Production , Characterization And Application Of Inulinase From Pseudozyma Sp . CCMB 300

The use of inulinases provides an alternative way to obtain fructose syrup through the hydrolysis of inulin. The objective of this work was to study the production, characterization and application of an inulinase from Pseudozyma sp CCMB 300 isolated from Brazilian semi-arid region. The higher production of inulinase in a medium containing 0.86 g L yeast extract and 14.54 g L glucose using response surface methodology (RSM). This statistical method was also used to access the optimum pH (8.28) and temperature (54oC) for fermentative inulinase production. The results showed that the inulinase has potential for inulin hydrolysis, where a conversion yields of roughly 87.39 % for an initial concentration of inulin of 1% (w/v).

Inulin is a naturally occurring polyfructan in plants consisting of linear chains of β (2,1)-linked fructose residues attached to a terminal sucrose molecule. It is produced naturally in over 36,000 plants world wide, including 1200 native grasses and cereals belonging to several genera. In addition, inulin has extensive documented historical human use through the consumption of edible plants and fruits [2].
Inulinase can be derived from plants and many microorganisms. Dandelion, chicory and Jerusalem artichoke have been proven to contain inulinase that can be purified, but according to Kochhar and Kanur [3], these sources are not as productive as the microbial one, which seem to be the only source capable of producing enough enzymes for industrial applications. For this reason, in the last three decades, significant efforts have been made to find the best microbial source for the extraction of inulinase [4].
Conventional fructose production from starch needs at least three enzymatic steps, including α-amylase, amyloglucosidades, and glucose isomerase action, yielding only 45 % fructose solutions. More concentrated fructose solution requires a dedicated chromatographic step. A viable alternative to this process is the hydrolysis of inulin by inulinases. Enzymatic formation of fructose from inulin has a single enzymatic step and yields up to 95% fructose [5].
The Brazilian semi-arid region represents a large area for bioprospection since naturally occurring microorganisms adapted to a tropical semi-arid environment, with high temperature and low humidity throughout the year, may possess some features of great industrial interest [6] In this study, we carried out a survey of the production, characterization and application of extracellular inulinases produced by Pseudozima sp. from 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 Pseudozyma sp. CCMB is from Culture Collection of Microorganisms of Bahia (CCMB) of the Universidade Estadual de Feira de Santana, Brazil (Uetanabaro and Góes-Neto 2006) and was identified in our previous work [7].

Inulinase production
The yeast was previously grown on YM agar at 28ºC for 48 h, as described in Oliveira (2007), diluted in sterile distilled water to a concentration of about 10 8 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; (NH4)2SO4, 3 g; KH2PO4, 4.5; MgSO4, 0.25g ), supplemented with yeast extract, 1 g; glucose, 10 g; pH 5.0, for fermentation. 8 After incubation at 28 ºC 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 ºC, 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 50ºC.

Enzyme assays
Inulinase activity was measured spectrophotometrically using the dinitrosalicylic reagent, as reported by Miller [8]. The reaction mixture consisted The mixture was incubated for 15 min at 50 ºC. After incubation, 1 mL dinitrosalicylic reagent was added, and the mixture was boiled at 100 ºC for 10 min and cooled with 10 mL distilled water. One unit of enzyme activity of inulinase was defined as the amount of the enzyme that catalyzed the formation of 1 mol fructose/min.

Protein determination
Total protein determination was performed according to Bradford [10], using bovine serum albumin as the standard. N o v e m b e r 30, 2 0 1 4

Doehlert experimental design for enzyme production
The Doehlert experimental design, with two variables (concentration of glucose and yeast extract) and three replicates at the centre of the domain leading to a total of 9 experiments (Table 1) was used to obtain the knowledge of the effect of glucose and yeast extract concentration on the production of enzyme. Thus, concentration of glucose was studied in 5 levels (7.5 to 12.5 g L -1) and yeast extract was studied in three levels (0.5 to 1.5 g L -1). The experimental errors were evaluated from replication of central point. The experimental data were processed by using the STATISTICA software. All the experiments in this step were carried out in random order.
To estimate the lack of fit of the model to the experimental data an analysis of variance (ANOVA) was performed ( Table  2), 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 inulinases obtained. This statistical technique for experimental design has advantages over methods that investigate only one variable at a time.
The experimental design, with two variables (temperature and pH) and three replicates at the centre of the domain leading to a total of 9 experiments (Table 3) was used to obtain the knowledge of the effect of temperature and pH of inulinase activity.   The pH was studied in 5 levels (5 to 9) and temperature was studied in three levels (30 to 70ºC). The experimental errors were evaluated from replication of central point. The experimental data were processed by using the STATISTICA software. All the experiments in this step were carried out in 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, Km and Vmax, 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 ºC for 15 min. The Km and Vmax for inulin was determined by the method of Lineweaver-Burk plots.

Hydrolysis of inulin
The extent of inulin hydrolysis (%) was calculated as 100 sugars total initial of amount released fructose of amount  [11]. Total sugars were determined by antrone method 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 centre of the domain leading to a total of 9 experiments (Table 4) was used to obtain the knowledge of the effect of enzyme concentration and fermentation time on the extent of inulin hydrolysis from a solution 1% (w / v) in citrate buffer 0.05 M.

Statistical analysis
All experiments were carried out in triplicate. One-way analysis of variance was used to compare the specific activity of inulinase among the strains. Where significant differences were detected between strains, the means were compared using Tukey's test. For all statistical analyses, the level of significance was set at 5%, and the analyses were performed using the standard statistical software SPSS for Windows (release 6.1.3, 1995; SPSS, Brazil, 1995).

Production of inulinases by yeasts
The The response surface graphics (Figures 01 and 02) shows the influence of glucose and yeast extract concentrations in the production of inulinase of CCMB 300. From the analysis of the area chart in Figures 01 and 02 it can be concluded that the production of inulinase by CCMB 300 strains clearly peaks for glucose concentrations in excess of 10 L -1 L glucose, for the whole range of concentration 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.
Different nitrogen sources were studied for Cryptococccus aureus inulinases. Yeast extract was the better and increased inulinase production [12]. According to these authors, vitamins and trace elements present in the yeast extract may enhance inulinase production.
The chart of Paretto ( Figure 03) shows that both linearly (L) and quadratically (Q), the variables glucose concentration and yeast extract concentration are significant for the production of inulinase by Pseudozyma CCMB 300, because both variables had a p value greater than 0.05. The statistical significance of the regression (Table 02) can be assessed by the ratio of the mean square regression and mean square of the residue and 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. It is obtained by the ANOVA table that calculated F (348.30) is higher than the F tabulated (9.01) showing that the function is well suited to the answers.

Response surface optimization of temperature and pH
The table 03 shows the Doehlert design applied to the optimization of temperature and pH inulinase obtained by Pseudozyma sp. CCMB 300.
The influence of temperature and pH on the activity of the enzyme was investigated for CCMB 300 by surface response methodology. The results are shown in Figures 04 and 05.

00101(P).(T). (Equation 2)
This equation illustrates the relationship of these two variables with enzyme activity (UA), where: P is the pH and T is the temperature ºC. Through the derivation of this equation, the points of maximum enzymatic activity can be obtained. According to this methodology, the optimal pH and temperature values were of 8.28 and 54°C, respectively.
Many works in the literature deal with the effects of temperature and pH on inulinase activity. It is certain that the response of the enzyme activity to these variables depends mainly on the strain used as a source for enzyme production. The studies of Pandey et al [13] showed that fungal inulinases exhibited an optimum pH between 4.5 and 7.0, yeasts inulinases between 4.4 and 6.5 and bacterial inulinases between 4.8 and 7.0. Inulinases preparations from A. niger strains have also been shown to have pH and temperature optima in the ranges of 4.35 to 5.35 and 45 to 60ºC [14] Information about the effect of temperature and pH on inulinase activity is very important for assessing the feasibility industrial application, and to the development of bioprocesses and to the selection of bioreactors.
The Paretto chart (Figure 06) shows that both linearly and quadratically, temperature and pH are exerting influence on the enzyme activity of inulinase Pseudozyma CCMB 300, because both variables had a p value greater than 0.05. The statistical significance (Table 04) of the regression can be assessed by the ratio of the mean square regression and mean square of the residue and 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. It is obtained by the ANOVA table that calculated F (35.67) is 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 are shown in Figure 07. The activity was maximal at a concentration of 0.15 mol/L of NaCl and KCl. Thconcentrations of 0.15 mol L -1 of NaCl and KCl increased the activity of inulinase from CCMB 300 by approximately 40%. When concentration higher than 0.15 mol L -1 were used, a decrease in the activity of the enzyme was observed.

Kinetics Parameters
The Lineweaver-Burk plot showed that the Km and Vmax values of the enzyme for inulin were 23 mg/mL and 28.33 µmol/mL.min, respectively (Fig. 08). These results demonstrate that the inulinase from CCMB 300 displayed affinity for inulin and are in agreement with other studies. The Km value for inulinase from Aspergillus ficuum JNPSP was 25.6 mg mL -1 [15].

Hydrolysis of inulin
The Table 05 shows the Doehlert design applied to optimize the hydrolysis in function of time of reaction and enzyme concentration of inulin by Pseudozyma sp. CCMB 300 inulinase.  The hydrolysis was studied in five levels (1 to 12.5 minutes) and enzyme concentration was studied in three levels (1 to 5 %) (Figure 09 and 10).The experimental data were processed by using the STATISTICA software. All the experiments in this step were carried out in random order. Through the derivation of this equation, the points of maximum enzymatic activity are obtained. In this work the highest productivity, based on the extent of hydrolysis, was observed for 3.26 % of enzyme and 7.7 minutes of incubation. The higher conversion yields was 87.39 %. There are few studies of inulin hydrolysis using the same conditions and SRM analysis, as performed in this work.
The Paretto chart ( Figure 11) shows that both linearly and quadratic, variables: enzyme concentration and time of hydrolysis are significant for the hydrolysis of inulin Pseudozyma sp CCMB 300, because both variables has a p value greater than 0.05. The statistical significance (Table 06) e of the regression can be assessed by the ratio of the mean square regression and mean square of the residue and 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. It is obtained by the ANOVA table that calculated F (103.46) is 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 the inulinase by Pseudozyme sp. CCMB 300 was effectively performed. The enzyme was afterwards used in the hydrolysis of inulin, where a conversion yields of roughly 90% 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 Pseudozyme sp. This corresponds to a medium containing 0.86 g L -1 yeast extract and 14.54 g L -1 glucose. This statistical method was also used to access the optimum pH (8.28) and temperature (54ºC 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 300 for fructose production at pilot scales.
This optimization through RMS can be used in future industrial scale fermentation in promoting the rational use of enzyme and substrate in the process.