Production , purification and characterization of a thermostable β1 , 3-glucanase ( laminarinase ) produced by Moniliophthora perniciosa

The enzyme glucanase from Moniliophthora perniciosa was produced in liquid medium and purified from the culture supernatant. A multivariate statistical approach (Response Surface Methodology – RSM) was employed to evaluate the effect of variables, including inducer (yeast extract) and fermentation time, on secreted glucanase activities M. perniciosa detected in the culture medium. The crude enzyme present in the supernatant was purified in two steps: precipitation with ammonium sulfate (70%) and gel filtration chromatography on Sephacryl S-200. The best inducer and fermentation time for glucanase activities were 5.9 g L−1 and 13 days, respectively. The results revealed three different isoforms (GLUI, GLUII and GLUIII) with purification factors of 4.33, 1.86 and 3.03, respectively. The partially purified enzymatic extract showed an optimum pH of 5.0 and an optimum temperature of 40◦C. The enzymatic activity increased in the presence of KCl at all concentrations studied. The glucanase activity was highest in the presence of 0.2 M NaCl. The enzyme showed high thermal stability, losing only 10.20% of its specific activity after 40 minutes of incubation at 90◦C. A purified enzyme with relatively good thermostability that is stable at low pH might be used in future industrial applications.


INTRODUCTION
The hemibiotrophic basidiomycete Moniliophthora perniciosa (Aime and Phillips-Mora 2005), the causative agent of witches' broom disease in Theobroma cacao, is responsible for major crop losses in South American and Caribbean cocoa plantations (Kilaru and Hasenstein 2005).
The disease shows two distinct stages: a biotrophic (intercellular monokaryotic mycelium) and a necro-trophic/saprotrophic phase (intracellular dikaryotic mycelium).The biotrophic mycelium depends on the host tissue for nutrients to sustain its growth and development.
A typical fungal cell wall is composed of chitin, glucans and proteins, and it can be degraded by chitinases and glucanases (Lin et al. 2007, Perez et al. 2002, Schirmbock et al. 1994).These enzymes are widely distributed among fungi, and some have been purified (Donzelli and Harman 2001).
Enzymes that catalyze depolymerization or selective modification of cereal β-glucans are of biotechnological and biomedical importance because they have shown a significant potential application in malting and brewing processes (Konig et al. 2002, Mannonen et al. 1997), biological control of plant pathogens (De La Cruz et al. 1995), fungal and yeast cell wall degradation (Pitson et al. 1993), animal feed (Konig et al. 2002) and medical fields (Pang et al. 2005, Li et al. 2007).
To evaluate M. perniciosa as an industrial source of glucanase, the present study evaluated the influence of fermentation time and inducer on the activity of secreted glucanase from the fungus, using response surface methodology (RSM).Furthermore, the study describes the production, purification and characterization of β-1,3-glucanase (laminarinase) from Moniliophthora perniciosa in saprotrophic phase mycelium.

CHEMICALS
Laminarim and bovine serum albumin were purchased from Sigma (Sigma Chemical Co., St. Louis, MO, USA).All other chemicals used were of high-quality analytical grade.

MICROORGANISM
Moniliophthora perniciosa (CCMB 0257) was obtained from the Collection of Cultures of Microorganisms of Bahia (CCMB, Feira de Santana, Brazil).The fungus was maintained on potato dextrose agar at 28 • C in an incubator (IGO 150 Cell Life -Jouan).

CULTURE CONDITIONS OF Moniliophthora perniciosa
Moniliophthora perniciosa was grown on potato dextrose agar for 10 days at 28 • C to produce β-1,3-glucanase.Then, discs of fungal mycelium, with 1 cm in diameter, were taken from the solid culture, transferred into 100 mL erlenmeyer flasks containing liquid culture media with the following components dissolved in distilled water: wheat bran, 40.0 g L −1 ; yeast extract, 3.0-9.0g L −1 ; K 2 HPO 4 3H 2 O, 1.0 g L −1 ; MgSO 4 7H 2 O, 0.2 g L −1 and KCl, 0.2 g L −1 .The incubation was carried out as described in the experimental design; flasks were heated at 28 • C for 7 to 21 days and shaken at 120 rev min −1 in a rotary shaker.The liquid culture medium, on which M. perniciosa was grown, was filtered and centrifuged (Centrifuge 5804R -Eppendorf, São Paulo, Brazil) at 8,000 g for 15 min at 4 • C, and the supernatant, containing the proteins from M. perniciosa, was used as a crude enzymatic extract (Hua et al. 2007).

DOEHLERT EXPERIMENTAL DESIGN FOR ENZYME PRODUCTION
Fermentation time and inducer concentration are among the most important parameters that affect the production of enzymes are fermentation time and inducer concentration (Giese et al. 2005).In this work, yeast extract was used as an inducer.To predict production of enzymes under any conditions of fermentation time and yeast extract concentration in the experimental domain, RSM was used.The Doehlert experimental design contained two variables, yeast extract and fermentation time, and three replicates at the center of the domain lead to a total of 9 experiments (Table I).Measurements were performed in triplicate to verify the best culture medium conditions to induce the production of β-1,3glucanase.Yeast extract was studied at five concentrations, ranging from 3 to 9 g/L, and fermentation time was studied at three time points, ranging from 7 to 21 days.The experimental errors were evaluated from replication of a central point.The experimental data was processed using STATISTICA software.All the experiments in this step were carried out in random order.Statistica version 7.0 was used to estimate the lack of fit of the model to the experimental data through analysis of variance (ANOVA).Cienc (2011) 83 (2) β-1,3-GLUCANASE PRODUCED BY Moniliophthora perniciosa

ENZYME PURIFICATION
The purification procedure for β-1,3-glucanase from M. perniciosa involved the following two steps: Step 1: Ammonium sulfate precipitation The crude enzymatic extract previously obtained was brought to 70% saturation by adding solid ammonium sulfate and centrifuged at 10,000 × g for 10 min after standing for 1 h.The precipitate, with high laminarinase activity, was resuspended in phosphate citrate buffer (pH 6.2) in the ratio of 1:3 (w/v) and stored at low temperature.
Step 2: Sephacryl S-200 chromatography The crude enzymatic extract was applied to a Sephacryl S-200 (Amersham Bioscience, USA) column (48 × 1.2 cm) previously equilibrated with sodium phosphate buffer (0.05 mol L −1 , pH 7.0), which was also used to elute the product.Fractions of 1.5 mL were collected and assayed for glucanase activity.Fractions showing β-1,3-glucanase activity were pooled and concentrated by ultrafiltration using an Amicon PM 10 membrane, which has a molecular weight cut-off of 10 kDa.

PROTEIN DETERMINATION AND ENZYME ANALYSIS
Protein concentration was determined by the method of Bradford (1976) using bovine serum albumin (BSA) as a standard.The enzyme activity of the purified β-1,3-glucanase was measured using laminarin (from Laminaria digitata, Sigma) as the substrate.Laminarin was dissolved in 0.05 mol L −1 citrate buffer (pH 6.2).
The reaction mixture contained 100μL of 0.1% laminarin and 100μL of enzyme solution.The mixture was incubated at 50 • C for 15 min.The reaction was stopped by the addition of 200μL of 1% 3,5-dinitrosalicylic acid, and the mixture was boiled for 15 min.The amount of reducing saccharides released from laminarin was measured spectrophotometrically (Cary 50 UV-Visible spectrophotometer, Varian Inc., São Paulo, Brazil) at 540 nm according to precedent (Miller 1959).
One unit (U) of β-1,3-glucanase activity was defined as the amount of the enzyme required to release one microgram of glucose per minute under the above conditions.
HEAT STABILITY AND OPTIMAL PH AND TEMPERATURE The optimal pH of β-1,3-glucanase activity was assayed at pH 5.0, 6.0, 7.0, 8.0, and 9.0.Blanks lacking glucanase were used in each determination.
To determine the optimum temperature, β-1,3-glucanase activity was tested under standard assay conditions in the temperature range of 30-90 • C. The temperatures were controlled using a circulating water bath.
To test the stability of the enzyme at elevated temperature, the samples of β-1,3-glucanase in test tubes of uniform size were incubated in water at different temperatures (50, 60, 70, 80 and 90 • C) and different amounts of time (0, 10, 20, 30, 40, 50 and 60 min).After heating, the tubes were cooled in an ice bath, and the residual activity measurement was carried out at a pH of 6.2 and at a temperature of 50 • C.

EFFECT OF CATIONS
The effect of Na + and K + on β-1,3-glucanase activity was investigated.The following concentrations of both NaCl and KCl were used: 0.2, 0.4, 0.6, 0.8 and 1 mol L −1 .β-1,3-glucanase activity was determined using the assay previously described, which uses laminarin as a substrate.An activity reading of 100% is obtained when no reagent is added.

REPLICATIONS AND STATISTICAL ANALYSIS
The extractions and chromatographic isolations were repeated at least three times.Average values of triplicates (which differed by <5%) were calculated.

PRODUCTION OF β-1,3-GLUCANASE
A Doehlert matrix for two variables was applied to find the optimum conditions for the β-1,3-glucanase production from Moniliophthora perniciosa.The experimental results obtained with the Doehlert design for these variables together with the coded and real values for the variables studied are shown in Table II.
Below, Equation 1 illustrates the relationship between these two variables and the response R, in which R is the enzyme activity, I C is the inducer concentration and T is the fermentation time: The corresponding surface response and level curves graphics for this equation are shown in Figures 1  and 2.
ANOVA has been applied using the experimental data to evaluate the quadratic model fitted.A test based on the Fisher distribution (F-test) has indicated that the fitted equation is statistically significant (F = 24.65 > 9.01).A lack-of-fit sum of squares (F = 16.02< 18.51) indicates that there is good agreement between the model's predicted response and the experimental values studied for each variable.Lagrange criterion applied to this equation indicates that the critical point is characterized as a maximum.The derivatization of this general equation as δ I C and δT results in the following two new equations: The results showed that fermentation times longer than 18 days and shorter than 7 days caused a reduction in the amount of β-1,3-glucanase produced by M. perniciosa.Concentrations of yeast extract greater than 9 g L −1 and less than 3 g L −1 caused a decrease in the production of β-1,3-glucanase.Concentrations of yeast extract between 4 and 8 g L −1 and fermentation times between 8 and 17 days showed the highest concentrations of β-1,3-glucanase.The optimum activity of 0.2 μmol min −1 mL −1 was obtained using yeast extract concentration of 5.9 g L −1 and a fermentation time of 13 days (Figs. 1 and 2).

HEAT STABILITY AND OPTIMAL PH AND TEMPERATURE
The β-1,3-glucanase from M. perniciosa was active between pH 5.0 and pH 9.0, with maximum specific activity at pH 5.0 (Fig. 4).The effect of temperature on the specific activity of laminarinase is shown in Figure 5.The maximum activity was obtained at 40 • C. The effect of temperature on the residual specific activity of glucanase is shown in Figure 5. β-1,3-glucanase retained 99.01, 95.61 and 92.38% of original activity after 50 min of incubation at 60, 70 and 80 • C, respectively, and lost 10.20% of its specific activity after 40 min of incubation at 90 • C (Fig. 6).

EFFECT OF CATIONS
The effects of Na + and K + ions on glucanase activity are shown in Figures 7 and 8.In the presence of KCl, the glucanase activity was higher in the concentration of 0.40 mol L −1 , showing an increase of 25% in the activity (Fig. 7), in relation to other analyzed concentrations.In the presence of NaCl, the glucanase activity was higher in a 0.2 mol L −1 solution, showing an in-    crease of 8.83% in the activity (Fig. 8), also in relation to other concentrations studied for the same compound.

DISCUSSION
β-1,3-glucanase is one of the most important cell wall lytic enzymes.It has been purified from a number of fungal species in which the enzyme differs in its phys-iological properties, molecular structure, and molecular weight (McQuilken and Gemmell 2004).It was also speculated that the activity of the enzyme may vary due to the β-glucan structure and the type of linkage (Vazquez-Garciduenas et al. 1998).β-1,3-glucanase has been purified from many plant species, usually by multistep procedures that employ ion exchange columns followed by gel filtration and hydrophobic interaction.No previous studies could be found that describe the purification or characterization of a β-glucan degrading enzyme from M. perniciosa.The extracellular β-1,3-glucanases in the supernatant of the cell culture of M. perniciosa were partially purified and obtained in three isoforms, in which the optimal pH and temperature were 4.0 and 40 • C, respectively.The isozymes showed high thermal stability, losing only 10.20% of their specific activity after 40 min of incubation at 90 • C. Murray et al. (2001) obtained a purification fold of 23.00 when they purified glucanase from Talaromy-ces emersonii by gel filtration, Hydrophobic Interaction Chromatography, and anion exchange.Boyce and Walsh (2007) obtained a purification fold of 9.00 when they purified glucanase using various purification steps.The β-1,3-glucanase from Periconia byssoides was purified by HiTrap Q XL, resulting in a purification fold of 4.50.
Studying the influence of metal ions on the activity of the α-1,3-glucanase showed that K + cations had an activating effect that was greater than that seen with Na + cations.Notario et al. (1976) found that the β-1,3-glucanases are acidic proteins, and our results agree with theirs.Similar values of pH have been reported for β-1,3-glucanases from other fungi, including Trichoderma harzianum (Marco and Felix 2007) and Trichoderma asperellum (Bara et al. 2003).
Optimal temperatures of 55-60 • C, 45 • C, and 65 • C were reported for β-glucanases from Bacillus polymyxa (Gosalbes et al. 1991), B. pumilis (Suzuki andKancho 1976) andB. brevis (Louw et al. 1993), respectively.Similar values have been reported, but the optimum temperature of M. perniciosa was lower when compared to The results of this study have shown that M. perniciosa could produce β-1,3-glucanase in the submerged culture, which contains three isoforms.The β-1,3-glucanases showed high thermostability and increased activity in the presence of KCl, with concentrations vary-ing from 0.1 to 1.0 M.More studies are necessary to investigate the production of glucanases from M. perniciosa, to explore the possibility of molecular targets as a disease control for witches' broom, and to apply this enzyme in industrial processes.

Fig. 7 -
Fig. 7 -The effect of KCl on activity in the range of 0.2-1 mol L −1 .All reactions were carried out in 0.05 mol L −1 citrate buffer (pH 6.2) at 50 • C for 15 min.

TABLE I Doehlert matrix used for the optimization of 1,3-glucanase production from Moniliophthora perniciosa.
(C): central point; coded values are presented in parentheses.

TABLE II Analysis of variance for data presented in Table I.
SS -sum of squares; df -degree of freedom; MS -mean square; F calcalculated F value; F tab -tabulated F value.