Bioprocess Biosyst Eng DOI 10.1007/s00449-015-1407-6

ORIGINAL PAPER

Immobilization of b-galactosidase from Lactobacillus plantarum HF571129 on ZnO nanoparticles: characterization and lactose hydrolysis E. Selvarajan1 • V. Mohanasrinivasan1 • C. Subathra Devi1 • C. George Priya Doss1

Received: 2 February 2015 / Accepted: 19 April 2015 Ó Springer-Verlag Berlin Heidelberg 2015

Abstract b-Galactosidase from Lactobacillus plantarum HF571129 was immobilized on zinc oxide nanoparticles (ZnO NPs) using adsorption and cross-linking technique. Immobilized b-galactosidase showed broad-spectrum pH optima at pH 5–7.5 and temperature 50–60 °C. Fourier transform infrared spectroscopy (FTIR) and field emission scanning electron microscopy (FESEM) showed that bgalactosidase successfully immobilized onto supports. Due to the limited diffusion of high molecular weight substrate, Km of immobilized enzyme slightly increased from 6.64 to 10.22 mM, while Vmax increased from 147.5 to 192.4 lmol min-1 mg-1 as compared to the soluble enzyme. The cross-linked adsorbed enzyme retained 90 % activity after 1-month storage, while the native enzyme showed only 74 % activity under similar incubation conditions. The cross-linked b-galactosidase showed activity until the seventh cycle and maintained 88.02 % activity even after the third cycle. The activation energy of thermal deactivation from immobilized biocatalyst was 24.33 kcal/mol with a half-life of 130.78 min at 35 °C. The rate of lactose hydrolysis for batch and packed bed was found to be 0.023 and 0.04 min-1. Keywords b-Galactosidase  Zinc oxide nanoparticles  Lactose hydrolysis  Glutaraldehyde  FTIR

Electronic supplementary material The online version of this article (doi:10.1007/s00449-015-1407-6) contains supplementary material, which is available to authorized users. & V. Mohanasrinivasan [email protected] 1

School of BioSciences and Technology, VIT University, Vellore, Tamil Nadu, India

Abbreviations FTIR Fourier transform infrared spectroscopy ZnO NPs Zinc oxide nanoparticles GRAS Generally recognized as safe SDS-PAGE Sodium dodecyl sulfate-polyacrylamide gel electrophoresis FESEM Field-emission scanning electron microscopy ONPG O-Nitrophenyl-b-D-galactopyranoside Kd Deactivation rate constants Vmax Maximum reaction velocity Km Michaelis–Menten constants DH Enthalpy DG Gibbs free energy DS Entropy

Introduction b-Galactosidase exists in a wide range of organisms and is well known for its catalytic activity. Microbial b-galactosidases have a prominent position in terms of their role in the production of various industrially relevant products like biosensor, lactose hydrolyzed milk, the production of galacto-oligosaccharides for use in probiotic foodstuffs, etc. The enzyme can be obtained from microbial cells such as bacteria, fungi or yeast with variable properties depending on the species [1–3]. Although, Escherichia coli produces the most studied b-galactosidase, possible toxic factors associated with coliforms make it unlikely that crude isolates of this enzyme may be permitted in food processes [4]. The major b-galactosidase enzymes of commercial interest are isolated mainly from the yeast

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Kluyveromyces lactic, K. fragilis, K. marxianus, Candida kefyr and Saccharomyces cerevisiae and the fungi Aspergillus niger and A. oryzae [5]. On the basis of amino acid similarities, b-galactosidases have been separated into four glycoside hydrolase (GH) families: 1, 2, 35 and 42 [6–9]. Recently, there have been more extensive applications of metal oxide nanoparticles, covering different fields such as optoelectronics, catalysis, medicine and sensor devices. Among the metal oxide nanostructures, ZnO NPs have been extensively investigated for several technological applications such as catalysis, gas sensing [10], cancer treatment, chemical absorbent [11], antibacterial and UV blocking functions [12–14] and in cosmetic and pharmaceutical industries [15]. In the recent past, metal oxide nanoparticles have been exploited as a potential candidate in immobilizing industrially important enzymes [16]. Apart from this, Zn compounds have been currently listed as GRAS, i.e., regarded as safe by the US Food and Drug Administration (21CFRI82.8991). Nanostructures are very attractive for enzymatic immobilization processes, since they possess the ultimate characteristics to equilibrate principal factors that determine biocatalyst efficiency, including specific surface area, mass transfer resistance and effective enzyme loading. Enzymes immobilized on nanoparticles exhibit high stability in a wide range of temperature and pH, compared to free enzymes. Many materials are used at nano size in processes of immobilization, such as silica, chitosan, gold, diamond and metals, including graphene and zirconium [17]. For this reason, nanomaterials have attracted more attention for utilization as support material for efficient enzyme immobilization. Nanomaterials can be used in the production of lactose-reduced dairy commodities at industrial scale, for harmless consumption by lactose-intolerant individuals. Packed bed reactors are habitually used for kinetic studies of heterogeneously catalyzed reactions. The employment of these reactors in biological processes could allow the application of innovative technology to hydrolyze the lactose present in milk and milk products at a commercial scale. For the reaction system with product inhibition, the process efficiency in a packed bed reactor is larger, because the inhibition effect decreases due to low difference between the substrate and product concentrations [18]. Lactose hydrolysis increases the solubility from 18 to 55 % (w/v) at 80 % conversion, and the sweetness goes up to 70 % related to sucrose. Thus, the production of self-sweetening products or products with less sucrose addition would be possible using lactose-hydrolyzed milk. In view of the scarcity of research in this area, we initiated this study to immobilize b-galactosidase on ZnO nanoparticles (NPs) or ZnO NPs and to develop technology for lactose hydrolysis.

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Experimental Materials and methods utilized O-Nitro phenyl-b-galactopyranoside, ethanolamine, starch and glutaraldehyde were obtained from SRL Chemicals (Mumbai, India). Sodium alginate was purchased from Thomas Baker Chemical Co. Mumbai, India. The other reagents and chemicals hired were of analytical grade and used without any further purification. ZnO NPs (biosynthesized) with size 19 nm were used in this study [19]. Enzyme preparation was carried out using a recent protocol [20]. Homogeneity of the purified preparation was checked by SDS-PAGE and gel permeation chromatography. The enzyme was highly purified with a specific activity of 143.7 IU/mg. Preparation and characterization of calcium alginate–starch beads An aqueous mixture of sodium alginate (2.5 %) and starch (2.5 %) was slowly extruded as droplets through a 10.0-mL syringe with an attached gauge needle No. 20 into 0.2 M calcium chloride solution. The formation of calcium alginate–starch beads was instantaneous and the mixture was gently stirred in calcium chloride solution for hardening (12 h) [21]. The beads were washed and stored in 0.1 M sodium phosphate buffer, pH 6.5 at 4 °C, until further use. The swelling ratio of the calcium alginate–starch beads was determined using a volumetric cylinder. The height of the dry beads (Hd) was measured, followed by adding of distilled water into the volumetric cylinder and mixed well at 50 rpm for 24 h. Then, the height of the swollen beads (Hs) was recorded. The following equation determined the swelling ratio. Equilibrium water swelling ratio = (Hs/Hd) Optimization of immobilization of b-galactosidase on bulk ZnO and ZnO NPs Soluble b-galactosidase (68.14 U) was independently adsorbed at varying concentrations of bulk ZnO and ZnO NPs (20–120 mg) overnight in 0.1 M sodium phosphate buffer, pH 6.5 at room temperature. Adsorption of b-galactosidase on ZnO NPs b-Galactosidase (170.35 U) was mixed with 100 mg ZnO NPs and continuously stirred overnight in sodium phosphate buffer, pH 6.5 at room temperature. Immobilized enzyme preparation was centrifuged at 30009g for 20 min. The resulting complex was washed thrice with

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0.1 M sodium phosphate buffer, pH 6.5, and finally suspended in the same buffer and stored at 4 °C for further use. Binding of adsorbed ZnO NPs b-galactosidase on the surface of calcium alginate–starch beads Calcium alginate–starch beads were incubated overnight with adsorbed ZnO NPs b-galactosidase (170.35 U) at room temperature with slight stirring. The bound enzyme was separated from unbound enzyme by repeatedly washing with 0.1 M sodium phosphate buffer, pH 6.5. Cross-linking of immobilized b-galactosidase ZnO NPs-layered calcium alginate–starch beads immobilized by b-galactosidase were cross-linked with 0.5 % (v/v) glutaraldehyde for 2 h at 4 °C. Ethanolamine was added to a final concentration of 0.01 % (v/v) to stop the crosslinking. Cross-linked beads were allowed to stand with ethanolamine for 90 min at 30 °C. No enzyme activity was released from the beads, indicating complete cross-linking of the immobilized enzyme. Scanning electron microscopy and FTIR spectra For electron microscopic studies, 20 lg of sample was sputter-coated on copper stub, and the images of immobilized b-galactosidase were studied using field emission scanning electron microscopy (FE-SEM JSM-6700, ZEISS, Japan). The Fourier transform infrared spectroscopy (FTIR) analysis of immobilized b-galactosidase was performed to confirm binding of the enzyme to nanoparticles. FTIR spectra of the immobilized b-galactosidase were recorded over a range of 500–4000 cm-1 on IRAffinity-1, Shimadzu, Japan. Effect of pH and temperature The activity of b-galactosidase (3.0 U) was assayed in the three buffers of different pH values (4.5–9.0). The buffers with 20 mM used were sodium acetate (pH 4.5–6.0), sodium phosphate (pH 6.0–7.5) and glycine (pH 7.5–9.0). The activity at pH 6.5 was taken as control (100 %) relative activity percentage. The effect of temperature on soluble and immobilized b-galactosidase (3.0 U) was determined by measuring the activity of the enzyme at different temperature ranges from 20 to 75 °C. Effect of galactose and CaCl2 The activity of soluble, immobilized and cross-linked bgalactosidase (3.0 U) was determined in the presence of

increasing concentrations of galactose (1.0–5.0 %, w/v) and CaCl2 (1.0–5.0 %, w/v) in 20 mM sodium phosphate buffer, pH 6.5 at 37 °C for 1 h. The activity of the enzyme without added galactose and CaCl2 was considered as control (100 %) for the calculation of remaining percent activity. Effect of salivary a amylase and trypsin b-Galactosidase (3.0 U), adsorbed with ZnO NPs bound on the surface of beads and cross-linked with glutaraldehyde, was incubated with increasing concentrations of salivary a amylase (20–140 U) and trypsin (0.02–0.10 mg/mL) in 20 mM phosphate buffer, pH 6.5 for 4 h and at 37 °C. The activity of the enzyme without the salivary a amylase and trypsin treatment was considered as 100 % for the calculation of remaining activity. Thermal denaturation for soluble and immobilized b-galactosidase The stability of soluble and immobilized enzyme was investigated by incubating at 60 °C in 20 mM sodium phosphate buffer (pH 6.5) several times. Aliquots of each preparation (3.0 U) were withdrawn at specific time intervals and kept on ice for 5 min. Once the incubation time was over, the enzyme was brought to room temperature. The enzyme activity without incubation at 60 °C was considered as control for the calculation of remaining percent activity. Storage stability and reusability Soluble, immobilized and cross-linked adsorbed b-galactosidase was stored at 4 °C in 20 mM sodium phosphate buffer, pH 6.5, for 1 month. The samples from each preparation (3.0 U) were taken at every 5-day intervals and analyzed for the remaining activity. For reusability, immobilized and cross-linked b-galactosidase (3.0 U) was taken for assaying its activity upon repeated usage. After each assay, the beads were taken from assay tubes and stored in 20 mM sodium phosphate buffer, pH 6.5, overnight at 4 °C for 7 days. The activity determined on the first day was treated as control for determining remaining percent activity. Determination of Km and Vmax The kinetic parameters of immobilized b-galactosidase were determined in the standard assay mixture at pH 6.5 by a Lineweaver–Burk plot. Different concentrations of ONPG (1–22 mM) were used as substrates.

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Lactose hydrolysis Milk lactose was prepared according to the methodology given by Dwevedi et al. [22]. Defatted milk powder (10 %) was dissolved in distilled water, to avoid the turbidity of the solution for uninterrupted spectroscopic analysis. The reaction started by adding 50 U of soluble, 400 U of immobilized and cross-linked enzyme in 5 ml of prepared milk at 50 °C. Aliquots of 20 lL were withdrawn at regular time intervals and glucose content released was estimated using the GOD–POD method. The percentage of unhydrolyzed lactose was calculated as % Lactose unhydrolyzed glucose present before b galactosidase treatment ¼ : glucose present after b galactosidase treatment A plot was generated with log % unhydrolyzed lactose versus time, and the rate constant of lactose hydrolysis was examined from the slope of the plot using the formula: k 100 Slope ¼ 2:303 ; whereas rate constant k ¼ 2:303 t log 100x : Calibration of lactose hydrolysis Calibration of lactose hydrolysis was done based on the varying lactose concentration (0.5–5 %) was prepared in distilled water. The calcium alginate–starch beads (bgalactosidase immobilized with ZnO NPs) were packed in the column. 5 mL of sample was introduced from the bottom to top. Glucose content was estimated using GOD– POD method. Hill plot was generated and determined Hills coefficient. All measurements were carried out at room temperature. Statistical analysis The test for soluble, cross-linked and immobilized bgalactosidase were conducted in duplicate and the data obtained were expressed as mean ± standard error and analyzed for ANOVA followed by Student’s t test. P values \0.05 were considered to be statistically significant.

Results and discussion Preparation and characterization of calcium alginate–starch beads Alginate obtained from brown algae was broadly used as polymer for the immobilization, encapsulation and entrapment of enzymes [23–25]. Though calcium alginate beads have been used for the entrapment of enzymes, they are not preferred due to their large pore size that

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may result in enzyme leakage. Starch is a high molecular weight polymer where the enzyme was immobilized on a large surface area of the support and could hydrolyze lactose. To overcome this problem, we developed a hybrid gel of calcium alginate–starch beads. These gel beads were layered with ZnO NPs b-galactosidase from L. plantarum HF571129. The optimum distance between the orifice of the needle and the surface of CaCl2 solution was found to be 6 cm. The calcium alginate–starch beads were spherical in shape with an area of 0.25434 cm2, volume of 0.038151 cm3 and porosity of 33.3 %, respectively. Optimization of immobilized b-galactosidase on bulk ZnO and ZnO NPs Optimization of adsorption of b-galactosidase on bulk ZnO and ZnO NPs is shown in Table 1. Maximum adsorption of the enzyme on bulk ZnO (20 U) and ZnO NPs (29 U) was achieved with 100 mg for both of the support. A similar behavior has also been observed by other investigators [26]. Adsorption of b-galactosidase on the surface of calcium alginate–starch beads and cross-linked with glutaraldehyde and epichlorohydrin Table 2 shows the activity yield of immobilized b-galactosidase without ZnO NPs. Adsorption of b-galactosidase on the surface of calcium alginate–starch beads retained nearly 82.6 % of the original activity. On the other hand, cross-linking with glutaraldehyde and epichlorohydrin resulted in a loss of 5 and 16 % enzyme activity. A number of authors have previously reported the use of glutaraldehyde as a cross-linking agent [27, 28]. b-galactosidase from A. oryzae adsorbed on Con A–cellulose and cross-linked [29] and b-galactosidase from Amygdalus communis adsorbed on Con A–cellulose and cross-linked [30] showed similar activities.

Table 1 b-Galactosidase adsorption on ZnO bulk and ZnO NPs Concentration of ZnO bulk/ZnO NPs (mg)

Enzyme activity adsorbed (U) ZnO bulk

ZnO NPs

20

8

17

40

11

24

60 80

15 18

27 28

100

20

29

120

20

29

Bioprocess Biosyst Eng Table 2 Activity yield of immobilized b galactosidase without ZnO NPs Enzyme preparation

Enzyme activity, X(U)

Enzyme activity in washes, Y(U)

Activity bound/g alginate beads Theoretical (X - Y = A)

Enzyme adsorbed on the surface of beads

170.35

24

Activity yield B/A 9 100

Actual (B)

146.35

121

82.6

Enzyme adsorbed on the surface of beads and cross-linked with glutaraldehyde

146.35

114

77.89

Enzyme adsorbed on the surface of beads and cross-linked with epichlorohydrin

146.35

97

66.27

Table 3 Activity yield of immobilized b-galactosidase with ZnO NPs Enzyme preparation

Enzyme activity, X(U)

Enzyme activity in washes, Y(U)

170.35

21

Activity bound/g alginate beads

Activity yield B/A 9 100

Theoretical (X - Y = A)

Actual (B)

149.35

130

87.04

Enzyme adsorbed with ZnO NPs and bind on the surface of beads and cross-linked with glutaraldehyde

149.35

125

83.67

Enzyme adsorbed with ZnO NPs and bind on the surface of beads and cross-linked with epichlorohydrin

149.35

108

72.31

Enzyme adsorbed with ZnO NPs and bind on the surface of beads

Adsorption of ZnO NPs and b-galactosidase on the surface of calcium alginate–starch beads and cross-linked with glutaraldehyde and epichlorohydrin

a smoother surface area for the immobilization of bgalactosidase. From the SEM image, it was evident that the morphology of b-galactosidase adsorbed with ZnO NPs was spherical and well distributed with tiny aggregation.

Table 3 shows the activity yield of immobilized b-galactosidase with ZnO NPs. Adsorption of ZnO NPs and bgalactosidase on the surface of calcium alginate–starch beads retained nearly 87.04 % of the initial activity. However, cross-linking with glutaraldehyde and epichlorohydrin resulted in an activity yield of 83.61 and 72.31 %, respectively. To prevent the leaching of enzyme from the surface of calcium alginate–starch beads, the immobilized preparation was cross-linked with glutaraldehyde and epichlorohydrin. These findings correlate with the observations reported by others [31–33]. The mechanism behind the immobilization process is given in Fig. S1.

FTIR spectra analysis

Scanning electron microscopy Scanning electron microscopy (SEM) was used to study the surface morphology of immobilized b-galactosidase (enzyme adsorbed with ZnO NPs). It was observed that the surface morphology of immobilized b-galactosidase changed after immobilization and these changes can easily be seen on scanning electron micrographs (Fig. 1). The SEM image of b-galactosidase adsorbed with ZnO NPs revealed

To understand better about the functional groups involved in the immobilization of the b-galactosidase process, FTIR analysis was performed in the range of 500–4000 cm-1 (Fig. 2). The peak observed at 528 cm-1 corresponds to the peak of ZnO NPs. In addition, the band located at 3439 cm-1 is due to the vibration mode of O–H and –NH groups present in the enzyme [34]. The shifting of peak values from 1068 to 1070 and 1631 to 1639 cm-1 correspond to the interaction of –CO group of enzyme with ZnO NPs, while the broadening of peak at 528 cm-1 also revealed uniform adsorption of b-galactosidase on the nanomatrix [35, 36]. The peak obtained at 1639 cm-1 was due to amide II of the enzyme, while the peak value observed at 1400 cm-1 confirmed CH vibrations [26]. Effect of pH The effect of different pH values on the catalytic activity of immobilized b-galactosidase was investigated by performing the enzyme assay in the reaction mixture having different pH ranging from 4.5 to 9.0 (Fig. 3). Soluble and

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Fig. 3 Activity profile for soluble, immobilized and cross-linked bgalactosidase on different pH Fig. 1 SEM image of b-galactosidase immobilized on ZnO NPs

60

1400.32

1639.49 1631.78

3008.95

80

1070.49

%T

991.41

100

3439.08 3417.86

40

4000 IMM

3000

2000

1500

1000

500 1/cm

Fig. 2 FTIR spectra of immobilized b-galactosidase

immobilized enzyme preparations exhibited the same optima at pH 6.5. Immobilized and cross-linked b-galactosidase retained very high activity at both acidic and alkaline than the soluble enzyme. However, immobilized enzyme exhibited a significant broadening of pH activity profile as compared to the soluble counterpart. Cross-linked adsorbed enzyme retained 82.87 and 39.5 % of the original enzyme activity at pH 4.5 and 8.5, because soluble enzyme showed 50.26 and 8.7 % original activity under similar experimental conditions, respectively. This kind of behavior was observed earlier in the case of concanavalin A zinc oxide nanoparticles-bound b-galactosidase from A. oryzae [31]. A similar result of optimum pH for immobilized enzyme was observed in many cases, such as when bgalactosidase immobilized on concanavalin A-layered aluminum oxide nanoparticles [16], immobilization of bgalactosidase on the surface of Con A–layered calcium alginate–starch beads [33], bioaffinity-based immobilization of almond (Amygdalus communis) b-galactosidase on Con A–layered calcium alginate-cellulose beads [30], bgalactosidase immobilized on surface functionalized AgNPs [37], b-galactosidase (A. oryzae) immobilized on Con A–celite 545 and cross-linked with glutaraldehyde

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Fig. 4 Activity profiles for soluble, immobilized and cross-linked bgalactosidase at different temperatures

[32] and immobilization of b-galactosidase on Con A– cellulose support [29]. A shunt in optimum pH from 6.5 to 7.0 upon immobilization of b-galactosidase from K. fragilis on cellulose beads [38] and immobilization of bgalactosidase from K. lactis on functionalized silicon dioxide nanoparticles have also been reported [39]. Effect of temperature Figure 4 illustrates the temperature activity profiles for soluble, immobilized and cross-linked enzyme. Soluble bgalactosidase showed temperature optima at 50 °C because enzyme adsorbed with ZnO NPs and cross-linked with glutaraldehyde exhibited broadening in temperature optima from 50 to 60 °C. This increase in temperature of immobilized b-galactosidase might be because the immobilized enzyme required greater amount of activation energy as compared to soluble enzyme that was readily available in an aqueous environment of the reaction. It was observed that enzyme adsorbed with ZnO NPs and cross-linked with glutaraldehyde exhibited 66.77 % activity at 70 °C, while enzyme adsorbed with ZnO NPs and soluble enzyme retained only 40.61 and 31.26 % activity under similar experimental conditions, respectively. In all the cases, the activity of the immobilized enzyme was greater than that of

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the soluble enzyme at high temperature, which suggested that the immobilization technique adequately improves the thermal stability of the enzyme. Such enhancement in enzyme stability was due to the rigidity provided by immobilization to the enzyme [40]. In addition, the greater stability of immobilized enzyme at higher temperature makes this preparation more suitable and valuable for lactose hydrolysis in recycling processes. The results obtained are comparable as earlier reported in case of concanavalin A zinc oxide nanoparticles-bound bgalactosidase from A. oryzae [31] and b-galactosidase immobilized on the surface-functionalized AgNPs [37]. In this regard, ZnO nanoparticles-adsorbed b-galactosidase from A. oryzae showed an optimum temperature pattern similar to that of b-galactosidase adsorbed with ZnO NPs in this study [26]. It was reported that the optimum temperature of b-galactosidase from K. lactis was increased up to 10 °C as compared to the soluble enzyme when immobilized on concanavalin A-layered aluminum oxide nanoparticles [16]. b-Galactosidase from A. oryzae immobilized via the diazotization on nylon membranes [41], and immobilization of b-galactosidase from K. lactis on functionalized silicon dioxide nanoparticles [39] had a higher temperature optimum than its soluble counterpart. In contrast to this study, bioaffinity-based immobilization of almond (A. communis) b-galactosidase on Con A–layered calcium alginate–cellulose beads [30] and b-galactosidase (A. oryzae) immobilized on Con A–celite 545 [32] showed that both immobilized and soluble enzyme showed an optimum activity at 50 °C. For this reason, the immobilized enzyme could work in wild environmental conditions with less activity loss compared to its soluble counterpart [42– 44]. Effect of galactose The activity of soluble, immobilized and cross-linked bgalactosidase was investigated in the presence of various concentrations (1.0–5.0 %, w/v) of galactose (Fig. 5). Incubation of soluble b-galactosidase with 5.0 % galactose for 1 h at 37 °C resulted in a loss of 32.51 % of initial activity, while the immobilized and cross-linked enzyme retained over 46.62 and 50.29 % activity under similar incubation conditions, respectively. The findings of this galactose study were in accordance with earlier results [16, 26, 29, 31, 37], which indicated that immobilized and cross-linked b-galactosidase was significantly more resistant to galactose inhibition. A similar result was obtained by b-galactosidase (A. oryzae) immobilized on Con A– celite 545 and cross-linked with glutaraldehyde [32] where the soluble enzyme with 5 % galactose for 1 h at 37 °C resulted in a loss of 70 % activity, while the cross-linked enzyme retained 67 % of activity.

Fig. 5 Effect of galactose on soluble, immobilized and cross-linked b-galactosidase

Fig. 6 Effect of CaCl2 on soluble, immobilized and cross-linked bgalactosidase

Effect of CaCl2 The enzyme activity of soluble, immobilized and crosslinked b-galactosidase was assayed in the presence of different concentrations of calcium chloride (Fig. 6). The pre-incubation of soluble b-galactosidase with 2.0 and 4.0 % CaCl2 resulted in the loss of 25 and 49 % activity for 1 h at 37 °C, respectively. However, immobilized bgalactosidase lost only 18 and 37 % of enzyme activity because cross-linked b-galactosidase retained high activity of 87.22 and 70.47 % under the same exposure conditions extensively. Milk has a good bioavailability of calcium (30–35 %). The person who does not consume enough calcium will have the risk of developing diseases such as osteoporosis and hypocalcemia. So, we have analyzed the effect of calcium chloride on the activity of soluble and immobilized b-galactosidase. In previous studies, the effect of calcium chloride had also been reported [29, 30] which was in full concurrence with our current results. Effect of salivary a amylase The effect of salivary a amylase was observed in the stability of soluble, immobilized and cross-linked b-galactosidase (Fig. 7). The soluble enzyme retained 90.92 %

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Propionibacterium freudenrechii, but not from Propionibacterium propionicum, A. oryzae and Amygdalus communis [30, 33, 46]. Thermal denaturation of soluble, immobilized and cross-linked b-galactosidase

Fig. 7 Effect of amylase on soluble, immobilized and cross-linked bgalactosidase

residual activity after treating with 120 U of amylase, because the cross-linked enzyme retained 97.18 % activity under identical amylase treatment. Similar behavior has been reported for b-galactosidase from A. oryazae adsorbed on the surface of beads and cross-linked with glutaraldehyde [45]. To remove the lactose present in the small intestine immobilized b-galactosidase may be given orally, which contains amylase. For this reason, the stability studies should be carried out for the immobilized enzyme against salivary a amylase. Effect of trypsin The effect of increasing concentration of trypsin on soluble, immobilized and cross-linked b-galactosidase was analyzed in Fig. 8. The activity of soluble enzyme increased to just about 114 % where trypsin was treated with 0.06 mg/ml for 1 h and further the activity reduced to 87 % at higher concentration (0.10 mg/ml). Moreover, the utmost activity of 131.7 % was achieved by the cross-linked b-galactosidase in the presence of trypsin at 0.10 mg/ml whereas the activity maintained by the immobilized enzyme was 121.77 % under the same experimental circumstances. The trypsin that is present in the intestinal fluid can inactivate b-galactosidase from strains of

Fig. 8 Effect of trypsin on soluble, immobilized and cross-linked bgalactosidase

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The thermal stability of soluble, immobilized and crosslinked b-galactosidase was analyzed by pre-incubation of enzyme at 60 °C for different time intervals. Results of thermal stability of soluble, immobilized and cross-linked b-galactosidase at 60 °C are shown in Fig. 9. The initial activity was defined as 100 %. b-Galactosidase adsorbed with ZnO NPs and cross-linked with glutaraldehyde retained 75 % of enzyme activity after 2 h exposure at 60 °C at the same time as soluble enzyme retained only 43 % under the same conditions. After 5 h, the cross-linked enzyme retained higher activity (about 57 % of its initial activity) than that of soluble enzyme (about 29 % of its primary activity). These findings were in agreement with earlier studies reported in the literature. In a study performed by Ansari and Husain [29], the thermal stability of b-galactosidase immobilized on Con A–cellulose and cross-linked with glutaraldehyde indicated much higher activity when compared to soluble 1. In addition, some other studies found that the thermal stability of ZnO nanoparticles-adsorbed b-galactosidase from A. oryzae retained 55 % of activity [26], b-galactosidase immobilized on surface-functionalized Ag NPs retained 70 % of activity [37] and concanavalin A zinc oxide nanoparticles-bound bgalactosidase from A. oryzae [31] retained 60 % of activity at 60 °C after 2 h. Higher thermal stability for the immobilized b-galactosidase from K. lactis onto a polysiloxane– polyvinyl alcohol magnetic (mPOS–PVA) composite [5] and immobilization of b-galactosidase from K. lactis on functionalized silicon dioxide nanoparticles [39] were observed as compared to soluble enzyme. With this stabilizing effects, immobilized and cross-linked b-galactosidase

Fig. 9 Stability of soluble, immobilized and cross-linked b-galactosidase was studied by incubating at 60 °C in 0.1 M phosphate buffer for various times

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exhibit advantage in continuous production of lactose-free product in the food industry. Storage stability Soluble, immobilized and cross-linked adsorbed b-galactosidase preparations were stored at 4 °C and the activity was checked at every 5-day intervals. As shown in Table 4, the soluble enzyme lost nearly 74 % of activity in 30 days, while cross-linked adsorbed enzyme preparation retained over 90 % activity under similar experimental conditions. The cross-linked adsorbed b-galactosidase showed practically no leaching of enzyme (\7 %) over a period of 20 days. Understanding the storage stability of an enzyme is a prerequisite to predicting its industrial applicability. The stability of an enzyme can normally be increased by cross-linking because intra- and intermolecular cross-links lead to a stiffer molecule that can withstand conformational changes [47]. Ansari and Husain [29] reported a storage stability of 93 % of remaining enzyme activity from crosslinked Con A–cellulose-bound b-galactosidase for 30 days that is similar to our work. Ansari et al. observed that bgalactosidase immobilized on the surface of functionalized Ag NPs retained over 98 % of activity for 30 days [37]. According to Ansari and Husain [30] 2011, the storage stability of b-galactosidase (A. oryzae) immobilized on Con A–celite 545 and cross-linked with glutaraldehyde retained 90 % of activity after 30 days while soluble enzyme showed 60 % of activity. Reusability of immobilized b-galactosidase Greater surface area of the immobilizing matrix permits higher local concentration of the enzyme in accordance with the Zulu effect, due to which less loss is observed over a number of washes [48]. The recycling efficiency of immobilized and cross-linked b-galactosidase was monitored by reusing the defined amount of immobilized b-galactosidase until seven batches of reactions (Fig. 10). It was observed

that the enzymatic activity of immobilized b-galactosidase started to decrease and there was approximately 6, 9, 16 and 17 % activity loss in first, second, third and fourth batch of reaction, respectively. After the fourth batch, a slight decrease in activity was observed and 75 % of its initial activity was retained after the seventh repeated batch. Crosslinked b-galactosidase retained 81 % of the initial activity after its seventh repeated use. The results obtained are analogous as earlier reported in case of cross-linked Con A– cellulose-adsorbed b-galactosidase from A. oryzae [29], entrapped cross-linked Con A–b-galactosidase [45], ZnO NPs-adsorbed b-galactosidase from A. oryzae [26], Con A– layered Al2O3-NPs-adsorbed b-galactosidase [30], immobilization of b-galactosidase from K. lactis on functionalized silicon dioxide nanoparticles [39] and higher than that obtained using Con A-layered calcium alginate–starch beads immobilized b-galactosidase from A. oryzae [33] and bgalactosidase immobilized on surface functionalized AgNPs [37]. In addition as reported by Ansari and Husain [30], the reusability of b-galactosidase from A. oryzae immobilized on Con A–celite 545 and cross-linked with glutaraldehyde retained 64 % and 71 % of the initial activity after the seventh repeated use. Determination of Km and Vmax Kinetic parameters of immobilized b-galactosidase were investigated using different concentrations (1–22 mM) of ONPG and (1–600 mM) of lactose. The Michaelis–Menten plots (rate of the reaction versus substrate concentration) are shown in Figs. 11 and 12. The reaction rate follows the rectangular hyperbolic shape as depicted in Figs. 11 and 12. The Lineweaver–Burk plot (double reciprocal plot) was also drawn to determine the Vmax and Km values for the immobilized enzymes. The obtained linear plot indicates the hydrolysis of ONPG and lactose by the tested immobilized b-galactosidase following Michaelis–Menten kinetics, which was reflected in the adjacent R2 values (close to unity). It was observed that the Vmax value (for both the substrate) of b-galactosidase slightly increased after

Table 4 Storage stability of soluble, immobilized and cross-linked b galactosidase Number of days

Control

Remaining activity (%) Soluble

Immobilized

Cross-linked

100

100

100

5

93

94

97

10

87

91

97

15

85

90

94

20

81

88

93

25

78

87

90

30

74

85

90

Fig. 10 The reusability of immobilized and cross-linked b-galactosidase was monitored for seven days

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immobilization as compared to soluble enzyme (Table 5), suggesting that the immobilized enzyme catalyzes more rapidly and serves as a better biocatalyst for the lactose hydrolysis. The Michaelis–Menten constants (Km) of immobilized enzyme also increased when compared to the soluble enzyme, which is an indicator that the substrate affinity is found to increase after the immobilization process. The increase in the Km value could be due to the change in the microenvironment of the enzyme molecules, which in turn depends on the tertiary structure of the enzyme [49]. A similar occurrence was observed in the case of b-galactosidase immobilized on the surface functionalized AgNPs [37] and immobilization of b-galactosidase from K. lactis on functionalized silicon dioxide nanoparticles [39]. For instance, researchers observed that the immobilization of b-galactosidase caused a decrease in both the Km and Vmax values [22, 50]. Higher Km and lower Vmax values for immobilized enzyme, when compared to those calculated for the soluble enzymes, have been registered by other authors [37, 51]. Thermal inactivation kinetics and estimation of the deactivation energy-immobilized b-galactosidase Thermal inactivation is an important limiting factor for prolonged use of enzymes in industrial processes. Fig. 11 Michaelis–Menten plot and Lineweaver–Burk plot of immobilized b-galactosidase from L. Plantarum HF571129 for ONPG

Fig. 12 Michaelis–Menten plot and Lineweaver–Burk plot of immobilized b-galactosidase from L. Plantarum HF571129 for lactose

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Inactivation rate constants (Kd) of immobilized b-galactosidase, presented in Fig. 13 at 35, 40, 45, 50, 55 and 60 °C, were calculated from the slope of the semilogarithmic plot of residual activity versus time. The immobilized enzyme showed better half-life time than the soluble enzyme at 35 and 40 °C. Immobilized b-galactosidase at 55 °C reveals approximately five times more half-life time than soluble enzyme. In the case of immobilized enzyme, the activation energy was lesser than that of the soluble enzyme. It is indicative of diffusion resistance of the substrate and product. Inactivation energies (Ed) of immobilized b-galactosidase were found to be 24.33 kcal mol-1. The inactivation energy estimated for this enzyme was 3.1 and 2.3 times higher than the commercial A. oryazae b-galactosidase [52] and b-galactosidase immobilization onto graphene [50], respectively, and two times lower than chitosan-immobilized b-galactosidase from K. lactis NRRL Y1564 [53]. It can be observed that DH slightly decreases with an increase in the temperature, while DS remains constant. In addition, the DS values were close to zero, recommending that thermal deactivation does not cause significant change to the tertiary structure of the enzyme [54]. Comparing the DS and DH values of the soluble and immobilized enzymes as shown in Table 6, it can be observed that immobilization was able to promote the thermal stability of b-galactosidase because the DS and the DH values proved to decrease upon immobilization.

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These observations support the findings reported by Lima et al. [53]. Reactor operational efficiency Volumetric activity is another vital parameter for packed bed reactor, because it allows decreased reactor volume and reduced production cost. A reduction in the operating cost of the process would result from the lower utilization for the further expenses related to the immobilization process [55]. The operational stability of packed bed reactor is an important parameter in the food industry, because it honestly affects the cost of the process [56]. In this section, the performance as stability of the immobilized bgalactosidase in the beads was evaluated in a continuous mode. Figure 14 shows the relationship between the adsorption efficiency and feed flow rate (i.e., residence time). Reduction in reactor firmness with time of the process can be detected. About 88 % of adsorption efficiency was achieved at the end of 2 h continuous operation with 1 mL/ min feed flow rate. Once the flow rate increased, the

adsorption efficiency decreases and reaches 69 % at 5 mL/ min feed flow rate for the same operation. This is because of the enzyme released into the buffer solution from the alginate beads. If the reactor volumetric activity increases (i.e., low residence time), enzyme leakage also increases that in turn decreases the adsorption efficiency. These results demonstrate that the immobilization process was effective in maintaining the operational stability of the immobilized enzyme. Comparable results with b-galactosidase from A. oryzae immobilized in silica are reported by Mariotti et al. [57] where the operational stability studies were carried out in packed bed reactor for a flow rate of 0.5 mL/min and the activity was reduced by 24 % after 130 days of operation. Klein et al. [58] reported that b-galactosidase from K. lactis immobilized on chitosan

Table 5 Kinetic parameters for immobilized b-galactosidase from L. plantarum HF571129 for the hydrolysis of ONPG and lactose Substrate

Vmax (lmol min-1 mg-1)

Km (mM)

ONPG

192.4

10.22

Lactose

16.85

25.61

Fig. 14 Effect of operation time on adsorption efficiency at different flow rates

Fig. 13 a Thermal inactivation of immobilized b-galactosidase at 35, 40, 45, 50, 55 and 60 °C. b Arrhenius plot of the inactivation rate constants for immobilized b-galactosidase

Table 6 Kinetic parameters for the thermal inactivation of the immobilized b-galactosidase

Temperature (°C)

Kd (min-1)

t1/2 (min)

DG (kJ/mol)

DH (kJ/mol)

DS (kJ/mol K)

35

0.0053

130.78

88.976

99.664

0.034

40

0.0058

119.50

90.211

99.623

0.030

45

0.0156

44.43

89.085

99.581

0.032

50

0.06767

10.24

86.591

99.540

0.040

55

0.6733

10.29

88.004

99.498

0.035

60

0.07300

9.49

89.13

99.457

0.030

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Bioprocess Biosyst Eng Fig. 15 Lactose hydrolysis by batch (a, b) and packed bed (c, d) b-galactosidase enzyme in milk lactose at 50 °C

Fig. 16 a Hill plot and b slope of the plot are used in the determination of Hill’s coefficient

macroparticles maintained 90 % of lactose hydrolysis in packed bed reactor. In a previous study, the operational stability of the immobilized b-galactosidase was studied for 30 days at a flow rate of 0.3 mL/min with 90 % of activity [59]. Lactose hydrolysis To explore the feasibility of using the immobilized enzyme in continuous mode, a packed bed reactor was constructed and lactose hydrolysis studied at 50 °C (Fig. S2). Hydrolysis to the extent of 97 % was noted at a flow rate of

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1 mL/min after 2 h. A similar finding for the continuous lactose hydrolysis in a packed column by calcium alginateentrapped b-galactosidase has been reported by Haider and Husain [51]. The rate of lactose hydrolysis for batch (Fig. 15a), packed bed (Fig. 15c) and t1/2 was determined from a plot of log % residual activity versus time for batch (Fig. 15b) and packed bed (Fig. 15d). The rate of lactose hydrolysis was found to be 0.023 and 0.04 min-1, whereas t1/2 was calculated to be 30.13 and 17.325 min for batch and packed bed, respectively. In the early stage, soluble b-galactosidase was available for hydrolyzing milk lactose when compared to

Bioprocess Biosyst Eng

immobilized enzyme. The rate of lactose hydrolysis decreased much faster after an extended incubation period; it can be clarified that the effect of product inhibition was higher in case of soluble enzyme when compared to immobilized b-galactosidase [60]. As a result, in view of greater lactose hydrolysis obtained in milk lactose and increased resistance to inhibition by galactose, ZnO NPsadsorbed b-galactosidase may be useful in lactose hydrolysis of dairy products in batch and also in packed bed reactors.

galactosidase can be further explored for commercialization at industrial scale.

Calibration of lactose hydrolysis

References

The present lactose hydrolysis was calibrated using a semilogarithmic plot of varying lactose concentration (%) versus glucose reading (mg dL-1) (Fig. 16a). According to the plot, it was found to be linear correspondingly from 0.1 to 2.0 % of lactose. Based on the range obtained, a verification plot of log Y (glucose reading) versus log Y/ Yss-1, where Yss is the reading at steady state, was generated (Fig. 16b), while Hill’s coefficient was found from the slope of the plot. In pharmacology, the Hill equation has been extensively used to analyze quantitative drug– receptor relationships. We have obtained Hill’s coefficient of 1.47 corresponding to lactose concentration ranging from 0.1 to 2 %. A similar result was obtained in a study [22] with the Hill’s coefficient of 1.71, when b-galactosidase from P. sativum was immobilized onto gold nanoparticles. Thus, based on results obtained, it can be concluded that the present immobilized b-galactosidase technology is best suited for estimation of lactose present in dairy samples with concentration \2 % (lactose content: 0.9–1.4 % as approved by the Food and Drug Administration, USA, in commercially available lactose hydrolyzed milk).

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Conclusion b-Galactosidase was immobilized onto ZnO NPs (biosynthesized) and pH, temperature, thermal stability and kinetic constraints (Km and Vmax) characterized with ONPG as substrate. The immobilized enzyme was reused for seven cycles, showing stability since it retained more than 70 % of its initial activity. The immobilized enzyme retained 85 % of its initial activity when it was stored at 4 °C and pH 6.5 for 30 days. The time required for 50 % lactose hydrolysis (t1/2) for batch and packed bed was calculated to be 30.13 min and 17.325 min. Finally, the immobilization of b-galactosidase on ZnO NPs not only improved enzyme stability, but also enhanced the performance of the biocatalyst in the hydrolysis of lactose, probably by altering the kinetic parameters of the enzyme. The immobilized b-

Acknowledgments We are greatly indebted to the VIT University for the constant encouragement, help and support for extending the necessary facilities. Conflict of interest of interest.

The authors declare that they have no conflict

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