CHAPTER-8 A PROCESS FOR ENHANCING ENZYME THERMAL STABILITY BY PREPARING THEIR NANOPARTICLES

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1 CHAPTER-8 A PROCESS FOR ENHANCING ENZYME THERMAL STABILITY BY PREPARING THEIR NANOPARTICLES Overview of the Chapter So far we have exploited nanotechnology for biotechnological application, i.e., biosensor fabrication by immobilization of enzyme on nanostructures. In the present chapter we have done hybridization of two fields and in true sense an example of nanobiotechnology is presented in the form of nanoparticles of enzyme itself. Major driving force behind present work being low shelf life of enzyme based electrodes due to fragile nature of enzymes. Long shelf life of biosensor in particular and any product in general, is of paramount importance for commercialization. The synthesized enzyme nanoparticles exhibit improved thermal stability and biocatalytic activity over a wide range of temperature relative to free enzyme in solution or immobilized over conventional or nanoparticles based matrices. 8.1 Introduction 8.2 Experimental Synthesis of enzyme nanoparticles Effect of desolvating agent Effect of crosslinking agent concentration Effect of functionalization agent on stability of enzyme nanoparticles Characterization of nanoparticles Activity analysis of enzyme nanoparticles Effect of temperature Effect of ph 8.3 Results and Discussions Characterization of synthesized enzyme nanoparticles TEM analysis UV-Visible Spectroscopy Analysis Activity analysis of free enzyme and enzyme nanoparticles 139

2 Effect of desolvating agent Effect of crosslinking agen Effect of functionalizing agent Thermal stability analysis Thermal stability of GOx NPs Thermal stability of HRP NPs Biosensing Efficiency Analysis Conclusion 8.1 Introduction Enzymes are extensively used biocatalyst in numerous industries like food, chemical, pharmaceutical and fermentation industries [195]. These biocatalyst are known for their selective substrate specificity and catalytic activity in most of the chemical reaction. Hence biosensors advancements in the field of food [196, 197] and beverage industry [198], fermentation [199] and pharmaceutical industry [200, 201] are expected to yield substantial returns. However, till date biosensor for medical applications continue to dominate the market, the major reason behind the dormancy of biosensor for other applications is the high fluctuations in operating environment i.e. exposure to temperature and ph farther away from optimum conditions for the enzymes used in these biosensors. The high temperature and ph variability and hence instability of enzyme, leading to low shelf life is a major concern for commercial viability. The low free energy difference (~40kJ/mol) between the native and denatured structure of enzyme makes it a fragile molecule to deal with and limits its unlimited applications. The appropriate folding of proteins leading to the complex three dimensional structure allows high catalytic activity of biocatalyst. Thus, any destruction or damage to this three-dimensional structure results in loss of catalytic activity of the biocatalyst. This crucial conformation of the protein is stabilized by a variety of interactions based on hydrophobic effects, electrostatic interactions, coordinative complexes and at times even covalent disulfide bonds. Different factors are known such as temperature, ph, etc., that may disturb the balance of weak non-covalent forces responsible for preserving the native conformation of enzyme [202]. It is well-known fact that thermal denaturation of many enzymes begins at temperatures exceeding 35 C, making the usage of enzyme difficult in any reaction system, where the substrate has a high melting point [203]. The presence of flexible regions in the structure of enzymes results in high kinetic energy of these regions from vibrations even at 140

3 room temperature. This enhanced kinetic energy of some region in structure of the enzyme molecule can rearrange weak non-covalent interactions and thereby inactivating the enzyme. Moreover, the enzyme is known to be highly unstable in solution. Chemical modifications can also lead to changes in the conformation of the protein. As reported by Manning et al. the solvent water for instance may react with proteins, resulting in deamidation of glutamine and asparagine residues, whereas oxidation modifies thiol groups [204]. Therefore, it is a must to provide an appropriate environment to enzyme in order to sustain their catalytic activity. Research efforts to analyze genetic makeup of thermophilic and hyper thermophilic microorganism in order to elucidate the factors responsible for their thermal stability like codon usage or specific amino acid sequence etc. are in progress. However, not much success has been achieved in converting a thermally labile enzyme into thermally stable enzyme. Different strategies have been reported in literature for improving the stability of the enzyme within an aqueous solution such as addition of stabilizers [205, 206]. Various available reports claims that different natural or synthetic substances like an amino acid [ ], a polyol [210, 211], sucrose [212, 213], dextran [ ] are often added to an enzyme solution for enhancing its thermal stability. However, the enzyme obtained by the conventional methods described above is stable only under very narrow temperature range, thus, is relatively unstable over broad temperatures fluctuations on either side of optimum values. Hence, it is imperative to develop methods to enhance the stability of enzymes. The same being the motivating factor behind the work presented in the present chapter. Synthesis of nanoparticles of enzymes itself has been able to achieve the desired aim. We report a novel process to synthesize nanoparticles of enzymes in general and results for two enzymes Glucose oxidase (GOx) and Horse radish peroxidase (HRP) are presented in this chapter. 8.2 Experimental Synthesis of Enzyme Nanoparticles Enzyme (GOx/HRP) was first dissolved in deionized water (1mg/ml) and stirred with a magnetic bar at room temperature. While stirring, a desolvating agent, was added at a constant rate. Following desolvation of protein, 0.005% glutaraldehyde solution was introduced into the enzyme solution while stirring overnight at 4 C. The enzyme nanoparticles thus formed were further functionalized using chemical X with constant stirring for

4 hours. The nanoparticles were purified from free enzyme and excess cross-linking agent using centrifugation, g for 10 minutes at 4 C. Pellets were redispersed to the original volume of phosphate buffer (0.1 M ph-7.4). Each redispersion step was performed in an ultra-sonication bath for 15 minutes. Figure 8.1 shows a simplified schematic representation of the synthesis process. Figure 8.1: Schematic illustration of nanoparticles synthesis and functionalization Effect of desolvating agent Different desolvating agents were used for the purpose of protein desolvation. Alcohols (ethanol, propanol) and ketones (acetone) were added to the enzyme solution at a constant concentration ranging from 50 to 100% at a constant rate of addition Effect of crosslinking agent concentration Following desolvation, the effect of concentration of crosslinking agent (glutaraldehyde) on the protein was studied. Varying concentration of glutaraldehyde from 0.001% to 0.005% were added to the desolvated protein. The crosslinking process was performed under continuous stirring of the suspension over a time period of 24 hours to ensure complete cross linking of the particles Effect of functionalization agent on stability of enzyme nanoparticles Functionalization of synthesised nanoparticles was performed using different functionalizing agents in order to analyze the effect of functionalizing agents on stability, functionality, and biocompatibility of synthesized enzyme nanoparticles. Functionalizing agent such as L- Cysteine and chemical X were added with constant stirring for 5-6 hours to the solution containing enzyme nanoparticles. 142

5 8.2.5 Activity analysis of enzyme nanoparticles The activity and stability of synthesized GOx nanoparticles (GOx NPs) and HRP nanoparticles (HRP NPs) were investigated and challenged by different environmental factors, such as the temperature and ph. Activity assay for GOx The activity and stability of synthesized glucose oxidase nanoparticles (GOx NPs) were investigated and compared with the activity of free enzyme. Activity analysis of enzyme was carried out by studying the rate of formation of red quinoeimine dye. The underlying principle behind the assay is discussed below, The highly specific glucose oxidase, catalyses oxidation of -D-glucose to D-glucono- lactone with the simultaneous release of hydrogen peroxide. The hydrogen peroxide produced reacts with phenol and 4-AAP in the presence of peroxidase enzyme to produce a red colored dye, measurable at 500 nm. Rate of formation of red colored quinoeimine dye is directly proportional to the concentration of glucose in the sample. Activity assay for HRP The activity assay was performed by following the protocol given by Trinder, Briefly, 4-aminoantipyrine act as a hydrogen donor and reaction rate is calculated by measuring an increase in absorbance at 510 nm resulting from the decomposition of hydrogen peroxide. According to Trinder, one unit of enzyme results in the decomposition of one μm hydrogen peroxide per minute at 25 C and ph 7.0 under the specified conditions Effect of temperature Assay mixture containing 500 U of horseradish peroxidase, mm of 4-aminoantipyrine (4- AAP), mm of phenol and 5 mm of glucose in 50mL of phosphate buffer solution (0.05 M. ph 7.4) resulting in a glucose concentration of 0.1 M was prepared. A solution of free enzyme and enzyme nanoparticles of similar molar concentration were used to assess the 143

6 activity of enzyme nanoparticles as compared to free enzyme. The temperature effect on activity was studied by determining the concentration of glucose left after incubating the enzyme NPs solution at various temperatures (25-95 C) while effect of ph was assessed between ph value of 5 to 9. For assessing activity of HRP, reaction cocktail containing 1.4 ml Phenol/aminoantipyrine solution ( M 4-Aminoantipyrine with 0.17 M phenol) and 1.5 ml Hydrogen peroxide solution ( M) was incubated at different temperatures. After incubating the reaction cocktail at different temperatures and 20mU of enzyme was added and absorbance was recorded at 510 nm. 8.3 Results and Discussions Enzyme nanoparticles synthesis was optimized in terms of choice of desolvating agent, rate of addition of the desolvating agent, agitation speed, temperature and ph values to achieve uniform size particles Characterization of synthesized enzyme nanoparticles TEM analysis The morphological analysis of enzyme nanoparticles was done using Transmission electron microscope. Figure 8.2: Transmission electron micrographs showing A) GOx NPs and B) HRP NPs Transmission electron microscope studies of the synthesized enzyme nanoparticles showed spherical, fairly uniformly sized nanoparticles of GOx with approximately nm diameter (Figure 8.2A) while HRP nanoparticles were of nm in size (Figure. 8.2B). 144

7 UV-Visible Spectroscopy Analysis Figure 8.3A represents UV-Visible spectra of free GOx as well as GOx nanoparticles while Figure 8.3B shows the absorption spectra of free HRP and HRP nanoparticles. As observed from figure 8.3A free enzyme GOx (5-6 nm) shows characteristic absorption peak at 278 nm while the nanoparticles formation (27-32 nm) of the same results in a red shift marked by an absorption peak at 291 nm. Similarly, the absorption maxima at 401 nm in case of HRP enzyme (2-3 nm in size) shows a red shift with absorption peak at 407 nm with formation of nanoparticles (10-12 nm in size) of the same (see Fig 8.2B). The red shift is indicative of increased size however, the effect on 3-D structural conformation of the enzymes after nanoparticles synthesis needs to be further analysed in terms of activity of the enzyme. Figure 8.3: UV-Visible absorption spectrum of A) free GOx and GOx NPs; B) HRP and HRP NPs Activity analysis of free enzyme and enzyme nanoparticles Effect of desolvating agent Desolvating agent changes the tertiary structure of the proteins by removing the in-between water molecules and thus reducing the distance between protein molecules. Here we have exploited the protein precipitation methodology for a controlled nanoparticles synthesis. It was observed that beyond certain critical concentration of desolvating agent, further increase in concentration lead to highly turbid solution with white precipitates at the bottom. Therefore, the efficacy of different desolvating agents such as alcohol or ketones was investigated in order to find the appropriate desolvating agent and its optimum concentration for preparation of enzyme nanoparticles. Figure 8.4 shows the thermal stability of the synthesized GOx nanoparticles using different desolvating agents. It was observed that 145

8 ethanol is the most suitable desolvating agent resulting in enhanced thermal stability of the enzyme nanoparticles while propanol and acetone probably destabilize the enzyme structure significantly, leading to much poorer enzymatic activity as compared to free enzyme GOx. The rate of addition of ethanol was controlled cautiously since it also plays a major role in the consequential particle size. The rate of addition of ethanol was maintained between ml/min while stirring the enzyme solution. Figure 8.4: Effect of desolvating agent on thermal stability of synthesized GOx enzyme nanoparticles Effect of crosslinking agent Glutaraldehyde was used as crosslinking agent for synthesis of enzyme nanoparticle as it can be used in aqueous solution under conditions close to physiological ph, ionic strength, and temperature. Figure 8.5 shows the effect of variaton in concentration of glutaraldehyde on activity of enzyme. It has been observed that the concentration of crosslinking agent plays a vital role in controlling the catalytic activity of enzyme. At concentration below certain critical concentration the probablility of reaction of functional groups of glutaraldehyde with the same enzyme molecule is higher than that of crosslinking different enzyme molecules. This results in enhanced intramolecular crosslinkings rather than intermolecular crosslinking. Further increase in concentartion of crossliniking agent facilitates intermolecular crosslinking. However, high concentrations of glutaraldehyde results in a tight structure of enzyme by excluding water molecules because of extensive crosslinking and distortion of the enzyme structure and eventual precipitation of enzyme rather than nanoparticle formation. 146

9 Figure 8.5: Effect of crosslinking agent concentration on enzyme NPs activity. The alteration of 3D strucutre of enzyme effects the binding efficinecy of the substrate to the active site of enzyme and hence reduction in catalytic activity of enzyme. Hence optimum concentration of glutaraldehyde is a must for enzyme nanoaparticle formation without losing the activity of the enzyme Effect of functionalizing agent Crosslinking enzyme molecules results in partial thermal stability for crosslinked enzyme. Figure 8.6: Effect of functionalizing agent on activity of GOx enzyme nanoparticles 147

10 particles (CLE) as reported by different research groups. However, functionalization of synthesized enzyme nanoparticles with suitable reagent imparts wider thermal stability. The aim of functionalization is to decorate nanoparticles with certain chemical groups so as to widen their scope of application without destroying the catalytic properties of enzyme nanoparticles. Figure 8.6 shows the effect of functionalizing agents - L-cysteine and Chemical X, on thermal stability of synthesized enzyme nanoparticles. It can be seen from the results that functionalization of enzyme nanoparticles with chemical X results in enhancement of catalytic activity as well as thermal stability of nanoparticles whereas enzyme nanoparticles functionalized with L-cysteine showed catalytic activity and thermal stability even lower than that of free enzyme Thermal stability analysis Optimized synthesis of enzyme nanoparticles was achieved by ethanol as desolvating agent, with 0.005% glutaraldehyde as crosslinking agent and chemical-x as functionalizing agent to monodispersed spherical enzyme nanoparticles of GOx and HRP as shown in Figure 8.1(A- B). Thermal stability of these enzyme nanoparticles was evaluated and compared with enzyme immobilized onto AuNPs, in addition to the free enzyme. Figure. 8.7 shows the stability of free enzyme in comparison to synthesized enzyme nanoparticles and enzyme immobilized onto gold nanoparticles over a temperature range of 25ºC - 85ºC Thermal stability of GOx NPs As observed in Figure 8.7 immobilization of enzyme onto amino functionalized AuNPs resulted in marginal improvement in enzymatic activity around optimum temperature. The optimum temperature shifted to 40 C in case of GOx immobilized onto AuNPs while in case of free GOx it was at 37 C. Thermal stability of GOx immobilized onto AuNPs was greater than 95% till 45 C beyond which thermal stability was same as that of free enzyme. However, enzyme nanoparticles showed a substantially improved thermal stability over a much wider temperature range. Enzyme nanoparticles retained more than 90% of the enzymatic activity after incubation at 55ºC while there was greater than 55% reduction in free enzyme activity in solution. At temperatures as high as 80 C, enzyme nanoparticles retained approximately 50% of catalytic activity at the same time free enzyme showed no activity after incubation at 80 C for 30 minutes. 148

11 Figure 8.7: Comparative thermal stability analysis of GOx NP, GOx immobilized onto AuNPs and Free GOx Thermal stability of HRP NPs HRP nanoparticles of nm shown in Figure 8.1B were analyzed for their thermal stability in comparison to free HRP enzyme (see Figure 8.8). HRP nanoparticles retained greater than 80% activity after incubation at 70ºC while free HRP enzyme showed only 50% Figure 8.8: Thermal stability analysis of HRP NPs and free enzyme HRP. 149

12 activity at the same temperature. In general, there was significant improvement in thermal stability over low as well as high temperature ranges. Though, the thermal stability effect in case of HRP NPs was not as pronounced as in case of GOx NPs, probably further optimizations needs to be explored Biosensing Efficiency Analysis We have fabricated the glucose biosensor using GOx NPs and HRP NPs and compared it with biosensors reported in previous chapters. There was a remarkable improvement in all the biosensor characteristics. (Results are not presented here since filing of patent is in process) Conclusion In this chapter we have presented a method/process for controlled synthesis of nanoparticles of enzymes retaining their activity. The process of synthesis is optimized to the extent that we could control the number of molecules of enzymes in a nanoparticle (GOx NP nm i.e. 5-6 GOx molecules; HRP NP nm i.e., 5-6 HRP molecules). Enzyme nanoparticles thus synthesized impart wide thermal stability and can be used for industrial applications requiring high operating temperature and hence biocatalysts that can maintain their catalytic activity over wide temperature range. We feel, these thermally stable enzyme nanoparticles would revolutionize the biotech field in general. It would not just be useful for the industries using/requiring enzymes under high temperature operating conditions but also the cost of enzymes would go down since these thermally stable enzyme NPs would not require low temperature storage and shipment conditions for their transportation. In addition to this the need for storage of medical diagnostic kits in refrigerators and the problem of non availability of many such kits in smaller rural hospitals can as well be addressed. 150