Induction and enhancement of the novel marine-derived Alternaria tenuissima KM 651985 laccase enzyme using response surface methodology : Application to Azo and Triphenylmethane dyes decolorization

Article history: Received on: 04/01/2016 Revised on: 05/02/2016 Accepted on: 13/03/2016 Available online: 30/04/2016 In our search for a laccase producer from unconventional sources, the marine derived fungus Alternaria tenuissima KM651985 was isolated from decayed wood. It was identified on the basis of morphological and molecular taxonomy and got the Genbank accession number KM651985. Two statistical experimental designs were employed to enhance laccase production. At first, a two level Plackett-Burman design was employed to screen the medium constituents and inducers that significantly affect the enzyme production. Second experiment was important for optimization of the most effective constituents and inducers using central composite design. Applying the above methods revealed that guaiacol (2 mM), copper sulphate (3 mM) and wheat bran (46.82 g/l) were the most effective factors affecting laccase production. A maximal enzyme activity of 91.84 U/ml was more than 6.33-folds the activity obtained using the basal medium. Application of A. tenuissima KM651985 culture medium containing laccase to decolorize two structurally different synthetic dyes was done successfully. This is the first report on the statistical optimization of the marine-derived A. tenuissima KM651985 laccase enzyme and its applications for dyes decolorization.


INTRODUCTION
Laccases (benzenediol: oxygen oxidoreductases, EC 1.10.3.2) are blue multicopper oxidases which catalyze the oxidation of an array of aromatic substrates with the reduction of molecular oxygen to water (Giardina et al., 2010).Laccases are known for their importance in the development of efficient biotechnology processes (degradation of different recalcitrant compounds, delignification of lingocellulosics, paper bleaching, bioremediation, dye decolorization and sewage treatment, etc) (Rodriguez et al., 2005;Osma et al., 2010;Shervedani and Amini, 2012).The majority of laccases are often found in whiterot fungi and higher plants such as the varnish tree Rhus vernicifera (Morozova et al., 2007).The production of laccases For this reason, it is useful to optimize the medium composition for production of laccase by fungi.Statistical experimental designs are known useful tools for medium optimization to screen the main variables rapidly from a multivariable system.The statistical experimental designs have many advantages, including more advanced results with less development time and less overall costs.Plackett-Burman (PB) and response surface methodology (RSM) have been successfully applied in the improvement of the medium conditions of laccase product from different microorganism such as Pleurotus sp. and Mycena purpureofusca (Bhattacharya et al., 2011;Zhang et al., 2012).
The main goal of the present paper was to find a new source for laccase production tolerant to saline and alkaline conditions.Optimization of medium composition for high laccase production using statistical experimental designs.The application of crude culture filtrate of laccase producing fungus to decolorize two chemically different synthetic dyes.

Microorganism and maintenance
The marine-derived fungal isolate used in this study was locally isolated from samples of marine decayed wood collected from Port-Said Governorate, Egypt, 2014(Abd El Aty et al., 2015).Stock cultures were maintained on malt extract agar (MEA) slant at 4 • C and sub-cultured every 3 months.The medium is composed of biomalt 20 g/L, agar 15g/L, 800 mL sterile sea water and 200 mL distilled water (Höller et al., 1999).

Identification of the marine-derived filamentous fungal isolate
From previous screening experiments, one selected isolate having the ability to produce laccase was used.It was identified morphologically in the department of Chemistry of Natural and Microbial Products, National Research Centre according to (Pitt and Hocking, 1985;Kohlmeyer and Kohlmeyer, 1979) to be Alternaria tenuissima.This was further confirmed using molecular taxonomy tools Molecular identification of the fungus was based on their internal transcribed spacer ribosomal DNA (ITS-rDNA) sequences.Mycelia were collected by centrifugation and DNA was extracted by using protocol of Gene Jet Plant genomic DNA purification Kit (Thermo # K0791).PCR was made by using Maxima Hot Start PCR Master Mix (Thermo# K0221), and PCR clean up to the PCR product made by using GeneJET™ PCR Purification Kit (Thermo# K0701) in Sigma Company of Scientific Services, Egypt (www.sigma-co-eg.com).Finally, sequencing to the PCR product performed in GATC Company, German, by use ABI 3730xl DNA sequencer with the new 454 technology.
Purified DNA was subjected to PCR amplification using a pair of ITS1 (5′-TCC GTA GGT GAA CCT GCG G-3′) and ITS4 (5′-TCC TCC GCT TAT TGA TAT GC-3′) primers for ITS-rDNA amplification (White et al., 1990).Sequence data was analyzed in the Gene Bank database by using the BLAST program available on the National Center for Biotechnology Information website (www.ncbi.nlm.nih.gov).The unknown sequence was compared to all of the sequences in the database to assess the DNA similarities (Altschul et al., 1997).Alignment and molecular phylogeny were evaluated using Bio Edit software.
The nucleotide sequence of the marine-derived fungal isolate determined in this study has been deposited in the GenBank under the accession number (KM651985).

Media and culture conditions for laccase production
Laccase producing ability of the marine-derived fungal isolate was tested in two different liquid fermentation media; Boyd & Kohlmeyer (B&K) synthetic culture medium containing (g/L), glucose 10, peptone 2, yeast extracts 1 in 50% sea water (D'Souza et al., 2006) and the natural culture medium containing, 3g/flask wheat bran without any additives.Fungus cultures were grown in 250 mL flasks containing 50 mL medium or only sea water with wheat bran medium at 28 ºC for 14 days under static conditions.
The spore suspension was prepared from a 7 days old culture grown on MEA slants prepared with seawater by adding 10 ml of sterile distilled water.One ml of the spore suspension containing about 1×10 7 spores/ml was used as inoculum.

Confirmation for laccase production (Guaiacol oxidation)
Laccase production in liquid fermentation medium was confirmed by addition of (0.7 ml) of culture filtrate containing about (10.16 U of crude enzyme) to Petri dish prepared with B&K supplemented with 4mM guaiacol, and incubated for 24h.at 28 ºC in dark.The production of an intense brown color around the hole containing the enzyme resulting from guaiacol oxidation was considered as a positive reaction for the presence of laccase activity.
The reaction mixture contained 600 µL sodium acetate buffer (0.1 M, pH 5.0 at 27 ºC), 300 µL ABTS (5 mM), 300 µL mycelial liquid fraction and 1400 µL distilled water.The mixture was then incubated for 2 min at 30 ºC.The absorbance was measured immediately in one-minute intervals.One unit of laccase activity was defined as activity of an enzyme that catalyzes the conversion of 1 µmole of ABTS per minute.The blank contained all the assay constituents except the active enzyme; it was replaced heated inactivated enzyme.
Tested factors represented in twelve trials Plackett-Burman experimental design with the response.Each row represented a trial run and each column represented an independent variable concentrations.All experiments were carried out in duplicate and the average laccase activity was taken as the response Table (1).Plackett-Burman experimental design is based on the first order linear model: (1) Y is the response (laccase production), B0 is the model intercept and Bi is the variables estimates.The effect of each variable was determined by the following equation, is the effect of the tested variable.M i + and M i − represent laccase production from the trials where the variable (Xi) measured was present at high and low concentrations, respectively and N is the number of trials in Eq. ( 2).Statistical analysis of PBD is performed by using Design-Expert® 8 software from Stat-Ease, Inc.Table (2).

Central composite design (CCD)
Based on the results of Plackett-Burman design, the optimal significant variables for laccase production were investigated using response surface methodology of central composite design (CCD).Using 3-factor-5-level central composite design (CCD) with twenty experiments, were carried out to determine the optimal values of copper sulphate (A), guaiacol (B) and wheat bran (C) and to develop a mathematical correlation between the three significant variables and laccase activity (Y).All three variables were investigated at low level (-1), zero level (0) and high level (+1), respectively, with α = 1.682.Codes and actual values of variables and matrix of CCD along with laccase activity of each trial are shown in Table (3).
Statistical analysis of CCD is shown in Table ( 4).The behavior of the system was explained by the following quadratic model equation.
where Y (activity) was the predicted production of laccase (U/mL), β0 intercept, β1, β2 and β3 linear coefficients, β11, β22 and β33 quadratic coefficients and β12, β13 and β23 interactive coefficients.A, B and C were the independent variables corresponding to the concentration of CuSO 4 , guaiacol and wheat bran respectively.Statistical analysis of the model was performed to evaluate the analysis of variance (ANOVA) and the quadratic models were represented as contour plots (3D) using Design-Expert 8 software from Stat-Ease, Inc. Poojary and Mugeraya (2012).

In vitro decolorization experiments
Two chemically different synthetic dyes: Congo red and Crystal violet were tested in the following study at two different concentrations 50 and 100 ppm.
Cultivation on solid media was carried out in Petri dishes (90 mm diameter) containing N-limited Kirk medium (Tien and Kirk, 1988) with the respective dye at the two different concentrations.The dishes were inoculated with mycelial plugs (10 mm diameter) cut from actively growing mycelia and incubated at 28 ºC in dark for 7 days.
The culture filtrate with laccase activity was used as a source of enzymes to test its efficiency in decolorization of dyes.This was carried out by incubating high concentration (64.29 U) and low concentration (27.55 U) of the enzyme in holes of agar plates supplemented with 50 and 100 ppm of the respective dyes for 24 h.The plates incubated under the same conditions.

Identification of the microorganism and guaiacol oxidation
The marine-derived fungus used in the present study was identified taxonomically by molecular and conventional methods.Data derived from morphology, and phylogenetic analyses identified it as Alternaria tenuissima KM651985 with 99% similarity Fig. (1&2).
Initial experiments concerning laccase enzyme production in two different fermentation media.B&K synthetic culture medium, which showed about (7.12±2.73U/ml) and wheat bran natural culture medium (14.51±3.41U/ml).These results indicated that the natural medium containing wheat bran was the most favorable as basal medium for laccase production.These results are consistent with our previous published work (Abd El Aty and Mostafa, 2013;Mostafa and Abd El Aty, 2013) where we found that wheat bran has a very strong potential for laccase production on comparison with other agro industry wastes.
The positive effect of using wheat bran may be due to its components.The wheat bran is a byproduct of the wheat processing industry usually composed of non-starch polysaccharides (αarabinoxylans, cellulose and β-(1,3) 1,4glucan), lignin, starch and crude protein (Songulashvili et al., 2011).

Statistical optimization of laccase production.
Two statistical designs were used for the optimization of medium components for laccase production.

Plackett-Burman design (PBD)
The medium components and inducers that are significantly influence A. tenuissima KM651985 laccase were determined by applying Plackett-Burman.
PB design experiments showed that, there were variations ranging from 1.3±0.497U/ml to 45.5±2.486U/ml in the production of laccase in 12 runs Table (1).This variation between obtained units reflected the importance of medium optimization to attain maximum yields.
Results obtained in the Pareto chart Fig. ( 4) showed the order of significance for the variables affecting laccase production.Among these variables, the inducers copper sulphate and guaiacol showed the highest significance by showing higher positive effect, followed by wheat bran, veratryl alcohol and glucose.Ammonium tartarate, phenol, ABTS and peptone contributed negatively.The positive effect of copper sulphate in our research is in agreement with previous published results ( Galhaup and Haltrich, 2001;Shutova et al., 2008;Zhang et al., 2012).The Cu 2+ is an extremely important metal ion and could induce laccase secretion.The role of Cu 2+ may be due to its induction effect on transcription of the laccase gene (Galhaup et al., 2002).Another reason is that Cu 2+ may be buried at the catalytic center and maintains the stability of the laccase.Also D' Souza et al. (2006) indicated that, CuSO 4 and guaiacol induced the maximum laccase production by the marine fungal isolate, NIOCC # 2a, individually as well as in combination.
According to the obtained results, the components of the following medium is expected to be near optimum: wheat bran 10 g/l, glucose 5 g/l, ammonium tartarate 0.2 g/l, peptone 0.2 g/l and veratryl alcohol 1 mM, with addition of copper sulphate 2 mM and guaiacol 1 mM after 6 days of incubation, in 250 ml Erlenmeyer flasks for 14 day incubation period.The enzyme activity measurement on this medium was 45.5±2.486U/ml.This result presented about 3.14-folds increase in the enzyme activity, when compared to (14.51±3.41U/ml), the results obtained in basal production medium containing only wheat bran and sea water.
The analysis of variance (ANOVA) for the experiment design showed that, The Model F-value of 60.02 implies the model is significant.Values of "Prob > F" less than 0.0500 indicate model terms are significant.In this case A, E, F, G, H, J, K are significant model terms.Values greater than 0.1000 indicate that the model terms are not significant.The "Pred R-Squared" of 0.8672 is in reasonable agreement with the "Adj R-Squared "of 0.9797."Adeq Precision" measures the signal to noise ratio, a ratio greater than 4 is desirable.Our ratio of 24.396 indicates an adequate signal.This model can be used to navigate the design space.Table (2).The first order model equation developed by PB design showed the dependence of laccase production on the medium constituents: R1 (laccase activity U/ml) = +13.06+4.11*A +2.06*C -2.39*D -4.57*E +7.48*F -3.03*G +6.47*H +2.82*J -2.51*KTherefore, the optimum combination of the variables (copper sulphate, guaiacol and wheat bran) which had the highest significant influence on laccase production was further analyzed by central composite design of response surface methodology.Other variables with less significant effect were not included in the next optimization experiment, but instead were used in all trials at their (-1) level and (+1) level, for the negatively contributing variables and the positively contributing variables, respectively.

Response Surface Methodology (RSM)
According to PB design, copper sulphate, guaiacol and wheat bran were further applied for optimization using RSM.A central composite design (CCD) was employed within a range ofα (-1.68) and +α (+1.68) in relation to laccase production to examine the combined effect of factors tested.The results presented in Table (3) showed the predicted and observed responses along with design matrix, the minimum response of 35.23 U/ml laccase activity was obtained with 4 mM copper sulphate, 3 mM guaiacol and 20 g/l wheat bran.The maximum response of 91.84 U/ml laccase activity was obtained with 3 mM copper sulphate, 2 mM guaiacol and 46.82 g/l wheat bran.These results indicated that, the high concentrations of copper sulphate and guaiacol were toxic for the fungus, while increasing the wheat bran concentration makes the medium near to semi-solid fermentation which is more useful for maximum laccase production.Wheat bran supply microorganisms with, zinc, hemicellulose, protein, manganese, iron and copper where its consider as a type of nutrient-richer by product of the wheat processing industry (Kalogeris et al., 2003;Oberoi et al., 2010), Wheat bran was also used for laccase production by (Marques de Souza et al., 2002) and Fomes sclerodermeus (Papinutti et al., 2003).The levels of laccase activity as a function of copper  4), which confirmed the statistical significance of the equation.ANOVA of regression model demonstrates that the model is highly significant as it is evident from the Fischer test with very low probability value.The value of lack of fit, Model F and model P>F were found to be 289.01,41.41and ˂0.0001 respectively, indicating that model was significant.The coefficient of regression (R 2 ) was calculated to be 0.9739 indicating the goodness of fit of the model.The adjusted regression coefficient (Adj R 2 = 0.9504) was also high, showed for high significance of the model.Low coefficient variation (CV=5.40%)confirm the reliability of the experimental design.
In this case B, C, AB, AC, BC, A2, B2 are significant model terms where "Prob > F" less than 0.0500.On the other hand A, C 2 seems to be insignificant where "Prob > F" greater than 0.1000, which doesn't affect the model and can be removed  The validation was carried out under the following optimum conditions of the medium.Wheat bran 46.82 g/l, glucose 5 g/l, ammonium tartarate 0.2 g/l, peptone 0.2 g/l and veratryl alcohol 1 mM, with addition of copper sulphate 3 mM and guaiacol 2 mM after 6 days of incubation, in 250 ml Erlenmeyer flasks at 28 ºC for 14 day incubation period.
The experimental laccase production of 91.84 U/ml was obtained which is closer to the predicted laccase production of 90.92 U/ml.This result indicated the validity and the effectiveness of the proposed model.The production of laccase by A. tenuissima KM651985 at the optimized conditions is much higher than that produced by the Brazilian marine-derived Aspergillus sclerotiorum CBMAI 849 (13.9 U/L), Cladosporium cladosporioides CBMAI 857 (203.70U/L) and Mucor racemosus CBMAI 847 (898.15U/L)(Bonugli-Santos et al., 2010).
It was obvious that, optimization of CCD resulted in 6.33-fold increase in laccase production.This reflects the necessity and value of optimization process.There is no report available on statistical optimization of the marine-derived A. tenuissima KM651985 laccase enzyme by using PBD and RSM, most reports available so far are on optimization of laccase by using white rot fungi (Poojary and Mugeraya, 2012;Zhang et al., 2012).

Application to synthetic dyes decolorization
Results of experiment Ӏ showed the ability of the marinederived A. tenuissima KM651985 to grow and decolorize the two structurally different (Azo and triphenylmethane) synthetic dyes on solid media at 50 and 100 ppm concentrations with different rates after 7 days, as shown in Table (5   Also Saparrat et al. (2008) indicated that, the white-rot fungus Grammothele subargentea LPSC no.436, was able to degrade 0.01% (100 ppm) of congo red more efficiently and in time less than taken for crystal violet.According to that mentioned by Parawira and Tekere (2013), the simple not very pure preparation of enzymes is more useful and economic for industrial applications of laccases.Therefore, crude A. tenuissima KM651985 laccase enzyme was used to decolorize the tested dyes at two different concentrations in experiment ӀӀ.
As shown in Table ( 5), significant differences were obtained, depending on the dye and crude enzyme concentrations.Crystal violet exhibited the least decolorization efficiency, while the azo dye congo red showed relatively high decolorization efficiency after 24 h Fig. (6, C&D).

CONCLUSION
The first goal of the search in finding the optimum conditions for laccase production by the marine-derived A. tenuissima KM651985 has been achieved by the combination of Plackett-Burman design and central composite design.Wheat bran can be used as substrate to produce laccase by A. tenuissima KM651985 decreasing the concentration of glucose level in the medium to 5 g/l.This is an economical and environment friendly way to reuse agricultural residues.In addition, copper sulphate and guaiacol as inducers have a positive effect and play a major role on laccase formation.A. tenuissima KM651985 was able to decolorize efficiently two different synthetic dyes, congo red and crystal violet on agar plates by mycelium plugs and by crude enzyme without any mediators.This is the first report on the applications of the marine-derived A. tenuissima KM651985 in dyes decolorization.This encourage further research for bioremediation of lignin-based derivatives and colored industrial pollutants that are alkaline and have high salt concentrations.

Fig. 2 :
Fig. 2: Phylogenetic tree based on 18S rRNA gene sequences showing closest relatives.Gen Bank sequence accession numbers are indicated in parentheses after the strain names.

Fig. 3 :
Fig. 3: Reddish brown zone formed around a pore containing (10.16 U of crude enzyme) after 24 h at 28 ºC in dark, this colour is due to the oxidative polymerization of guaiacol by A. tenuissima KM651985 laccase enzyme.When this culture filtrate added to B&K plates supplemented with 4 mM guaiacol, a reddish brown zone formed around a hole containing crude enzyme, due to the oxidative
sulphate, guaiacol and wheat bran can be presented in terms of coded factors as in the following second-order regression equation: laccase activity (U/ml) = +65.52-0.60*A +3.22*B +15.92*C -3.72*A*B +2.89*A*C +2.67*B*C -3.30*A2 -2.52*B2 -0.49*C2 F-test and analysis of variance for response surface quadratic model is shown in Table ( . The three dimensional (3D) response surface plots of the three factors are showed in Fig. (5).

Fig. 5 :
Fig. 5: Three dimensional response surface plot for the effect of (a)copper sulphate and guaiacol, (b) wheat bran and copper sulphate, (c) wheat bran and guaiacol.
) and Fig. (6, A&B).A positive correlation between the radial growth and the decolorization rate was found.Congo red, azo dye used in textile industries, decolorized almost completely by the marine-derived A. tenuissima KM651985 after 14 days incubation.The results are nearly consistent with the reports of Mtui and Nakamura (2008) and Yang et al. (2003) that demonstrate significant decolorization of aromatic dyes by Flavodon flavus and terrestrial fungi while Raghukumar (2005) reported the same trends for facultative marine fungi.

Table 5 :
Decolorization of synthetic dyes by the marine-derived A. tenuissima KM651985 and its crude enzyme on solid medium.Radial growth rate measured as a diameter of mycelial colony and decolorization measured as a diameter of decolorized zone on a Petri dish on the 7th day of cultivation on Kirk N-limited medium containing 50 and 100 ppm of the respective dyes ** Experiment ӀӀ: Decolorization rate of two different concentrations of the crude enzyme measured as a diameter of decolorized zone around the hole on a Petri dish containing agar supplemented with 50 and 100 ppm of the respective dyes after 24h.(All the plates incubated at 28 ºC in dark).

Table 1 :
Twelve trials Plackett-Burman experimental design with the response.

Table 3 :
Coded levels and actual values of variables, along with matrix of central composite design (CCD) showing experimental and predicted values of laccase activity.

Table 4 :
Analysis of variance (ANOVA) for response surface quadratic model.