Galactose oxidase nanoaggregates: Preparation and characterization

Introduction

The best excellency and biocompatibility of various nanoparticles with biomolecules possess unique structural, magnetic, optical, catalytic properties and also enhance the conductivity of biosensor in terms of sensitivity[1]. Enzyme molecules were precipitated as insoluble aggregates with their native confirmation and cross linked by bifunctional cross linkers [2,3].Cross linking of enzyme via glutaraldehyde with reactive NH₂ groups on protein surface was developed more than 40 year ago. Cross-linked enzymes are produced by mixing both solution of enzyme and  glutaraldehyde [4,5]. Enzyme nanoaggregates could be reused over again and again and also have a longer shelflife and stability over denaturation by heat and organic solvents and problems related to enzyme leaching is not associated with these aggregates [6,7].Cross linked enzyme NP aggregates could be immobilized on specific support using high proportion of active enzyme. Enzyme nanoaggregates are supramolecular structure held by non covalent interactions[8].These enzyme nanoaggregates play  important role as industrial and biomedical catalyst. Variations have been found in terms of pH, temperature in biosensors which results in leaching and denaturation of enzyme [9]. Because of these problems, the enzyme nanaparticles based biosensor achieve superior design as it is not time consuming and non-laborious process [10].

Although various aggregates of enzyme NPs were formed by this method like of glucose oxidase, trypsin, lipase, laccase, phytase, penicillin amylase, tyrosinase, cholesterol oxidase, horseradish peroxidase and they have wide applications like in pharmaceutical, food, detergent ,bioreactore, biofuels, biomedical devices[8-10]. The galactose oxidase nanoaggregates are more stable to denaturation by solvents, heat and proteolysis in comparision to soluble form of enzyme[11].So, the present work describes the preparation of galactose oxidase nanoaggregates by simple method and its characterization.

Experimental

Materials and methods

Reagents- Galactose oxidase, horseradish peroxidase and Glutarldehyde (25%) were from sigma-aldrich, USA. Dextrose, cysteamine dihydrochloride, ethanol, methanol were from Himedia laboratory chemicals, India. All other reagents were of analytical reagent (AR) grade. Double distilled water (DW) was used in all experiments.

Instruments-UV-visible spectroscopy, Fourier transform infrared (FTIR) spectrophotometer ( Bruker) was carried out at Department of Zoology,  M.D University, Rohtak , Spectronic 20 (Thermo, USA) for measuring A₄₂₅nm. Transmission electron microscopy (TEM) carried out at  PU, Chandigarh.

Assay for Free Galactose oxidase (GalOx)

GalOx assay was carried out as per Aries et.al.,1992 [14] with modifications. It was carried out in 15ml test tubes wrapped with black paper. Assay mixture contained 1.8ml of 0.1M potassium phosphate buffer(PB, pH 6.0),0.1ml enzyme (1mg/ml) and 0.1ml galactose (360mg/ml) as substrate. After incubating the reaction mixture at 37^oC for 2 min, 1 ml of colour reagent was added and kept at room temperature for 15min to develop the color. A₄₂₅ nm was read in spectronic -20 and H₂O₂ generated was determined from standard curve between H₂O₂  versus A_425. The color reagent consisted of 50mg of 4-aminophenazone, 100mg phenol(solid),and 1mg horseradish peroxidase in 100ml of 0.4MPB, pH 7.0 and stored in amber colored bottle at 4C.

Preparation of Galactose oxidase Nanoparticles (GalOxNP) Aggregates.

The GalOx-NP aggregate havebeen prepared as per Liu et al., 2005[1] by desolvation method with modification . In this method first of all 6ml of absolute ethanol was added to 3ml of enzyme solution (1mg/ml) at a droping rate of 0.1-0.2 ml/min under constant stirring at 500 rpm resulting into aggregation of enzyme/protein molecules into smaller particles/nanoparticles. After then,1.8 ml of 2.5% glutaraldehyde was added to these NPs suspensions with continous stirring at 500rpm at 4^oC for 24hr, thus ensuring the crosslinking of  enzyme nanoarticles. Amino group were introduced on GalOxNPs by addition of 0.12g of cysteamine dihydrochloride with 5-6hr of continous stirring. Synthesized GalOxNP were then separated from free enzyme solution by centrifugation at 12000rpm at 4^oC for 10 min followed by dispersion in 0.1M PB (pH 6.0) with  sonication for 5min. It is assumed that –NH₂ group of cysteamine reacts with –CHO group of glutaraldehyde cross linked galactose nanoparticles to form Schiff base. These nanoparticles aggregates further characterized by UV spectroscopic, TEM and FTIR spectroscopic studies. GalOxNPs were stored at 4^oC, for further use. The scheme is shown below.

Scheme 1: Scheme of cross-linked GalOx nanoparticles aggregates by Desolvation method.  Click here to View scheme

 

Figure 1: Synthesized Galactose oxidase nanoparticles.  Click here to View figure

 

Characterization of Galactose oxidase nanoparticles

The aggregated galactose oxidase nanoparticles were studied by Transmission electron microscopy (TEM) at Punjab University, Chandigarh, FTIR spectroscopy (BRUKER) ,UV visible spectroscopy(Spectronic-20) at Maharshi Dayanand University, Rohtak. Samples are prepared by dispersing drop of colloid on copper grid, covered with the carbon film and the solvent is evaporated. UV visible spectroscopy of both free Galactose oxidase and GalOxNPs were measured in spectronic-20 at different wavelengths.

Results and Discussion

Transmission electron microscopy

TEM image of prepared GalOxNPs showed their spherical shape with diameter in the range of upto 20nm (Fig.2). GalOxNPs have a hollow spherical structures on the electron microscopic images. The TEM image showed  GalOxNPs aggregates with an average diameter of 100nm

Fourier Transform infrared spectroscopy (FTIR)

FTIR spectra of free Galactose oxidase showed the vibration bands at 1090 and 1405cm^-1 confirm the presence of amide I and amide II infrared bands. These bands are used in conformational changes monitoring. The amide I band is due to C=O stretching of peptide linkages while amide II band is due to combination of N-H bending and C-N stretching of peptide linkages.whereas the FTIR spectra of GalOxNPs aggregates obtained a vibration band at 1634cm^-1 due to C=N stretching  formed after glutaraldehyde reaction,confirming the formation of GalOxNPs aggregates also with the vibration bands of amideI and amideII having its enzymatic activity. There is also a broadening of vibration band at 2000-2500cm^-1 in case of GalOxNPs confirming the presence of more free amine(NH₂) groups, introduced by cysteamine dihydrochloride (Fig.3).

Figure 2: Transmission electron microscopic image of prepared GalOxNPs aggregatesrevealing their synthesis.  Click here to View figure

 

UV Visible spectroscopy

UV visible spectra of free galactose oxidase and galactose oxidase nanoparticles were studied separately. Free galactose oxidase showed a characterstic absorption peak at 220nm(Fig.4A) and Galactose oxidase nanoparticles aggregates showed a absorption peak at 237nm(Fig.4B).This shift in absorption maxima is the indicative of structural conformation of enzyme after nanoparticles synthesis. The peak observed in enzyme nanoparticles synthesis is due to the absorption by free amino acid groups whereas the peak shown in case of free enzyme is due to absorption by peptide bonds,which indicates the synthesized galactose oxidase nanoparticles.

Figure 3: FTIR spectra of GalOxNPs  Click here to View figure

 

Figure 4A: UV visible spectroscopy of free galactose oxidase  Click here to View figure

 

Figure 4B: UV visible spectroscopy of galactose oxidase nanoparticles  Click here to View figure

 

Conclusion

Galactose oxidase nanoparticles can improve the prospects of clinical applications of enzyme drugs

Use of these synthesized enzyme nanaoparticles  can offer advantage in genetic engeeneering and clinical trials for diagnose the disorders related to galactose metabolism. These enzyme nanoaggregates also have various application in context of industrial and biomedical biocatalyst. They play a major role in biosensing platform to increase shelflife and operational stability. Direct immobilization of enzyme nanoparticles with proper choice of functionalization seems to be a promising strategy for biosensor development with high sensitivity, affinity, response time.

Author Contributions

Minakshi Sharma Conceived the Plan, Mamta Sharma performed the experiment and data analysis. Authors have no competing financial interests.

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