Chapter 8 Immobilization of β-glucosidase on magnetic nanoparticles 8.1. Introduction Safe and sustainable methodologies for hydrolysis rely up on biocatalysis which offer mild reaction conditions, environmental safety, high efficiency and selectivity. Reuse of enzymes reduces the cost of production and thus researches aim at developing suitable technologies to immobilize enzymes. Enzyme immobilization provides many advantages over free enzymes namely enzyme reuse, continuous operation, controlled product formation, simple processing and enhanced stability under both storage and operational conditions (Gargouri et al., 2004; Sheldon, 2007). Easy separation of enzyme from the product simplifies enzyme applications and supports a reliable and efficient reaction technology. Also, reuse of enzymes provides a cost advantage which is a prerequisite for establishing enzyme catalyzed process (Tischer and Wedekind, 1999). The technique of immobilization may be by binding to a support (carrier), entrapment (encapsulation) and cross linking. The choice of immobilization method depends on the enzyme, support, chemical reagents and reactor (Gargouri et al., 2004). Support binding can be physical by means of hydrophobic or van der Waals interactions, ionic or covalent in nature. Physical means of immobilization is not industrially applicable owing to possible detachment of enzymes under change in ionic strength and ph. Covalent binding prevents leaching of enzymes, but chances of enzyme deactivation during immobilization occur at a high rate. Entrapment of enzyme in polymer network or microcapsules is another method of immobilization employed for industrial purposes like in a hollow fiber reactor. Cross linking of enzymes using bi-functional reagents offer highly concentrated enzyme activity in the catalyst, high stability and low production cost owing to exclusion of carriers (Sheldon, 2007). Particle size of carriers plays an important role in the success of immobilization. Nanoparticles offer high surface area to which large number of enzyme molecules can 185
be immobilized. Nanoparticles are defined as particulate dispersions or solid particles with a size in the range of 10-1000 nm (Mohanraj and Chen, 2006). A number of inorganic carriers can be used, including alumina, silica and zeolites. However, recovery of nanoparticles becomes difficult especially when biomass is used as substrate for catalysis. Magnetic nanoparticles are a new technology whereby immobilized enzymes can easily be recovered under strong magnetic fields, even from viscous reactants/products. Magnetic nanoparticles have been synthesized with different compositions and phases, including iron oxides, such as Fe 3 O 4 and γ-fe 2 O 3, pure metals, such as Fe and Co, spinel-type ferromagnets, such as MgFe 2 O 4, MnFe 2 O 4 and CoFe 2 O 4, as well as alloys, such as CoPt 3 and FePt. The common methods of synthesis employed include co-precipitation, thermal decomposition and/or reduction, micelle synthesis, hydrothermal synthesis, and laser pyrolysis techniques (Lu et al., 2007). In the present study, an attempt was made to prepare magnetic nanoparticles and to improve its stability by silica coating. Purified β-glucosidases were successfully immobilized onto magnetic nanoparticles and the immobilized enzymes were recovered using a strong magnet. 8.2. Materials and Methods 8.2.1. Synthesis of magnetic nanoparticles Magnetic nanoparticles were synthesized by a modified protocol of Berger et al (1999). Four milliliters of 2.0 M FeCl 2 was mixed with 20 ml of 2.0 M FeCl 3 in a 500 ml Erlenmeyer flask. Using a burette, 250 ml of 0.7 M ammonium hydroxide was added to this mixture drop by drop under vigorous agitation. Stirring was stopped once a black precipitate of magnetite (Fe 3 O 4 ) was formed, and the precipitate was allowed to settle. The supernatant was decanted and the rest of the suspension was centrifuged at 2000 rpm for 5 min to obtain the dark sludge like magnetite. The magnetite was washed thrice with distilled water and recovered as pellet after centrifugation (2000 rpm, 5 min). The pellet was dried at 50 C overnight. 186
8.2.2. Silica coating of magnetic nanoparticles For enhancing stability of magnetic nanoparticles, silica coating was performed as per the procedure of Bo et al (2008). 10.98 g of sodium silicate was dissolved in deionized water. The ph of the solution was adjusted to 12.5 by addition of 2.0 M HCl. 500 mg of magnetic nanoparticles, prepared as outlined in section 8.2.1 were added to this solution and was ultra-sonicated for 30 min. The suspension was then kept in a shaking water bath at 80 C. Using a burette, 2.0 M HCl was added drop wise into the solution so as to attain a ph of 6-7 in a time span of 3 h. The precipitate was washed several times with deionized water and recovered as pellet by centrifugation at 2000 rpm for 5 min. The brown yellow precipitate was dried at 50 C overnight. 8.2.3. Silica coating of magnetic nanoparticles under N 2 atmosphere Silica coated nanoparticles produced as outlined in section 8.2.2 showed oxidation as evident from brown coloured supernatant. Hence a modification of the procedure was performed. In this method, silica coating using sodium silicate was done at 60 C with stirring by a magnetic stirrer under nitrogen containing atmosphere. The precipitate was recovered as outlined under section 8.2.2. 8.2.4. Functionalization of magnetic nanoparticles Functionalization of magnetic nanoparticles were performed using APTES (3- amino propyl-triethoxysilane) as per the protocol of Can et al (2009). Four gram of silica coated magnetic nanoparticles was sonicated in 150 ml 50 % ethanol for 30 min. To this suspension, 16.16 g of APTES was added and the solution was stirred at 40 C under N 2 atmosphere for 2 h. The suspension was cooled to room temperature and the APTES modified nanoparticles were collected by centrifugation at 2000 rpm for 5 min. The pellet was washed thrice with deionized water and dried at 70 C. 187
8.2.5. Immobilization of β-glucosidase Immobilization protocol as outlined by Can et al (2009) was used with minor modifications. 1.53 g of silica coated nanoparticles was dispersed in 60 ml 5 % glutaraldehyde. The suspension was kept overnight with stirring at 4 C. The nanoparticles were recovered using a magnet and washed thrice with deionized water. It was dispensed in 20 ml citrate buffer (ph 4.8, 0.05 M). To this, purified β-glucosidase equivalent to 2000 U in citrate buffer (ph 4.8, 0.05 M) was added and incubated at 4 C for 5 h with mild agitation. Purified β-glucosidase isoforms- BGL 2, BGL 3 and BGL 6 were immobilized individually onto nanoparticles. After incubation, the nanoparticles were separated by a magnet and washed thrice with citrate buffer (ph 4.8, 0.05 M). The immobilized nanoparticles were stored at 4 C in citrate buffer (ph 4.8, 0.05 M). The particles were diluted and β-glucosidase activity was determined as outlined in section 2.5.1. 8.2.6. Polyelectrolyte coating of enzyme immobilized magnetic nanoparticles The enzyme immobilized magnetic nanoparticles were coated with polyelectrolyte. PAH Poly (allylamine hydrochloride) was taken as the +ve polyelectrolyte and PS (Polystyrene) was used as the negative polyelectrolyte. 10 mg/ml stock solution of each polyelectrolyte was prepared in deionized water. The enzyme immobilized nanoparticles, dispersed in 10 ml citrate buffer (ph 4.8, 0.05 M) corresponding to total activity of 211 U of BGL 2, 469 U of BGL 3 and 1150 U of BGL 6 were used for polyelectrolyte coating. To the immobilized enzyme, 100 µl stock solution of PAH was added and was shaken at 100 rpm for 10 minutes. The suspension was centrifuged at 5000 rpm, 4 C for 5 min and washed once with citrate buffer (ph 4.8, 0.05 M), recovered by centrifugation (5000 rpm, 4 C for 5 min) and reconstituted to 10 ml using the same buffer. To this 100 µl stock solution of PS was added and was shaken at 100 rpm for 10 min. The suspension was centrifuged at 5000 rpm, 4 C for 5 min and was washed once with citrate buffer (ph 4.8, 0.05 M), recovered by 188
centrifugation (5000 rpm and 4 C for 5 min) and reconstituted to 10 ml using the same buffer. The process was repeated twice with alternating the polyelectrolyte and the immobilized enzyme was recovered, the activity was assayed as outlined in section 2.5.1 and stored at 4 C in citrate buffer (ph 4.8, 0.05 M). 8.2.7. Analysis of magnetic nanoparticles by Scanning Electron Microscopy (SEM) The nanoparticles and silica coated nanoparticles were spread on clean cover slips and dried at 50 C for 16 h. The cover slips were coated with gold particles and were visualized under scanning electron microscope (NIIST SEM). 8.2.8. Analysis of magnetic nanoparticles by Dynamic Light Scattering Spectrophotometry (DLS) The nanoparticles, silica coated nanoparticles, enzyme immobilized nanoparticles and polyelectrolyte coated enzyme immobilized nanoparticles were filtered through 0.22 µ filter. The size of nanoparticles and their behavior in aqueous suspension in presence and absence of ionic detergent polysorbate 80 (0.01 % v/v) was analyzed using Zeta Potential Analyzer (Malvern Instruments). The suspension was poured in to a plastic cuvette and the light scattering of nanoparticles were measured against distilled water as blank. 8.2.9. Analysis of magnetic nanoparticles by Atomic Force Microscopy (AFM) To study the size and surface morphology of nanoparticles, atomic force microscopy was employed. The nanoparticles were filtered through 0.22 µ filter and the filtrate was serially diluted to 10-6 dilutions. 100 µl of the filtrate as well as 10-6 dilution were spread across clean cover slips and were dried at 50 C for 16 h. The samples were then analyzed using atomic force microscope. 189
8.3. Results and Discussion 8.3.1. Synthesis of magnetic nanoparticles Magnetic nanoparticles (magnetite-fe 3 O 4 ) were successfully synthesized by co-precipitating FeCl 2 and FeCl 3 in presence of ammonia. 2FeCl 3 + FeCl 2 + 8NH 3 + 4H 2 O Fe 3 O 4 + 8NH 4 Cl The nanoparticles were washed with deionized water and dried at 50 C and stored in an air tight container. The magnetite so obtained was black in colour (Fig 8.1). The magnetic nature of the particles was ascertained by their movement under an external magnetic field. Figure 8.1: Magnetic nanoparticles SEM analysis of magnetic nanoparticles revealed irregular shaped particles with uneven margins (Fig 8.2). The size, shape and composition of magnetic nanoparticles depend on the Fe 2+ /Fe 3+ ratio, type of salt, reaction temperature, ph and ionic strength of the media (Lu et al., 2007). 190
Figure 8.2: SEM image of magnetic nanoparticles Dynamic light scattering analysis showed four peaks of size intensity corresponding to 2.4 nm, 25.7 nm, 140 nm and 8000 nm (Fig 8.3). Nanoparticles owing to their small size and large surface area can lead to particle-particle aggregation (Mohanraj and Chen, 2006). This problem aggravates when nanoparticles is magnetic due to magnetic attraction between the particles. Figure 8.3: DLS analysis of magnetic nanoparticles 191
Addition of tween 80 (0.01 % v/v) to the nanoparticles reduced the number of peaks to one with an average size intensity of 8.7 nm (Fig 8.4). Tween 80 (Polysorbate 80) is a common surfactant added during hydrolysis experiment. It reduces the enzyme adsorption to the lignocellulosic substrate (Eriksson et al., 2002; Tu et al., 2007). Addition of polysorbate 80 to nanoparticles were found to reduce aggregation of silver nanoparticles (Li et al., 2012). Addition of tween 80 increased the stability of Fe 3 O 4 magnetic nanoparticles by the hydrophilic block of tween 80 that stabilizes the nanoparticles in water (Wang et al., 2011). Figure 8.4: DLS analysis of magnetic nanoparticles in presence of 0.01 % v/v tween 80 The AFM images of magnetic particles synthesized in the present study confirmed their nanoscale size (Fig 8.5). AFM analyses indicated that the undiluted nanoparticles showed a tendency to aggregate (Fig 8.6). The aggregated particles appeared in the form of branched flakes with irregular or serrated margins. Similar self aggregation has been reported with lipase immobilized magnetic nanoparticles (Dyal et al., 2003). However, in diluted samples (10-6 dilution) the aggregation was highly 192
decreased as evident from the size distribution graph (Fig 8.7). The particle size along the reference line was below 10 nm. Figure 8.5: Surface morphology of magnetic nanoparticles with corresponding 3D size range Figure 8.6: Aggregation of magnetic nanoparticles and size distribution graph of particles along the reference line 193
Figure 8.7: Surface morphology of diluted magnetic nanoparticles (10-6 ) with corresponding size distribution graph of particles along the reference line 8.3.2. Preparation of silica coated magnetic nanoparticles Magnetic nanoparticles are very sensitive to oxidation and agglomeration due to large surface area, high chemical reactivity and magnetic dipole interactions. Under ambient conditions, rapid oxidation occurs resulting in the deposition of oxide layers than can change the particle properties. Agglomeration in to larger clusters also affects the use of magnetic nanoparticles. To protect magnetic nanoparticles from oxidation and agglomeration, encapsulation using carbon, silica, metal oxides, organic polymers or surfactants can be carried out (Faraji et al., 2010). Silica coating of magnetic nanoparticles have several advantages like protection of magnetic core, stability under aqueous conditions, easy surface modifications, easy control of inter-particle interactions by variation of shell thickness and creation of abundant silanol groups which can be easily activated by functional groups (Deng et al., 2005; Lu et al., 2007). Silica coated nanoparticles prepared by the procedure as outlined in section 8.2.2 showed oxidation as evident from the brown supernatant. Exposure to high temperature without a reducing atmosphere might have caused oxidation of magnetite during the encapsulation. Hence silica coating of magnetic nanoparticles were done by the modified protocol as mentioned in 8.2.3. The magnetic properties of nanoparticles were maintained even after 194
silica coating as evident from by their spiking effect under an external magnetic field (Fig 8.8). SEM analysis indicated silica coating to decrease serrations in the nanoparticles, creating smooth surfaces (Fig 8.9). Figure 8.8: Spiking effect of silica coated magnetic nanoparticles under the influence of an external magnetic field. Spiking effect Figure 8.9: SEM image of silica coated magnetic nanoparticles Silica coating of magnetic nanoparticles caused increase in particle size. DLS analysis reveals three peaks of size intensity, the major one corresponding to a size of 309 nm (Fig 8.10). However, compared to uncoated nanoparticles, the highest average size got reduced from 8000 nm to 309 nm indicating the decreased agglomeration due to silica coating. 195
Figure 8.10: DLS analysis of silica coated magnetic nanoparticles With addition of tween 80, agglomeration got reduced and major peak of size got shifted to 9.4 nm. However, an additional peak of size intensity corresponding to 175 nm was present (Fig 8.11). Figure 8.11: DLS analysis of silica coated magnetic nanoparticles in presence of 0.01 % v/v tween 80 196
8.3.3. Immobilization of β-glucosidase on to silica coated magnetic nanoparticles The silica coated magnetic nanoparticles were functionalized using APTES and β-glucosidase enzyme was immobilized by covalent binding with glutaraldehyde reagent. The enzyme immobilized nanoparticles were separated by a magnet and washed thrice with citrate buffer and stored at 4 C in citrate buffer (ph 4.8, 0.05 M). The immobilization efficiency varied with the isoforms. Among the three isoforms, BGL 5 showed high immobilization rate with a total of 1149 U activity against the initial 2000 U provided for immobilization (Table 8.1).The immobilized enzymes showed 100 % storage stability at 4 C for 1 month. Lipases immobilized on magnetic nanoparticles had a constant activity over one month of storage. The enzyme-nanoparticle composites showed only 15 % activity loss over one month, probably by desorption or denaturation (Dyal et al., 2003). Beta-glucosidase from Trichoderma reesei immobilized on to magnetic nanoparticles showed 98 % activity retention with a stability of at least 45 days (Valenzuela et al., 2011). Beta-glucosidase from Agaricus arvensis immobilized on silicon oxide nanoparticles showed improved thermal stability with a 288 fold enhancement of t 1/2 at 65 C (Singh et al., 2011). On immobilization to magnetic aluminum nitride nanoparticles, β-glucosidase showed an activity retention of 78.4 % (Pan et al., 2008). Table 8.1: Efficiency of immobilization Enzyme used Total BGL activity in magnetic nanoparticles (U) Efficiency of immobilization (% activity retention after immobilization) BGL 2 211.6 10.58 BGL 3 469.02 23.45 BGL 6 1149.58 72.48 DLS analysis of enzyme immobilized nanoparticles revealed a major peak of size corresponding to 139 nm and a minor peak of 2 nm (Fig 8.12). Addition of tween 80 197
resulted in the shift of peak towards 9.6 nm, still having a second peak corresponding to a size of 133 nm (Fig 8.13). Figure 8.12: DLS analysis of enzyme immobilized magnetic nanoparticles Figure 8.13: DLS analysis of enzyme immobilized magnetic nanoparticles in presence of 0.01 % v/v tween 80 198
8.3.4. Polyelectrolyte coating of immobilized enzyme Surfactants or polymers are often employed to passivate the surface of nanoparticles to avoid agglomeration. It creates a electrostatic or stearic repulsion that disperses the nanoparticles and keeps them in a colloidal state (Lu et al., 2007). Polyelectrolyte coating of immobilized enzymes can enhance enzyme stability and also reduces the attack of proteolytic enzymes. Polymer coated catalase was found to be stable against protease degradation, retaining 100 % activity after 100 minutes incubation, while uncoated catalase losing more than 90 % of activity under similar conditions (Caruso et al., 2000).The immobilized isoforms were coated thrice with polyelectrolyte with alternating +ve and ve electrolyte. The total activity of immobilized enzymes after polyelectrolyte coating is given in table 8.2. Isoforms varied to their activity retention after polyelectrolyte coating. Maximum retention of activity was shown by BGL 3. Table 8.2: Enzyme activity after polyelectrolyte coating Enzyme used Total BGL activity in magnetic nanoparticles (U) % activity retention after polyelectrolyte coating compared with initial activity in immobilized nanoparticles BGL 2 141.34 66.80 BGL 3 442.64 94.38 BGL 6 927.36 80.67 Dynamic light scattering analysis showed two peaks of size intensity corresponding to 92 nm and 681 nm (Fig 8.14).Polymer coating has thus increased the average size of nanoparticles. Addition of tween 80 reduced agglomeration as evident from the reduced average size of peaks to 9.3 and 82 nm (Fig 8.15). 199
Figure 8.14: DLS analysis of polymer coated enzyme immobilized magnetic nanoparticles Figure 8.15: DLS analysis of polymer coated enzyme immobilized magnetic nanoparticles in presence of 0.01 % v/v tween 80 200
8.4. Conclusions The recovery and reuse of enzymes by immobilization has been a matter of interest in catalysis research. Gel entrapment is the most widely used technique but has the demerit of diffusional resistance that can result in accumulation of products causing enzyme inhibition. Nanoparticles offer greater surface area for enzyme immobilization and remain in colloidal or near colloidal state. Magnetic nanoparticles have the greatest advantage of easy recovery using an applied magnetic field. The present study was successful in synthesizing magnetic nanoparticles. The stability of the produced nanoparticles was enhanced by silica coating and β-glucosidase was effectively immobilized on to the nanoparticles. Additional protection in the form of polyelectrolyte covering was provided which might significantly reduce proteolytic cleavage by proteases present in crude cellulase preparations used in hydrolysis. 201
Chapter 9 Anti β-glucosidase antibody production and its applications