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Immobilization of pectinase onto chitosan magnetic nanoparticles by macromolecular cross-linker
Accepted Manuscript Title: Immobilization of pectinase onto chitosan magnetic nanoparticles by macromolecular cross-linker Author: Uttam V. Sojitra Shamraja S. Nadar Virendra K. Rathod PII: DOI: Reference:
S0144-8617(16)31177-8 http://dx.doi.org/doi:10.1016/j.carbpol.2016.10.018 CARP 11638
To appear in: Received date: Revised date: Accepted date:
5-8-2016 5-10-2016 6-10-2016
Please cite this article as: Sojitra, Uttam V., Nadar, Shamraja S., & Rathod, Virendra K., Immobilization of pectinase onto chitosan magnetic nanoparticles by macromolecular cross-linker.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2016.10.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Immobilization of pectinase onto chitosan magnetic nanoparticles by macromolecular cross-linker
Uttam V. Sojitraa, Shamraja S. Nadara, Virendra K. Rathoda*
a
Department of Chemical Engineering, Institute of Chemical Technology, Matunga (E)
Mumbai-400019, India.
Corresponding Author: Dr. Virendra K. Rathod E-mail: [email protected], Phone: +91-22-33612020, Fax: 91-22-33611020.
Highlights:
Pectinase immobilized onto chitosan MNPs by dextran polyaldehyde cross-linker.
Biocompatible, biodegradable functionalize and cross-linker used for immobilization.
Hydrolysis of pectin improve quality, stability and yield of fruit juice.
Thermal stability of immobilized pectinase was expressed as half-life.
Prepared magnetic pectinase nanobiocatalyst employed for apple juice clarification.
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Abstract Pectinase was immobilized onto chitosan magnetic nanoparticles (CMNPs) by dextran polyaldehyde as a macromolecular cross-linking agent. The parameters like cross-linking concentration, time and CMNPs to enzyme ratio were optimized. Further, prepared magnetic pectinase nanobiocatalyst was characterized by FT-IR and XRD. The thermal kinetic studies for immobilized pectinase showed two folds improved thermal stability in the range of 55-75°C as compared to free form. The Vmax and Km values of immobilized pectinase were found to be nearly equal to native form which indicated that conformational flexibility of pectinase was retained even after immobilization. The residual activity of immobilized pectinase was 85% after seven successive cycles of reuse, while it retained upto 89% residual activity on storage of fifteen days which exhibited excellent stability and durability. The conformational changes in pectinase after immobilization were evaluated by FT-IR spectroscopy data analysis tools. Finally, magnetic pectinase nanobiocatalyst was employed for apple juice clarification which showed turbidity reduction upto 74% after 150 min treatment.
Keywords: Immobilization, chitosan magnetic nanoparticles, pectinase, dextran cross-linker, juice clarification,
Introduction Enzymes are highly effective, versatile and eco-friendly catalysts which operate under mild reaction conditions (Kim et al., 2008; Ge et al., 2009; Wang 2006). They have become a potential tool in making benign industrial manufacturing processes which attract more and more researchers to exploit it. However, industrial applicability of enzymes is always obstructed due to several limitations such as low operational stability, difficulty in recovery and reusability, which make them unaffordable (Sheldon et al., 2013). To overcome these shortcomings, enzyme immobilization onto solid carrier is one of the effective techniques which not only stabilizes enzymes under operational conditions but also allows easy recovery and reusability for multiple cycles (Cao et al., 2005). Generally, immobilization of enzyme on the carrier involves synthesis of functionalized carrier and covalent cross-linking of enzyme on its surface. Over the past decade, a number of 2
nano-carriers have been prepared and used as a support for immobilization of enzyme (Ansari et al., 2012). Among different types of carriers, magnetic nanoparticles (MNPs) are significantly used as a support due to their unique characteristics such as their tailored surface chemistry, unique physicochemical properties, biocompatibility, biodegradability, low toxicity and low cost (Caterina et al., 2013, Seenuvasan et al., 2014a). Also, magnetic nanoparticles allow easy, quick and efficient separation of enzyme from the reaction mixture by using external magnet (Laurent et al., 2008, Karthikeyan et al., 2014). Most commonly, functionalized nano-carrier for enzyme immobilization is prepared in two-steps (synthesis of carrier and functionalization), which creates difficulty in handling, longer reaction time, loss of product at each step and also requires high temperature conditions for the functionalization (Kim et al., 2013). Further, during cross-linking enzymes on nano-carriers, cross-linking agent plays crucial role as it has direct influence on activity recovery and operational stability (Talekar et al., 2013b). Amongst available protein cross linking agents, glutaraldehyde is the preferred choice as the cross-linker. Because it is inexpensive, easily available, easy to manipulate and has the ability to make covalent bonds with most of the enzymes (Barbosa et al., 2014). However, glutaraldehyde cannot be universally employed as a cross-linker since it possesses some inherent drawbacks. In some cases, use of glutaraldehyde as cross-linker resulted in complete loss of enzyme activity which might be due to small size of cross-linker getting easily penetrated into the active site and cross-linking catalytically important amino acids residues leading to inactivation of enzyme (Mateo et al., 2004). Moreover, cross-linking of enzyme by glutaraldehyde forms lumps of enzyme molecules hindering the active sites. Hence, it suffers from serious mass transfer limitations for macro-substrates resulting in lower catalytic efficiency (Zhen et al., 2013). Additionally, leaching of glutaraldehyde from prepared biocatalyst causes adverse effect on human and aquatic lives because of its toxic nature (Arsenault et al., 2011). Therefore, in past years, polysaccharides based cross linkers have received increasing attention as a potential cross-linking agent for proteins over glutaraldehyde (Talekar et al., 2014). More recently, Nadar et al. (2016c) employed pectin based cross-linker to immobilize glucoamylase onto 3-aminopropyl triethoxysilane (APTES) modified MNPs which was found to be more effective as compared to traditional glutaraldehyde. In recent years, fruit juice technologies have attracted considerable attention for the improvement of juice quality due to increase in natural fruit juice consumption (Cassano et al., 2013). The freshly pressed fruit juices are cloudy and turbid in appearance due to colloidal dispersion of pectin (0.9-1.5%) present in the forms of disrupted cell wall and cell materials of 3
fruit, which is one of the major hurdle in fruit juice processing (Tapre et al., 2014). Thereby, pectinase is most extensively used enzyme in fruit juice clarification to decrease cloudiness, improve quality and yield of fruit juice (Kashyap et al., 2001). Considering food based application of pectinase (enzyme acting on macromolecular substrate), the materials used for immobilization, that is, functionalizing agent for magnetic nanoparticles and cross-linking agent for anchoring enzyme, must be biocompatible and non-hazardous (Nadar et al., 2016b). Thus, in this work, we report for the first time a non-hazardous, biocompatible, eco-friendly and efficient immobilization strategy. Here, we synthesized magnetic nanoparticles in the presence of chitosan biopolymer which not only provides stability and amino group to magnetic nanoparticles but also makes them highly biocompatible. Further, dextran polyaldehyde was prepared via controlled periodate induced oxidation which is used as a non-toxic and biodegradable cross-linker to anchor enzyme onto CMNPs. The different parameters like cross-linker concentration, cross-linking time and ratio of CMNPs to enzyme were optimized. The prepared immobilized pectinase was characterized by Fourier transform infrared spectroscopy (FT-IR) and crystal structures were determined by X-ray powder diffraction (XRD). The kinetic parameters (Vmax and Km) were evaluated for prepared immobilized pectinase. In addition to that, the thermal stability of prepared magnetic pectinase nanobiocatalyst was evaluated in terms of deactivation rate constants (kd), half-life (t1/2) and deactivation energy (Ed). Finally, reusability and storage stability of immobilized pectinase were investigated to determine its industrial applicability. At the end, immobilized pectinase was employed for clarification of apple juice and the relative clarity of juice was determined.
Materials and methods Materials Pectinase (126 U/mL, protein content 10 mg/mL) was procured from Enzymes India Pvt. Ltd, Chennai, India. Apple pectin (Mw 100-120 kDa, degree of esterification 85–90%, galacturonic acid min. 65%), dextran (Mw 10000 kDa) and chitosan (Mw 40–60 kDa, DDA ≥75%) were purchased from HiMedia Laboratories Pvt. Ltd. Mumbai, India. 3,5-Dinitrosalicylic acid (DNSA), ferrous sulphate (FeSO4·4H2O) and ferric chloride (FeCl3·6H2O) were purchased from S. D. Fine Chemicals, Mumbai, India. 2, 4-dinitrophenylhydrazine (DNPH) was purchased from HiMedia Laboratories Pvt. Ltd. Mumbai, India. All other chemicals were of analytical grade and procured from reliable sources. Synthesis of chitosan magnetic nanoparticles
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Chitosan magnetic nanoparticles (CMNPs) were synthesized by single step method as reported by Gregorio-Jauregui et al., (2012). Briefly, chitosan (1%) was dissolved completely in acetic acid (1 % v/v, 45 mL) in a 50 mL capacity glass vessel equipped with impeller. Ferrous sulphate (FeSO4·4H2O, 0.2 M) and ferric chloride (FeCl3·6H2O, 0.4 M) (in molar ratio of 1:2) were dissolved in above chitosan solution. The mixture was permitted to mix for 30 min at room temperature (30±2 °C) and sodium hydroxide (1 M) was added slowly with continuous agitation (1000 rpm) by overhead laboratory stirrer (RQ - 40 Plus, Remi, India). After 30 min of reaction, magnetic nanoparticles were recovered by magnet and washed five times with deionized water. The particles were kept for overnight drying at room temperature and further analyzed by using FT-IR, XRD and TEM. Preparation of dextran macromolecular cross-linker Dextran polyaldehyde cross-linker was prepared by the method as reported by Jadhav et al. (2013). Briefly, sodium metaperiodate (50-400 mM) was prepared in sodium phosphate buffer and used as an oxidizing agent. The dextran was oxidized with oxidizing solution for 30-150 min in the dark condition. After oxidation, ethylene glycol (0.3 mL) was added to the reaction mixture to stop the oxidation. Oxidized dextran solution was dialyzed against sodium phosphate buffer at 4°C overnight. The extent of oxidation of dextran was determined by 2, 4-dinitrophenylhydrazine (DNPH) test (Gupta et al., 2013). In this test, oxidized dextran (0.3%, 3×10-4 g/L) was added to freshly prepared solution of DNPH (10 mL, 1% w/v). The mixture was permitted to stand for 1 h and then centrifuged at 8000 rpm for 10 min. The amount of unreacted DNPH in supernatant was determined by using UV-Vis spectrophotometer (Jasco V-630, Japan) at 357 nm. The amount of generated aldehyde was calculated according to: Aldehyde content (mmol/g) =
(Reacted DNP (mg/L)/198.14) Amount of oxidized dextran (g/L)
Where, 198.14 is the molecular weight of DNPH. Finally, the oxidized dextran was dissolved in phosphate buffer (100 mM, pH 6) and used as macromolecular cross-linker. Pectinase activity assay The activity of pectinase was assessed using pectin as a substrate. Pectinase (0.5 mL) was mixed with pectin solution (1 % prepared in phosphate buffer of pH 6, 100 mM) and incubated at 50°C for 20 min and then DNSA reagent (2 mL) was added to the reaction mixture, boiled for 15 min, cooled and absorbance was measured at 575 nm using UV-Vis spectrophotometer 5
(Jasco V-630, Japan) (Miller et al., 1959). One unit of enzyme activity (U) was defined as the amount of the enzyme liberating one µmole of ß-galacturonic acid per minute at optimum conditions (50°C, pH 6.0). The protein concentration in the supernatant was measured by the Bradford’s reagent using bovine serum albumin (0-1 mg/mL) as the standard (Bradford et al., 1978). Immobilization of pectinase onto CMNPs The CMNPs were mixed with pectinase (10 mg protein content) in sodium phosphate buffer (100 mM, pH 6) in the ratio of 1:1 to 5:1 (w/w). The reaction mixture was kept for shaking at 175 rpm for 30 min. Then, dextran cross-linker was added (0.5-4 % with respect to total solution mixture) and incubated for 3-24 h. After that, sodium borohydrate (5 mg) was added and kept for 30 min to terminate cross-linking. The immobilized pectinase was separated magnetically, washed four times with buffer and assayed for the enzyme activity. Characterization of immobilized pectinase onto CMNPs The immobilized pectinase onto CMNPs were confirmed by Fourier transform infrared (FT-IR) spectroscopy using Shimadzu IR Affinity-1 FT-IR Spectrophotometer. Crystal structure of CMNPs before and after immobilization was investigated by Powder X-ray diffraction (XRD) (Philips PW 1830 X-ray Diffraction). Also, the size of CMNPs was determined by using transmission electron microscopy (TEM) (JEOL JEM 2100, USA) and hydrodynamic diameter was analysed by using DLS (Zetasizer Nano ZS, Malvern Instruments, UK). Thermal stability study of free and immobilized pectinase Thermal kinetics of pectinase in free and immobilized form was studied by incubating them in phosphate buffer (100 mM, pH 6.0) at 55, 65 and 75°C. The sample was collected after 10 min interval for 60 min, and activity was determined in terms of residual activity. The inactivation rate constant (kd) was estimated by plotting residual activity (%) against time in semi-log. The half-life (t1/2) is time required to decrease the activity to half of its original and was calculated as 0.693/kd. Further, deactivation energy (Ed) of the free and immobilized pectinase was determined from Arrhenius plot. Kinetics parameters Kinetics parameters (Km and Vmax) of free and immobilized pectinase were determined using different pectin concentrations in the range of 0.1-3.0 mg/mL in phosphate buffer (100 mM, pH 6.0) at 50°C. Km and Vmax values of free and immobilized pectinase were calculated from 6
non-linear regression fitting of the initial rates of reaction corresponding to different pectin concentrations by Graph Pad Prism software. Secondary structure analysis of free and immobilized pectinase The changes in secondary structure of free and immobilized pectinase were examined by using FT-IR method (Secundo et al., 2013). The FT-IR spectra of free pectinase and immobilized pectinase were noted by using FT-IR (Shimadzu IR Affinity-1) from 400 to 4000 cm-1 with sample dispersed in the KBr pellets. Secondary derivative of peak frequencies in amide-I region (1600–1700 cm−1) were identified and smoothened with a 13-point Savitzky–Golay by Essential FTIRTM 3.00. Then, multicomponent peak areas under amide I bands of free and immobilized pectinase were analysed using multi-peak fitting program with Gaussian function by Origin 9.0. The fractions of secondary structure were estimated as per our previous work (Nadar et al., 2016a). Reusability studies Reusability of immobilized pectinase was carried out at optimum condition of pH and temperature. The reaction was carried out for 30 min using pectin as a substrate in batch mode. After each cycle, immobilized pectinase was separated magnetically, washed with phosphate buffer (100 mM, pH 6) and re-suspended in fresh substrate solution to determine its activity. This process was carried out for seven consecutive cycles. The activity was determined after each cycle in terms of residual activity, considering 100 % residual activity for the first cycle. Storage stability studies Storage stability of the free and immobilized form of pectinase were determined by incubating enzyme in sodium phosphate buffer (100 mM, pH 6) without substrate. After every three days of interval till fifteen days, enzyme samples were separated from the buffer, washed by deionized water (DI) and then assayed for residual activity using pectin as the substrate. Application of immobilized pectinase in fruit juices clarification The ability of magnetic pectinase nanobiocatalyst was studied for clarification of apple (Malus domestica) juice. The fresh apple juice was prepared and centrifuged at 5000 rpm for 20 min, and then supernatant was used for the clarification process. The immobilized pectinase (equivalent amount of free pectinase) was mixed with diluted juices (20:80 v/v of juice to distilled water) and treated for 150 min at 50 °C. After enzymatic treatment, clarity of the juice was determined in terms of turbidity which was evaluated by spectrophotometric method (Magro et al., 2016).
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Results and discussion Characterization of chitosan magnetic nanoparticles The CMNPs were simultaneously synthesized and functionalized (with -NH2 group) via co-precipitation method. The Fe2+ and Fe3+ ions (molar ratio 1:2) was reduced by sodium hydroxide solution in the presence of chitosan. The chemical reaction can be written as follows (Maity et al., 2007): Fe2+ + 2 Fe3+ + 8 OH−
Fe3O4 + 4 H2O
Further, the prepared CMNPs were characterized by using XRD and FT-IR. The crystalline structure of CMNPs was identified by using XRD analysis. In Figure 1a, X-ray powder diffraction profile exhibited peaks at 2θ = 30.31°, 35.67°, 41.18°, 57.24°, 62.84° which are characteristics of Fe3O4. According to the joint committee on powder diffraction standards (JCPDS) database, the crystal structure of Fe3O4 (reference code 01-075-0449) is inverse spinel in nature with 100% purity. On the basis of full width half maximum (FWHM), the average crystallite diameter of CMNPs was found to be 29.6 nm by Debye-Scherrer’s equation. Also, the size of CMNPs was found to be ~ 45 nm by using transmission electron microscopy (TEM), while hydrodynamic diameter was 107.6 nm by using DLS (Figure S1a-b). Further, the prepared CMNPs were confirmed by using FT-IR (Figure 1c). The FT-IR spectrum showed a characteristic peaks at 586.36 cm-1 which is corresponding to intrinsic stretching vibrations of Fe–O. The peaks at 3385.07 and 1068.56 cm-1 were corresponding to O–H, N–H and C–O–C respectively from chitosan. The peak at 2883.58 and 1643.35 cm-1 were for C–H and N-H stretching respectively which confirmed successful preparation of CMNPs (Gregorio-Jauregui et al., 2012, Liu et al., 2014). Periodate mediated controlled oxidation of dextran cross-linker Macromolecular dextran cross-linker was prepared by metaperiodate mediated controlled oxidation of native dextran. Periodic acid and its salts can oxidize dextran specifically to yield dialdehyde groups after breaking carbon bond at C2-C3 of glucose unit according to the Malaprade mechanism (Jadhav et al., 2013). The extent of oxidation is very important to generate maximum amount of aldehyde group which is dependent on sodium metaperiodate concentration and reaction time. Hence, effect of sodium metaperiodate concentration and time of reaction was studied in terms of generated aldehyde group and analysed using DNPH test. As shown in Figure 2a, increase in the sodium metaperiodate concentration increased the
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aldehyde content, which was maximum at 150 mM concentration for 120 min. However, at higher concentrations of 200-400 mM sodium metaperiodate, generated aldehyde group of dextran was found to be reduced. This might be due to oxidatory inhibition of dextran at higher concentration of metaperiodate. Also, longer reaction time did not give a significant increase in oxidation. Similar results were observed for dextran cross-linker preparation by Jadhav et al., (2012). Finally, oxidized dextran was dissolved in sodium phosphate buffer (100 mM, pH 6.0) and used as dextran macromolecular cross-linker. Immobilization of pectinase onto CMNPs by dextran macromolecular cross-linker The pectinase was covalently immobilized onto CMNPs by using polyaldehydic dextran as macromolecular cross-linker. The reactive aldehyde groups of oxidized dextran can cross-link amino groups present in terms of lysine or hydroxyl lysine side groups of enzyme and chitosan to form Schiff's base which prevents leaching of enzyme during reaction (Jaydee et al., 2014). Cross-linking parameters (cross-linker concentration and cross-linking time) were the key steps in immobilization which have direct influence on enzyme loading, activity recovery and operational stability of prepared magnetic biocatalyst (Talekar et al., 2013b). The extent of cross-linking between enzyme and CMNPs is needed to be optimized to get maximum activity recovery. The effect of cross-linker concentration on the activity recovery of pectinase in immobilized form was studied by varying concentration in the range of 0.5-4% (v/v) (Figure 2b). At low concentration of cross-linker (0.5-1.0%), less activity recovery was observed due to inadequate cross-linking of enzyme. The activity recovery of pectinase increased with increase in cross-linker concentration. The maximum activity recovery was found at 2.5% (v/v) dextran polyaldehyde. Further increase in the concentration of cross-linker resulted into higher rigidification of enzymes, thereby consecutive loss of activity (Zhen et al., 2013). Cross-linking time is another important factor in immobilization of enzyme. As the cross-linking time was increased, the activity recovery of enzyme also increased. It can be seen that almost 50% activity was recovered in initial 6 h itself, due to rapid cross-linking (Figure 2c). The maximum activity recovery in immobilized pectinase was found at 15 h of cross-linking time. Further increase in cross-linking time, decreased the activity recovery of pectinase gradually. This might be due to excessive cross-linking, which is speculated to cause the chemical modification of enzyme (Wang et al., 2013) To maximize pectinase immobilization, the amount of CMNPs (non-catalytic support) should be optimized. This was done by varying the ratio of CMNPs to enzyme (1:1 to 5:1) (Figure 2d).
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At lower ratio (1:1-2:1), the amount of CMNPs was not sufficient to load pectinase, thereby lowering the activity recovery. As the ratio increased, the activity recovery also increased significantly upto 95% till the ratio of 4:1. With further increase in the CMNPs to enzyme ratio, the activity recovery remained constant, and it might be due to the maximum loading of enzyme on carrier (Yu et al., 2013). Characterization of immobilized pectinase onto CMNPs The prepared magnetic pectinase nanobiocatalyst was further characterized by XRD and FT-IR. X-ray powder diffraction profile exhibited peaks at 2θ = 30.31°, 35.67°, 41.18°, 57.24°, 62.84° which are similar to the peaks obtained for Fe3O4. XRD analysis of immobilized magnetic pectinase nanobiocatalyst showed almost similar XRD pattern as that of CMNPs which implies that there was no phase change after pectinase immobilization (Figure 1b). By using Debye-Scherrer’s equation, the average crystallite diameter of immobilized pectinase onto CMNPs was calculated to be 38.4 nm. The FT-IR spectrum of magnetic pectinase nanobiocatalyst showed prominent peaks at 1026.13 and 601.79 cm−1 which are characteristic for stretching vibration of the C–O–C and Fe-O bond from CMNPs (Figure 1d). Also, the peaks observed at 1527.62 and 1334.74 cm−1 were corresponding to N–H deformation and C–O vibration from chitosan. The characteristic peak at 1689.64 cm−1 reveals the presence of amide-I region of pectinase which confirmed the successful immobilization of pectinase onto CMNPs via dextran polyaldehyde cross-linker (Seenuvasan et al., 2014b, Nadar et al., 2016c). Thermal stability study of free and immobilized pectinase The extent of thermal stability of pectinase in free and immobilized form was expressed in terms of deactivation rate constants (kd), and half-life (t1/2). These parameters were determined separately by incubating each form of enzyme in phosphate buffer at different temperatures (55, 65 and 75°C) and the residual activity was determined after equal interval of 10 min till 60 min (Figure 3a-b). The kd and t1/2 values are summarized for pectinase in different forms in Table 1. After cross-linking enzymes with macromolecular dextran cross-linker onto CMNPs, the kd values of immobilized pectinase were lower than that of free form in the range of 55-75°C. Also, the half-life of immobilized pectinase was increased by 2.3 folds with respect to free form. It seems that covalent cross-linking between amino residues present on the surface of enzymes and CMNPs via dextran polyaldehyde maintained the active tertiary structure of
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enzyme in immobilized form against native forms (Talekar et al., 2013a, Seenuvasan et al., 2013b). Further, the deactivation energy (Ed) was determined as a measure of thermal stability of immobilized pectinase by using linear fit of the Arrhenius plot (Figure 3c). The higher deactivation energy is an index of high thermal stability of protein indicating more thermal stability. The Ed for denaturation of immobilized and free form of pectinase was 51.36 and 35.07 kJ mol−1 respectively. These results imply that immobilization of enzymes onto CMNPs provide protection from thermal denaturation due to covalent cross-linking with macromolecular cross-linker. Hence, it requires much more energy to break down their active conformation over the free form (Magro et al., 2016, Talekar et al., 2012). Therefore, magnetic pectinase nanobiocatalyst has good potential for industrial applications due to its high thermal stability.
Kinetic parameters Kinetic parameters (Vmax and Km) of free and immobilized pectinase were determined by measuring initial reaction rates for each form by varying amounts in the range of 0.1-3.0 mg/mL. The values of Vmax and Km are summarized in Table 2. Km values of immobilized form were nearly same as that of free pectinase. This indicated that substrate affinity remained unchanged after enzyme immobilization through dextran cross-linker. This could be probably due to retention of conformational flexibility even after cross-linking which maintained accessibility for macro-molecular substrate (Nadar et al., 2016b, Zhen et al., 2013).
The Vmax values of immobilized pectinase via dextran cross-linker were found to be nearly equal to free forms, which indicated that the rate of pectin hydrolysis did not change after immobilization. It could be possibly explained on the basis of macromolecular dextran cross-linker which covalently binds pectinase along its linear chain. This prevents lump formation of enzyme molecules, resulting in unchanged mass transfer of substrate and product (Nadar et al., 2016c, Talekar et al., 2014, Zhen et al., 2013). Secondary structure analysis of free and immobilized pectinase The secondary structure of enzyme plays important role in catalytic activity and its stability. FT-IR spectroscopy is considered as a useful data tool to examine structural changes in enzyme
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after immobilization in powder form (Secundo et al., 2013). The amide I band (1700–1600 cm-1) is most sensitive spectral region of protein structural components, hence considered for the determination of secondary structure of immobilized enzyme using FT-IR spectroscopy data analysis tool. However, due to close proximity of bands and extensive overlapping of structural component bands, the secondary derivative of amide I band was used to get improved resolution and identify structural component peak frequencies. Then, the peak areas under multicomponent of amide I bands were quantified using multi-peak fitting done by Gaussian function.
The second derivative FT-IR spectra for free pectinase and immobilized pectinase were obtained (Figure S2 a-b).The relative contents of β-sheet structure (1610-1640 cm-1) (red), α-helix structure (1650-1658 cm-1) (blue), random coil structure (1640-1650 cm-1) (green) and β-turn structure (1660-1700 cm-1) (yellow) based on the modelled multi-component peak areas were calculated according to our previous work by using software (Nadar et al., 2016a). The altered frequencies of secondary structures were due to the cross-linking between amino group present in the enzyme and CMNPs via dextran polyaldehyde which affects molecular geometry and hydrogen bonding pattern of immobilized pectinase. The fraction of secondary structure of free and immobilized pectinase is summarized in Table 3. It was found that the reduction in α-helical component was 6.41% in immobilized form; while β-turn content increased by 6.87% as compared to the free form. However, there were slight changes in β-turns and random coils after immobilization. The favourable changes in secondary structure after immobilization onto CMNPs resulted in significant improvement in stability. Reusability studies For any industrial applications, reusability of immobilized enzyme is a key factor to make process economically feasible. The residual activity of immobilized pectinase was found to be 85% even after seven consecutive cycles of reuse in batch mode (Figure 4a). The enzyme (protein content) was not leaching in the reaction mixture after multiple cycles of use which indicates significant reduction in purification (of protein/enzymes contamination) cost. The reduction in residual activity upto seventh cycle might be due mechanical damage and deactivation of enzyme during recycling assay. Since it is convenient to separate immobilized enzyme easily by external magnetic field, it can be reused repeatedly decreasing the overall cost (Nadar et al., 2016c).
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Storage stability studies Storage stability of the free and immobilized pectinase was evaluated by incubating them at room temperature (28±2°C) in aqueous buffer. It was determined till 15 days by monitoring the enzyme activity after three days interval. The residual activity of free pectinase was found to be 68%, whereas, immobilized form retained 89% residual activity (Figure 4b). The improved storage stability of immobilized pectinase might be due to chemical cross-linking by dextran polyaldehyde onto CMNPs which prevents possible distortion effects on the active sites of enzyme caused by the buffer solution (Sojitra et al., 2016, Seenuvasan et al., 2013). Clarification of apple fruit juice by pectinase nanobiocatalyst The turbid and hazy appearance of fruit juices is due to the presence of pectin and pectic components. Therefore, breakdown of these polysaccharides by pectinase has helped to improve quality characteristics and storage stability (Sojitra et al., 2016, Magro et al., 2016). For the clarification studies, apple (Malus domestica) fruit juice was treated using the immobilized and free pectinase. The clarification of juice was measured in terms of turbidity reduction by spectroscopic method (Table 4). At the start of clarification process, the rate of turbidity reduction by free pectinase was found to be 8-9%, whereas, immobilized pectinase exhibited 6-7% turbidity reduction in 30 min. As time progressed, free pectinase treatment reduced turbidity of apple juice upto 70%, while, by immobilized pectinase treatment, turbidity was reduced upto 74% after 150 min of treatment.
Conclusion Pectinase was immobilized on CMNPs by using dextran polyaldehyde as a macromolecular cross-linker for fruit juice clarification. The magnetic pectinase nanobiocatalyst exhibited superior thermal stability (55-75°C) as t1/2 increased by 2.3 folds as compared to the free enzymes. The Vmax and Km values of magnetic pectinase nanobiocatalyst were found to be nearly equal to free form due to retention of conformational flexibility even after immobilization. Moreover, immobilized pectinase showed 87% residual activity even after seven cycles of recyclability which emphasizes its high stability and durability thereby industrial applicability. Furthermore, immobilized pectinase employed for clarification of 13
apple juice showed rapid reduction upto 74% turbidity while, free form showed turbidity reduction upto 70% till 150 min of treatment. These results highlight the potential use of dextran as the cross-linking agent for the immobilization of pectinase onto CMNPs. As this immobilization technique involves renewable and biocompatible natural biopolymer for functionalizing and cross-linking agent, it has considerable advantages from environmental and worker safety point of view over the traditional chemicals.
Acknowledgements The authors also would like to acknowledge the University Grants Commission (UGC) of India for financial assistance in the research work.
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Liu M., Dai X., Guan R., Xu X., (2014). Immobilization of Aspergillus niger xylanase: A on Fe3O4-coated chitosan magnetic nanoparticles for xylooligosaccharide preparation, Catalysis Communications. 55, 6–10. Magro D., Hertz P. F., Fernandez-Lafuente R., Kleinac M. P., Rodrigues R. C., (2016). Preparation and characterization of a Combi-CLEAs from pectinases and cellulases: a potential biocatalyst for grape juice clarification, RSC Advances. 6, 27242-51. Maity D., Agrawal D.C., (2007). Synthesis of iron oxide nanoparticles under oxidizing environment and their stabilization in aqueous and non-aqueous media, Journal of Magnetism and Magnetic Materials. 308, 46–55 Mateo B., Palomo J. M., Van Langen L. M., Van Rantwijk F., Sheldon R.A., (2004). A new, mild cross-linking methodology to prepare cross-linked enzyme aggregates, Biotechnology and Bioengineering. 86, 273–276. Miller G., (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar, Analytical Chemistry. 31, 426–428. Nadar S. S., Gawas S. D., Rathod V. K., (2016a). Self-assembled organic-inorganic hybrid glucoamylase nanoflowers with enhanced activity and stability, International Journal of Biological Macromolecules. 92, 660-669. doi:10.1016/j.ijbiomac.2016.06.071 Nadar S. S., Muley A. B., Ladole M. R., Joshi P. U., (2016b). Macromolecular cross-linked enzyme aggregates (M-CLEAs) of α-amylase, International Journal of Biological Macromolecules. 84, 69–78. Nadar S. S., Rathod V. K., (2016c). Magnetic macromolecular cross linked enzyme aggregates (CLEAs) of glucoamylase, Enzyme and Microbial Technology. 83, 78–87. Secundo F., (2013). Conformational changes of enzymes upon immobilisation, Chemical Society Reviews. 15, 6250-61. Seenuvasan M., Malar G.C.G., Preethi S., Balaji N., Iyyappan J., Kumar M.A., Kumar K.S., (2013b). Immobilization of pectinase on to co-precipitated magnetic nanoparticles for its enhanced stability and activity. Research journal of biotechnology, 8, 24-30. Seenuvasan M., Kumar K. S., Malar C. G., Preethi S., Kumar A. K., Balaji N., (2014b). Characterization, Analysis and Application of Fabricated Fe 3O4-Chitosan-Pectinase Nanobiocatalyst, Applied Biochemistry and Biotechnology. 172, 2706-19. 16
Seenuvasan M, Malar C., Preethi S., Balaji N., Iyyappan J., Anil Kumar M., Sathish Kumar K., (2013a). Fabrication, characterization and application of pectin degrading Fe 3O4–SiO2 nanobiocatalyst, Materials Science and Engineering C. 33, 2273–2279 Seenuvasan M., Sathish Kumar K., Anil Kumar M., Iyyappan J., Suganthi J., (2014a). Response surface estimation and canonical quantification for the pectin degrading Fe3O4-SiO2-nanobiocatalyst fabrication, International Journal of ChemTech Research. 6, 3618-3627. Sheldon R. A., Van Pelt S., (2013). Enzyme immobilisation in biocatalysis: why, what and how, Chemical Society Reviews. 42, 6223-6235. Sojitra U. V., Nadar S. S. and Rathod V. K., (2016). A magnetic tri-enzyme nanobiocatalyst for fruit juice clarification, Food Chemistry. 213, 296-305. doi: 10.1016/j.foodchem.2016.06.074 Talekar S., Desai S., Pillai M., Nagavekar N., Ambarkar S., Surnis S., Ladole M., Nadar S., Mulla M., (2013a). Carrier free co-immobilization of glucoamylase and pullulanase as combi-cross linked enzyme aggregates (combi-CLEAs), RSC Advances, 3, 2265-71 Talekar S., Ghodake V., Ghotage T., Rathod P., Deshmukh P., Nadar S., Mulla M., Ladole M., (2012). Novel magnetic cross-linked enzyme aggregates (magnetic CLEAs) of alpha amylase, Bioresource Technology. 123, 542–547. Talekar S., Joshi A., Joshi G., Kamat P., Haripurkar R. & Kambale S., (2013b). Parameters in preparation and characterization of cross linked enzyme aggregates (CLEAs), RSC Advances. 3, 12485–12511. Talekar S., Nadar S., Joshi A., Joshi G., (2014). Pectin cross-linked enzyme aggregates (pectin-CLEAs) of glucoamylase, RSC Advances. 4, 59444–59453. Tapre A. R., Jain R. K., (2014). Pectinases: Enzymes for fruit processing industry, International Food Research Journal, 21, 447-453. Wang B., Cheng F., Lu Y., Ge W., Zhang M., Yue B., (2013). Immobilization of pectinase from Penicillium oxalicum F67 onto magnetic corn starch microspheres: Characterization and application in juice production, Journal of Molecular Catalysis B: Enzymatic. 97, 137–43. Wang P., (2006). Nanoscale biocatalyst systems, Current Opinion in Biotechnology. 17, 574– 579.
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Yu D., Ma Y., Xue S. J., Jiang L., Shi J., (2013). Characterization of immobilized phospholipase A1 on magnetic nanoparticles for oil degumming application, LWT - Food Science and Technology. 50, 519-525. Zhen Q., Wang M., Qi W., Su R., He Z., (2013). Preparation of β-mannanase CLEAs using macromolecular cross-linkers Journal cover: Catalysis Science & Technology Catalysis Science & Technology. 3, 1937-1941.
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Figure 1a
Figure 1b
19
Figure 1c
Figure 1d Figure 1 XRD patterns of the prepared CMNPs (a) and immobilized pectinase onto CMNPs cross-linked by dextran polyaldehyde (b). FT-IR spectrum of the prepared CMNPs (c), and pectinase immobilized onto CMNPs cross-linked by dextran polyaldehyde (d).
20
18
Aldehyde content (mmol/g)
16 14 12 10 8 6 4 2 0
10
50
100
150
200
300
400
Periodate concentration (mM) 30 min
60 min
90 min
120 min
150 min
2
2.5
3
Activity recovery (%)
Figure 2a
100 90 80 70 60 50 40 30 20 10 0 0.5
1
1.5
3.5
4
Cross-linker concentration (%)
Figure 2b
21
100
Activity recovery (%)
90
80 70 60 50 40 30 20 10 0 3
6
9
12
15
18
21
24
Cross-linking time (h)
Figure 2c 100 90
Activity recovery i(%)
80
70 60 50 40 30
20 10 0 1:1
2:1
3:1
4:1
5:1
Ratio of CMNPs to enzyme (protein) (w/w)
Figure 2d Figure 2 (a) Aldehyde content generated in dextran after oxidation with metaperiodate (50-400 mM) determined by DNPH method. Effect of cross-linker concentration (b), cross-linking time (c) and ratio of CMNPs to enzyme (protein) (w/w) (d) on activity recovery of pectinase in magnetic nanobiocatalyst. The 100% activity recovery corresponds to pectinase (126 U). The measurements were performed in triplicate and the error bar represents the percentage error.
22
5 4.5 4
ln (A/Ao)*100
3.5 3 2.5 2 y = -0.054x + 4.6
1.5 1 y = -0.1132x + 4.6
y = -0.0749x + 4.6
0.5 0 0
10
20
30 40 Time (min) 55°C
65°C
50
60
70
75°C
Figure 3a 5 4.5
ln (A/Ao)*100
4 y = -0.0166x + 4.6
3.5 3
y = -0.0315x + 4.6
2.5 2 1.5
y = -0.0492x + 4.6
1 0.5 0 0
10
20
30 40 Time (min) 55°C
65°C
50
60
70
75°C
Figure 3b
23
1/ Temperature (K-1) -1.5 0.00285
0.0029
0.00295
0.003
0.00305
0.0031
-2
ln k
-2.5 y = -4218.2x + 9.9243
-3 -3.5 -4
y = -6210x + 14.861
-4.5 Free pectinase
Immobilized pectinase
Figure 3c Figure 3 Thermal kinetics profile of free pectinase (a) and immobilized pectinase by dextran polyaldehyde (b). Arrhenius plot for inactivation of free and immobilized pectinase (c).
24
100
Residual activity (%)
95
90
85
80
75 1
2
3
4
5
6
7
Cycles
Figure 4a
Residual activity (%)
100 80 60 40 20 0 0
3
6
9
12
15
Time (days) Immobilized Pectinase
Free Pectinase
Figure 4b Figure 4 (a) Percentage residual activity (upto seven cycle) of immobilized pectinase onto CMNPs. The 100% residual activity of respective enzyme corresponds to activity at first cycle. (b) The storability studies of free and immobilized pectinase at room temperature in phosphate
25
buffer (100 mM, pH 6). The measurements were performed in triplicate and the error bar represents the percentage error.
26
Table 1. Thermal deactivation kinetic parameters: Thermal deactivation constant (Kd), half-life (t1/2) and deactivation energy (Ed) of free and immobilized pectinase. Kd (min-1)
t1/2 (min)
Ed (kJ/mol)
Temperature 55
65
75
55
65
75
55-75
0.0540
0.0749
0.1132
12.83
9.25
6.12
35.07
0.0166
0.0351
0.0492
41.75
22.01
14.10
51.63
(°C) Free Pectinase Immobilized Pectinase
Table 2. Kinetic parameters of free and immobilized pectinase Form of pectinase
Km (µM)
Vmax (µmol/min)
Free Pectinase
0.6803 ± 0.045
5.9816 ± 0.32
0.708 ± 0.026
5.7391 ± 0.48
Immobilized Pectinase
27
Table 3. Fractions of secondary structures for free and immobilized pectinase. Form
α-helix (%)
β-sheet (%)
β-turn (%)
Random Coils (%)
21.93
6.04
49.90
22.13
15.52
4.24
56.77
23.47
Free Pectinase Immobilized Pectinase
28
Table 4 Clarification (in terms of turbidity) of apple juices by free and immobilized pectinase. Turbidity (%) of Apple juice
Time (min)
Free pectinase
Immobilized pectinase
0
100 ± 1.28
100 ± 1.43
30
91 ± 2.93
93 ± 1.33
60
84 ± 3.8
86 ±2.12
90
79 ± 2.25
82 ± 2.25
120
74 ± 3.40
78 ± 3.40
150
70 ± 2.24
74 ± 2.84
29
S0144-8617(16)31177-8 http://dx.doi.org/doi:10.1016/j.carbpol.2016.10.018 CARP 11638
To appear in: Received date: Revised date: Accepted date:
5-8-2016 5-10-2016 6-10-2016
Please cite this article as: Sojitra, Uttam V., Nadar, Shamraja S., & Rathod, Virendra K., Immobilization of pectinase onto chitosan magnetic nanoparticles by macromolecular cross-linker.Carbohydrate Polymers http://dx.doi.org/10.1016/j.carbpol.2016.10.018 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Immobilization of pectinase onto chitosan magnetic nanoparticles by macromolecular cross-linker
Uttam V. Sojitraa, Shamraja S. Nadara, Virendra K. Rathoda*
a
Department of Chemical Engineering, Institute of Chemical Technology, Matunga (E)
Mumbai-400019, India.
Corresponding Author: Dr. Virendra K. Rathod E-mail: [email protected], Phone: +91-22-33612020, Fax: 91-22-33611020.
Highlights:
Pectinase immobilized onto chitosan MNPs by dextran polyaldehyde cross-linker.
Biocompatible, biodegradable functionalize and cross-linker used for immobilization.
Hydrolysis of pectin improve quality, stability and yield of fruit juice.
Thermal stability of immobilized pectinase was expressed as half-life.
Prepared magnetic pectinase nanobiocatalyst employed for apple juice clarification.
1
Abstract Pectinase was immobilized onto chitosan magnetic nanoparticles (CMNPs) by dextran polyaldehyde as a macromolecular cross-linking agent. The parameters like cross-linking concentration, time and CMNPs to enzyme ratio were optimized. Further, prepared magnetic pectinase nanobiocatalyst was characterized by FT-IR and XRD. The thermal kinetic studies for immobilized pectinase showed two folds improved thermal stability in the range of 55-75°C as compared to free form. The Vmax and Km values of immobilized pectinase were found to be nearly equal to native form which indicated that conformational flexibility of pectinase was retained even after immobilization. The residual activity of immobilized pectinase was 85% after seven successive cycles of reuse, while it retained upto 89% residual activity on storage of fifteen days which exhibited excellent stability and durability. The conformational changes in pectinase after immobilization were evaluated by FT-IR spectroscopy data analysis tools. Finally, magnetic pectinase nanobiocatalyst was employed for apple juice clarification which showed turbidity reduction upto 74% after 150 min treatment.
Keywords: Immobilization, chitosan magnetic nanoparticles, pectinase, dextran cross-linker, juice clarification,
Introduction Enzymes are highly effective, versatile and eco-friendly catalysts which operate under mild reaction conditions (Kim et al., 2008; Ge et al., 2009; Wang 2006). They have become a potential tool in making benign industrial manufacturing processes which attract more and more researchers to exploit it. However, industrial applicability of enzymes is always obstructed due to several limitations such as low operational stability, difficulty in recovery and reusability, which make them unaffordable (Sheldon et al., 2013). To overcome these shortcomings, enzyme immobilization onto solid carrier is one of the effective techniques which not only stabilizes enzymes under operational conditions but also allows easy recovery and reusability for multiple cycles (Cao et al., 2005). Generally, immobilization of enzyme on the carrier involves synthesis of functionalized carrier and covalent cross-linking of enzyme on its surface. Over the past decade, a number of 2
nano-carriers have been prepared and used as a support for immobilization of enzyme (Ansari et al., 2012). Among different types of carriers, magnetic nanoparticles (MNPs) are significantly used as a support due to their unique characteristics such as their tailored surface chemistry, unique physicochemical properties, biocompatibility, biodegradability, low toxicity and low cost (Caterina et al., 2013, Seenuvasan et al., 2014a). Also, magnetic nanoparticles allow easy, quick and efficient separation of enzyme from the reaction mixture by using external magnet (Laurent et al., 2008, Karthikeyan et al., 2014). Most commonly, functionalized nano-carrier for enzyme immobilization is prepared in two-steps (synthesis of carrier and functionalization), which creates difficulty in handling, longer reaction time, loss of product at each step and also requires high temperature conditions for the functionalization (Kim et al., 2013). Further, during cross-linking enzymes on nano-carriers, cross-linking agent plays crucial role as it has direct influence on activity recovery and operational stability (Talekar et al., 2013b). Amongst available protein cross linking agents, glutaraldehyde is the preferred choice as the cross-linker. Because it is inexpensive, easily available, easy to manipulate and has the ability to make covalent bonds with most of the enzymes (Barbosa et al., 2014). However, glutaraldehyde cannot be universally employed as a cross-linker since it possesses some inherent drawbacks. In some cases, use of glutaraldehyde as cross-linker resulted in complete loss of enzyme activity which might be due to small size of cross-linker getting easily penetrated into the active site and cross-linking catalytically important amino acids residues leading to inactivation of enzyme (Mateo et al., 2004). Moreover, cross-linking of enzyme by glutaraldehyde forms lumps of enzyme molecules hindering the active sites. Hence, it suffers from serious mass transfer limitations for macro-substrates resulting in lower catalytic efficiency (Zhen et al., 2013). Additionally, leaching of glutaraldehyde from prepared biocatalyst causes adverse effect on human and aquatic lives because of its toxic nature (Arsenault et al., 2011). Therefore, in past years, polysaccharides based cross linkers have received increasing attention as a potential cross-linking agent for proteins over glutaraldehyde (Talekar et al., 2014). More recently, Nadar et al. (2016c) employed pectin based cross-linker to immobilize glucoamylase onto 3-aminopropyl triethoxysilane (APTES) modified MNPs which was found to be more effective as compared to traditional glutaraldehyde. In recent years, fruit juice technologies have attracted considerable attention for the improvement of juice quality due to increase in natural fruit juice consumption (Cassano et al., 2013). The freshly pressed fruit juices are cloudy and turbid in appearance due to colloidal dispersion of pectin (0.9-1.5%) present in the forms of disrupted cell wall and cell materials of 3
fruit, which is one of the major hurdle in fruit juice processing (Tapre et al., 2014). Thereby, pectinase is most extensively used enzyme in fruit juice clarification to decrease cloudiness, improve quality and yield of fruit juice (Kashyap et al., 2001). Considering food based application of pectinase (enzyme acting on macromolecular substrate), the materials used for immobilization, that is, functionalizing agent for magnetic nanoparticles and cross-linking agent for anchoring enzyme, must be biocompatible and non-hazardous (Nadar et al., 2016b). Thus, in this work, we report for the first time a non-hazardous, biocompatible, eco-friendly and efficient immobilization strategy. Here, we synthesized magnetic nanoparticles in the presence of chitosan biopolymer which not only provides stability and amino group to magnetic nanoparticles but also makes them highly biocompatible. Further, dextran polyaldehyde was prepared via controlled periodate induced oxidation which is used as a non-toxic and biodegradable cross-linker to anchor enzyme onto CMNPs. The different parameters like cross-linker concentration, cross-linking time and ratio of CMNPs to enzyme were optimized. The prepared immobilized pectinase was characterized by Fourier transform infrared spectroscopy (FT-IR) and crystal structures were determined by X-ray powder diffraction (XRD). The kinetic parameters (Vmax and Km) were evaluated for prepared immobilized pectinase. In addition to that, the thermal stability of prepared magnetic pectinase nanobiocatalyst was evaluated in terms of deactivation rate constants (kd), half-life (t1/2) and deactivation energy (Ed). Finally, reusability and storage stability of immobilized pectinase were investigated to determine its industrial applicability. At the end, immobilized pectinase was employed for clarification of apple juice and the relative clarity of juice was determined.
Materials and methods Materials Pectinase (126 U/mL, protein content 10 mg/mL) was procured from Enzymes India Pvt. Ltd, Chennai, India. Apple pectin (Mw 100-120 kDa, degree of esterification 85–90%, galacturonic acid min. 65%), dextran (Mw 10000 kDa) and chitosan (Mw 40–60 kDa, DDA ≥75%) were purchased from HiMedia Laboratories Pvt. Ltd. Mumbai, India. 3,5-Dinitrosalicylic acid (DNSA), ferrous sulphate (FeSO4·4H2O) and ferric chloride (FeCl3·6H2O) were purchased from S. D. Fine Chemicals, Mumbai, India. 2, 4-dinitrophenylhydrazine (DNPH) was purchased from HiMedia Laboratories Pvt. Ltd. Mumbai, India. All other chemicals were of analytical grade and procured from reliable sources. Synthesis of chitosan magnetic nanoparticles
4
Chitosan magnetic nanoparticles (CMNPs) were synthesized by single step method as reported by Gregorio-Jauregui et al., (2012). Briefly, chitosan (1%) was dissolved completely in acetic acid (1 % v/v, 45 mL) in a 50 mL capacity glass vessel equipped with impeller. Ferrous sulphate (FeSO4·4H2O, 0.2 M) and ferric chloride (FeCl3·6H2O, 0.4 M) (in molar ratio of 1:2) were dissolved in above chitosan solution. The mixture was permitted to mix for 30 min at room temperature (30±2 °C) and sodium hydroxide (1 M) was added slowly with continuous agitation (1000 rpm) by overhead laboratory stirrer (RQ - 40 Plus, Remi, India). After 30 min of reaction, magnetic nanoparticles were recovered by magnet and washed five times with deionized water. The particles were kept for overnight drying at room temperature and further analyzed by using FT-IR, XRD and TEM. Preparation of dextran macromolecular cross-linker Dextran polyaldehyde cross-linker was prepared by the method as reported by Jadhav et al. (2013). Briefly, sodium metaperiodate (50-400 mM) was prepared in sodium phosphate buffer and used as an oxidizing agent. The dextran was oxidized with oxidizing solution for 30-150 min in the dark condition. After oxidation, ethylene glycol (0.3 mL) was added to the reaction mixture to stop the oxidation. Oxidized dextran solution was dialyzed against sodium phosphate buffer at 4°C overnight. The extent of oxidation of dextran was determined by 2, 4-dinitrophenylhydrazine (DNPH) test (Gupta et al., 2013). In this test, oxidized dextran (0.3%, 3×10-4 g/L) was added to freshly prepared solution of DNPH (10 mL, 1% w/v). The mixture was permitted to stand for 1 h and then centrifuged at 8000 rpm for 10 min. The amount of unreacted DNPH in supernatant was determined by using UV-Vis spectrophotometer (Jasco V-630, Japan) at 357 nm. The amount of generated aldehyde was calculated according to: Aldehyde content (mmol/g) =
(Reacted DNP (mg/L)/198.14) Amount of oxidized dextran (g/L)
Where, 198.14 is the molecular weight of DNPH. Finally, the oxidized dextran was dissolved in phosphate buffer (100 mM, pH 6) and used as macromolecular cross-linker. Pectinase activity assay The activity of pectinase was assessed using pectin as a substrate. Pectinase (0.5 mL) was mixed with pectin solution (1 % prepared in phosphate buffer of pH 6, 100 mM) and incubated at 50°C for 20 min and then DNSA reagent (2 mL) was added to the reaction mixture, boiled for 15 min, cooled and absorbance was measured at 575 nm using UV-Vis spectrophotometer 5
(Jasco V-630, Japan) (Miller et al., 1959). One unit of enzyme activity (U) was defined as the amount of the enzyme liberating one µmole of ß-galacturonic acid per minute at optimum conditions (50°C, pH 6.0). The protein concentration in the supernatant was measured by the Bradford’s reagent using bovine serum albumin (0-1 mg/mL) as the standard (Bradford et al., 1978). Immobilization of pectinase onto CMNPs The CMNPs were mixed with pectinase (10 mg protein content) in sodium phosphate buffer (100 mM, pH 6) in the ratio of 1:1 to 5:1 (w/w). The reaction mixture was kept for shaking at 175 rpm for 30 min. Then, dextran cross-linker was added (0.5-4 % with respect to total solution mixture) and incubated for 3-24 h. After that, sodium borohydrate (5 mg) was added and kept for 30 min to terminate cross-linking. The immobilized pectinase was separated magnetically, washed four times with buffer and assayed for the enzyme activity. Characterization of immobilized pectinase onto CMNPs The immobilized pectinase onto CMNPs were confirmed by Fourier transform infrared (FT-IR) spectroscopy using Shimadzu IR Affinity-1 FT-IR Spectrophotometer. Crystal structure of CMNPs before and after immobilization was investigated by Powder X-ray diffraction (XRD) (Philips PW 1830 X-ray Diffraction). Also, the size of CMNPs was determined by using transmission electron microscopy (TEM) (JEOL JEM 2100, USA) and hydrodynamic diameter was analysed by using DLS (Zetasizer Nano ZS, Malvern Instruments, UK). Thermal stability study of free and immobilized pectinase Thermal kinetics of pectinase in free and immobilized form was studied by incubating them in phosphate buffer (100 mM, pH 6.0) at 55, 65 and 75°C. The sample was collected after 10 min interval for 60 min, and activity was determined in terms of residual activity. The inactivation rate constant (kd) was estimated by plotting residual activity (%) against time in semi-log. The half-life (t1/2) is time required to decrease the activity to half of its original and was calculated as 0.693/kd. Further, deactivation energy (Ed) of the free and immobilized pectinase was determined from Arrhenius plot. Kinetics parameters Kinetics parameters (Km and Vmax) of free and immobilized pectinase were determined using different pectin concentrations in the range of 0.1-3.0 mg/mL in phosphate buffer (100 mM, pH 6.0) at 50°C. Km and Vmax values of free and immobilized pectinase were calculated from 6
non-linear regression fitting of the initial rates of reaction corresponding to different pectin concentrations by Graph Pad Prism software. Secondary structure analysis of free and immobilized pectinase The changes in secondary structure of free and immobilized pectinase were examined by using FT-IR method (Secundo et al., 2013). The FT-IR spectra of free pectinase and immobilized pectinase were noted by using FT-IR (Shimadzu IR Affinity-1) from 400 to 4000 cm-1 with sample dispersed in the KBr pellets. Secondary derivative of peak frequencies in amide-I region (1600–1700 cm−1) were identified and smoothened with a 13-point Savitzky–Golay by Essential FTIRTM 3.00. Then, multicomponent peak areas under amide I bands of free and immobilized pectinase were analysed using multi-peak fitting program with Gaussian function by Origin 9.0. The fractions of secondary structure were estimated as per our previous work (Nadar et al., 2016a). Reusability studies Reusability of immobilized pectinase was carried out at optimum condition of pH and temperature. The reaction was carried out for 30 min using pectin as a substrate in batch mode. After each cycle, immobilized pectinase was separated magnetically, washed with phosphate buffer (100 mM, pH 6) and re-suspended in fresh substrate solution to determine its activity. This process was carried out for seven consecutive cycles. The activity was determined after each cycle in terms of residual activity, considering 100 % residual activity for the first cycle. Storage stability studies Storage stability of the free and immobilized form of pectinase were determined by incubating enzyme in sodium phosphate buffer (100 mM, pH 6) without substrate. After every three days of interval till fifteen days, enzyme samples were separated from the buffer, washed by deionized water (DI) and then assayed for residual activity using pectin as the substrate. Application of immobilized pectinase in fruit juices clarification The ability of magnetic pectinase nanobiocatalyst was studied for clarification of apple (Malus domestica) juice. The fresh apple juice was prepared and centrifuged at 5000 rpm for 20 min, and then supernatant was used for the clarification process. The immobilized pectinase (equivalent amount of free pectinase) was mixed with diluted juices (20:80 v/v of juice to distilled water) and treated for 150 min at 50 °C. After enzymatic treatment, clarity of the juice was determined in terms of turbidity which was evaluated by spectrophotometric method (Magro et al., 2016).
7
Results and discussion Characterization of chitosan magnetic nanoparticles The CMNPs were simultaneously synthesized and functionalized (with -NH2 group) via co-precipitation method. The Fe2+ and Fe3+ ions (molar ratio 1:2) was reduced by sodium hydroxide solution in the presence of chitosan. The chemical reaction can be written as follows (Maity et al., 2007): Fe2+ + 2 Fe3+ + 8 OH−
Fe3O4 + 4 H2O
Further, the prepared CMNPs were characterized by using XRD and FT-IR. The crystalline structure of CMNPs was identified by using XRD analysis. In Figure 1a, X-ray powder diffraction profile exhibited peaks at 2θ = 30.31°, 35.67°, 41.18°, 57.24°, 62.84° which are characteristics of Fe3O4. According to the joint committee on powder diffraction standards (JCPDS) database, the crystal structure of Fe3O4 (reference code 01-075-0449) is inverse spinel in nature with 100% purity. On the basis of full width half maximum (FWHM), the average crystallite diameter of CMNPs was found to be 29.6 nm by Debye-Scherrer’s equation. Also, the size of CMNPs was found to be ~ 45 nm by using transmission electron microscopy (TEM), while hydrodynamic diameter was 107.6 nm by using DLS (Figure S1a-b). Further, the prepared CMNPs were confirmed by using FT-IR (Figure 1c). The FT-IR spectrum showed a characteristic peaks at 586.36 cm-1 which is corresponding to intrinsic stretching vibrations of Fe–O. The peaks at 3385.07 and 1068.56 cm-1 were corresponding to O–H, N–H and C–O–C respectively from chitosan. The peak at 2883.58 and 1643.35 cm-1 were for C–H and N-H stretching respectively which confirmed successful preparation of CMNPs (Gregorio-Jauregui et al., 2012, Liu et al., 2014). Periodate mediated controlled oxidation of dextran cross-linker Macromolecular dextran cross-linker was prepared by metaperiodate mediated controlled oxidation of native dextran. Periodic acid and its salts can oxidize dextran specifically to yield dialdehyde groups after breaking carbon bond at C2-C3 of glucose unit according to the Malaprade mechanism (Jadhav et al., 2013). The extent of oxidation is very important to generate maximum amount of aldehyde group which is dependent on sodium metaperiodate concentration and reaction time. Hence, effect of sodium metaperiodate concentration and time of reaction was studied in terms of generated aldehyde group and analysed using DNPH test. As shown in Figure 2a, increase in the sodium metaperiodate concentration increased the
8
aldehyde content, which was maximum at 150 mM concentration for 120 min. However, at higher concentrations of 200-400 mM sodium metaperiodate, generated aldehyde group of dextran was found to be reduced. This might be due to oxidatory inhibition of dextran at higher concentration of metaperiodate. Also, longer reaction time did not give a significant increase in oxidation. Similar results were observed for dextran cross-linker preparation by Jadhav et al., (2012). Finally, oxidized dextran was dissolved in sodium phosphate buffer (100 mM, pH 6.0) and used as dextran macromolecular cross-linker. Immobilization of pectinase onto CMNPs by dextran macromolecular cross-linker The pectinase was covalently immobilized onto CMNPs by using polyaldehydic dextran as macromolecular cross-linker. The reactive aldehyde groups of oxidized dextran can cross-link amino groups present in terms of lysine or hydroxyl lysine side groups of enzyme and chitosan to form Schiff's base which prevents leaching of enzyme during reaction (Jaydee et al., 2014). Cross-linking parameters (cross-linker concentration and cross-linking time) were the key steps in immobilization which have direct influence on enzyme loading, activity recovery and operational stability of prepared magnetic biocatalyst (Talekar et al., 2013b). The extent of cross-linking between enzyme and CMNPs is needed to be optimized to get maximum activity recovery. The effect of cross-linker concentration on the activity recovery of pectinase in immobilized form was studied by varying concentration in the range of 0.5-4% (v/v) (Figure 2b). At low concentration of cross-linker (0.5-1.0%), less activity recovery was observed due to inadequate cross-linking of enzyme. The activity recovery of pectinase increased with increase in cross-linker concentration. The maximum activity recovery was found at 2.5% (v/v) dextran polyaldehyde. Further increase in the concentration of cross-linker resulted into higher rigidification of enzymes, thereby consecutive loss of activity (Zhen et al., 2013). Cross-linking time is another important factor in immobilization of enzyme. As the cross-linking time was increased, the activity recovery of enzyme also increased. It can be seen that almost 50% activity was recovered in initial 6 h itself, due to rapid cross-linking (Figure 2c). The maximum activity recovery in immobilized pectinase was found at 15 h of cross-linking time. Further increase in cross-linking time, decreased the activity recovery of pectinase gradually. This might be due to excessive cross-linking, which is speculated to cause the chemical modification of enzyme (Wang et al., 2013) To maximize pectinase immobilization, the amount of CMNPs (non-catalytic support) should be optimized. This was done by varying the ratio of CMNPs to enzyme (1:1 to 5:1) (Figure 2d).
9
At lower ratio (1:1-2:1), the amount of CMNPs was not sufficient to load pectinase, thereby lowering the activity recovery. As the ratio increased, the activity recovery also increased significantly upto 95% till the ratio of 4:1. With further increase in the CMNPs to enzyme ratio, the activity recovery remained constant, and it might be due to the maximum loading of enzyme on carrier (Yu et al., 2013). Characterization of immobilized pectinase onto CMNPs The prepared magnetic pectinase nanobiocatalyst was further characterized by XRD and FT-IR. X-ray powder diffraction profile exhibited peaks at 2θ = 30.31°, 35.67°, 41.18°, 57.24°, 62.84° which are similar to the peaks obtained for Fe3O4. XRD analysis of immobilized magnetic pectinase nanobiocatalyst showed almost similar XRD pattern as that of CMNPs which implies that there was no phase change after pectinase immobilization (Figure 1b). By using Debye-Scherrer’s equation, the average crystallite diameter of immobilized pectinase onto CMNPs was calculated to be 38.4 nm. The FT-IR spectrum of magnetic pectinase nanobiocatalyst showed prominent peaks at 1026.13 and 601.79 cm−1 which are characteristic for stretching vibration of the C–O–C and Fe-O bond from CMNPs (Figure 1d). Also, the peaks observed at 1527.62 and 1334.74 cm−1 were corresponding to N–H deformation and C–O vibration from chitosan. The characteristic peak at 1689.64 cm−1 reveals the presence of amide-I region of pectinase which confirmed the successful immobilization of pectinase onto CMNPs via dextran polyaldehyde cross-linker (Seenuvasan et al., 2014b, Nadar et al., 2016c). Thermal stability study of free and immobilized pectinase The extent of thermal stability of pectinase in free and immobilized form was expressed in terms of deactivation rate constants (kd), and half-life (t1/2). These parameters were determined separately by incubating each form of enzyme in phosphate buffer at different temperatures (55, 65 and 75°C) and the residual activity was determined after equal interval of 10 min till 60 min (Figure 3a-b). The kd and t1/2 values are summarized for pectinase in different forms in Table 1. After cross-linking enzymes with macromolecular dextran cross-linker onto CMNPs, the kd values of immobilized pectinase were lower than that of free form in the range of 55-75°C. Also, the half-life of immobilized pectinase was increased by 2.3 folds with respect to free form. It seems that covalent cross-linking between amino residues present on the surface of enzymes and CMNPs via dextran polyaldehyde maintained the active tertiary structure of
10
enzyme in immobilized form against native forms (Talekar et al., 2013a, Seenuvasan et al., 2013b). Further, the deactivation energy (Ed) was determined as a measure of thermal stability of immobilized pectinase by using linear fit of the Arrhenius plot (Figure 3c). The higher deactivation energy is an index of high thermal stability of protein indicating more thermal stability. The Ed for denaturation of immobilized and free form of pectinase was 51.36 and 35.07 kJ mol−1 respectively. These results imply that immobilization of enzymes onto CMNPs provide protection from thermal denaturation due to covalent cross-linking with macromolecular cross-linker. Hence, it requires much more energy to break down their active conformation over the free form (Magro et al., 2016, Talekar et al., 2012). Therefore, magnetic pectinase nanobiocatalyst has good potential for industrial applications due to its high thermal stability.
Kinetic parameters Kinetic parameters (Vmax and Km) of free and immobilized pectinase were determined by measuring initial reaction rates for each form by varying amounts in the range of 0.1-3.0 mg/mL. The values of Vmax and Km are summarized in Table 2. Km values of immobilized form were nearly same as that of free pectinase. This indicated that substrate affinity remained unchanged after enzyme immobilization through dextran cross-linker. This could be probably due to retention of conformational flexibility even after cross-linking which maintained accessibility for macro-molecular substrate (Nadar et al., 2016b, Zhen et al., 2013).
The Vmax values of immobilized pectinase via dextran cross-linker were found to be nearly equal to free forms, which indicated that the rate of pectin hydrolysis did not change after immobilization. It could be possibly explained on the basis of macromolecular dextran cross-linker which covalently binds pectinase along its linear chain. This prevents lump formation of enzyme molecules, resulting in unchanged mass transfer of substrate and product (Nadar et al., 2016c, Talekar et al., 2014, Zhen et al., 2013). Secondary structure analysis of free and immobilized pectinase The secondary structure of enzyme plays important role in catalytic activity and its stability. FT-IR spectroscopy is considered as a useful data tool to examine structural changes in enzyme
11
after immobilization in powder form (Secundo et al., 2013). The amide I band (1700–1600 cm-1) is most sensitive spectral region of protein structural components, hence considered for the determination of secondary structure of immobilized enzyme using FT-IR spectroscopy data analysis tool. However, due to close proximity of bands and extensive overlapping of structural component bands, the secondary derivative of amide I band was used to get improved resolution and identify structural component peak frequencies. Then, the peak areas under multicomponent of amide I bands were quantified using multi-peak fitting done by Gaussian function.
The second derivative FT-IR spectra for free pectinase and immobilized pectinase were obtained (Figure S2 a-b).The relative contents of β-sheet structure (1610-1640 cm-1) (red), α-helix structure (1650-1658 cm-1) (blue), random coil structure (1640-1650 cm-1) (green) and β-turn structure (1660-1700 cm-1) (yellow) based on the modelled multi-component peak areas were calculated according to our previous work by using software (Nadar et al., 2016a). The altered frequencies of secondary structures were due to the cross-linking between amino group present in the enzyme and CMNPs via dextran polyaldehyde which affects molecular geometry and hydrogen bonding pattern of immobilized pectinase. The fraction of secondary structure of free and immobilized pectinase is summarized in Table 3. It was found that the reduction in α-helical component was 6.41% in immobilized form; while β-turn content increased by 6.87% as compared to the free form. However, there were slight changes in β-turns and random coils after immobilization. The favourable changes in secondary structure after immobilization onto CMNPs resulted in significant improvement in stability. Reusability studies For any industrial applications, reusability of immobilized enzyme is a key factor to make process economically feasible. The residual activity of immobilized pectinase was found to be 85% even after seven consecutive cycles of reuse in batch mode (Figure 4a). The enzyme (protein content) was not leaching in the reaction mixture after multiple cycles of use which indicates significant reduction in purification (of protein/enzymes contamination) cost. The reduction in residual activity upto seventh cycle might be due mechanical damage and deactivation of enzyme during recycling assay. Since it is convenient to separate immobilized enzyme easily by external magnetic field, it can be reused repeatedly decreasing the overall cost (Nadar et al., 2016c).
12
Storage stability studies Storage stability of the free and immobilized pectinase was evaluated by incubating them at room temperature (28±2°C) in aqueous buffer. It was determined till 15 days by monitoring the enzyme activity after three days interval. The residual activity of free pectinase was found to be 68%, whereas, immobilized form retained 89% residual activity (Figure 4b). The improved storage stability of immobilized pectinase might be due to chemical cross-linking by dextran polyaldehyde onto CMNPs which prevents possible distortion effects on the active sites of enzyme caused by the buffer solution (Sojitra et al., 2016, Seenuvasan et al., 2013). Clarification of apple fruit juice by pectinase nanobiocatalyst The turbid and hazy appearance of fruit juices is due to the presence of pectin and pectic components. Therefore, breakdown of these polysaccharides by pectinase has helped to improve quality characteristics and storage stability (Sojitra et al., 2016, Magro et al., 2016). For the clarification studies, apple (Malus domestica) fruit juice was treated using the immobilized and free pectinase. The clarification of juice was measured in terms of turbidity reduction by spectroscopic method (Table 4). At the start of clarification process, the rate of turbidity reduction by free pectinase was found to be 8-9%, whereas, immobilized pectinase exhibited 6-7% turbidity reduction in 30 min. As time progressed, free pectinase treatment reduced turbidity of apple juice upto 70%, while, by immobilized pectinase treatment, turbidity was reduced upto 74% after 150 min of treatment.
Conclusion Pectinase was immobilized on CMNPs by using dextran polyaldehyde as a macromolecular cross-linker for fruit juice clarification. The magnetic pectinase nanobiocatalyst exhibited superior thermal stability (55-75°C) as t1/2 increased by 2.3 folds as compared to the free enzymes. The Vmax and Km values of magnetic pectinase nanobiocatalyst were found to be nearly equal to free form due to retention of conformational flexibility even after immobilization. Moreover, immobilized pectinase showed 87% residual activity even after seven cycles of recyclability which emphasizes its high stability and durability thereby industrial applicability. Furthermore, immobilized pectinase employed for clarification of 13
apple juice showed rapid reduction upto 74% turbidity while, free form showed turbidity reduction upto 70% till 150 min of treatment. These results highlight the potential use of dextran as the cross-linking agent for the immobilization of pectinase onto CMNPs. As this immobilization technique involves renewable and biocompatible natural biopolymer for functionalizing and cross-linking agent, it has considerable advantages from environmental and worker safety point of view over the traditional chemicals.
Acknowledgements The authors also would like to acknowledge the University Grants Commission (UGC) of India for financial assistance in the research work.
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Figure 1a
Figure 1b
19
Figure 1c
Figure 1d Figure 1 XRD patterns of the prepared CMNPs (a) and immobilized pectinase onto CMNPs cross-linked by dextran polyaldehyde (b). FT-IR spectrum of the prepared CMNPs (c), and pectinase immobilized onto CMNPs cross-linked by dextran polyaldehyde (d).
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18
Aldehyde content (mmol/g)
16 14 12 10 8 6 4 2 0
10
50
100
150
200
300
400
Periodate concentration (mM) 30 min
60 min
90 min
120 min
150 min
2
2.5
3
Activity recovery (%)
Figure 2a
100 90 80 70 60 50 40 30 20 10 0 0.5
1
1.5
3.5
4
Cross-linker concentration (%)
Figure 2b
21
100
Activity recovery (%)
90
80 70 60 50 40 30 20 10 0 3
6
9
12
15
18
21
24
Cross-linking time (h)
Figure 2c 100 90
Activity recovery i(%)
80
70 60 50 40 30
20 10 0 1:1
2:1
3:1
4:1
5:1
Ratio of CMNPs to enzyme (protein) (w/w)
Figure 2d Figure 2 (a) Aldehyde content generated in dextran after oxidation with metaperiodate (50-400 mM) determined by DNPH method. Effect of cross-linker concentration (b), cross-linking time (c) and ratio of CMNPs to enzyme (protein) (w/w) (d) on activity recovery of pectinase in magnetic nanobiocatalyst. The 100% activity recovery corresponds to pectinase (126 U). The measurements were performed in triplicate and the error bar represents the percentage error.
22
5 4.5 4
ln (A/Ao)*100
3.5 3 2.5 2 y = -0.054x + 4.6
1.5 1 y = -0.1132x + 4.6
y = -0.0749x + 4.6
0.5 0 0
10
20
30 40 Time (min) 55°C
65°C
50
60
70
75°C
Figure 3a 5 4.5
ln (A/Ao)*100
4 y = -0.0166x + 4.6
3.5 3
y = -0.0315x + 4.6
2.5 2 1.5
y = -0.0492x + 4.6
1 0.5 0 0
10
20
30 40 Time (min) 55°C
65°C
50
60
70
75°C
Figure 3b
23
1/ Temperature (K-1) -1.5 0.00285
0.0029
0.00295
0.003
0.00305
0.0031
-2
ln k
-2.5 y = -4218.2x + 9.9243
-3 -3.5 -4
y = -6210x + 14.861
-4.5 Free pectinase
Immobilized pectinase
Figure 3c Figure 3 Thermal kinetics profile of free pectinase (a) and immobilized pectinase by dextran polyaldehyde (b). Arrhenius plot for inactivation of free and immobilized pectinase (c).
24
100
Residual activity (%)
95
90
85
80
75 1
2
3
4
5
6
7
Cycles
Figure 4a
Residual activity (%)
100 80 60 40 20 0 0
3
6
9
12
15
Time (days) Immobilized Pectinase
Free Pectinase
Figure 4b Figure 4 (a) Percentage residual activity (upto seven cycle) of immobilized pectinase onto CMNPs. The 100% residual activity of respective enzyme corresponds to activity at first cycle. (b) The storability studies of free and immobilized pectinase at room temperature in phosphate
25
buffer (100 mM, pH 6). The measurements were performed in triplicate and the error bar represents the percentage error.
26
Table 1. Thermal deactivation kinetic parameters: Thermal deactivation constant (Kd), half-life (t1/2) and deactivation energy (Ed) of free and immobilized pectinase. Kd (min-1)
t1/2 (min)
Ed (kJ/mol)
Temperature 55
65
75
55
65
75
55-75
0.0540
0.0749
0.1132
12.83
9.25
6.12
35.07
0.0166
0.0351
0.0492
41.75
22.01
14.10
51.63
(°C) Free Pectinase Immobilized Pectinase
Table 2. Kinetic parameters of free and immobilized pectinase Form of pectinase
Km (µM)
Vmax (µmol/min)
Free Pectinase
0.6803 ± 0.045
5.9816 ± 0.32
0.708 ± 0.026
5.7391 ± 0.48
Immobilized Pectinase
27
Table 3. Fractions of secondary structures for free and immobilized pectinase. Form
α-helix (%)
β-sheet (%)
β-turn (%)
Random Coils (%)
21.93
6.04
49.90
22.13
15.52
4.24
56.77
23.47
Free Pectinase Immobilized Pectinase
28
Table 4 Clarification (in terms of turbidity) of apple juices by free and immobilized pectinase. Turbidity (%) of Apple juice
Time (min)
Free pectinase
Immobilized pectinase
0
100 ± 1.28
100 ± 1.43
30
91 ± 2.93
93 ± 1.33
60
84 ± 3.8
86 ±2.12
90
79 ± 2.25
82 ± 2.25
120
74 ± 3.40
78 ± 3.40
150
70 ± 2.24
74 ± 2.84
29