Magnetic separation and high reusability of chloroperoxidase entrapped in multi polysaccharide micro-supports

Highlights

• Magnetic micro-supports were developed based on a multi polysaccharide shell.

• All supports were characterized in terms of chemical stability under reaction conditions.

• Chloroperoxidase was successfully entrapped in multi polysaccharide magnetic supports.

• Leakage of the enzyme was observed with single-shell polysaccharide coating.

• High CPO reusability was measured with a polymeric double-shell magnetic micro-support.

Abstract

Enzyme immobilization on magnetic supports represents a great advantage for the industrial application of enzymatic catalysis since it allows an easy recovery of the catalyst, avoiding any contamination of the product by residual enzyme. Iron oxide nanoparticles are very useful for this purpose. Using a polymer to diminish the interaction between the magnetic cores themselves, can improve the colloidal stability of the support and prevent any interaction with the environment that would affect both support properties and enzyme stability. For this reason, in this work different magnetic micro-supports, based on polydopamine-coated iron oxide nanoparticles with a multi polysaccharide shell, have been developed. These supports have been used to immobilize chloroperoxidase, a very interesting enzyme, able to catalyze many reactions of large-scale interest, but whose application is limited by its sensitivity to reaction conditions. The multi polysaccharide shells of the supports were obtained through a combination of chitosan and alginate. An in-depth analysis of physicochemical and catalytic properties of all the developed magnetic supports is reported. CPO was successfully immobilized with an efficiency of entrapment between 92% and 100% in the case of supports with chitosan in the interior or outer shell respectively. A very good chemical stability of the support under reaction conditions was observed in the case of an interior shell of alginate and an outer coating of chitosan, together with an excellent reusability of the immobilized enzyme, that was recycled to catalyze up to 25 consecutive reaction cycles.

Introduction

Enzymatic catalysis represents a valuable tool for industrial applications. Nevertheless, in some cases its use on large scale could be a complicated issue due to catalyst sensitivity to reaction conditions and high costs of enzyme production [1]. These limitations can be overcome through enzyme immobilization on solid supports to allow the recovery and reuse of the catalytic system [[2], [3], [4], [5], [6], [7], [8]]. The improved stability of the enzyme to environmental conditions, i.e. presence of organic solvents, temperature and pH, allows a longer lifetime of the catalyst with respect to the non-immobilized enzyme. The easier the handling and recovery of the enzyme, the simpler is the downstream process and the better the economy of the process. Moreover, in the case of using a supported catalyst, the reaction product is not contaminated by any residual of the enzyme, an aspect that gained a great importance in food and pharmaceutical industrial applications.

The choice of a suitable support allows maintaining the catalytic properties of the enzyme after its immobilization and maximizing the efficiency of the conversion process [4].

Enzyme immobilization on nanomaterials represents one of the most forefront research fields in biocatalysis, biosensoring and biomedical applications thanks to the unique properties that the small size confers to these supports. An improvement in enzyme stability and catalytic performances can be obtained through enzyme immobilization on nanoparticles, probably rigidifying them and preventing their denaturation in the recycling process. Among all kinds of nanoparticles, magnetic ones offer a great advantage for the applicability of the catalytic system on large scale, since they can be easily separated from the medium at the end of reaction process and recovered for their reuse thanks to their fast response to an applied magnetic field [[2], [3], [4], [5]]. In the last few decades, they have acquired importance as carrier for binding proteins, enzymes, antibodies and drugs, for biomedical and biotechnological applications [2,[9], [10], [11]].

In many cases it has been found that coating and surface modification of these inorganic nanoparticles allow to control the interaction between the magnetic cores themselves and interactions with the environment, improving a better colloidal stability of the support and so a higher activity of the immobilized biomolecule [10,[12], [13], [14]]. Polymeric coatings are widely used to increase stability and biocompatibility of magnetic nanoparticles and also to preserve their magnetic properties. In fact they can avoid aggregation effects due to magnetic or surface interactions and they can also protect the magnetic core from direct exposure to reaction conditions that could lead to any degradation of the inorganic core. Natural polymers, as polysaccharides, are used as coatings for inorganic nanoparticles since they have the advantage over synthetic ones of being abundant in nature, biodegradable and cheap [12].

Natural polymers were also widely used for the coating of magnetic nanoparticles for enzyme immobilization. Chitosan-coated magnetic nanoparticles were employed to immobilize glucose-6-phosphate dehydrogenase [15] and pectinase [16], while chitosan-collagene or alginate coating were used for lipase from Candida rugosa immobilization [17,18].

Among natural polymers, both chitosan and sodium alginate are biodegradable, non-toxic, biocompatible polysaccharides [[19], [20], [21]]. While chitosan is rich in amino groups, which confer positive charges to the polymeric chains at pH values below 6.0, sodium alginate is a negatively charged polymer due to the presence of carboxyl groups. The presence of such functional groups on their structure allows the interaction with biomolecules of interest. Both polysaccharides have been used for the coating of magnetic nanoparticles to obtain biocompatible supports.

Chloroperoxidase from Caldaromyces fumago (CPO), a highly glycosylated heme enzyme, possesses a great industrial interest since it is able to catalyze many reactions of large-scale interest, i.e. sulfooxidation, epoxidation and oxidative halogenation. Nevertheless, this enzyme is very sensitive to high concentrations of oxidants, which limit its industrial applications [22].

These drawbacks can generally be overcome by immobilization of the enzyme. Different supports have been employed to improve CPO catalytic efficiency. Silica materials [[23], [24], [25]], iron oxide magnetic nanoparticles [26,27], Au@Fe₃O₄ nanoparticles [28], nanostructured titanium oxide materials [29,30], ZnO nanowire/silica composite [31], alumina nanorods [32], an acrylic-based material (Eupergit^® C) [33] and graphene oxide nanosheets [34] are some of the most recently used carrier from the literature.

In our previous papers, hybrid polymer-silica matrices, synthesized by sol-gel method, were found to be excellent supports for CPO immobilization by entrapment [35,36]. In particular, chitosan-silica composites were found to be the most effective supports to improve biocatalyst reusability, up to 18 consecutive reaction cycles [36]. To further investigate the applicability of chitosan-based supports on CPO reusability, in this work, a biodegradable magnetic support for CPO immobilization has been developed through the combination of magnetic iron oxide nanoparticles and a multi-shell support of chitosan and sodium alginate.

A nanoemulsion-based core further coated with polysaccharide multiple shells has been developed to encapsulate magnetic nanoparticles to obtain a magnetic support for CPO entrapment [37]. Magnetic nanoparticles were firstly coated with polydopamine (PDA), to stabilize them and favor the following interaction with the encapsulating system [18,38]. Different magnetic supports were obtained for CPO immobilization in polymer shells by combining chitosan and sodium alginate. After physicochemical characterization and catalytic tests, the optimal support for catalyst immobilization in terms of easy recovery and high reusability was selected in view of large-scale applications.