Production , characterization and application of inulinase from fungal endophyte CCMB 328

Inulinase (β-2,1-Dfructan fructanohydrolase), EC 3.2.1.7, targets the β-2,1 linkage of inulin, a polyfructan consisting of linear β-2,1 linked fructose, and hydrolyzes it into fructose. This use provides an alternative to produce fructose syrup through the hydrolysis of inulin. The objective of this work was to study the production, characterization and applications of inulinases from the fungal endophyte CCMB 328 isolated from the Brazilian semi-arid region. Response Surface Methodology (RSM) was employed to evaluate the effect of variables (concentration of glucose and yeast extract), on secreted inulinase activities detected in the culture medium and also in the inulin hydrolysis. The results showed that the best conditions for inulinase production by CCMB 328 are 9.89 g / L for glucose and 1.09 g / L for yeast extract. The concentration of 0.20 mol/L of NaCl and KCl increased the activity of inulinase from CCMB 328 by approximately 63% and 37%, respectively. The results also showed that the inulinase has potential for inulin hydrolysis, whose conversion yields roughly 72.48 % for an initial concentration of inulin at 1% (w/v).

Inulinase can be derived from many microorganisms.In the last three decades, significant efforts have been made to find the best microbial source for the extraction of inulinase (Ricca et al. 2007, Vijayaraghavan et al. 2009).
Conventional fructose production from starch needs at least three enzymatic steps, including α-amylase, amyloglucosidades, and glucose isomerase action, yielding only 45% of fructose solutions.An alternative to this process is the hydrolysis of inulin by inulinases.The enzymatic DIEGO S. NASCIMENTO et al. formation of fructose from inulin has a single enzymatic step and yields up to 95% of fructose (Figueiredo-Ribeiro et al. 2007).The use of microbial inulinases (EC 3.2.1.7),which yield up to 95% of pure fructose in a single-step enzymatic reaction, is therefore a viable alternative for the production of high-fructose syrup (Gill et al. 2006).
Inulinases have also been used for the production of inulooligosaccharides-low caloric saccharides by acting as a growth factor for beneficial microorganisms in the intestinal flora.They are considered a prebiotic agent (Skowronek and Fiedurek 2006).
The Brazilian semi-arid region represents a large area for bioprospection since it fosters naturally occurring microorganisms adapted to a tropical semi-arid environment, with high temperature and low humidity throughout the year that may possess some features of great industrial interest (Uetanabaro and Góes-Neto 2006).
In this study, we analyzed the production, characterization and application of extracellular inulinases obtained from fungal endophyte (CCMB 328) isolated in the Brazilian semi-arid region.

CHEMICALS
Inulin, bovine serum albumin, and 3,5-dinitrosalicylic acid were purchased from Sigma Chemical Co.(St Louis, MO, USA).All the other chemicals used were also of high-quality analytical grade.

MICROORGANISMS
The yeast strains fungal endophyte (CCMB 328) is from Culture Collection of Microorganisms of Bahia (CCMB) of Universidade Estadual de Feira de Santana, Brazil (Uetanabaro and Góes-Neto 2006), and was identified in our previous work (Lima et al. 2009).

INULINASE PRODUCTION
The yeast was previously grown on YM agar at 28°C for 48 h, as described in Oliveira (2007), and diluted in sterile distilled water to a concentration of about 108 colony-forming units/ml.10% (v/v) of the diluted growth medium was inoculated in flasks containing (per 1 L) mineral medium (CaCl2, 0.25 g; (NH 4 ) 2 SO 4 , 3 g; KH 2 PO 4 , 4.5; MgSO 4 , 0.25g ) and supplemented with yeast extract, 1 g; glucose, 10 g; pH 5.0, for fermentation (Patching and Rose 1969).After the incubation at 28°C for 48 h in an orbital shaker at 150 rpm, the cells were separated by centrifugation at 10,000 g for 10 min at 48°C, and the supernatant liquid media was used as the extracellular fraction.

BIOMASS QUANTIFICATION
The cells, which were previously separated by centrifugation, were used to determine the dry weight at 80°C.

ENZYME ASSAYS
The inulinase activity was measured spectrophotometrically using the dinitrosalicylic reagent, as reported by Miller (1959).The reaction mixture consisted of 900 µL of 2 % (w/v) inulin in 0.05 M acetate buffer, pH 5.5, and 100 µL of culture supernatant.The mixture was incubated for 15 min at 50°C.After incubation, 1 mL dinitrosalicylic reagent was added, and the mixture was boiled at 100°C for 10 min and cooled with 10 mL of distilled water.One unit of enzyme activity of inulinase was defined as the amount of the enzyme that catalyzed the formation of 1 µmol fructose/min.INULINASE FROM fungal endophyte PROTEIN DETERMINATION Total protein determination was performed according to Bradford (1976), using bovine serum albumin as the standard.

DOEHLERT EXPERIMENTAL DESIGN FOR ENZYME PRODUCTION
The Doehlert experimental design, with two variables (concentration of glucose and yeast extract) and three replicates at the center of the domain leading to a total of 9 experiments (Table I), was used to assess the effect of glucose and yeast extract concentration on the production of the enzyme.
Thus, the concentration of glucose was studied in 5 levels (7.5 to 12.5 g/L), and the concentration of yeast extract was studied in three levels (0.5 to 1.5 g/L).The experimental errors were evaluated from the replication of the central point.The experimental data were processed by using the STATISTICA software.All the experiments in this step were carried out in a random order.
To estimate the lack of fit of the model to the experimental data, an analysis of variance (ANOVA) was performed, using Design Statistics, version 7.0.

RESPONSE SURFACE OPTIMIZATION OF TEMPERATURE AND PH
Response surface modeling was applied to cultures of yeast to determine the optimum temperature and pH for the inulinases obtained.This statistical technique for experimental design has advantages over methods that investigate only one variable at a time (Hanrahan and Lu 2006).
The experimental design, with two variables (temperature and pH) and three replicates at the center of the domain leading to a total of 9 experiments (Table II) was used to get the effect of temperature and pH of the inulinase activity.
The pH was studied in 5 levels (5 to 9), and temperature was studied in three levels (30 to 70°C).The experimental errors were evaluated from the replication of the center point.The experimental data were processed by using the STATISTICA

Experiment
Concentration of glucose (g/L)  software.All the experiments in this step were carried out in a random order.
To estimate the lack of fit of the model to the experimental data, an analysis of variance (ANOVA) was performed using Design Statistica, version 7.0.

DETERMINATION OF KINETICS PARAMETERS
To obtain the parameters of the Michaelis-Menten kinetics of inulinase for the hydrolysis of inulin, K m and V max , 0.9 µL inulin (1, 1.5, 2.0, 2.5 and 3.0% w/v) was added to 100 µL of inulinase in 0.05 M acetate buffer (pH 5.5) and incubated at 50 °C for 15 mim.The K m and V max for inulin were determined by the method of Lineweaver-Burk plots.

HYDROLYSIS OF INULIN
The extent of inulin hydrolysis (%) was calculated as (Nakamura et al. 1995).Total sugars were determined by the anthrone method (Yemm and Willis 1954), and reducing sugars were determined by the dinitrosalicylic acid method (Miller 1959).The Doehlert experimental design, with two variables (concentration of enzyme and fermentation time) and three replicates at the center of the domain leading to a total of 9 experiments (Table IV), was used to get the effect of enzyme concentration and fermentation time on the extent of inulin hydrolysis from a solution of 1% (w / v) in citrate buffer 0.05 M.

THERMOSTABILITY OF INULINASE
Samples of inulinases in test tubes (selected to be equal in weight, volume and size) were incubated in buffer at different temperatures (50°C, 60°C, 70°C, 80°C and 90°C) and for various times (0,10,20,30,40,50   process, the tubes were cooled in melting ice and the residual activity measurement was carried out at pH 6.0 and at a temperature of 50°C.

PRODUCTION OF INULINASES BY YEASTS
Table I shows the Doehlert design applied to optimize the production of inulinase by fungal endophyte CCMB 328.The first column describes the number of planning, with C representing the central compound.In the second and third columns we have the values of glucose concentration and the concentration of yeast extract, respectively.The fourth column represents the enzymatic activity.
The last column represents the predicted values.
The response surface graphics (Figures 1 and 2) shows the influence of glucose and yeast extract concentrations in the production of inulinase of CCMB 328.From the analysis of the area chart in Figures 1 and 2 it can be concluded that the production of inulinase by CCMB 328 strains clearly peaks for glucose concentrations in excess of 10 g/L glucose for the whole range of concentrations of yeast extract tested.
Equation 1 illustrates the relation of these two variables and the enzyme activity (UA), where G is the glucose concentration g/L and YE is the yeast extract concentration g/L:   Through the derivation of this equation, the media composition, regarding glucose and yeast extract concentrations, which are expected to allow for the production of maximum enzymatic activity, can be obtained.For CCMB 328 the conditions are 9.89 g / L for glucose and 1.09 g /L for yeast extract.
Kalil (2004) used the technique of factorial design and response surface analysis to optimize the culture medium for inulinase production by Kluyveromices marxianus According to his results, the best concentration of yeast extract was 10 g / L. Schneider (1996) studied the production of inulinase by Kluyveromices marxianus and found that the presence of yeast extract is essential for the growth of microorganisms and enzyme production.
The chart of Paretto (Figure 3) shows that the variables glucose concentration and yeast extract concentration are linearly and quadratically significant for the production of inulinase fungal endophyte CCMB 328, because both variables have a P value greater than 0.05.
The statistical significance of the regression can be assessed by the ratio of the mean square regression and mean square of the residue, as well as by comparing these sources of variation using the Fisher distribution (F test) (Table III).Thus, a statistically significant value of this ratio must be greater than the tabulated value for F. The calculated F value from the ANOVA table (712.55) was higher than the F tabulated (9.01), showing that the function is well suited to the answers.

RESPONSE SURFACE OPTIMIZATION OF TEMPERATURE AND PH
Table III shows the Doehlert design applied to the optimization of temperature and pH inulinase obtained by fungal endophyte CCMB 328.
The influence of temperature and pH on the activity of the enzyme was investigated for CCMB 328 by the surface response methodology.The results are shown in Figures 4 and 5.The regression model provided for the enzyme activity in relation to pH and temperature in the experimental design is expressed by Equation 2: UA = -4.4934+1.9239(pH)-0.1367(pH) 2 + 0.0356(T) -0.0004(T) 2 +0.0004

(pH).(T)
This equation illustrates the relationship of these two variables with the enzyme activity (UA) where P is the pH and T is the temperature (°C).Through the derivation of this equation, the points of maximum enzymatic activity could be obtained.According to this methodology, the optimal pH and temperature values were 7.11 and 48.35°C, respectively.
Regarding to the pH of the enzymatic reaction, although the microbial inulinases are described as stables from pH 3.5 to 6.5 ours results indicate a maximum inulinase activity at pH 7.11.In relation to the reaction temperature, it was observed that the maximum enzymatic activity at 48.35°C.Similar values were found in Cazetta et al. (2005).The Paretto chart (Figure 6) shows that the temperature concentration and pH variables are independent for the production of inulinase from fungal endophyte CCMB 328 because this had a p value less than 0.05.
The statistical significance of the regression can be assessed by the ratio of the mean square regression and the mean square of the residue, as well as by comparing these sources of variation using the Fisher distribution (F test) (Table IV).Thus, a statistically significant value of this ratio must be greater than the tabulated value for F. The F value calculated from the ANOVA table (75.71) was higher than the F tabulated (9.01), showing that the function is well suited to the answers.

EFFECT OF CATIONS
The effect of salts NaCl and KCl on the activity of inulinase is presented in Figure 7.The concentration DIEGO S. NASCIMENTO et al. of 0.20 mol/L of NaCl and KCl increased the activity of inulinase from CCMB 328 by approximately 63% and 37%, respectively.A KCl concentration higher than 0.20 mol/L decreases the activity of the enzyme (Figure 7).
Our results were similar to those observed by Sheng (2007) who reported an increase in the enzymatic activity at 62.2% in the presence of KCl.Ohta et al. (2002), using inulinases produced by Rhizous, noted that the presence of KCl did not change the enzyme activity.

KINETICS PARAMETERS
The Lineweaver-Burk plot showed that the K m and V max values of the enzyme inulinase were 7.53 mg/mL and 2.34 µmol/mL.min - , respectively (Figure 8).While assessing the kinetic characterization of the inulinase from A. niger 20 OSM, Showronek and Fiedureki (2006) reported K m and V max values of 6.7 g/L and 0.0476 mg/mL/min, respectively.The hydrolysis time was studied in five levels (1 to 15 minutes), and enzyme concentration was studied in three levels (1 to 5%) (Figures 9

and 10). INULINASE FROM fungal endophyte
The experimental errors were evaluated from the replication of the central point.The experimental data were processed by using the STATISTICA software.All the experiments in this step were carried out in a random order.
Equation 3 illustrates the relationship of these two variables with the extent of hydrolysis (% H), where C is the Enzyme Concentration (% v/v) and T is time (min).Through the derivation of this equation, the points of maximum enzymatic activity were obtained.Through the derivation of this equation, the points of maximum enzymatic activity were obtained.In this work the highest productivity, based on the extent of hydrolysis, was observed for 4.90 % of enzyme and at 6.0 minutes of incubation.There are few studies of inulin hydrolyses using the same conditions and RSM analysis, as performed in this work.
The Paretto chart (Figure 11) shows that the variables temperature concentration and pH are independent for the production of inulinase CCMB 328, because s had a p value less than 0.05.
The statistical significance of the regression (Table VI) can be assessed by the ratio of the mean square regression and mean square of the residue, as well as by comparing these sources   of variation using the Fisher distribution (F test).Thus, a statistically significant value of this ratio must be greater than the tabulated value for F. The F value calculated from (201.2) was higher than the F tabulated (9.01), showing that the function is well suited to the answers.

CONCLUSION
In this work the production and characterization of inulinase by fungal endophyte CCMB 328 was effectively performed.The enzyme was afterwards used in the hydrolysis of inulin whose a conversion yields of roughly 72.48 % for an initial concentration of inulin of 1% (w/v).Therefore, the inulinase from this yeast is a potential candidate for inulin hydrolysis in the food industry.
Using the response surface methodology it was possible to determine the best media composition for obtaining inulinase from fungal endophyte CCMB 328.This corresponds to a medium containing 9.89 g / L for glucose and 1.09 yeast extract.This statistical method was also used to access the optimum pH (7.11) and temperature (48.38°C) for fermentative inulinase production.
The combination of results obtained in this work (enzymatic characterization and hydrolysis of inulin) can provide useful guidelines for the utilization of inulinase from CCMB 328 for fructose production at pilot scales.

Figure 1 .
Figure 1.Response surface for yeast extract

Figure 9 .
Figure 9. Response surface for hydrolysis of inulin

Figure 10 .
Figure 10.Level curves for hydrolysis of inulin

TABLE I Results of the Doehlert design for inulin production from CCMB 328
DIEGO S. NASCIMENTO et al.
and 60 min).After the heating

TABLE II Results of the Doehlert design for temperature and pH on inulinase from CCMB 328 amount
of fructose released × 100 amount of initial total sugars INULINASE FROM fungal endophyte

TABLE IV Analysis of variance (ANOVA) for the quadratic model fitted to data from the Table II
. INULINASE FROM fungal endophyte

TABLE V Results of the Doehlert design for inulin hydrolysis
DIEGO S. NASCIMENTO et al.

TABLE VI Analysis of variance (ANOVA) for the quadratic model fitted to data from the Table V.
SS: Sum of squares; df: degree of freedom; MS: mean of square.INULINASE FROM fungal endophyte