Adsorption and hydrolytic activity of lysozyme on diamond nanocrystallites

Abstract

Oxidative-acid-treated nanodiamonds exhibit high affinity for proteins, a property well suited for immobilization of enzymes for biotechnological application. Using lysozyme as an example, this work demonstrates that the enzyme can retain much of its activity after physical adsorption to the surfaces of 100-nm diamond crystallites. The activity relative to that of free lysozyme in solution is ∼ 60% at the maximum surface coverage of 50% and pH 5. While the enzymatic activity decreases as the surface coverage is lowered, it can be recovered by blocking the empty sites on the surface with supplementary proteins such as cytochrome c to create a more “crowded” environment. A relative activity up to 70% can be attained at a partial coverage of 20%.

Introduction

Diamond is a material widely used in industry because of its superior physical and chemical properties including ultrahigh hardness, high thermal conductivity, low friction coefficient, and excellent resistance against corrosive chemicals. Interestingly, it also holds many unique properties well suited for biotechnological application. The material is biocompatible [1], [2], [3], chemically inert, and its surface can be easily functionalized with carboxyl and amino groups [4], [5], [6]. Nano-sized diamond crystallites, in particular, have a large specific surface area and after treatment in strong oxidative acids, they show an exceptionally high affinity for proteins in aqueous solution [7], [8]. A recent experiment further demonstrated that nanodiamonds containing isolated nitrogen-vacancy defect centers can be used as a non-toxic, non-photobleaching fluorescent probe of biological cells [9].

Previously, we reported that oxidative-acid-treated nanodiamond (ND) is a superb solid phase support for extraction of proteins and peptides in highly dilute solution [7], [8]. Polylysine-coated ND powders also serve well as an enrichment device for DNA oligonucleotides [10]. Covalent immobilization of proteins (such as cytochrome c) can further be made on polylysine-coated NDs with heterobifunctional crosslinkers [6]. The last work was motivated by the notion that enzymes covalently linked to ND are potentially useful as biocatalysts, which have the benefit of reusability. A number of techniques regarding enzyme immobilization have been developed in solid-phase protein chemistry, including noncovalent adsorption, covalent attachment, and physical entrapment of enzymes in a polymeric gel, membrane, or capsule [11], [12]. In most cases, maintaining the high catalytic activity of the immobilized enzyme is the key issue. Noncovalent immobilization offers the simplest approach, while others usually involve complicated chemistry to produce covalent linkages between enzyme and surface.

Here, we address the feasibility of using ND as a carrier for noncovalent enzyme immobilization and examine the activity of such immobilized enzymes with different loading densities. The model enzyme chosen to study is hen egg white lysozyme, which is an antimicrobial protein widely used in food and pharmaceutical industries. The enzyme attacks bacterial cell walls and degrades the cell wall through hydrolysis of the sugar backbone of the peptidoglycan component. The enzyme molecule is roughly ellipsoidal in shape with a crystallographic dimension of 3.0 × 3.0 × 4.5 nm [13]. It is composed of a single peptide chain containing 129 amino acid residues that form five helical segments as well as a three-stranded antiparallel β sheet with 4 disulfide bridges. In this work, in addition to examination for the hydrolytic activity of the surface-bound lysozyme in buffer solution at pH 5, the adsorption behavior of this globular protein at a wider pH range was also investigated spectrophotometrically.