Appl Microbiol Biotechnol (2010) 87:1983–1992 DOI 10.1007/s00253-010-2658-4
BIOTECHNOLOGICAL PRODUCTS AND PROCESS ENGINEERING
Magnetically recyclable, antimicrobial, and catalytically enhanced polymer-assisted “green” nanosystem-immobilized Aspergillus niger amyloglucosidase Rocktotpal Konwarh & Dipankar Kalita & Charulata Mahanta & Manabendra Mandal & Niranjan Karak
Received: 25 January 2010 / Revised: 16 April 2010 / Accepted: 1 May 2010 / Published online: 21 May 2010 # Springer-Verlag 2010
Abstract The present work reports the integration of polymer matrix-supported nanomaterial and enzyme biotechnology for development of industrially feasible biocatalysts. Aqueous leaf extract of Mesua ferrea L. was used to prepare silver nanoparticles distributed within a narrow size range (1–12 nm). In situ oxidative technique was used to obtain poly(ethylene glycol)-supported iron oxide nanoparticles (3–5 nm). Sonication-mediated mixing of above nanoparticles generated the immobilization system comprising of polymer-supported silver–iron oxide nanoparticles (20–30 nm). A commercially important enzyme, Aspergillus niger amyloglucosidase was coupled onto the immobilization system through sonication. The immobilization enzyme registered a multi-fold increment in the specific activity (807 U/mg) over the free counterpart (69 U/mg). Considerable initial activity of the immobilized enzyme was retained even after storing the system at room temperature as well as post-repeated magnetic recycling. Evaluation of the commendable starch saccharification rate, washing performance synergy with a panel of commercial R. Konwarh : N. Karak (*) Department of Chemical Sciences, Tezpur University, Napaam, Tezpur, Assam 784028, India e-mail:
[email protected] D. Kalita : C. Mahanta Department of Food Processing Technology, Tezpur University, Napaam, Tezpur, Assam 784028, India M. Mandal Department of Molecular Biology and Biotechnology, Tezpur University, Napaam, Tezpur, Assam 784028, India
detergents, and antibacterial potency strongly forwards the immobilized enzyme as a multi-functional industrially feasible system. Keywords Amyloglucosidase immobilization . Polymeric nanocomposite . Antimicrobial potency . Magnetic recyclability . Starch saccharification . Detergent compatibility
Introduction Inactivation and poor reusability of the labile biocatalysts are major challenges to the enzyme-based industries (Gupta 1992). To address these issues, various immobilization strategies have been explored time and again. The amount of enzyme lost from the immobilization systems, diffusion limitations, and the net increment in the enzyme activity are critical issues in the various immobilization protocols (Arica et al. 2000; Bai et al. 2009). Amyloglucosidase (AMG) (1, 4-α-D glucan glucanohydrolase, EC 3.2.1.3.) is an industrially important biocatalyst (Crabb and Mitchinson 1997; Kaur and Satyanarayana 2004; Vihinen and Mantsala 1989; Vijayakumar et al. 2005). AMG is a multi-domain, exo-acting enzyme that catalyzes the hydrolysis of starch and related substrates from the non-reducing ends mainly by cleaving α-1,4glycosidic linkages and a few α-1,6 linkages, but at a very slow pace (Pandey et al. 2000). More than two decades ago, various methods for immobilization of AMG were evaluated (Ivanova et al. 1985). The nature of the carrier and degree of purity of the enzyme dictate the binding efficiency and properties of the immobilized AMG. Immobilization onto polyaniline polymer (Silva et al. 2005) and mesoporous and
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hydrophilic novel bead carriers containing epoxy groups (Bai et al. 2009), chemical ligation onto carbon nanotubes (CangRong and Pastorin 2009), etc., are some of the recently reported immobilization studies on AMG. Immobilization of a wide range of biomolecules onto various nanosystems is being reported. Coupling onto polymer-assisted magnetic nanoparticles (MNPs) with large active surface area seems to be an attractive avenue to improve performance of biocatalysts addressing industrial sustainability requirements like magnetic recyclability and thermostability along with activity augmentation (Konwarh et al. 2009). On the other hand, with myriad of synthesis protocols (Guzman et al. 2008) and numerous applications (Nair and Laurencin 2007), silver nanoparticles have carved a unique niche of their own in the domain of nanobiotechnology. The antimicrobial action (Sharma et al. 2009) of silver nanoparticles justifies their inclusion in a wide variety of products. Silver nanoparticle-based systems are gaining special significance in the context of the emerging concept of “green technology” (Sharma et al. 2009). The present work reports the tapping of a bioresource namely Mesua ferrea L. (locally known as “Nahar” in North-East India) leaf aqueous extract for the preparation of nanoparticles of this noble metal. The extract from the various parts of this plant are traditionally known to have a variety of medical uses. Several in vivo studies have validated these ethnopharmaceutical practices (Dweck and Meadows 2002). In this work, we report the immobilization of AMG onto poly(ethylene glycol) (PEG) matrix-supported silver–iron oxide nanoparticles. This is an attempt to combine the innate advantages of polymeric nanocomposite, an advanced class of material with the catalytic potential of an industrial enzyme. The study has been focused on enzyme activity enhancement and assessment of its magnetic recyclability, stability, antimicrobial potency, and applicability prospects for detergent formulation inclusion and starch saccharification.
Materials and methods Preparation of polymer-assisted silver–iron oxide nanoparticles for AMG immobilization PEG-supported MNPs were prepared as described previously by us (Konwarh et al. 2009) with a minor variation. Instead of mechanical stirring, a sonication step (2 min, 60% amplitude, and 0.5 cycle) was introduced for mixing the iron chloride (FeCl2·4H2O; ≥99.0%, BDH) and PEG (number average molecular weight, Mn =6,000 gmol−1, GS Chemicals, coded as PEG6000). Ultrasonication was carried out with the standard sonotrode (tip-diameter
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3 mm) in a high-intensity ultrasonic processor (UP200S, 24 kHz, and acoustic power density 460 W/cm2, Hielscher Ultrasonics GmbH, Germany). Other steps remained essentially the same. For the preparation of the silver nanoparticles, about 20 matured leaves of M. ferrea (collected from the Tezpur University campus in the month of September) were washed with water. These were crushed and boiled in 50 mL of water for 10 min. The leaf extract was filtered through a muslin cloth. Two hundred microliters of 0.1 M silver nitrate (AgNO3; Qualigens®) was taken in 7.5 mL of 5% (w/v) PEG solution, prepared in MilliQ water and 6 mL of the aforestated M. ferrea leaf extract was added as the reducing agent. The pH of the reaction mixture was adjusted to 11.5 with sodium hydroxide (NaOH; Merck). The nanoparticles, well-distributed within a narrow size range (1–12 nm, with average diameter of 5 nm), were obtained under statistically optimized sonication parameters of 3 min, 60% amplitude, and 0.5 cycle by using response surface methodology (not shown, where the response was either red or blue shift in the λmax position in UV-visible spectrum, taken as the indicator of the size of silver nanoparticles). Equal-volume (250 µL) suspensions of the silver and iron oxide nanoparticles were taken in an Eppendorf tube for sonication for 3 min at 60% amplitude and 0.5 cycles. The polymer-assisted nanosystem before and after enzyme immobilization was analyzed using different characterization tools as described below. Immobilization of amyloglucosidase For immobilization, 0.5 mL of Aspergillus niger amyloglucosidase (Fluka, Sigma) (1 mg mL−1) in acetate buffer (0.05 M, pH4.5) was added to 50 mg of PEG-supported silver–iron oxide magnetic nanoparticles followed by sonication for 15 min at 277 K (ice-bath). This was followed by centrifugation at 3,000 rpm for 10 min after which the mass was magnetically decanted. The postprecipitate supernatant was collected in fresh microfuge tube while the precipitate was washed twice and finally suspended in 0.5 mL of acetate buffer (0.05 M, pH4.5). The percentage binding was estimated by determining the amyloglucosidase activity for both the post-precipitate supernatant and the immobilized enzyme fraction. Bio-physical characterization UV-visible spectra were analyzed in Hitachi (Tokyo, Japan) U2001 UV spectrophotometer. Fourier transform infrared spectroscopy (FTIR) spectra were recorded in a Nicolet (Impact 410, Madison, WI) FTIR spectrophotometer by using KBr pellets. Lakeshore vibrating sample magnetometer (VSM; Model 668) was used to study the magnetic
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behavior of the samples. Size and distribution of polymerassisted nanoparticles were studied in transmission electron microscope (TEM; JEOL, JEMCXII) at operating voltage of 100 kV.
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optimum temperature for the free and the immobilized AMG for 24–48 h. Reducing sugars in the starch hydrolysate were estimated by DNSA reagent (Bernfeld 1955). The percent starch saccharification was calculated according to Mishra and Maheshwari (1996).
Enzyme activity determination 3,5-Dinitrosalicylic acid reagent (DNSA; Merck) was used for determination (Bernfeld 1955) of the AMG activity. One unit of AMG activity was defined as the amount of enzyme required to produce 1 μmol of glucose in 5 min at 298 K. Protein concentration was determined by Lowry's method (Lowry et al. 1951) with bovine serum albumin as a standard. The specific activity of the immobilized enzyme was defined as the number of micromoles of glucose which were liberated in 1 min by 1 g of the immobilizate at its optimum temperature. Determination of optimum temperature, pH, Km, and Vmax of the immobilized AMG The effect of temperature on the enzyme activity of the free and immobilized samples was studied in the temperature range from 303 K to 358 K with 1% (w/v) soluble starch in acetate buffer (50 mM pH4.5). The effect of pH on the enzyme activity was determined with 1% (w/v) soluble starch in 50 mM sodium citrate-phosphate buffer of an appropriate pH at 298 K. The Michaelis constant, Km and maximal rate, Vmax were determined by measuring the initial production rate from 0.1–4% (w/v) soluble starch in 50 mM acetate buffer at respective pH and temperature optima of the free and immobilized AMG. Determination of thermostability and reusability Thermal stability of free and bound AMG was checked by measuring their residual activities after incubation for 15 min in the temperature range of 303–358 K. Residual activity of the free and the coupled enzyme was also assayed after incubating at their respective optimum temperature for about 4 h. Activities of free and bound AMG were determined post-storage at 277 K or 298 K. The measurements were performed at intervals of 5 days within a period of 30 days. The reusability of the bound AMG was assessed by evaluating the activity at the optimum temperature postrepeated magnetic separation and washing with phosphate buffer. Starch saccharification Saccharification of 20% (w/v) soluble starch prepared in 0.1 M phosphate buffer was studied at the respective
Determination of potency for detergent formulation inclusion For the starch de-staining test (washing performance), cotton cloth pieces (size, 4.5×4.5 cm2) were stained with 0.2 mL of aqueous starch solution. The stock solutions of four commercially available detergents, namely Surf Excel Blue (Hindustan Unilever Ltd.), Active Wheel Gold (Hindustan Unilever Ltd.), Ariel OxyBlue (Procter and Gamble), and Tide (Procter and Gamble) were prepared in distilled water (2.0 gL−1). Two stained cloth pieces were taken for each detergent. One piece was washed with detergent alone, while another piece was washed with detergent in the presence of immobilized AMG at the optimum temperature for 20 min with continuous stirring. Similarly, one stained piece was washed with distilled water and immobilized AMG. Each washed cloth piece was manually rinsed twice with distilled water. The washing performance in each case was determined according to a method reported previously by Dhingra et al. (2006). Minimum amount of starch content indicated effective washing. Determination of antimicrobial potency PEG-assisted silver–iron oxide nanoparticles with and without AMG were tested against a panel of microorganisms including Staphylococcus aureus MTCC96, Bacillus subtilis MTCC736, and Pseudomonas aeruginosa PN1. The antibacterial tests were performed as described elsewhere (Turkoglu et al. 2007) after some modification. Antimicrobial activity of the samples was determined by the agar-well diffusion method. All the microorganisms mentioned above were incubated (310 K, 24 h) by inoculation into Mueller Hinton broth (Jorgensen et al. 1999). The McFarland 0.5 standard (Andrews 2005) was used as turbidity standard for the preparation of the culture suspensions. Mueller Hinton Agar (20 mL) was poured into each sterilized Petri dish (10 × 90 mm) followed by inoculation with 100 μl of bacterial cultures. To test the antibacterial potency, the samples were filter-sterilized through a 0.22-μm membrane filter. The spherical silver nanoparticles, poly(ethylene glycol)-assisted silver–iron oxide nanoparticles and AMG-immobilized polymersupported nanoparticles at various concentrations were introduced directly into the wells (6 mm) in agar plates. The plates were incubated at 310 K for 24 h. At the end of
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the incubation period, minimum inhibitory concentration (MIC) for the respective samples was determined. The experiments were performed in triplicate with ampicillin (50 μg) as positive control.
Results Preparation and characterization of the polymer-assisted nanosystem with and without AMG The preparation and characterization of the immobilization system, i.e., the polymer-supported nanoparticles and subsequent coupling with the enzyme were the prime objectives of the present work. The characteristic surface Plasmon resonance peak at around 400 nm for the spherical silver nanoparticles alone shifted to higher wavelength with a broad featureless absorption band at around 330–450 nm when iron oxide nanoparticles were present in the system. The characteristic UV peak for the free enzyme shifted from 280 to about 230 nm upon immobilization. Thus, the UV-visible spectroscopic analysis gave the first indication of enzyme immobilization onto the polymer-assisted nanosystem as evident from the shifting of the silver surface Plasmon peak and enzyme's peak from the lower energy levels to higher energy levels (Fig. 1). In the FTIR spectra (image not shown), there was a broadening of the band at 3,470 cm−1 upon immobilization of the enzyme onto the PEG-assisted silver–iron oxide nanoparticles, compared with the system without enzyme. This was indicative of hydrogen bonding between the NH2 group of the enzyme and the hydroxyl group of PEG. In
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addition to the above, the carbonyl peak of the free enzyme at 1,639 cm−1 split into two peaks at 1,642 and 1,563 cm−1 upon coupling. This splitting was illustrative of the free and hydrogen-bonded carbonyl for the immobilized enzyme. The above observations indicated PEGylation of the enzyme. The strategy adopted to prepare the immobilization system is presented in Fig. 2. The “green” method of preparing silver nanoparticles generated spherical particles within a distribution range of 1–12 nm and average diameter of 5 nm (Fig. 2a). In the present protocol for generation of iron oxide nanoparticles, the step involving sonication had a profound effect as can be seen from the dense population of particles in the range of 2–5 nm (Fig. 2b). This is in sharp contrast to the size range of the nanoparticles (5–17 nm) which were obtained earlier (Konwarh et al. 2009). Now, when the two sets of nanoparticles were subjected to sonication for immobilization of the enzyme (Fig. 2c), the nanoparticles obtained were of greater shape–size diversity (Fig. 2d) with maximal particles of triangular shape (20–30 nm), a critical factor to decide the antimicrobial potency. The magnetic property, antimicrobial property, and the synergy of the immobilized system with commercial detergents are graphically represented in Fig. 2d. The results of the particle size distribution from TEM analysis is presented in the histograms (Fig. 2e–g). Lakeshore VSM was used to study the magnetic behavior of the PEG-assisted silver–iron oxide nanoparticles before and after enzyme coupling in comparison to the PEGassisted MNPs. The M–H hysteresis loops of the various systems were measured at room temperature (Fig. 3). The Ms (saturation magnetization) was found to be highest (76.065 emu g−1) for the PEG-assisted MNP alone (Fig. 3a) while it decreased by almost 50% for the PEG-assisted AgMNP system (Ms =37.785 emu g−1; Fig. 3b) and the enzyme-immobilized system (36.093 emu g−1) (Fig. 3c). The remnant magnetization (Mr) showed a similar trend. The coercivity values for the various systems did not show a significant variation. Carefully designed immobilization system can be instrumental in devising strategies to minimize the loss of enzyme by desorption. The AMG activity estimation in the post-precipitate supernatant as described in the experimental section revealed about 86% binding of the enzyme onto the immobilization system. Kinetic parameters of the immobilized enzyme
Fig. 1 UV-visible spectra of a AMG in free state, b silver nanoparticles with characteristic surface plasmon peak at around 400 nm and c AMG-conjugated PEG-assisted nanoparticles. au arbitrary units
The immobilized enzyme showed multi-fold enhancement in activity compared with the free counterpart. The free enzyme showed a specific activity of 69 Umg−1 while the immobilized enzyme recorded a specific activity of 807 Umg−1 (Fig. 4). Table 1 enlists few of the kinetic parameters of the
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Fig. 2 Sonication-mediated strategy for immobilization of AMG a TEM micrograph of the spherical silver nanoparticles, b TEM micrograph of the PEG-assisted iron oxide magnetic nanoparticles, c structure of amyloglucosidase, d TEM micrograph of the AMG-
immobilized system with illustrations of its multiple attributes. Histogram showing the particle size distribution of e silver nanoparticles, f PEG-assisted iron oxide magnetic nanoparticles and g AMG-conjugated PEG-assisted nanoparticles
free and the immobilized system. The Michaelis constant, Km indicates the substrate concentration at which half the active sites are filled. Furthermore, a low Km indicates strong enzyme–substrate binding and vice versa. The Km value depends on the substrate used and the environmental conditions such as pH, temperature, and ionic strength (Rodwell and Kennelly 2003). The maximal rate, Vmax, reveals the turnover number of an enzyme. The Km value decreased by almost 50% for the immobilized enzyme compared with the free counterpart. The increment in the Vmax indicated greater efficiency of the immobilized enzyme. There was no apparent change in the optimum pH of 4.5 for the enzyme before and after immobilization. However, the immobilization process had shifted the optimum temperature to 343 K, an increment of 10 K over that of the free enzyme (333 K; Fig. 5).
the immobilized enzyme and the free enzyme. As shown in Fig. 5, the enzyme activity of the free AMG declined to almost 34% when incubation temperature was raised to 358 K. However, the immobilized AMG still had a residual activity of 52% at the same temperature. Interestingly, the immobilized enzyme and the free enzyme showed an activity of about 92% and 76%, respectively, after an incubation period of 4 h at their corresponding optimum temperature (343 K and 333 K). As depicted in Fig. 6, a rapid decline was observed in the activity of the free AMG just after 5 days of storage at 298 K while it retained 79% of its activity after 30 days storage at 277 K. In contrast, the immobilization led to a considerable retention of initial activity (about 95%) of both the systems stored at 277 K and 298 K, respectively, during the analysis for a 30-day storage period. The results justify that the storage stability of the enzyme improves upon immobilization. Repetitive use of the enzyme under identical experimental conditions showed very little bioleaching with retention of initial activity up to 96% after eight cycles of use
Thermostability, storage stability, and reusability After predetermined thermal inactivation, the residual activities were determined to check the thermostability of
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Fig. 3 Hysteresis plot of a PEG-supported MNPs, b PEG-supported Ag-MNP, and c enzyme-immobilized system
(Fig. 7). This can be safely attributed to the structural stabilization of the enzyme in the system. The shifting of the optimum temperature to higher values with greater activity even at higher temperature is indicative of the potentials of the system for various industrial applications.
Starch saccharification–application for food industry An attempt was made to probe into the possible utility of the immobilized system in starch industry. The free enzyme showed about 62% saccharification after 24 h. This increased by another 8% after 48 h. The immobilized system had yielded about 79% saccharification after 24 h. This increased up to 91% after 48 h. Furthermore, after magnetic recycling of the immobilized system, 90% saccharification was registered during the second and third rounds of use. Table 1 Comparative data on the kinetic parameters of the free and immobilized AMG
Fig. 4 Specific activity of free and immobilized enzyme
Parameters
Free
Immobilized
pH optimum Temperature optimum (K) Km (mM) Vmax (nM min−1 )
4.5 333 6 0.03
4.5 343 3 0.08
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Fig. 5 Thermostability plot of free and immobilized enzyme
Fig. 7 Recyclability efficiency plot of the immobilized enzyme
Washing performance synergy of the immobilized AMG with commercial detergents
Antibacterial efficacy of the immobilized system
Starch-stained pieces of cloth were washed with the selected detergents alone and in the presence of immobilized AMG, followed by the determination of the residual starch. For the interpretations, the minimal residual starch indicated better washing. The washing performance of the detergents was in the following order: Surf Excel Blue> Tide>Ariel OxyBlue>Active Wheel Gold>distilled water when used alone (Table 2). However, better washing performance was noted when the detergents were used in combination of the immobilized AMG. The immobilized enzyme helps in the rapid removal of the starch stain, thereby indicating a synergistic effect with the various components of the detergents. It is to be noted that, when individual results are compared, the difference may be attributed to the unique washing performance of the detergent alone.
Fig. 6 Storage stability plot of free and immobilized enzyme at two different temperatures
The antimicrobial action of the immobilized system is primarily attributed to the presence of the silver nanoparticles (Table 3). The MIC values registered a dip for the nanocomposite (with and without the enzyme) in comparison to the silver nanoparticles alone. The presence of the enzyme did not alter the MIC value. Furthermore, antimicrobial action was more pronounced against the Gram-positive bacteria than the Gram-negative bacteria.
Discussion The preparation of PEG-supported iron oxide and silver nanoparticles and combining the properties attributed to the individual classes of nanoparticles into a single system was the primary objective of the work. Various components of the leaf extract like triedelin and triterpenes of the friedelin group, namely canophyllal, canophyllol, and canophyllic acid (Dweck and Meadows 2002) can be envisaged as active components for the generation of the silver nanoparticles. The shifts in the UVvisible spectra peak position of the enzyme and the silver nanoparticles indicated the PEG-assisted metal-oxide– enzyme-metal interaction. The broadening of the silver surface resonance peak was also indicative of larger and greater particle shape diversity of the nanoparticles in the immobilization system. Interaction of the M. ferrea extract and poly(ethylene glycol) together with the influence of sonication might be responsible for the diversity in the shape and size of the nanoparticles in the immobilization system. Unlike covalent coupling (using cyanamide for keratinase) as reported previously by us (Konwarh et al. 2009), we had tried to explore the efficacy of sonication for bioconjugation
1990 Table 2 Colorimetric determination of residual starch content for the assessment of the washing performance of the detergents with and without the immobilized enzyme a
Detergent alone
b
Detergent with immobilized AMG
Appl Microbiol Biotechnol (2010) 87:1983–1992 Detergent used
Surf Excel Blue Active Wheel Gold Ariel OxyBlue Tide
Residual starch content System Aa (x1)
System Bb (x2)
ðx1x2Þ x1
0.557 0.976 0.763 0.665
0.311 0.779 0.520 0.442
44.165 20.18 31.84 33.53
in this case. The sonication induced collision between the polymer-supported nanoparticles, and the enzyme moiety in solution is likely to have a profound effect on the morphology of the nanoparticles. This is also likely to facilitate in the establishment of H-bonding between the hydroxyl groups of the PEG chains and the surface hydroxyl group of the iron oxide nanoparticles with the enzyme moeity. The FTIR spectra are supportive of possible PEGylation of the immobilized enzyme. The preponderence of H-bonding, among other possible secondary interactions is an important factor in the reported bioconjugation. The binding of magnetic particles to bioactive substances also involves the chelating interactions between the biological moiety and the metal's centers. Such bindings pave the way for the coupling of biomolecular entities with enhanced stability (Konwarh et al. 2009). The decrease recorded in few of the magnetometric values for the enzyme-immobilized system in comparison to the iron oxide nanoparticles is attributed to the presence of diamagnetic components like polymer-supported silver nanoparticles and the biocatalyst in the system. It is to be noted that the immobilization of the enzyme onto the PEGassisted silver–iron oxide nanosystem did not alter the magnetic quality of the immobilization system to a great extent. Magnetic field susceptibility and consequently the recyclability of the biocatalyst make the reported system attractive in terms of industrial economy. The changes in kinetic properties of the enzyme after immobilization are determined by four factors: change in enzyme conformation and its microenvironment, steric, bulk, and diffusional effects (Kennedy and White 1985). Recently, modulation of the binding affinity of αchymotrypsin with N-succinyl-alanine-alanine-prolinephenylalanine-p-nitroanilide substrate by tetraethylene glycol functionalized gold nanoparticles via an excluded Table 3 Antibacterial potency of the nanoparticles with and without enzyme immobilization
Test organism
S. aureus MTCC96 B. subtilis MTCC736 P. aeruginosa PN1
Ranking of detergent
100
System A
System B
First Fourth Third Second
First Fourth Third Second
volume mechanism has been reported to increase the enzyme activity (Jordan et al. 2009). In a similar way, it has been proposed that macromolecular crowding potential of PEG is responsible for increasing the effective concentrations of the enzyme and the substrate (Dominak and Keating 2008; Vergara et al. 2006). The greater surface area of the immobilization system in the nano-dimension with possible structural modulation may be viewed as the reason for better substrate–enzyme interaction, through exposure of the active site. The immobilized enzyme showed better thermostability, storage stability, and multiple cycle use in contrast to the free enzyme. It is quite likely that PEGylation had induced structural modulation of the enzyme as a result of immobilization. PEGylation has been instrumental for protecting a host of biocatalysts against denaturation (Castellanos et al. 2005; Garcia-Arellano et al. 2002). Stabilization was proposed to originate from solvation of the bound PEG chains with water molecules, thereby restricting local molecular mobility which, in turn, led to a decrease in the unfolding rate of the protein (Hernáiz et al. 1999). Improvement of the storage stability is another attractive feature of the system. With a possibility of holding the immobilized enzyme in a more stable position than the free counterpart, the coupling reduces the distortion effects imposed from aqueous ambience on the active site of the enzyme. At this point, it is notable that polymeric nanocomposite offers numerous advantages compared to the nanomaterials alone when used for immobilization. Encapsulation or entrapment of inorganic particles in organic polymers is known to impart novel properties to the particles (Ziolo et al. 1992). Improvement in dispersibility and stability of the particles are reported to be due to the enhanced compatibility, their reduced leaching, and protection of the surfaces
MIC (µg/mL) Spherical silver
PEG-assisted Ag-iron oxide
Immobilized AMG
6.32 6.32 25.3
3.16 6.32 15.1
3.16 6.32 15.1
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from damage when coated with polymers (Bourgeat-Lami and Lang 1998). Here, the biocompatible poly(ethylene glycol) not only stabilized the nanoparticles within a narrow-size spectrum but also protected the biocatalyst. This may be viewed as the result of probable PEGylation of surface-exposed residues like cysteines known for their involvement in oxidative denaturation of proteins. The chelation-type interaction between the nanoparticles' metal center and the biomolecule further stabilized the whole system that helped in reducing the bioleaching. A number of anti-oxidants are naturally pooled in leaf extracts to prevent thermal, photo, or oxidative denaturation of various biochemical entities. These components may be instrumental in improving the storage stability of the enzyme. The domain of enzyme biotechnology has opened up new possibilities for advanced application-oriented products in the food industry. There are several reports on the use of amylase and amyloglucosidase for starch saccharification (Soni et al. 1995; Mishra and Maheshwari 1996; Kaur and Satyanarayana 2004). The increased percent saccharification and the magnetic recyclability (without losing the enhanced action) of the immobilized system make it a better candidate (compared with the free enzyme) for application. Detergents normally contain surfactants (anionic, cationic, non-ionic, and amphoteric), builders, co-builders, bleach, bleach activators, and special additives, such as fluorescent brightener, filler, corrosive inhibitors, antifoaming agents, and enzymes (in case of only enzymic detergents), and perfumes (Tyebkhan 2002). However, due to ethical obligations and professional confidentiality, the chemical composition of these marketed detergents and the overall ionic state of the surfactants used are not generally stated. The synergy of the detergents and the immobilized enzyme has been reflected clearly in the starch de-staining test. The reported system not only protects the enzyme from the inhibitory effects of ionic surfactants but also is expected to prevent the degradation due to proteases—the likely constituents in enzymatic detergent formulations. This is supported by the repeated separation of the nanosystems even after five washing cycles with a simple laboratory magnet and its reuse without losing the enhanced enzyme activity. Various mechanisms have been put forward on explanation for the antimicrobial action of Ag nanoparticles. These include alteration of the cell membrane permeability primarily by immediate dissipation of the proton motive force and interaction with sulfur and phosphorus-containing macromolecules like DNA and thereby affecting the replication machinery. The shape–size correlation between the silver nanoparticles is responsible for their antimicrobial potency. It has also been reported that triangular silver nanoparticles are more efficient in counteracting the bacterial proliferation (Sharma et al. 2009). The {111}
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facets have high-atom-density, which is favorable for the reactivity of Ag. A triangular nanoplate has a high percentage of {111} facets whereas spherical and rodshaped Ag nanoparticles predominantly have {100} facets along with a small percentage of {111} facets (Wiley et al. 2005). In the present system, the morphology directing action of the various components is responsible for generation of relatively more nanoparticles of triangular shape. This leads to a decrease in the MIC values compared with that of the spherical nanoparticles. The differential interaction of the nanoparticles with the molecular moieties at the surface of the two groups of the bacterial species— Gram-positive and Gram-negative—might be responsible for the observed differences in the antimicrobial effect. The basic architectural differences between the cell walls in the two groups of bacteria may also have a role in determining the antimicrobial activities of the nanoparticles. The antimicrobial potential of the PEG-assisted silver–iron oxide nanosystem is retained even after the enzyme coupling. This further supports its inclusion in commercial detergent formulation. The excellent thermostability, storage stability, magnetic field recyclability, and catalytic action (evaluated for starch saccharification) of the antimicrobial immobilized system is complemented by the washing performance synergy with commercial detergents. Thus, the immobilized system represents the multi-faceted advantages of coupling an industrially important biocatalyst onto polymer-supported nanomaterials, prepared through greener route. Acknowledgements Mr. Rocktotpal Konwarh sincerely acknowledges the receipt of his Junior Research Fellowship from the Department of Biotechnology, New Delhi. RSIC, NEHU, Shillong and CIF, and IIT Guwahati are thankfully acknowledged for the TEM imaging and magnetometric studies respectively.
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