DSpace at VNU: Biochemical studies on the immobilized lactase in the combined alginate-carboxymethyl cellulose gel

Biochemical Engineering Journal 74 (2013) 81–87 Contents lists available at SciVerse ScienceDirect Biochemical Engineering Journal journal homepage: www.elsevier.com/locate/bej Regular article Biochemical studies on the immobilized lactase in the combined alginate–carboxymethyl cellulose gel Thi Hai Anh Mai , Van Nguyen Tran, Van Viet Man Le ∗ Department of Food Technology, Ho Chi Minh City University of Technology, 268 Ly Thuong Kiet Street, District 10, Ho Chi Minh City, Viet Nam a r t i c l e i n f o Article history: Received June 2012 Received in revised form 24 February 2013 Accepted March 2013 Available online 14 March 2013 Keywords: Alginate Carboxymethyl cellulose Enzyme activity Immobilization Immobilized enzyme Lactose a b s t r a c t Addition of carboxymethyl cellulose to alginate gel significantly improved the yield of lactase immobilization and the total and specific activity of the fixed enzyme It was due to a decrease in protein loss during the gel bead formation in the enzyme immobilization procedure and an increase in specific surface area of the gel beads When the weight ratio of carboxymethyl cellulose to sodium alginate in the gel support was 1.0:1.5, the yield of lactase immobilization achieved 58.2% and this value was 14.2% higher than the yield of lactase immobilization in alginate gel The immobilized lactase in the combined alginate–carboxymethyl cellulose gel exhibited higher thermal and pH stability than the fixed enzyme in the conventional alginate gel However, the Michaelis–Menten constant (Km ) increased from 99.57 mM (fixed lactase in alginate gel) to 107.24 mM (fixed lactase in alginate–carboxymethyl cellulose gel) while the apparent turnover number (Kcat ) and the specificity constant (Kcat /Km ) of the both immobilized biocatalysts were statistically similar © 2013 Elsevier B.V All rights reserved Introduction Lactose is the main sugar in milk and whey, and its utilization is rather limited due to its low solubility and indigestibility in many people [1] The hydrolysis of lactose is a promising process in the food industry for the development of new products with no lactose in their composition [2] The ␤-galactosidase or lactase (E.C 3.2.1.23) of Aspergillus oryzae has received special interest for use in hydrolyzing lactose in milk and whey due to its optimum acidic pH and high thermal stability [3,4] ␤-Galactosidase can be used in two forms: in the soluble enzyme form, which is normally used in batch processes and the immobilized form, which is used preferably in continuous operation [5,6] During the last decades, immobilized lactase has been attracted great attention [7] Immobilization allows the reutilization of enzymes, provides control of the catalytic process, provides high selectivity, allows large quantities of substrate to be processed, increases enzyme stability and permits continuous operation [8,9] Among different techniques for lactase immobilization, enzyme entrapment in calcium alginate gel offers certain advantages due to its simplicity and non-toxic character The main operation of the ∗ Corresponding author Tel.: +84 38 64 62 51; fax: +84 38 63 75 04 E-mail addresses: lvvman@hcmut.edu.vn, lvvman2003@yahoo.com (V.V.M Le) Present address: Faculty of Agriculture and Forestry, Tay Nguyen University, 567 Le Duan Street, Buon Ma Thuot City, Daklak Province, Viet Nam 1369-703X/$ – see front matter © 2013 Elsevier B.V All rights reserved http://dx.doi.org/10.1016/j.bej.2013.03.003 lactase immobilization procedure in alginate gel is dropping the mixture of alginate and enzyme preparation into calcium chloride solution for formation of gel beads [7,10–12] During this operation, high protein loss was reported [10] It should be noted that low entrapment efficiency increases the production cost of the immobilized lactase For the first time, Dashevsky [10] investigated water loss during the formation of gel beads for lactase immobilization in alginate gel When the mixture of alginate and enzyme preparation was extruded into calcium chloride solution, the carboxylate groups of the guluronate monomers in alginate interacted with calcium cations and the weight of the formed gel beads decreased due to “syneresis” The contraction phenomenon by the alginate crosslinking and subsequent water leakage was the reason for the loss of water soluble protein [10] For improvement in protein entrapment efficiency in alginate gel, reduction in water loss during the gel formation is essential Dashevsky added high water absorbent like bentonite to alginate solution for reduction in water loss during the gel formation and that significantly decreased the protein loss during lactase immobilization in alginate gel [10] In addition, some researchers combined alginate with other hydrophilic polymers like gelatin [13], hydroxypropyl methyl cellulose [14] as new matrixes for lactase or protein immobilization However, none of these studies focused on the loss of protein during the formation of the gel beads Carboxymethyl cellulose is a water soluble cellulose derivative Due to ability of gel formation, the presence of carboxymethyl 82 T.H.A Mai et al / Biochemical Engineering Journal 74 (2013) 81–87 cellulose in gel products provides excellent water retention properties This polymer has been used as an additive in food industry [15–17] In this study for the first time, the combination of alginate and carboxymethyl cellulose was used as a matrix for lactase immobilization The objective of this research was: (i) to evaluate the yield of lactase immobilization in the combined alginate–carboxymethyl cellulose gel and (ii) to determine the temperature profile, thermal stability, pH profile, pH stability and kinetic parameters of the immobilized and free lactase Materials and methods 2.1 Materials ␤-Galactosidase G5160 from A oryzae was purchased from Sigma–Aldrich (The United States) The preparation was in the form of lyophilised powder with the lactase activity of U mg−1 One unit (U) of enzyme activity was defined as the amount of enzyme able to form ␮mol o-nitrophenol from ortho-nitrophenyl-␤-dgalactopyranoside (ONPG) at 30 ◦ C and pH 4.5 in Sodium alginate and carboxymethyl cellulose were provided by Kanto Chemical Co (Japan) Bovine serum-albumin, Coomassie Brilliant Blue, lactose monohydrate and Merckoquant Glucose Test Strips used for glucose analysis were obtained from Merck (Germany) 2.2 Experimentation 2.2.1 Evaluation of the yield of lactase immobilization in the combined alginate–carboxymethyl cellulose gel Carboxymethyl cellulose and sodium alginate with the weight ratio varied from 0.0:1.0 (control sample) to 1.0:3.0, 1.0:2.0, 1.0:1.5 and 1.0:1.0 were together dissolved in water; the total concentration of the matrix solution obtained was fixed at 30 (g/L) Lactase preparation was also dissolved in acetate buffer (pH 4.5) to form an enzyme solution of 50 mg protein/L The matrix solution and lactase solution were then mixed with the volume ratio of 2:1 and homogenized by magnetic stirring during 30 The mixture was slowly extruded as droplets through a mL syringe into 0.2 M CaCl2 solution The formed beads were retained in CaCl2 solution during h for gel hardening The obtained gel beads were 2–3 mm in diameter Finally, the gel beads were separated by using a screen with pore size of 0.5 mm and washed with distilled water times The biocatalyst was ready for use The yield of lactase immobilization Y was calculated by the following equation [18]: Y= (A − B) × 100, A where A is the protein content (mg) in the enzyme solution which was mixed with the matrix solution for lactase immobilization and B is the total protein content (mg) in the CaCl2 solution for gel hardening and in the washing solution of the gel beads in the immobilization procedure 2.2.2 Determination of temperature profile and thermal stability of the immobilized lactase The temperature profile was tested by change of the ␤galactosidase assay conditions in which the temperature was varied from 30 to 70 ◦ C The thermal stability of the free lactase, immobilized lactase in the combined alginate–carboxymethyl cellulose gel and immobilized lactase in the alginate gel were determined by measuring the residual activity of the enzyme exposed to three different temperatures (55, 60 and 65 ◦ C) in acetate buffer (0.1 M, pH 4.5) for 180 After every 30 min, a sample was taken and assayed for enzymatic activity The inactivation rate constant, k, and the half-life, t1/2 , were calculated with the following equation [19]: [A] = [Ao ] × e−kt , where k is the inactivation rate constants (min−1 ), Ao is the initial activity (U/mg protein enzyme) and A is the activity after a time t (U/mg protein enzyme) 2.2.3 Determination of pH profile and pH stability of the immobilized lactase The pH profile was examined by change of the ␤-galactosidase assay conditions in which pH was varied from 4.5 to 6.5 The pH stability of the free lactase, immobilized lactase in the combined alginate–carboxymethyl cellulose gel and immobilized lactase in the alginate gel were determined by measuring the residual activity of the enzyme exposed to three different pH values in acetate buffer (0.1 M, pH 4.5 and 5.5), in Tris–HCl buffer (pH 6.5) for 180 After every 30 min, a sample was also taken and assayed for enzymatic activity The inactivation rate constant, k, and the half-life, t1/2 , were calculated by the same equation mentioned in Section 2.2.2 2.2.4 Determination of kinetic parameters Apparent Km and Vmax values of the free lactase, immobilized lactase in the combined alginate–carboxymethyl cellulose gel and immobilized lactase in alginate gel were determined by Lineweaver–Burk method using various lactose concentration (from 0.06 to 0.6 M) The volume of each sample was fixed at 25 mL The total enzyme activity in each sample was 20 U The experiment was carried out at 55 ◦ C and pH 4.5 using acetate buffer (0.1 M) The turnover number (Kcat ) of the free and fixed biocatalysts was calculated from Vmax ; the molecular weight of lactase from A oryzae was refered from the research of Tanaka et al [20] 2.3 Analytical methods 2.3.1 Protein Protein was quantified by Bradford method; bovine serum album was used as a standard protein [21] 2.3.2 Lactase One unit (U) of enzyme activity was defined as the amount of enzyme able to form ␮mol glucose per from lactose under the defined conditions Lactose was dissolved in acetate buffer to form a 4.5% lactose solution The pH and temperature were varied according to each experiment The free and immobilized enzyme was added to lactose solution for lactose hydrolysis The reaction time was fixed at 15 For lactase inactivation, the reaction mixture was boiled in a water bath for Glucose released was measured by the enzymatic method with glucose kit [22] using Merckoquant Glucose Test Strips and Reflectometer RQflex plus 10 2.3.3 Moisture Moisture content of the gel beads was determined by drying method using a moisture analyzer (AND–P1014258) [23] 2.3.4 Morphology of gel beads Morphology of gel beads was evaluated with scanning electron microscopy (SEM) The gel beads were freeze-dried and observed under scanning electron microscope (HITACHI-9154-02) [24] 2.3.5 Specific surface area of gel beads Specific surface area of the gel beads was determined by MultiPoint BET method and NOVA 1200e equipment (Quantachrome02090-AG-1) [25] T.H.A Mai et al / Biochemical Engineering Journal 74 (2013) 81–87 64 Yield of immobillized lactase (%) 2.3.6 Mechanical properties of gel beads Mechanical properties including hardness, cohesiveness and gumminess of the gel beads with the immobilized lactase were evaluated by using a texture analyzer (Brookfield CT3) In texture profile analysis, trigger was 4.5 G compressed twice into each gel sample at a defined rate of mm s−1 to a depth of mm A delay period was 15 s between the two compressions At least five replicate analyses were performed for each formulation at room temperature using a fresh sample in each case Data collection and calculation were performed using the Texture Exponent 3.0.5.0 software package of the instrument Hardness, cohesiveness and gumminess were defined from the resultant force–time plot [26] 56 52 48 44 40 Fig 1A shows that the addition of carboxymethyl cellulose to the sodium alginate solution significantly increased the yield of lactase immobilization The higher level of carboxymethyl cellulose added to the matrix solution, the higher yield of lactase inclusion in the combined alginate–carboxymethyl cellulose gel The maximum yield of lactase immobilization achieved when the weight ratio of alginate to carboxymethyl cellulose was 1.0:1.0 However, the gel beads obtained were irregular in shape The alginate concentration in these gel beads was low (0.015 g/cm3 ) Panesar et al fixed lactase in alginate gel and stated that low alginate level in the gel beads reduced the gel mechanical stability [27] When the weight ratio of carboxymethyl cellulose to sodium alginate changed from 1.0:3.0 to 1.0:2.0 and 1.0:1.5, the gel bead shape was uniform The ratio of 1.0:1.5 was therefore selected for lactase fixation in the combined gel At this ratio, the yield of lactase immobilization achieved 58.2% and this value was 14.1% higher than that in the conventional alginate gel It was observed that the presence of carboxymethyl cellulose in alginate gel enhanced the moisture content of the gel beads and that was due to water retention properties of this hydrophilic polymer [15–17] Our results proved that the higher the carboxymethyl cellulose level in the combined gel, the higher the moisture content in the gel beads obtained With the weight ratio of carboxymethyl cellulose to alginate of 1.0:1.5, the moisture content of the gel beads obtained was 98.1% while the conventionally alginate gel beads in the control had the moisture content of 92.2% Due to high water solubility of enzyme protein, carboxylmethyl cellulose decreased water loss as well as protein loss during the gelation in the enzyme immobilization procedure In this study, the protein level in the combined alginate–carboxymethyl cellulose gel was 14.2% higher than that in the alginate gel Similar observation was previously noted when bentionite was added to alginate gel for lactase immobilization [10] Fig 1B presents the total activity of the biocatalysts All samples with the immobilized lactase in the combined alginate–carboxymethyl cellulose gel exhibited higher catalytic activity than the control It was mainly due to higher protein level in the combined gel beads 1.0:1.5 1.0:2.0 1.0:3.0 0.0:1.0 1.4 AcƟvity of immobillized lactase (U/g of support) 3.1 Evaluation of the yield of lactase immobilization in the combined alginate–carboxymethyl cellulose gel 1.0:1.0 Weigh raƟo of carboxylmethyl cellulose to alginate (w/w) B 1.2 1.0 0.8 0.6 0.4 1.0:1.0 1.0:1.5 1.0:2.0 1.0:3.0 0.0:1.0 Weigh raƟo of carboxylmethyl cellulose to alginate (w/w) 90.0 AcƟvity of lactase (U/mg of immobillized protein) Results and discussion A 60 2.4 Statistical analysis All experiments were carried out in triplicate Means values were considered significantly different when P value was less than 0.05 One-way analysis of variance was performed using the software Statgraphic Centurion, version XV 83 C 80.0 70.0 60.0 50.0 40.0 30.0 20.0 10.0 0.0 1.0:1.0 1.0:1.5 1.0:2.0 1.0:3.0 0.0:1.0 Weigh raƟo of carboxylmethyl cellulose to alginate (w/w) Fig Effect of weight ratio of carboxymethyl cellulose to sodium alginate to the yield of lactase immobilization (A) and the total and specific activity of the biocatalyst (B and C) It should be noted that the addition of carboxymethyl cellulose to alginate gel also enhanced the specific activity of the immobilized lactase in comparison with the control (Fig 1C) According to Bosley and Clayton, mass transfer limitation was the reason for reduction in specific activity of immobilized enzyme [28] Fig shows the morphology of the gel beads with the immobilized lactase The outer surface of the combined alginate–carboxymethyl cellulose gel bead (Fig 2B and D) was visibly rougher and more wrinkled than that of the alginate gel beads (Fig 2A and C) In addition, the cross-section of the combined gel bead (Fig 2F) was apparently more porous than that of the alginate gel bead (Fig 2E) Our experimental results revealed that the specific surface area of the combined alginate–carboxymethyl cellulose gel beads and alginate gel beads was 5.47 m2 /g and 2.58 m2 /g, respectively Increase in specific surface area of the gel beads would 84 T.H.A Mai et al / Biochemical Engineering Journal 74 (2013) 81–87 Fig Scanning electron micrograph of the surface (A and C) and the cross-section (E) of alginate gel bead, of the surface (B and D) and the cross-section (F) of the combined alginate–carboxymethyl cellulose gel bead (the weight ratio of carboxymethyl cellulose to sodium alginate in the combined gel was 1.0:1.5) improve the mass transfer during the enzymatic reaction This also led to an increase in both total and specific activity of the fixed lactase in the combined gel as seen in Fig 1B and C It is worth noting that the total and specific activity of the immobilized lactase changed by the same way when the weight ratio of carboxymethyl cellulose and alginate in the gel beads was altered Mechanical properties of the gel beads with the fixed lactase are presented in Table The addition of carboxymethyl cellulose to alginate gel reduced the hardness 22.5% in comparison with the conventional alginate gel However, the cohesiveness and gumminess of the combined alginate–carboxymethyl cellulose gel and alginate gel were statistically similar These data predicted that mechanical stability of the two gels was approximately equivalent 3.2 Determination of temperature profile and thermal stability of the immobilized lactase Our results showed that the activity of all three enzyme samples achieved maximum at the temperature of 55 ◦ C (data not shown) Consequently, entrapment of lactase in the alginate gel or in the combined alginate–carboxymethyl cellulose gel did not change the enzyme temperature optimum Similar observation was also T.H.A Mai et al / Biochemical Engineering Journal 74 (2013) 81–87 85 Table Mechanical properties of the gel beads with the immobilized lactase (n = 5) Alginate–carboxymethyl cellulose gel beads* Alginate gel beads Hardness (g) Cohesiveness Gumminess (g) 88.24 ± 1.73a 113.90 ± 2.00b 0.03 ± 0.01a 0.03 ± 0.01a 3.36 ± 0.35a 3.61 ± 0.80a Different small letters in the same column mean significant difference at P < 0.05 * The weight ratio of carboxymethyl cellulose to sodium alginate in the gel beads was 1.0:1.5 90 90 Lactase acƟvity (% of iniƟal acƟvity) 100 Lactase acƟvity (% of iniƟal acƟvity) 100 80 70 60 50 40 30 20 10 80 70 60 50 40 30 20 10 0 30 60 90 120 150 180 Time (min) Fig Thermal denaturation of the soluble and entrapped lactase The enzyme activity measured at the initial moment was taken as 100%: (· · ·) free lactase; (—) lactase entrapped in alginate–carboxymethyl cellulose gel; (- - -) lactase entrapped in alginate gel: ( ) 55 ◦ C, (᭹) 60 ◦ C, and ( ) 65 ◦ C reported by Haider and Husain who immobilized lactase from A oryzae via immunoaffinity support [29] Fig shows the heat inactivation curves of the free and immobilized lactase at three temperature (55, 60 and 65 ◦ C) In addition, it should be noted that the protein leakage from the alginate and combined alginate–carboxymethyl cellulose gel beads was not observed during the investigated period In all cases, the fixed enzymes always exhibited higher temperature resistance than the free enzyme According to Bailey and Ollis, immobilization could stabilize enzyme activity due to the existence of a local environment for the immobilized enzyme which is less damaging than bulk solution conditions [19] The half-life values and the thermal inactivation rate constants for the free and immobilized lactase are presented in Table The higher temperature, the lower half-life value and the higher thermal inactivation rate constant for both free and immobilized lactase This result was in accordance of the findings of Guidini et al who immobilized lactase in an ion exchange resin by the ionicbinding and crosslinking methods [30] It is worthy to note that addition of carboxymethyl cellulose in alginate gel improved 54.6% the half-life value as well as reduced 33.2% the thermal inactivation rate constant for the new immobilized biocatalyst in comparison with the lactase entrapped in alginate gel at 65 ◦ C 30 60 90 Time (min) 120 150 180 Fig pH denaturation of soluble and entrapped lactases The enzyme activity measured at the initial moment was taken as 100%: (· · ·) free lactase; (—) lactase entrapped in alginate–carboxymethyl cellulose gel; (- - -) lactase entrapped in alginate gel: pH ( ) 4.5, (᭹) 5.5, and ( ) 6.5 3.3 Determination of pH profile and pH stability of the immobilized lactase Similarly to the temperature profile, our results also revealed that all three enzyme samples had the optimum pH of 4.5 (data not shown) Grosová et al also reported that immobilization of lactase in polyvinylalcohol hydrogel did not change the optimal pH value in comparison with the free enzyme [31] The inactivation curves of the free and immobilized lactase at three pH value (4.5, 5.5 and 6.5) are given in Fig The activity of the immobilized lactase in the combined gel always remained higher than that of the free enzyme and that of the enzyme entrapped in the alginate gel for the same incubation time Moreover, the liquid fraction of all samples with immobilized lactase exhibited no protein leakage during the examined period It can be concluded that the presence of carboxymethyl cellulose in the combined gel bead decelerated the reduction in lactase activity during the storage of enzyme preparation However, a study on change in enzyme tridimentional structure should be carried out to clearly understand the phenomenon observed When the pH change from 4.5 to 6.5, the half-life value of the lactase fixed in the combined gel was 100.0–133.3% and 31.8–35.9% higher than that of the free lactase and the lactase fixed in the Table Half-lives (t1/2 ) and inactivation rate constant (k) of the free and immobilized lactase in alginate–carboxymethyl cellulose gel and alginate gel at three different temperatures Temperature (◦ C) 55 60 65 Free lactase Lactase entrapped in alginate– carboxymethyl cellulose gel Lactase entrapped in alginate gel t1/2 (min) k (min−1 ) t1/2 (min) k (min−1 ) t1/2 (min) k (min−1 ) 139 21 5.0 × 10−3 3.2 × 10−2 12.2 × 10−2 315 45 17 2.2 × 10−3 1.5 × 10−2 4.1 × 10−2 248 36 11 2.8 × 10−3 2.0 × 10−2 6.2 × 10−2 86 T.H.A Mai et al / Biochemical Engineering Journal 74 (2013) 81–87 Table Half-lives (t1/2 ) and inactivation rate constant (k) of the free and immobilized lactase in alginate–carboxymethyl cellulose gel and alginate gel at three different pH values pH Free lactase k (min−1 ) t1/2 (min) 4.5 5.5 6.5 Lactase entrapped in alginate gel Lactase entrapped in alginate– carboxymethyl cellulose gel 5.1 × 10 × 10−3 8.31 × 10−2 135 87 k (min−1 ) t1/2 (min) −3 −3 2.2 × 10 3.9 × 10−3 4.3 × 10−2 315 178 16 t1/2 (min) k (min−1 ) 239 131 12 2.9 × 10−3 5.3 × 10−3 5.8 × 10−2 Table Apparent Km and Kcat values of the free and immobilized lactase at pH 4.5 and temperature 55 ◦ C Km (×10−3 M) Kcat (min−1 ) Kcat /Km (M−1 min−1 ) Free lactase Immobilized lactase in alginate–carboxymethyl cellulose gel Immobilized lactase in alginate gel 73.48 ± 2.40a 7.66 ± 0.03a 104.24 ± 3.67a 107.24 ± 0.18b 0.61 ± 0.02b 5.66 ± 0.22b 95.57 ± 4.24c 0.63 ± 0.02b 6.29 ± 0.53b Different small letters in the same row mean significant difference at P < 0.05 alginate gel, respectively (Table 3) In addition, the thermal inactivation rate constant of the immobilized lactase in the new combined support was 48.9–56.9% and 24.1–26.4% lower than that for the free enzyme and the enzyme in alginate gel, respectively In summary, the biocatalyst in the new combined gel exhibited high resistance to pH change from 4.5 to 6.5 and that would be applied to lactose hydrolysis in whey (pH 4.6) and milk (pH 6.6) [32] 3.4 Determination of kinetic parameters Table presents some kinetic parameters of the free and fixed lactase Immobilization of lactase in the gels increased apparent Km values while reduced apparent Kcat values Increase in apparent Km value reduced affinity of the enzyme for its substrate and that led to a decrease in enzymatic reaction rate This was due to the lower accessibility of the substrate to the active site of the immobilized lactase or lower transporting of the substrate and products into and out the gel beads [19,33] Many researchers stated that immobilization in alginate–gelatin fibers [13] or polyvinyl alcohol gel [31] increased Michaelis–Menten constant for lactase The results in Section 3.1 of this study affirmed that the specific surface area of the combined alginate–carboxymethyl cellulose gel beads was significantly higher than that of the alginate gel beads That means the accessibility of the substrate to the active site of the immobilized lactase in the combined gel would be better than that in the alginate gel Nevertheless, the apparent Km of the fixed lactase in the combined alginate–carboxymethyl cellulose gel was 12.2% higher than that of the enzyme entrapped in the conventional alginate gel In addition, the apparent turnover number (Kcat ) and the specificity constant (Kcat /Km ) for the both immobilized biocatalysts were statistically similar (Table 4) This was probably due to high level of galactose accumulated in the gel beads and that would reduced the rate of enzymatic reaction This hypothesis will be investigated in the next study Conclusions The higher level of carboxymethyl cellulose added to the alginate solution, the 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the fixed lactase in the combined alginate–carboxymethyl cellulose gel was 12.2%... 22.5% in comparison with the conventional alginate gel However, the cohesiveness and gumminess of the combined alginate–carboxymethyl cellulose gel and alginate gel were statistically similar These