Natural montmorillonite as support for the immobilization of inulinase from
Kluyveromyces marxianus NRRL Y-7571
Chaline C. Coghetto
a, Robison P. Scherer
a, Marceli F. Silva
a, Simone Golunski
a, Sibele B.C. Pergher
b,
De´bora de Oliveira
c,1, J. Vladimir Oliveira
c,1, Helen Treichel
d,e,na
Universidade Regional Integrada do Alto Uruguai e das Miss ~oes—URI—Campus de Erechim Departamento de Engenharia de Alimentos Av. 7 de Setembro, 1621, 99700-000 Erechim, RS, Brazil
b
Universidade Federal do Rio Grande do Norte, Centro de Ciˆencias Exatas Departamento de Quı´mica, Av. Salgado Filho, 3000, Lagoa Nova, 59078-970 Natal, RN, Brazil
c
Universidade Federal de Santa Catarina, Departamento de Engenharia Quı´mica e de Alimentos, Campus Universita´rio, Floriano´polis 88800-000, SC, Brazil
dUniversidade Federal do Rio Grande, Escola de Quı´mica e Alimentos Rua Alfredo Huch, 475, 96201-900 Rio Grande, RS, Brazil e
Universidade Federal da Fronteira Sul—Campus de Erechim, Av. Dom Jo ~ao Hoffmann, 313, 99700-000 Erechim, RS, Brazil
a r t i c l e
i n f o
Article history: Received 24 March 2012 Received in revised form 20 June 2012
Accepted 22 June 2012 Available online 11 July 2012 Keywords:
Kluyveromyces marxianus NRRL Y-7571 Inulinase
Inorganic support Immobilization
a b s t r a c t
The objective of this study was to investigate the process of immobilization of inulinases using natural montmorillonite as inorganic support. The enzyme to buffer ratio of 3:10 and 10 min of immobilization led to the highest specific activity, 375.07 U/mg protein. The immobilized inulinase kept its activity after 1968 h under storage at low temperatures and after 456–1826 h at high temperatures. The pH value of 3.5 led to the highest specific activity. Kmvalues of 1.46 and 0.38 mM, and vmaxof 0.2487 and
0.2396 mol/L min, were obtained, respectively, for sucrose and inulin.
&2012 Elsevier Ltd. All rights reserved.
1. Introduction
The fructooligosaccharides (FOS) are storage carbohydrates found in many vegetable species. The exo and endo-inulinases act hydrolyzing the inulin, yielding fructose and FOS, which can be used as functional ingredient, replacing sugar and fat in foods. Thus, large attention has been drawn to carbohydrate diet, with special focus on the oligosaccharides, particularly fructooligosac-charides (Yun, 1996;Treichel et al., 2011).
The inulinases can be produced by submerged fermentation, using synthetic and agro-industrial media (Contiero, 2004;
Manzoni and Cavazzoni, 1992). Microorganisms of the genus Kluyveromyces are recognized as good inulinases producers (Mazutti et al., 2010). The yeast Kluyveromyces marxianus NRRL Y-7571 presents high production, since it can reach high cell densities in a short time, as well as high enzyme yields, mainly in media composed of agro-industrial residues. Studies on the production of inulinases by solid state fermentation (SSF) are rather recent and bring perspectives for industrial application for
valorization of agro-industrial residues and by-products (Mazutti et al., 2010;Mazutti et al., 2006).
Enzymes are subject to inactivation by chemical, physical and biological factors, during the use or when stored. To make the catalysis efficient in a determined process, the enzymes need to be protected from the interaction with the solvent, since it can cause its inactivation. Enzyme immobilization techniques have been used with this main purpose, to enhance the enzyme stability and also to make easier its recuperation and reutilization (Villeneuve et al., 2000;Dalla-Vechia et al., 2004).
Aiming at utilizing the catalytic potential of enzymes in industrial processes, several forms of making these catalysts insoluble in the reaction medium have been studied. In this sense, the immobilization has become a matter of great interest. A process that allows achievement of an active and stable enzyme, with good specificity by the substrate generally elim-inates most of disadvantages of the use of enzymes, making possible their use in industrial processes (Sobral et al., 2003).
The major contribution to achieve a good performance of immobilized catalyst is primarily provided by the strategy employed for immobilization (Cardias et al., 1999) and by the characteristics of the support. According toGomes et al. (2006), the choice of the appropriate support can enhance the half-life of the enzyme and increase the global performance of the process. Several organic, Contents lists available atSciVerse ScienceDirect
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n
Corresponding author at: Universidade Federal da Fronteira Sul-Campus de Erechim, Av. Dom Jo ~ao Hoffmann, 313, 99700-000 Erechim, RS, Brazil.
E-mail address: helentreichel@gmail.com (H. Treichel).
inorganic and natural materials with different characteristics have been used for inulinases immobilization. However, appraisal of literature information on the subject showed that just a few works regarding the use of low-cost inorganic supports for inulinase immobilization are available (Bajpai and Margaritis (1987);
Betancor et al., 2006; Catana et al., 2005; Ettalibi and Baratti, 2001;Gaspari et al., 1999;Gill et al., 2006;Kochhar et al., 1998;
Yun et al., 2000;Wenling et al., 1999).
As just a few works are presented in the literature about this subject, one believes that the present work can be seen as a good contribution to this field of knowledge (Richetti et al., 2012;
Nguyen et al., 2011; Zhao et al., 2011; Kuhn et al., 2011;
Melliawati et al., 2003). The immobilization of inulinase from K. marxianus NRRL Y-7571 was evaluated by our research group using sodium alginate, glutaraldehyde and activated coal as support. The experimental condition of 20 g/L of sodium alginate, 50 mL/L of glutaraldehyde and 30 g/L of activated coal led to the highest specific activity (2063.5 U/mg of protein), corresponding to an enhancement of about 26 times compared to the activity of the free enzyme (79.1 U/mg of protein). The effect of pH and temperature on the immobilized enzyme activity was also eval-uated, showing optimal activities at pH of 5.5 and 55 1C. The study of storage of immobilized inulinase in different temperatures showed that the extract kept its initial activity after 43 days of storage at 40 1C and 50 1C and after 138 days of storage either at 4 1C or 25 1C (Richetti et al., 2012).
In this sense, taking into account the lack found in the literature about the use of low-cost clays as support for immobi-lization of inulinases, the main objective of this work was to investigate the process of immobilization of inulinases from K. marxianus NRRL Y-7571 using natural montmorillonite as inorganic support.
2. Material and methods
2.1. Production of inulinase from K. marxianus NRRL Y-7571 The yeast K. marxianus NRRL Y-7571 was kindly provided by the Laboratory of Bioprocesses Engineering of State University of Campinas (UNICAMP, Campinas, SP, Brazil) and was kept at 4 1C in agar YM (3 g/L yeast extract, 3 g/L malt extract, 5 g/L peptone, 10 g/L glucose and 20 g/L agar).The medium for pre-inoculum was constituted of 5 g/L yeast extract, 20 g/L sucrose, 5 g/L K2HPO4, 1.5 g/L NH4Cl, 1.15 g/L KCl and 0.65 g/L MgSO47H2O. Each tube of YM broth was transferred to Erlenmeyer of 500 mL containing 100 mL of medium and incubated at 30 1C and 150 rpm by 24 h. The solid medium used as substrate for the production of inulinase was composed by sugarcane bagasse supplemented by 15 wt% of pre-treated sugarcane molasses (SCM), 30 wt% of corn steep liquor (CSL) and 20 wt% of soybean meal(SM) (Mazutti et al., 2010; Mazutti et al., 2006). The CSL was purchased from Corn Products International (S ~ao Paulo, Brazil), the SCM from Refinaria E´ster (S~ao Paulo, Brazil) and the SM from Olfar (Rio Grande do Sul, Brazil). The initial moisture content of the substrates was set to 65%, defined in previous works by our research group (Mazutti et al., 2006).
The production of inulinase was carried out by solid state fermentation in batch mode (BF) in a fixed bed bioreactor, using 2 kg of dry sugarcane bagasse supplemented as described before. After correction of the moisture content, the medium was sterilized at 121 1C for 20 min. The fermentation medium was inoculated using an initial cell mass of 14 g (Mazutti et al., 2010). The fermentation time was 24 h and the inulinase was extracted from the medium by the addition of 100 mL of sodium acetate buffer (0.1 M, pH 4.8) and incubated for 30 min at 50 1C and
150 rpm. Crude enzymatic extract was immediately frozen at 80 1C, freeze-dried for 24 h and called as lyophilized preparation.
2.2. Experimental procedure for inulinase immobilization
The lyophilized preparation was solubilized in acetate buffer (pH 4.8) (2 g of enzyme and 60 mL of buffer) and submitted to preferential immobilization by physical adsorption on the support (2 g). Immobilization was carried out using magnetic stirring in an ice cooler according the time defined by the experimental design. After this, samples were filtered under vacuum and the retentate was kept in desiccator for 24 h. Then, the enzyme activity and the protein content were determined, following the methodology described further.
2.3. Optimization of immobilization process
The effects of the immobilization time (10–90 min) and enzyme to support ratio (1:10–3:10) on the immobilization process were evaluated using a central composite design (CCD) 22. During the immobilization process, aliquots of 1 mL were withdrawn for measurement of enzyme activity and protein content.
2.4. Analytical methodology 2.4.1. Protein content
The protein content of each sample was determined by a Qubit Fluorimeter (Kit Quant-iT Protein Assay), following the metho-dology proposed by the manufacturer. The kit provides concen-trated assay reagent, dilution buffer, and pre-diluted BSA standards. The reagent was diluted to 1:200, load 200
m
L into the wells of a microplate, added 1–20m
L sample volumes, mixed, then the fluorescence was read. The assay is highly selective for protein. In the range of 0.25–5m
g of protein, the response curve is sigmoidal (pseudo-linear from 0.5–4m
g) and exhibits low pro-tein-to-protein variation. The assay was performed at room temperature, and the signal was stable for 3 h. Common con-taminants, such as salts, solvents, or DNA, but not detergents, are well tolerated in the assay.2.4.2. Determination of inulinase activity
The inulinase activity was assayed using 0.5 g of immobilized enzyme and 4.5 mL of 2% (w/v) sucrose or inulin solution in sodium acetate buffer (0.1 M pH 4.8) at 50 1C and 150 rpm for 30 min. Reducing sugars released were measurement by the 3,5-dinitrosalicylic acid method (Miller, 1959). A separate blank was set up for each sample to correct the non-enzymatic release of sugars. One unit of inulinase was defined as the amount of enzyme that released 1
m
mol of reducing sugars per minute under the standard assay conditions. The inulinase activity of non-immobilized enzyme was measured following the same procedure. The specific activity of inulinase was determined by dividing the enzyme activity by the protein content and expressed as U/mg of protein.2.4.3. Determination of the yield of immobilization The yield of immobilization was calculated by Eq. (1).
Z
ð%Þ ¼Pa Po100 ð1Þ
where
Z
¼yield (%), Parepresents the amount of protein adsorbed, Po denotes the amount of protein used in the immobilization (inlet solution).2.4.4. Determination of the percentage of retention The percentage of retention was calculated by Eq. (2) Rað%Þ ¼AR 100
Ar ð2Þ
where Ra ¼percentage of retention (%), AR¼ real activity (immo-bilized enzyme) (U); Ar¼theoretical activity (U).
2.5. Partial characterization of immobilized inulinase
The partial characterization of immobilized inulinase of K. marxianus NRRL Y-7571 was performed in terms of optimal temperature and pH and thermal stability. The kinetic parameters kmand vma´xwas also determined under two substrates, inulin and sucrose.
2.5.1. Thermal stability
The thermal stability of immobilized inulinase was evaluated by incubation of the enzyme in acetate buffer (0.1 M, pH 4.8) at 40, 50, 60 and 70 1C. Samples were withdrawn at pre-determined intervals of time for inulinase activity measurement.
The stability of immobilized inulinase at low temperatures was performed using sodium acetate buffer (0.1 M, pH 4.8). Samples (20 g) were stored at 4, 10 and 80 1C and the enzyme activity was measured at pre-determined intervals of time to follow stability of the immobilized catalyst during the storage. 2.5.2. Effect of the pH on the stability
The stability of the immobilized inulinase against different pH values was tested by incubation of the catalyst in sodium acetate buffer (pH values of 3.5, 4.0, 4.5, 5.0 and 5.5) at 50 1C. Samples were withdrawn at determined times and the enzyme activity was determined as described previously.
2.5.3. Determination of the kinetic parameters kmand vmax Enzyme assays using 0.5 g of immobilized inulinase were carried out in sodium acetate buffer (0.1 M, pH 4.8) at 50 1C, using different sucrose and inulin concentrations: 0.5, 0.75, 1, 1.5, 2, 3, 4, 5, 10, 15, 20, 25, 30 and 35 g/L. The reaction time was 10 and 30 min, respectively for sucrose and inulin. The kinetic parameters km and vmax were determined by the Lineweaver-Burk method.
2.6. Characterization of the support and immobilized inulinase The free and immobilized enzymes and supports were par-tially characterized in terms of specific surface area and small-angle X-ray powder diffraction (SAXRD). The textural character-ization of the samples was carried out using an Autosorb-1 (Quantachrome Nova-2200e) by the low-temperature N2 adsorption–desorption experiments. The specific surface area was determined by the BET method. The SAXRD analyses were carried out in a Diffraktometer model D5000 (Siemens) using filter of Ni and radiation Cu-k
a
(l
¼1.54 ˚A).3. Results and discussion 3.1. Characterization of the support
The analysis of the support structure for enzyme immobiliza-tion is necessary to evaluate the crystallinity, kind of structure, morphology, size of crystals, pore diameter and specific super-ficial area (Macario et al., 2005). In the present work, the analyses employed for characterization of the support were small-angle
X-ray powder diffraction (SAXRD) and textural analysis by N2 adsorption.
Fig. 1 presents the X-Ray diffractogram of natural montmor-illonite, used as support for the inulinase immobilization. From this one can observe that the clay has quartz as impurity (2
y
¼271) and a peak at 2y
¼201, characteristic of natural mon-tmorillonite. Actually, this clay has a basal spacing (Bragg’s law) of 9.7 ˚A (calcined) and of 15.12 ˚A (hydrated).From the adsorption isotherms the following properties of the support was determined: mean pore diameter (determined by the BJH method) of 38.60 ˚A and superficial area (determined by the BET isotherm) of 62.70 m2/g. The determined pore diameter and superficial area showed that this material can be considered a promising support for enzyme immobilization (Scherer et al., 2011).
3.2. Preliminary tests for inulinase immobilization
Preliminary experiments were carried out to evaluate the enzyme to buffer ratio (1:10; 2:10; 3:10; 4:10; 5:10) on the immobilization process. The inulinase activity and the protein content were determined in all experimental conditions. The results obtained in this step are presented as specific enzyme activity. The activity and the protein content of the crude inulinase activity were measured before each immobilization test.
Table 1presents the specific activity of free inulinase and the immobilized one for each enzyme to buffer ratio tested. The use of a ratio of 1:10 led to a loss of enzyme activity. Among the other ratios, 2:10 and 3:10 led to higher specific activities. These results can be considered interesting since, besides the immobilization time, the enzyme concentration is also an important parameter for the process. However, it is important to mention that the support presents a load capacity that should be respected, not to use an excess of enzyme which could cause or even hinter the enzyme stabilization (Goulart et al., 2008).
10 20 30 40 50 60 0 100 200 300 400 500 600 Intensidade (u.a) 2θ
Fig. 1. SAXRD of the natural montmorillonite.
Table 1
Specific inulinase activity: free and immobilized catalyst.
Free inulinase Immobilized inulinase
Dilution Specific activity (U/mg) Dilution Specific activity (U/mg)
2:10 325.24 2:10 343.78
3:10 307.52 3:10 375.07
4:10 201.16 4:10 250.92
Richetti et al. (2012)immobilized the inulinase from K. marx-ianus NRRL Y-7571 using sodium alginate, glutaraldehyde and activated coal as supports. At the optimized conditions, a specific activity of 2063.52 U/mg protein was obtained. (Paula et al., 2008) studied the immobilization of an inulinase from a microorganism var. bulgaricus using activated coal as support, reaching a specific activity of 0.34 UA/mg protein. No works were found in the current literature about the immobilization of inulinases using inorganic supports, such as clays.
Based on the preliminary results presented inTable 1, one can conclude that the inulinase presented affinity for the support. This result can be considered relevant since the enzyme was obtained from agroindustrial residues and the support tested is a low-cost one.
3.3. Maximization of the immobilization process
After the preliminary experiments, in an attempt to maximize the immobilization process, a CCD 22 was used to evaluate the effect of the immobilization time and the enzyme to buffer ratio on the process yield, as presented inTable 2. Data were statisti-cally treated and the results are presented in the Pareto chart of
Fig. 2. It was possible to observe that the immobilization time did no influence significantly (po0.05) the immobilization process. Ten minutes were enough to the complete adsorption of the enzyme to the support. After setting the parameters the yield, retention and enzyme activity was determined: 63.67%, 42.08% and 56.84 U/g, respectively.Astolfi et al. (2011) determined the inulinase activity of the free enzyme obtained by fed-batch fermentation and found an activity of 484 U/mL.
Kochhar et al. (1998) immobilized an inulinase from A. versicolor and obtained a percentage of retention of 56% using chitin and 10% using casein as supports. (Paula et al., 2008) studied the immobilization of an inulinase from K. marxianus var. bulgaricus in different supports and obtained an efficiency of immobilization of 2.25% using activated coal, 3.04% with activated coal treated with ethanol, 16.98% with silica with controlled porosity and partially purified extract and 82.60% of efficiency of immobilization using gelatin as support.
Some examples of using inorganic compounds as support for enzyme immobilization are found in the literature. (Yesiloglu, 2005) used bentonite clay for the immobilization of lipase from Candida rugosa by adsorption process. The immobilized catalyst presented a residual activity of 30% compared to the free enzyme.
Spagna et al. (1995)immobilized a commercial pectin-lyase and obtained an immobilization yield of 16% and enzyme activity of 67 U/g.Scherer et al. (2011), using different inorganic supports for the immobilization of porcine pancreatic lipase, observed that the immobilization process was more efficient when the clays KSF, natural montmorillonite and K-10 were used as supports, with immobilization yields higher than 50%.
3.4. Characterization of the immobilized inulinase
From Fig. 3 it is possible to observe that the free inulinase presents an amorphous behavior. After the immobilization pro-cess, using montmorillonite as support, no change on the struc-ture was observed, making possible to conclude that the enzyme was fixed in the interlamellar region, since no peaks referred to the free enzyme were observed.
The comparison of the superficial area of the support (62.70 m2/g) and the immobilized enzyme (4.66 m2/g) permits us to verify a significant reduction on this parameter, indicating the adherence of the enzyme on the cavities of the support. An increase on the pore diameter was observed after the immobili-zation process (38.60 and 55.51 ˚A for the support and immobi-lized enzyme, respectively), probably caused by the entrance of the enzyme on the structure of the clay.
3.5. Partial characterization of the immobilized enzyme 3.5.1. Thermal stability
The thermal stability of the biocatalyst is of fundamental impor-tance for the effective characterization of a bioconversion reaction system (Catana et al., 2007). The thermal stability of the immobilized inulinase from K. marxianus NRRL Y-7571 was determined and,
Table 2
Variables and levels studied in the full 22experimental design (coded and real
values) with the responses in terms of inulinase activity, protein content and specific activity. Assay Time (min) Enzyme to buffer ratio (mL/mL) Inulinase activity (U/mL) Protein content (mg/mL) Specific activity (U/mg protein) 1 10 ( 1) 1:10 ( 1) 0 0 0 2 90 (1) 1:10 ( 1) 0 0 0 3 10 ( 1) 3:10 (þ 1) 19.28 0.1045 184.43 4 90 (1) 3:10 (þ 1) 20.22 0.0867 233.17 5 50 (0) 2:10 (0) 18.17 0.0854 222.87 6 50 (0) 2:10 (0) 21.51 0.0901 232.72 7 50 (0) 2:10 (0) 17.58 0.0928 210.48
Fig. 2. Pareto chart of the effect of the enzyme to support ratio and the time on the immobilization of inulinase.
10 20 30 40 50 60 Intensity u.a. 2θ Natural montmorillonite Immobilized inulinase Free inulinase
Fig. 3. X-Ray diffractograms of free inulinase, natural montmorillonite and immobilized inulinase.
within the range evaluated, the immobilized enzyme did not present significant loss of activity. At 40 1C, a loss of 9% was observed after 1826 h, at 50 1C was 19% at 1792 h, 60 1C of 37% (678 h) and at 70 1C a loss of inulinase activity of 30% was verified after 456 h of storage. The thermal stability of a free inulinase of K. marxianus NRRL Y-7571 was determined byAstolfi et al. (2011)in the range from 40 to 70 1C. The authors observed that higher stability was achieved at 40 1C. (Santos and Maugeri, 2007) obtained the temperature of 50 1C as that correspondent to the highest stability for the free inulinase from K. var. bulgaricus ATCC 16045. Cazetta et al. (2005) and Mazutti et al. (2007) also obtained high stability at 50 1C for the free inulinase from K. marxianus.Mazutti et al. (2010)studied the thermal stability of inulinases obtained from the same microorganism both by submerged and solid state fermentation and observed that the extracts presented high stability at 50 1C.
3.5.2. Stability to low temperatures
The stability of the immobilized inulinase to low temperatures was evaluated at 4, 10 and 80 1C. The enzyme activity was measured periodically until 82 days. Independent from the temperature, the inulinase immobilized in montmorillonite kept its original activity after 82 days of storage.
3.5.3. Effect of pH on the stability
The influence of the pH on the stability of the immobilized inulinase was verified in the range from 3.5 to 5.5. The analysis of the results obtained demonstrated that the immobilized catalyst presented higher stability at pH values of 3.5, after 448 h of incubation, showing a loss of activity of about 37%, comparing to the original activity. At pH values of 4.0, 4.5, 5.0 and 5.5 a more significant loss of activity was observed (78, 77; 75 and 74%, respectively), after 304 h of incubation.
The comparison of these results with those obtained byAstolfi et al. (2011), using the free inulinase obtained from the same microorganism, permits us to verify that different values were found, demonstrating a possible interference of the support on the stability of the enzyme to the pH. The immobilized catalyst showed higher stability to acid pHs, compared to its free form.
The effect of the pH under the activity of inulinase from K. marxianus var. bulgaricus immobilized in gelatin was evaluated by
Paula et al. (2008). The authors verified that, from pH values of 2.5–8.0, higher inulinase activities were obtained at 3.5. (Ettalibi and Baratti, 2001) obtained an optimal pH value of 5.0 for the activity of inulinase from A. ficuum immobilized in porous glass.
Wenling et al. (1999)found an optimal pH value of 5.0 at 50 1C for an immobilized form of inulinase from Kluyveromyces sp. Y-85. 3.5.4. Kinetic parameters kmand vmax
The effect of substrates concentration ([S]) (sucrose and inulin) on inulinase activity was evaluated in the range of 0.5–35 g L1. The correlation between 1/v and 1/[S] for each substrate allowed determining the kinetic parameters km and vmax. The following regression equations were obtained for each substrate
1/v¼ 0.0059 (1/[S]) þ4.0205; R2¼0.96, for sucrose (1) 1/v¼ 0.4963 (1/[I]) þ4.1726; R2¼0.98, for inulin (2) The km values for sucrose and inulin were 1.46 mM and 0.38 mM, respectively, and the vmax values were 0.249 mol L min1and 0.239 mol L min1. These parameters permitted us to verify that the low value for km(0.38 mM) and high value for vmax(0.239 mol L min1) demonstrates the highest affinity of the catalyst under inulin compared to sucrose.
Astolfi et al. (2011) evaluated the kinetic parameters of the crude free enzymatic extract of inulinase from K. marxianus NRRL Y-7571 and obtained km values for sucrose and inulinase of 2.94 mM and 8.71 mM, respectively, and the vmax values were 0.053 mol L min1and 0.017 mol L min1. These parameters per-mitted us to verify that the free inulinase presented higher affinity under sucrose, differently of the immobilized catalyst, demonstrating the change on the enzyme affinity by the substrate after the immobilization process.
(Ettalibi and Baratti, 2001) immobilized inulinases from Asper-gillus ficuum and obtained values of 0.060 M for kmand 192 U g1 for vmax, using sucrose as substrate. Nguyen et al. (2011), evaluating the kinetic parameters km and vmax for free and immobilized inulinase using chitin as support, found values of 2.04% (w/v) and 80.88 U/mg, and 2.19% (w/v) and 291.58 U/g, respectively, using sucrose as substrate.
4. Conclusions
New experimental data for the immobilization of inulinase from K. marxianus NRRL Y-7571 using montmorillonite as support, indicating the technical feasibility of the process, are presented in this work. Besides, at maximized experimental conditions for enzyme immobilization, the extract was partially characterized. The information presented here is not available in the literature, showing a promising perspective for the purpose of implementing the scale up of the process of inulinase immobilization.
Results obtained for inulinase immobilization showed that the best experimental condition for the process was at an enzyme to buffer ratio of 3:10 and immobilization time of 10 min, reaching a specific activity of 375.07 U/mg of protein, percent of retention of 42.08% and a yield of immobilization of 63.67%. The partial characterization of the immobilized extracted showed that the immobilized inulinase kept its activity after 1968 h under storage at low temperatures and after 456–1826 h at high temperatures. The pH value of 3.5 led to the highest specific activity. The kinetic constants were determined using sucrose and inulin as substrates. Kmvalues of 1.46 and 0.38 mM, and vmaxof 0.2487 and 0.2396 mol/ L min, were obtained, respectively, for sucrose and inulin. References
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