Preparation and Characterization of Pectinase bound Co-precipitated Magnetic Nanoparticles

A. Ramankannan¹, J. Rini Gnana Suganthi¹, N.Balaji², M. Seenuvasan²*

¹IV Year B.Tech-Biotechnology, Department of Biotechnology, Madha Engineering College, Chennai, India.

²Assistant Prof & Head, Department of Biotechnology, Madha Engineering College, Chennai, India.

*Corresponding Author E-mail: seenuchem786@gmail.com

ABSTRACT:

The binding of pectinase onto co-precipitated magnetic magnetic nanoparticles (MNPs) via glutaraldehyde activation was investigated.The transmission electron microscopy (TEM), X-ray diffraction (XRD) analysis and Fourier transform infrared (FT-IR) spectroscopy were studied to characterize size, structure, morphology and binding of enzyme onto the nanoparticles. Debye-Scherrer relation was analysed based on the XRD results, reports that binding process did not cause any significant change in size of MNPs. The maximum activity of immobilized pectinase was obtained at its weight ratio of about 16.2 x10-3 mg bound pectinase/mg MNPs. The stability and activity of the bound pectinase was analyzed using various parameters like pH, temperature, reusability, storage ability and kinetic studies. The same was compared with the free pectinase for showing its enhanced stability and activity.

KEYWORDS: Nanoparticles, pectinase, immobilization, pectin, MNPs.

1. INTRODUCTION:

The pectinase is used in clarification of fruit juice is gaining more importance because they depolymerise the pectins which create turbidity is directly through cleavage of glycosidic linkages¹. The problems encountered in the enzymatic reactions are enzyme recovery and recycling and this can be counteracted by the use of immobilized enzymes, thereby decreasing the overall cost^2-6. The immobilization of enzymes onto the support is an important tool as it provides distinct advantages including enhanced stability, easy separation, improved catalytic properties and arrest of microbial growth⁷. The binding of enzymes and protein on to the carrier is accomplished by adsorption, covalent bonding or encapsulation⁸.

The immobilization of enzymes on the nanoparticles offers high surface area-to-volume ratio. The enzyme bound nanoparticles posses Brownian movement, when dispersed in aqueous solutions showing that the enzymatic activities are comparatively better than that of the unbound enzyme^9-10. An efficient and economical means of enzyme recovery and recycling relies on the use of magnetic nanoparticle¹¹.

The magnetic nanoparticles are biocompatible, super-paramagnetic material which finds wide applications in drug delivery, and enzyme and protein immobilization as they possess small size, high specific surface area, low toxicity, strong magnetic properties, chemical stability, and uniformity in size and dispersion in aqueous phase^12-14.

The various methodologies such as co-precipitation, micro emulsion, thermal decomposition and hydrothermal synthesis have been extensively used for the synthesis of magnetic nanoparticles Among them, co-precipitation is a convenient method to synthesize iron oxides from aqueous salt solutions in the presence of a base with high yield and relatively narrow size distribution depending on the temperature, pH, type of salt etc.^14-15.

The present investigations are to synthesize the MNPs by co-precipitation method and to characterize the synthesized MNPs using TEM and XRD analytical techniques. The extent of pectinase immobilization on to the magnetic nanoparticles and its activity was confirmed using FT-IR analysis. The kinetic parameters, pH and thermal stability were studied. The reusability and storage stability was done to find the extent of stability loss.

2. MATERIALS AND METHODS:

2.1. Chemicals and instruments

Pectinase enzyme (EC No.3.2.1.15) is the product of sigma, D-(+)-galacturonic acid monohydrate the product of Fluka was obtained from Sigma Aldrich (Bangalore, India). Ferric chloride hexa hydrate (FeCl₃.6H₂O) and ferrous chloride tetra hydrate (FeCl₃.4H₂O), ammonium hydroxide (NH₄OH, 29.6%) are the product of Thomas Baker Chemicals (Mumbai, India). 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde, 25% (w/v) were purchased from Alfa Aesar (Hyderabed, India) and Nice Chemicals (Kochi, India) respectively. DNS (3, 5-dinitrosalicylic acid) was the guaranteed reagent of LobaChemiePvt Ltd, (Mumbai, India). Pectin was obtained from S&D fine chemicals (Mumbai, India). Deionized water was used throughout the experiment. All the chemicals used were of analytical grade and of highest purity. The absorbances were measured on Jasco V-630 double beam spectrophotometer. The size and morphology of the nanoparticles were determined by Transmission Electron Microscopy (TEM) using Technai 10, Philips. The X-Ray Diffraction XRD measurement were performed on X-ray diffractometer using Philips X'pert Pro Materials Research Diffractometer (MRD) in the receiving slit operation mode with a single Cu Kα radiation (λ=0.154 nm) and the XRD patterns were recorded at high angles (10-70 degree). The binding of pectinase onto the Fe₃O₄ nanoparticles were confirmed by Fourier Transform Infra Red (FT-IR) spectroscopy (Perkin Elmer spectrum RX 1) using the potassium bromide pellet method in the range of 400-2400 cm^-1.

2.2 MNP’s synthesis: co-precipitation method

The magnetic nanoparticles [16] are synthesised using FeCl₂.4H₂O and FeCl₃.6H₂O (molar ratio, 1:2) in deionized water. About 75 mL of NH₄OH solution was added under vigorous stirring in the presence of N₂ and the solution pH was maintained at 10.0. The formed black precipitate was heated at 80°C for 30 min and then cooled to room temperature. The particles were magnetically decanted and were washed several times with water, and one time with aqueous ethanol. The obtained MNPs showed strong magnetic response and were dried in vacuum oven to remove moisture.

2.3 MNP’s activation process

The synthesized magnetic nanoparticles (~2g) were dispersed in ethanol and sonicated for about 10 min for getting the complete dispersion. After sonication 1.3 mL of APTES was added and incubated at 30⁰C for overnight under shaking conditions. The magnetically decanted APTES-bound nanoparticles were then washed with ethanol several times and cured at 115°C for 2 h. Glutaraldehyde solution (10%) was added to the above particles and was incubated at room temperature for about 2 h. The particles were then washed with water to remove the excess glutaraldehyde.

2.4 Immobilisation of pectinase by MNP’s

A 500 µL of varying concentration of pectinase solution (50.8-509.9 µg in 500 µL of 0.1 M acetate buffer, pH 4.0) was added to the activated MNPs (5.0 mg) and sonicated for 5 min. The sonicated mixture was stored at 4°C for 1 h and again sonication was done for complete dispersion. This cycle was continued for two times and finally the content was brought to room temperature. The pectinase-bound MNPs were then decanted using permanent magnet and washed twice in water. The supernatants collected in each wash were assayed for protein analysis using bovine serum albumin (BSA) as standard¹⁷.

2.5 Activity assay of pectinase

The pectinase activity was determined by measuring the reducing sugar (galacturonic acid) as a result of the reaction between the pectinase and the pectin. An equal volume (500 µL) and same concentration of free pectinase and pectinase bound MNPs was added to 1.0 mL of pectin solution (0.1 M acetate buffer, pH 4.0) containing 2.0 mg of pectin separately and incubated for 1h at 50°C under shaking condition.. The concentration of reducing sugar in the supernatant was estimated using DNS method¹⁸. The standard compound used for the calibration curve for determining pectinase activity was D-(+)-galacturonic acid monohydrate and the absorbance was spectrometrically measured at 540 nm. One unit of enzyme activity (IU/mg) is defined the amount of galacturonic acid produced (µmol) per mg of enzyme used.

2.6 Stability studies

To find the optimum condition for maximum activity and maximum binding of the pectinase, the weight ratio (mg bound pectinase/ mg MNPs) for every enzyme loading was determined. The pH stability on the pectinase activity was evaluated by measuring the activity of free and immobilized pectinase at varying pH levels ranging from 2.0 to 8.0. The thermal stability on the enzyme activity was evaluated by measuring the activity of free and immobilized pectinase at temperature levels ranging from 30°C to 80°C at its determined optimal pH.

2.7 kinetic parameter calculations

Michelis-Menten kinetics was used to evaluate the enzymatic activities of both free pectinase and pectinase bound MNPs using different concentrations of pectin solution (2.0 – 8.0 mg/mL),

V_max (S)

Velocity V = ---------------- (1)

K_m+S

Where, S is the substrate concentration (mg/mL), V_maxis the maximum reaction rate attained at infinite substrate concentration (μmol of galacturonic acid /mg.min) and K_m is the Michaelis-Menten constant (mg/mL). For the purpose of establishing the kinetic parameters of both free pectinase and fabricated nanobiocatalyst, Lineweaver-Burk (LB) and Michaelis-Menten (MM) plots were used.

2.8 Reusability assay

The reusability is known by conducting the activity measurement of bound pectinase added with the pectin solution was incubated at its optimized stable conditions for 24 h. After each cycle, the supernatant was assayed for activity measurements and the enzyme bound particles were magnetically separated. Fresh substrate (pectin solutions) was added to the particles and this was continued upto half of its maximum activity.

3. RESULTS AND DISCUSSION:

3.1. TEM analysis

The morphologies of the MNPs without and with bound pectinase were shown in Fig.1. It is clear that the MNPs were almost spherical in shape before and after immobilization. The naked MNPs seem to be aggregated due to its dipole-dipole interactions (Fig. 1a) and the immobilization not significantly results in agglomeration (fig .1).

3.2. XRD analysis

The XRD pattern of the MNPs without and with bound pectinase (Fig.2) depicts the series of characteristic peaks occurred at 2Ө of 30.39°, 35.78°, 43.1°, 57.5°, 62.99° and their indices (2 2 0), (3 1 1), (4 0 0), (5 2 0) and (4 4 1) . The average size of the particles is calculated from the XRD pattern using the well known Debye-Scherrer relation with the most intense peak (3 1 1) and the corresponding full width at half maximum (FWHM). The average size of the particles was found to be 10.39 and 10.69 nm for naked MNPs and pectinase bound MNPs. It can be inferred that the immobilization of pectinase did not cause any size, phase change of nanoparticles and the immobilization had occurred only on the surface and did not alter the morphology of the particles.

3.3. FT-IR analysis

The FT-IR spectra of naked MNPs with, without pectinase and the free pectinase were shown in Fig.3. The spectrum shows the characteristic absorption peak at 585 cm^-1 (Fe-O). The characteristic frequency at 1626 cm^-1in the naked Fe₃O₄ may be due to N-H stretching of the amine functional group. After pectinase binding, this characteristic band disappeared. Thus, the binding may be accomplished through the reaction between the amine group on magnetic nanoparticles and carboxyl group of pectinase after being activated by glutaraldehyde. The amine group might be present due to the use of ammonia solution during the co-precipitation of Fe²⁺ and Fe³⁺ ions. It could be noted that the peak at 1653 cm^-1 in the free pectinase shifted to 1570 cm^-1 in bound MNPs showing the successful binding of pectinase.

3.4. Effect of pectinase loading on immobilization

The effects of various loadings of pectinase (50.8-509.9 µg) over 5.0 mg of MNPs on percentage immobilization are shown in Table 1. The maximum percentage of immobilized pectinase was found to be around 90.5% and it was found to be decreasing in the case of high pectinase loadings. The percentage relative activity of pectinase bound MNPs was found to be saturated at around 250 µg of initial pectinase loading and immediately the weight ratio (16.2x10^-3 mg pectinase/mg MNPs) also found to be saturated in very next dosage of pectinase amount. This may be attributed to the reason that the surface of MNPs was saturated with the excess of pectinase loading which in turn cause a steric hindrance between the enzyme molecules thereby blocking the active sites over MNPs.

3.5. Effect of pH

The pH stability for both the free and immobilized pectinase was studied in the range of pH (2.0-8.0) as shown in the Fig.4(a). The optimum pH value for immobilized pectinase is usually the point at which the free pectinase is most active. There was a remarkable change in the percentage relative activity of the immobilized pectinase over the free pectinase; this may be due several reasons. At first, in the acidic region the MNPs provide the favorable environment for the pectinase to act against pectin i.e., the increased affinity towards the pectic substrates to the pectinase. Secondly, for free pectinase the change in pH may not affect the shape of pectinase but it may change the shape or charge properties of the pectin so that either pectin cannot bind to the active site or it cannot undergo catalysis. After immobilization the particles provide the large surface area for the pectinase to act against the pectin so that the above said effect can be minimized.

3.6. Effect of temperature

The thermal stability of the free and immobilized pectinase were studied in the range of (30-80˚C) as shown in Fig.4(b). The temperature on which the pectinase works fast is at its optimum for both the free and immobilized pectinase. The enzymatic activity increases with greater temperature (20-50˚C) due to the greater kinetic energy possessed by molecules, thereby increasing the possibilities of collision between the pectinase and pectin and the pectin fitting into the pectinase. However, with the temperature exceeding the optimum limit (>50˚C), the pectinase starts to degrade due to the breaking of chemical bonds thereby resulting in the loss of active sites. Comparatively, the increase in percentage relative activity of immobilized pectinase over free pectinase at high temperatures. This may be attributed to the reason that there exists a strong covalent bond between the MNPs and the pectinase. Due to this the rigidity was increased and the MNPs protect the pectinase from the unconditional disturbances.

Effect of initial amount of pectinase on percentage immobilization, weight ratio and percentage relative activity: MNPs=5.0 mg, pH 4.0 and temperature=50˚C

Pectinase added (µg)	Pectinase immobilized (%)	Weight ratio (mg bound pectinase/mg MNPs)x10^-3	Relative activity (%)
50.8	90.5	9.2	56
101.6	67.4	13.7	81
178.4	45.4	16.2	100
250.2	33.5	16.8	91
324.3	26.5	17.2	85
402.8	21.6	17.4	79
509.9	17.1	17.5	78

Fig.4. The (a) pH and (b) thermal stability of the pectinase bound MNPs and the free pectinase

Fig.5. (a) Residual activity of the pectinase bound MNPs and (b) storage stability of the pectinase bound MNPs and free pectinase

Kinetic parameters for free pectinase and fabricated nanobiocatalyst at pH 4.0 and temperature 50˚C: pectin- 2.0 to 8.0 mg/mL of 0.1M acetate buffer

Parameters	Michaelis-Menten		Lineweaver-Burk
Parameters	Free pectinase	Immobilized pectinase	Free pectinase	Immobilized pectinase
V_max(μmol of galacturonic acid /mg.min)	0.6385	0.845	0.8079	0.871
K_m(mg/mL)	3.278	2.542	5.462	2.74

3.7. Effect of Kinetic parameters

The maximal activities (V_max) and the Michaelis-Menten constant (K_m) are calculated from the Lineweaver-Burk graph plotted for activities of free and immobilized pectinase for varying substrate concentrations are shown in the Table 2. It is clear from the results that there was not much steric hindrance in the active sites of pectinase on immobilization. The immobilization of pectinase onto the magnetic nanoparticles did not affect the pectinase-pectin reaction and there was less diffusional resistance. The K_m value of free pectinase was greater revealing that there was a higher affinity of immobilized pectinase to substrate, more available active sites due to the expansion of pectinase over the small and nonporous nanoparticles surfaces. The increases in V_maxconforms that there was an improvement in catalytic activity of immobilized pectinase over free pectinase.

3.8. Reusability assay

The reusability of immobilized pectinase is of more importance as it finds wide applications in the economical point of view. In this study, number of recycles was performed until the activity was reduced to half of its maximum. For each cycle, the corresponding activity was determined and approximately 50% remains at the end of the eighth day and the activity was observed to decrease in every cycle (Fig.5). This loss of enzymatic activity may be caused due to many reasons like protein denaturation, end-product inhibition. It was found from Fig. 5(b) that the improvement in storage stability of immobilized pectinase over free pectinase. The immobilized pectinase conserved 60 % of their initial and the free pectinase conserved 51 % of their initial after storing them upto 24 d.

4. CONCLUSIONS:

The immobilization of pectinase onto co precipitated MNPs were done by glutaraldehyde activation. The average diameter of the MNPs was found to be 10.39 nm by XRD analysis and there is no significant difference in size after the pectinase immobilization. The reduced agglomeration and good dispersability was achieved after the immobilization of pectinase. FT-IR analysis confirmed the binding of pectinase onto the nanoparticles. The effect of parameters like pH, temperature was studied and the optimum pH and temperature were found to be similar for both free and immobilized pectinase but overall, immobilized pectinase over MNPs shows better stability and activity than free pectinase. The study on the kinetic parameters confirmed that the affinity of the enzyme towards the substrate increased after immobilization. The reusability and storage ability of the immobilized pectinase was assessed and it was found that it is capable of withstanding multiple recycles and long period of storage.

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Received on 14.09.2013 Accepted on 01.10.2013

Asian J. Pharm. Tech. 2013; Vol. 3: Issue 4, Pg 175-180