Evaluation of whole lysosomal enzymes directly immobilized on titanium (IV) oxide used in the development of antimicrobial agents

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Abstract

Lysosomal enzymes isolated from egg white were directly immobilized on titanium (IV) oxide (TiO2) particles using shaking methods (150 rpm, room temperature, 10 min), and the immobilization efficiency, activity, and stability of lysosomal enzymes immobilized on TiO2 were evaluated. Of the various mass ratios (w/w) of lysosomal enzymes to TiO2 tested, we found that 100% immobilization efficiency was observed at a ratio of 1:20 (enzymes:TiO2; w/w). Furthermore, the antimicrobial activities of the immobilized lysosomal enzymes were confirmed using viable cell counts against Escherichia coli. Our results showed that the antimicrobial activity of immobilized lysosomal enzymes is stable and can be maintained up to one month, but the antimicrobial activity of free enzymes without immobilization completely disappeared after five days in storage. In addition, enhanced immobilization efficiency was shown in TiO2 pretreated with a divalent, positively charged ion, Ca2+, and the antimicrobial activity for E. coli increased as a function of increasing ratio of immobilized enzymes. However, K+, a monovalent, positively charged ion, did not have any positive effect on immobilization or antimicrobial activity. Finally, we suggest that activity and stability of immobilized lysosomal enzymes can be maintained for a longer time than those properties of free lysosomal enzymes.

Highlights

► Lysosomal enzymes isolated from egg immobilized on TiO2 using shaking method were evaluated immobilization efficiency with reaction time and various mass ratios. ► Antimicrobial activity of lysosomal enzymes immobilized on TiO2 was confirmed using viable cell counts against Escherichia coli and was stable and can be maintained up to one month. ► In addition, enhanced immobilization efficiency was shown in TiO2 pretreated with a divalent, positively charged ion, Ca2+, and the antimicrobial activity for E. coli increased as a function of increasing ratio of immobilized lysosomal enzymes.

Introduction

Lysosomes, which contain 50–60 hydrolases that constitute the cellular site for bulk macromolecule degradation, function to mediate several processes in cell feeding and antimicrobial defense [1], [2], [3] One of the various functions of lysosomes isolated from egg white is to kill bacteria using lysozymes, which are enzymes in lysosomes [4]. Moreover, lysozyme, which has applications in pharmaceutical products and food because of its antimicrobial characteristics, is used as an antimicrobial agent in lysosomes [3], [5], [6]. However, the functional properties of each lysosomal enzyme fraction in lysosomes have been neither identified nor characterized [7]. To date, the long-term activity of lysosomal enzymes in lysosome has not been reported.

There have been many attempts to improve enzyme stability via enzyme immobilization, enzyme modification, protein engineering, and medium engineering. Enzyme immobilization represents the attachment or incorporation of enzyme molecules onto or into large structures (e.g., mesoporous media [8], carbon nanotubes [9], nanofibers [10], or nanoparticles [11]) via simple adsorption, covalent attachment, electrostatic binding, or encapsulation [12], [13]. The use of nanophase materials offers many advantages due to their small size and physical properties [13]. During the last decade, the biomaterial applications of titanium (IV) dioxide (TiO2) have increased in numerous fields [14]. Due to a high dielectric constant, TiO2 undergoes further modifications upon binding to various surrounding ions. These may include calcium, phosphate, and hydrogen and result in the generation of OH-radicals in the oxide [15], [16]. The oxide layer may adsorb biomolecules because the metal Ti is covered by an oxide layer, which has a negative charge at physiological pH [17].

Hen's egg whites are more and more widely used not only for direct consumption, but also as important materials in food production and in pharmaceutical industries [18]. Biologically active substances contained in egg white, e.g. lysozyme, cystatin, and avidin, have recently drawn much attention of researchers, which were originated from cell organelles, lysosomes and composed up to 37% in hen's egg white [19]. Therefore, in this study, to maintain and protect the antimicrobial activity of whole lysosomal enzymes isolated from hen egg whites, the lysosome was immobilized on two types of titanium (IV) oxide particles: anatase and rutile.

To optimize the conditions for immobilizing lysosomal enzymes on TiO2 several reaction conditions (e.g., mixing mass ratio (w/w) of lysosomal enzymes to particles, reaction time) were examined. In addition, we evaluated the antimicrobial activity of immobilized lysosomal enzymes on TiO2 via treatment of Escherichia coli, and the stability of lysosomal enzymes immobilized on TiO2 was measured. In addition, TiO2 pretreated with divalent positive ions was compared to untreated TiO2 to assess immobilization efficiency and antimicrobial activity.

Section snippets

Isolation of lysosomal enzymes from hen egg white

The lysosomal enzymes were extracted by removing the membranes of lysosomes, which were isolated from egg white using the cell fraction method [3]. The yolk was separated from the white component of the eggs and discarded. The white component of the hen eggs was homogenized until the viscosity of the egg white decreased significantly. Two particulate fractions were obtained by centrifuging the homogenate successively for 5 min at 500 × g (unbroken cells and nuclei) and 30 min at 20,000 × g (lysosomal

Results and discussion

Typically, lysozyme is used in pharmaceutical products because of its antimicrobial properties [3], [4]. However lysozyme is a single enzyme localized in lysosomes, thus, in this study, whole lysosomal enzymes, not the single lysozyme was used to evaluate antimicrobial activity. The antimicrobial activity was compared using the whole lysosomal enzymes in lysosome that was isolated from hen egg white and commercialized single lysozyme (Fig. 1). The cell mortality of E. coli after treatment with

Conclusions

One of the most important aims of enzyme immobilization is to protect and stabilize the activity of an enzyme for extended use. The extent of stabilization depends on the method for immobilizing materials, type of the materials, and characteristics and structure of the enzyme. In this study lysosomal enzymes were immobilized onto TiO2. As a result of the immobilization of whole lysosomal enzymes onto TiO2, the optimized immobilization efficiency of the ratio of lysosomal enzymes to particles

Acknowledgements

This work was supported by Mid-career Researcher Program through NRF grant funded by the MEST (No. R01-2008-000-20773-0). The authors are grateful for their support.

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