the need to find resistant biological methods for the removal of different dyes and chemical pollutants (Sarayu & Sandhya 2012; Singh et al. 2015). Different enzymes with the ability to decolorize dyes are peroxidases, azoreductases, and laccases (Singh et al. 2015). Besides the advantages of using laccases in wastewater treatments, the industrial production costs of the enzyme can be significantly problematic. Immobilization of laccase is a promising approach to overcome this problem by reducing the costs due to the reusability of the immobilized enzyme (Fernández-Fernández et al. 2013) and at the same time, protecting the enzyme from the harsh environment.

Fungi are the well-known laccase producers and most of the laccase reports have related to fungi until now; however, bioinformatics efforts provide evidence for their distribution in prokaryotes (Alexandre & Zhulin 2000). Some extracellular bacterial laccases have been isolated from different species of Bacillus such as Bacillus sp. strain WT (Siroosi et al. 2016). Moreover, CotA protein, which is present in spores from Bacillus strains, is responsible for spore laccase activity, as the first spore laccase was reported in 1997 and then from other Bacillus species.

In the present study, a screening procedure for laccase producing bacterial strains was carried out and spores of B. safensis sp. strain S31 that were isolated from chromite mine soil exhibited laccase activity. After spore purification steps, the spore laccase from B. safensis sp. strain S31 was characterized and its decolorization activity against three different dyes was examined. Besides promising dye decolorization results after encapsulation of the spores in alginate beads, the good tolerance of the spores to NaN3 (a common laccase inhibitor found in textile effluents) could make the enzyme a good choice for bioremediation processes. Previously, an extracellular laccase was reported from B. safensis DSKK5 (Singh et al. 2014), but there is no report on the laccase activity from B. safensis spores so far.

METHODS

Chemicals

ABTS, syringaldazine (4-Hydroxy-3,5-dimethoxybenzaldehyde azine), sodium alginate, and guaiacol were purchased from Sigma-Aldrich (St Louis, MO, USA). Dyes were obtained from the Ciba-Geigy GmbH representative in Iran (CIBA). Culture media ingredients and salts were Merck products (Darmstadt, Germany). Polymerase chain reaction (PCR) reagents were purchased from SinaClon BioScience (Iran) and other chemicals were of analytical grade.

Screening bacteria for laccase production

About 50 different bacterial strains isolated from chromite mine soil samples in Iran were screened for laccase production on a nutrient agar medium (0.5%; yeast extract, 0.3%; agar, 1.5% (w v−1); pH 7.5) supplemented with 3% (w v−1) NaCl, 0.2 mM CuSO4.5H2O, and 0.02% (v v−1) guaiacol. After about two weeks of incubation at 35 °C, the reddish-brown zone beneath the colonies due to oxidative polymerization of guaiacol was the sign of bacterial laccase activity.

Strain identification

To identify the laccase producing strain through 16S rRNA gene sequence analysis, the genomic DNA of the strain was extracted by Marmur's method (Marmur 1961). The 16S rDNA of the strain was amplified using the universal bacterial forward (F27 (5′-AGAGTTTGATCATGGCTCAG-3′)) and reverse primers (1492R (5′-CACGGATCCTACGGGTACCTTGTTACGACTT-3′)). After PCR product sequencing, the phylogenic relationship of this strain was determined by comparing the sequencing data with the related 16S rRNA genes using the EzTaxon-e server (Kim et al. 2012) and the phylogenic tree was constructed using MEGA software version 6. Also, the methods used to determine the temperature, pH, and NaCl concentration ranges for the strain growth were similar to those described previously (Siroosi et al. 2016).

Determination of laccase activity

The laccase producing strain was cultured on nutrient agar sporulation medium and incubated at 35 °C for 4 days. After appropriate incubation time, spore suspension was prepared and spore laccase activity was determined at 30 °C using 5 mM ABTS or 1 mM syringaldazine as the substrates. The spore laccase activity was detected by measuring the increase in absorbance at 420 nm (ɛ = 36,000 mol−1 cm−1) or 525 nm (ɛ = 65,000 mol−1 cm−1), according to the oxidation of ABTS (final concentration of 0.1 mM in the reaction mixture) in 50 mM sodium-acetate buffer (pH 5.0) or syringaldazine (final concentration of 20 μM in the reaction mixture) in 50 mM sodium-phosphate buffer (pH 6.0), respectively. One unit of the enzyme activity was defined as the amount of enzyme required to oxidize 1 μmol of substrate per minute under standard assay conditions.

ENZYME CHARACTERIZATION

Effect of temperature and pH on laccase activity

Temperature profile of the spore laccase activity was determined between 25 and 75 °C. The effect of pH on the spore laccase activity was studied in a range of 2.5–6.5, using the following buffers at a concentration of 50 mM: pH 2.5–5.5, sodium acetate and pH 6.0–6.5, sodium phosphate. The highest spore laccase activity was considered as 100%.

Effect of inhibitors, NaCl, metal ions, and organic solvents on laccase activity

Effect of inhibitors and SDS on the spore laccase activity were investigated in separated reaction mixtures contained 0.1 and 1 mM EDTA (a chelating agent), NaN3 (a common inhibitor of metalloenzymes), L-Cys, and SDS (a detergent). The effect of NaCl, one of the laccase inhibitors, on the spore laccase activity was tested by measuring the enzyme activity in reaction mixtures containing different NaCl concentrations from 0 to 1 M. To determine the effect of metal ions on the spore laccase activity, 0.1 and 1 (mM, final concentration) of different metal ions (Mg2+, Ca2+, Zn2+, Mn2+, Ba2+, Ni2+, Fe2+, Cu2+, Na1+, and Ag2+) were added to separated reaction mixtures. The effect of organic solvents on the spore laccase activity was studied by adding 5 and 10% (v v−1, final concentration) of methanol, ethanol, propanol, and acetone to separated reaction mixtures. All the activity measurements were performed under standard assay conditions using ABTS as the substrate. The spore laccase activity without any additives was considered as 100%.

Dye decolorization by spore laccase

Decolorization of three dyes including malachite green (a triphenylmethane dye), toluidine blue (a heterocyclic dye), and reactive black 5 (an azo dye) was investigated by the spore laccase in the presence or absence of ABTS (mediator) at different pH values. Each dye decolorization reaction mixture (2 ml) contained 50 mM sodium acetate buffer (for pH values of 3.0–6.0) or 50 mM Tris buffer (for pH values of 7.0 and 8.0), the spore laccase suspension (8 U l−1), and dye (final concentration of 10 mg l−1). The effect of ABTS as a redox mediator on dye decolorization was also examined by adding ABTS to the reaction mixtures in a final concentration of 15 μM. The reaction mixtures were incubated at 30 °C under mild shaking condition in a dark place for 2 h. Heat-inactivated spore laccase suspension was used in parallel as control under identical conditions. The absorption spectrum of each dye between 400 and 800 nm was measured and maximum absorption visible wavelengths were determined. Decolorization was expressed in terms of percentage by measuring the decrease in absorbance (after centrifugation of reaction mixtures) under the maximum visible wavelength of each dye.

Immobilization of spore laccase

Sodium alginate (2%, w v−1) was dissolved in distilled water and the spore laccase suspension (500 μl, 0.065 U) was added to the alginate solution (2 ml) and mixed completely. Alginate beads containing encapsulated spores were prepared by dropping the mixture into cold CaCl2 solution (200 mM) and the beads (approximate diameter of 1–2 mm) remained in this solution for 1 h to harden. Then, the CaCl2 solution was removed and the alginate beads containing encapsulated spores were stored at 4 °C. Control alginate beads without spores were prepared in parallel. Surface morphology of both types of beads was studied using a scanning electron microscope.

Dye decolorization by immobilized spore laccase

In order to study the ability of encapsulated spores to decolorize reactive black 5, 0.6 ml of encapsulated spores (0.016 U) was added to 1 ml decolorization assay mixture containing 50 mM sodium acetate buffer (pH 4.0) and dye (final concentration of 25 mg l−1). The reaction mixture was incubated at 30 °C under mild shaking condition in a dark place for 1 h. Alginate beads without encapsulated spores were used in parallel as control under identical conditions. After the appropriate incubation time, the supernatant of assay mixtures was separated from the beads and decolorization was expressed in terms of percentage by measuring the decrease in absorbance under the maximum visible wavelength of the dyes.

RESULTS AND DISCUSSION

Strain identification

Laccases are enzymes with a low level of substrate specificity, which makes them valuable biocatalysts for various biotechnological applications. Although fungal laccases are well known in the literature, studies on bacterial ones need to be continued to achieve more organized and detailed information about the biochemistry of bacterial laccases (Chandra & Chowdhary 2015).

In the present study, about 50 bacterial strains were screened for laccase activity on nutrient agar medium supplemented with NaCl, guaiacol, and CuSO4.5H2O. The laccase producing strain S31 showed reddish-brown color beneath its colony after about two weeks of incubation at 35 °C, which was the sign of guaiacol oxidation by laccase. According to 16S rRNA gene sequence analysis (GenBank accession number: KR063004), the strain was identified as B. safensis sp. strain S31. The phylogenic relationship between B. safensis sp. strain S31 and other related bacteria is shown in Figure 1. The phylogenic tree was constructed according to the neighbour-joining algorithm.

Figure 1

Neighbour-joining tree based on 16S rRNA gene sequences, showing the phylogenetic relationship between B. safensis sp. strain S31 and related taxa.

Figure 1

Neighbour-joining tree based on 16S rRNA gene sequences, showing the phylogenetic relationship between B. safensis sp. strain S31 and related taxa.

The strain showed the best growth at 35 °C, pH 7.5, and 5% (w v−1) NaCl. The strain was also able to grow at temperatures between 20–45 °C, pH 6.0–10.0, and 0–10% (w v−1) NaCl.

Effect of temperature and pH on laccase activity

In order to study the spore laccase activity, B. safensis sp. strain S31 was cultured on sporulation medium. After appropriate incubation time, spores were purified and the laccase activity using two common laccase substrates, syringaldazine and ABTS, was determined. Specific activity of the spore laccase was 0.06 and 0.05 U g−1 of dry spores toward syringaldazine and ABTS, respectively. Further enzymatic studies were carried out using ABTS as the substrate, because the purple color of the oxidized syringaldazine after enzyme catalysis was unstable and needed rapid measurements that might cause difficulties.

The effect of temperature and pH on the spore laccase activity are shown in Figure 2. Results showed that the spore laccase was active in the range of 25 to 40 °C with optimum activity at 30 °C (Figure 2(a)). Further increase in temperature reduced the enzyme activity; the enzyme retained only 11% of its maximum activity at 75 °C.

Figure 2

Effect of temperature (a) and pH (b) on the spore laccase activity. Error bars represent standard deviations of three independent experiments.

Figure 2

Effect of temperature (a) and pH (b) on the spore laccase activity. Error bars represent standard deviations of three independent experiments.

The effect of pH on the spore laccase activity from B. safensis sp. strain S31 was examined at pH values ranging from 2.5 to 6.5 (Figure 2(b)). The enzyme showed its optimum activity at pH 5.0 and retained about 30% and 40% of its maximum activity at pH values of 2.5 and 6.5, respectively.

Effect of inhibitors, NaCl, metal ions, and organic solvents on laccase activity

Effects of potential laccase inhibitors, SDS, several metal ions, and organic solvents on the spore laccase activity are summarized in Table 1. L-Cys, with its reducing activity on oxidized substrates of laccase; EDTA, with its chelating nature that negatively affects metalloenzymes; and azides and halides, with their interfering effect on the internal electron transfer in the laccase molecule by binding to copper atoms in the laccase active site, are the most common laccase inhibitors. Also, SDS is a detergent with a protein denaturing effect, and metal ions may influence protein stability (Siroosi et al. 2016).

Table 1

Effect of different potential laccase inhibitors, SDS, metal ions, and organic solvents on the spore laccase activity.

Effectors Final concentration (mM or % (v v−1)) Relative activity (%) 
– – 100 
Detergent Values are given as mM  
SDS 0.1 8.1 ± 0.4 
1.3 ± 0.1 
Inhibitors Values are given as mM  
EDTA 0.1 15.8 ± 1.2 
2.5 ± 0.2 
L-Cys 0.1 0.2 ± 0.2 
0.1 ± 0.1 
NaN3 0.1 94.5 ± 0.0 
92.5 ± 1.6 
Metal ions Values are given as mM  
Fe2+ 0.1 14.7 ± 4.3 
12.4 ± 0.3 
Cu2+ 0.1 100.6 ± 0.4 
102.4 ± 0.3 
Ca2+ 0.1 100.1 ± 10.2 
102.5 ± 3.6 
Zn2+ 0.1 100.6 ± 3.8 
103.7 ± 6.4 
Na+ 0.1 99.2 ± 6.4 
107.7 ± 4.0 
Mn2+ 0.1 34.3 ± 1.4 
13.2 ± 0.5 
Ag+ 0.1 102.0 ± 1.5 
107.4 ± 2.0 
Mg2+ 0.1 109.2 ± 12.3 
110.4 ± 2.0 
Ni2+ 0.1 101.9 ± 1.2 
104.4 ± 1.9 
Ba2+ 0.1 101.4 ± 1.0 
102.3 ± 3.9 
Solvents Values are given as % (v v−1 
Methanol 63.8 ± 3.5 
10 63.4 ± 5.1 
Ethanol 64.4 ± 3.2 
10 53.9 ± 6.1 
Propanol 54.3 ± 1.7 
10 26.6 ± 5.5 
Acetone 69.9 ± 6.1 
10 54.5 ± 4.2 
Effectors Final concentration (mM or % (v v−1)) Relative activity (%) 
– – 100 
Detergent Values are given as mM  
SDS 0.1 8.1 ± 0.4 
1.3 ± 0.1 
Inhibitors Values are given as mM  
EDTA 0.1 15.8 ± 1.2 
2.5 ± 0.2 
L-Cys 0.1 0.2 ± 0.2 
0.1 ± 0.1 
NaN3 0.1 94.5 ± 0.0 
92.5 ± 1.6 
Metal ions Values are given as mM  
Fe2+ 0.1 14.7 ± 4.3 
12.4 ± 0.3 
Cu2+ 0.1 100.6 ± 0.4 
102.4 ± 0.3 
Ca2+ 0.1 100.1 ± 10.2 
102.5 ± 3.6 
Zn2+ 0.1 100.6 ± 3.8 
103.7 ± 6.4 
Na+ 0.1 99.2 ± 6.4 
107.7 ± 4.0 
Mn2+ 0.1 34.3 ± 1.4 
13.2 ± 0.5 
Ag+ 0.1 102.0 ± 1.5 
107.4 ± 2.0 
Mg2+ 0.1 109.2 ± 12.3 
110.4 ± 2.0 
Ni2+ 0.1 101.9 ± 1.2 
104.4 ± 1.9 
Ba2+ 0.1 101.4 ± 1.0 
102.3 ± 3.9 
Solvents Values are given as % (v v−1 
Methanol 63.8 ± 3.5 
10 63.4 ± 5.1 
Ethanol 64.4 ± 3.2 
10 53.9 ± 6.1 
Propanol 54.3 ± 1.7 
10 26.6 ± 5.5 
Acetone 69.9 ± 6.1 
10 54.5 ± 4.2 

Each value represents the mean ± SD of three independent experiments.

According to Table 1, L-Cys showed the most potent inhibitory effect on the spore laccase activity. It is notable that 1 mM NaN3 caused only 7.5% reduction in the laccase activity from B. safensis sp. strain S31, because NaN3 is a strong laccase inhibitor and a complete or nearly complete laccase inhibitory effect of 1 mM NaN3 has been reported many times (Wang et al. 2010; Wu et al. 2010). The reason for the resistance of laccase toward NaN3 in this study could be the special structure of its active site, which inhibits azide molecules to bind copper atoms in copper centers located in the active site (Dubé et al. 2008).

In the case of metal ions, Mn2+ and Fe2+ decreased the spore laccase activity from B. safensis sp. strain S31 similarly to previous studies (Niladevi et al. 2008; Wang et al. 2010; Wu et al. 2010). Other metal ions (Ca2+, Zn2+, Ba2+, Ni2+, Cu2+, Na1+, Mg2+, and Ag2+) had no or little increasing effect on the enzyme activity, which indicates high tolerance of the spore laccase from B. safensis sp. strain S31 toward most metal ions, which can be an important characteristic for its applications.

As seen in Table 1, all organic solvents reduced the spore laccase activity by about 30–40%. The relative resistance of the spore laccase against solvents is a specific property leading to some potential applications. The laccase might be used as a biocatalyst in organic synthesis reactions containing organic solvents (Chandra & Chowdhary 2015).

The effect of NaCl (0–1 M) on the enzyme activity is shown in Figure 3. The highest activity was observed when no NaCl was added to the reaction mixture.

Figure 3

Effect of NaCl on spore laccase activity. The enzyme activities were measured in the presence of different concentrations of NaCl. Error bars represent standard deviations of three independent experiments.

Figure 3

Effect of NaCl on spore laccase activity. The enzyme activities were measured in the presence of different concentrations of NaCl. Error bars represent standard deviations of three independent experiments.

The spore laccase from B. safensis sp. strain S31 showed a good tolerance to many effectors, especially NaN3. NaN3 is a toxic substance found in textile effluents (Robson et al. 2007) and, as noted before, it has a strong inhibitory effect on many other laccases that limits their usage in treatment of textile and dyeing industries' wastewaters.

Dye decolorization

Decolorization of wastewaters containing synthetic dyes (produced by textile and dye industries) by using chemical methods is more expensive and environmentally harmful compared to microbial decolorization approaches (Singh et al. 2011). In addition, microbial or enzymatic decolorization procedures consume less water and therefore generate a lower amount of sludge with non-toxic end products, compared to other decolorization methods. Because of these advantages, research on the subject of bioremediation is a great field in environmental sciences (Saratale et al. 2011).

Decolorization activity of the spore laccase from B. safensis sp. strain S31 in the presence or absence of ABTS was examined by measuring a decrease in the absorbance of different reaction mixtures at the maximum visible wavelength of each dye (620 nm, malachite green; 630 nm, toluidine blue; 597 nm, reactive black 5), and the values were expressed as a percentage. The spore laccase showed decolorization activity toward all three dyes, with the maximum decolorization percentage in the presence of ABTS, as shown in Figure 4. The optimal pH values for decolorization of malachite green, toluidine blue, and reactive black 5 by the spore laccase alone were 5.0, 6.0, and 4.0, respectively. The maximum decolorization of malachite green, toluidine blue, and reactive black 5 by the spore laccase in the presence of ABTS as a mediator was at pH 5.0, 6.0, and 3.0, respectively. The spore laccase led to higher decolorization percentage in the presence of ABTS than the reactions without the mediator. The enzyme showed its highest decolorization efficiency toward malachite green (87%) in the presence of ABTS, and toward reactive black 5 (75%) in the absence of ABTS. Spore laccase from B. amyloliquefaciens showed no decolorization activity against reactive black 5 (25 mg l−1) even in the presence of ABTS; the best result (75% decolorization) was achieved in the presence of acetosyringone as another mediator (Lu et al. 2012). Resistance to NaN3 along with dye decolorization activity of the spore laccase from B. safensis sp. strain S31 are notable features of the enzyme that make it an appropriate option for bioremediation activities. For example, effluents contaminated with dyes and phenols might be the targets. Also, laccase with such decolorization activity could be considered as an appropriate replacement for harsh chemical compounds used in the stone washing process (Chandra & Chowdhary 2015), and can be applied to lighten the colors in a controlled manner.

Figure 4

Effect of the spore laccase on decolorization of dyes. (a) malachite green; (b) toluidine blue; (c) reactive black 5. Black columns: samples treated with the spore laccase, Gray columns: samples treated with the spore laccase and ABTS. Error bars represent standard deviations of three independent experiments.

Figure 4

Effect of the spore laccase on decolorization of dyes. (a) malachite green; (b) toluidine blue; (c) reactive black 5. Black columns: samples treated with the spore laccase, Gray columns: samples treated with the spore laccase and ABTS. Error bars represent standard deviations of three independent experiments.

Immobilization of spore laccase and its decolorization activity

Immobilization of enzymes has different advantages such as protecting them against inhibitors and providing the possibility of reusing the enzymes (Asgher et al. 2017). Encapsulation is a preferable method to immobilize enzymes and there is a little chance to change the enzyme structure by using this method (Daâssi et al. 2014). Purified spores from B. safensis sp. strain S31 were immobilized within calcium alginate polymers to study the decolorization ability of encapsulated spore laccase. The surface morphology of control alginate beads and alginate beads containing encapsulated spores were analyzed by SEM (Figure 5); encapsulated spores are seen in Figure 5(b). As the free spore laccase showed the best decolorization activity toward reactive black 5 in the absence of ABTS, the study into the effect of encapsulated spore laccase was conducted on this azo dye (final concentration of 25 mg l−1). After 1 h incubation, 95% of reactive black 5 was decolorized by encapsulated spore laccase without the addition of ABTS. This observation was considered as a promising result because this dye has a high redox potential, making it a poor substrate for laccases (Camarero et al. 2005). Our findings are in agreement with Daâssi et al. (2014), who found encapsulated laccase from the fungus Coriolopsis gallica in calcium alginate beads decolorized about 30% of reactive black 5 (36 mg l−1) after 8 h incubation without any addition of mediator. Furthermore, encapsulated spore laccase from B. amyloliquefaciens showed 72% decolorization toward reactive black 5 (25 mg l−1) after 1 h incubation in the presence of acetosyringone as a redox mediator (Lu et al. 2012).

Figure 5

Surface morphology of beads. Scanning electron micrograph of beads: (a) without encapsulated spores; (b) with encapsulated spores.

Figure 5

Surface morphology of beads. Scanning electron micrograph of beads: (a) without encapsulated spores; (b) with encapsulated spores.

CONCLUSIONS

Spores from B. safensis sp. strain S31 showed spore laccase and dye decolorization activities. High tolerance of the spore laccase toward NaN3, metal ions, and solvents, along with its dye decolorization ability (in free or immobilized form) without any addition of mediators, make the enzyme a potential option for applied purposes, especially decolorization of dye containing effluents. As the enzyme showed the best decolorization activity at acidic or near acidic pH values, acidic effluents or alkaline effluents after adjusting the pH could be the targets for decolorization. Furthermore, the enzyme could be used in fabric factories (stone washing) and organic synthesis processes.

ACKNOWLEDGEMENT

The authors would like to acknowledge the research council of Tarbiat Modares University and financial support of University of Tehran Science and Technology Park for this research under grant number 94012.

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