In situ preparation of magnetic Fe₃O₄-chitosan nanoparticles for lipase immobilization by cross-linking and oxidation in aqueous solution

Abstract

A new and simple method has been proposed to prepare magnetic Fe₃O₄-chitosan (CS) nanoparticles by cross-linking with sodium tripolyphosphate (TPP), precipitation with NaOH and oxidation with O₂ in hydrochloric acid aqueous phase containing CS and Fe(OH)₂, and these magnetic CS nanoparticles were used to immobilize lipase. The effects on the sequence of adding NaOH and TPP, the reaction temperature, and the ratio of CS/Fe(OH)₂ were studied. TEM showed that the diameter of composite nanoparticles was about 80 nm, and that the magnetic Fe₃O₄ nanoparticles with a diameter of 20 nm were evenly dispersed in the CS materials. Magnetic measurement revealed that the saturated magnetisation of the Fe₃O₄-CS nanoparticles could reach 35.54 emu/g. The adsorption capacity of lipase onto nanoparticles could reach 129 mg/g; and the maximal enzyme activity was 20.02 μmol min⁻¹ mg⁻¹ (protein), and activity retention was as high as 55.6% at a certain loading amount.

Introduction

Lipase (EC 3.1.1.3) is a very important kind of enzyme with a broad variety of applications in the food industry, fine chemistry and pharmaceutical industry due to the multiplicity of reactions it catalyzes (Chang et al., 2008, Kose et al., 2002). Application of lipase can be achieved more economically and efficiently by immobilization to enhance its activity, selectivity, and operation stability. Therefore, a lot of effort has been made on the preparation of lipases in immobilized forms, which involve a variety of both support materials and immobilization methods.

The most notable feature of lipase is that the reactions they catalyze occur at the interface of oil and water. If lipase is immobilized onto the nanoparticles, which have high specific surface area and low diffusion resistance, it would be very effective for the catalysis. Nanophase materials have many advantages due to their unique size and physical properties. However, the nanoparticles are difficult to separate from the solution, except though the use of high-speed centrifugation. Using the magnetic property is a good solution to this problem. With the rapid development of nanotechnology, magnetic nanoparticles are now being studied all over the world. In recent years, magnetic nanoparticles have been synthesized through different approaches like chemical co-precipitation process (Wang et al., 2009), by sol–gel self-propagation (Yang et al., 2008) and in the tiny pools of water-in-oil micro-emulsion (Wang et al., 2008), and they have been applied in the removal of heavy metals, magnetic resonance imaging (MRI) contrast agents, biosensor, and embolotherapy (Wang et al., 2009, Li et al., 2008, Kim et al., 2005, Chana et al., 2006). Superparamagnetic iron oxide (Fe₃O₄ and γ-Fe₂O₃) nanoparticles have attracted researchers’ attention due to their multifunctional characteristics, including small size, superparamagnetism and low toxicity, and most importantly it is very easy to separate from the reaction system (Chen et al., 2005, Cui et al., 2006). The nano-Fe₃O₄ particles have been used in enzyme immobilization too. Lee et al. (2008) used SDS-bound NSM particles to immobilize lipase, enabling the protein immobilized on the support to reach 52.1 mg/g, but lost 50% of the initial activity after used three times. Yong et al. (2008) used polymer-grafted magnetic nanoparticles for lipase immobilization, with the loading ability of the particles reaching 105.2 mg/g, and losing 30% of the initial activity after used 5 times.

Chitosan has been applied in many fields, such as metal adsorption, enzyme immobilization, protein adsorption and the controlled release of drugs because of its excellent properties such as non-toxicity, biocompatibility, mucus-adhesion and biodegradation. Compared with that of the CS particles in micrometer size, the adsorption equilibrium was achieved much faster, and the maximum adsorption loading highly increased (Jiang et al., 2005, Zhi et al., 2005, Agnihotri et al., 2004).

Zhi et al. prepared magnetic CS-Fe₃O₄ nanoparticles in situ with tiny pools of water-in-oil microemulsion containing CS and ferrous salt as micro-reactors by adding the basic precipitant of NaOH into the micro-emulsion (Zhi et al., 2006, Wang et al., 2003). The magnetic CS-Fe₃O₄ particle size varied from 10 nm to 80 nm. The problem with this method is that productivity is low; in addition, it uses a lot of surfactant and co-surfactant compounds that are harmful to the environment. The products were used to adsorb BSA, etc., but there are very few reports about enzyme immobilization onto magnetic CS-Fe₃O₄ nanoparticles.

It is well known that the NH₂ group on CS molecules may interact with Fe²⁺ in aqueous solution. In this study, the CS nanoparticles were firstly prepared by cross-linking with TPP in HCl solution, the CS nanoparticles was gel and porous, then Fe²⁺ was added into the solution and adsorbed by the CS nanoparticles. NaOH was used to adjust pH and precipitate Fe(OH)₂, and a small amount of O₂ was used to oxidise the Fe(OH)₂ into Fe₃O₄. As the Fe₃O₄ nanoparticles were present in the pores of the CS gel, the monodispersion was good. The whole procedure was completed in an aqueous solution, so the method is simple and effective, and can be used for industrial production. After the particles were synthesized, they were used to immobilize lipase to test their immobilization characteristic.