Elsevier

Process Biochemistry

Volume 49, Issue 11, November 2014, Pages 1920-1928
Process Biochemistry

Optimized immobilization of peracetic acid producing recombinant acetyl xylan esterase on chitosan coated-Fe3O4 magnetic nanoparticles

https://doi.org/10.1016/j.procbio.2014.08.008Get rights and content

Highlights

Abstract

Recombinant acetyl xylan esterase (rAXE) from Aspergillus ficcum, which mediated the production of peracetic acid (PAA), was covalently immobilized with magnetic Fe3O4–chitosan nanoparticles (Fe3O4–CSN) using glutaraldehyde. Fe3O4–CSN were prepared by co-precipitation of Fe2+ and Fe3+ ions in ammonia solution followed by coating of chitosan with sodium tripolyphosphate (TPP), and were characterized by FESEM, SEM, FTIR and XRD. The immobilization rAXE on Fe3O4–CSN was optimized using response surface methodology (RSM) by examining immobilization cross-linking time, enzyme concentration, and glutaraldehyde (GA) concentration. Based on the experimental values the predicted variables for the maximum immobilization of rAXE in terms of specific activity (0.042 U) were found to be 13.65 μg of rAXE protein and 0.265% of GA concentrations, where the optimum cross-linking time was 11.33 h. The immobilized rAXE onto Fe3O4–CSN nanoparticles shows better stability at thermal and pH ranges than soluble free rAXE. The immobilized rAXE was stable for around 90% of activity in the aqueous phase, whereas it retains only 60% of its activity in the semi-aqueous phase after 10 cycles of reuse.

Introduction

Peracetic acid (PAA) has been widely used as an excellent oxidant in the degradation of lignin substructures in biomass, especially the acid soluble lignin [1] by cleaving aromatic nuclei in lignin, generating dicarboxylic acids and their lactones [2]. It is also used for the selective degradation of common industrial pollutants found around the world, like synthetic dyes from textile industries, and polycyclic aromatic hydrocarbons (PAHs) from chemical and pesticide manufacturing industries. They are commonly composed of two or more fused benzene rings which are ubiquitous in nature [3], [4]. PAA is also a strong disinfectant, with a wide spectrum of antimicrobial activity, as it has been used as a disinfectant for waste water effluents [5]. PAA has been produced using acetic acid and hydrogen peroxide in chemical industries by conventional methods. The process is a tedious and expensive method of production. PAA can be explosive in its concentrated form and this safety hazard increases the cost of storage and transport of PAA across countries. Transportation and storage hazards can, of course, be limited by in situ generation of peracetic acid using enzymes, which reduces the overall cost of production. Acetyl xylan esterases (AXE) are esterases which catalyze the perhydrolysis of ester to peracetic acid [6]. It is possible to produce large amounts of AXE, which is a major eco-friendly biocatalyst, with the help recombinant DNA technologies [7]. However, its use is limited by economic considerations, due to their lack of reusability. Immobilization of enzymes as biocatalysts would circumvent the economic limitation and efficient utilization of enzymes. The immobilized enzyme can be easily separated from the reaction medium [8]. With the advent of nanotechnology, it is possible to immobilize various kinds of therapeutical proteins for target site delivery, and various enzyme proteins to carry out biotransformation reactions. Immobilization of proteins on Fe3O4 magnetic nanoparticles has been receiving increasing attention, due to their large specific surface area and ease of separation from reaction mixture through the use of a magnetic field [9]. Such kind Fe3O4 nanoparticles need to be modified or coated with a few atomic layers of chemically active polymer, to provide functional groups for linkage and immobilization of enzymes such as alcohol dehydrogenase [10], β-d-galactosidase [11], or laccase [12].

Chitosan is an excellent polymer for the chemical incorporation of cations for nanoparticles, as it is a biodegradable polysaccharide derived from the partial deacetylation of chitin. Because of the presence of free amine groups chitosan has the greater solubility and reactivity than that of chitin and cellulose polymers [13], [14]. The deacetylated chitosan backbone of glucosamine units has a high density of amine groups, which provides linkage for the covalent immobilization of proteins by simple and inexpensive cross linking methods, such as through the use of cross linking agents like glutaraldehyde [15]. Glutaraldehyde (GA), 5-carbon dialdehyde, is a clear, colorless to pale straw-colored oily liquid that is soluble in all proportions in water as well as many organic solvents. Among several dialdehydes, GA possesses unique characteristics that render it one of the most effective proteins cross linking reagent. GA exists in 13 different forms depending on the solution conditions. All of these forms can react with proteins in different ways, which causes the immobilization of proteins. It may react with proteins by several types of reactions such as Michael-type addition or aldol condensation. The protocol for using GA should be specified for each particular enzyme and application. Due to the discrepancies and unique characteristics of GA, cross linking procedures with each enzyme are largely developed through empirical statistical observation [16], [17]. Two various methods of glutaraldehyde mediated cross linking may be used to activate the support and immobilize the enzyme, as a ‘glutaraldehyde-activated support’ (in this case the immobilization is promoted by ionic exchange) or adsorption of the proteins onto the aminated supports, and treat the immobilized preparation with glutaraldehyde to cross-link both the enzyme and the support. Both possibilities have their own advantages and disadvantages when applied to protein, as higher concentrations inhibit enzyme activity, as described by Guisán [18], [19]. Insolubilization of the enzyme with high concentration of GA may inactivate the enzyme by distorting its structure. In order to retain its full catalytic activity the choice of the enzyme GA ratio is a critical factor for each enzyme. Also time was also taken into the context as it is an important factor for the effective cross linking of the enzyme with the support as well as the stability of enzyme molecules in GA solutions [20].

Scheme 1 shows the step-wise synthesis of chitosan coated Fe3O4 magnetic nanoparticles and the immobilization of rAXE using glutaraldehyde as a crosslinker. To minimize the amount of enzyme usage and the enzyme inactivation during GA mediated cross linking, optimization was performed using response surface methodology (RSM). The three factors were: enzyme concentration, GA concentration, and time of cross linkage.

Section snippets

Chemicals

Chitosan powder (MW 140,000 Da/mol) with 90% deacetylated, ferric chloride hexahydrate (FeCl3·6H2O) and ferrous sulfate heptahydrate (FeSO4·7H2O), sodium tripolyphosphate (TPP), and bovine serum albumin were purchased from Sigma–Aldrich (St. Louis, MO). Ammonium hydroxide (28%) was purchased from Katayama Chemical Co. Ltd. (Osaka, Japan). Unless otherwise noted, all reagents and chemicals were analytic grade.

Preparation of chitosan coated-Fe3O4 nanoparticles

Fe3O4 nanoparticles were prepared by the co-precipitating method, as described by

Synthesize of Fe3O4–chitosan nanoparticles

The first step was the co-precipitation of Fe2+ and Fe3+ ions to form Fe3O4 nanoparticles. Chitosan (CS) was coated on the Fe3O4 nanoparticles by adjusting the pH of the mixture containing Fe3O4 nanoparticles and chitosan. In acidic conditions, the amino group of CS would protonate into cationic charges, which facilitates the adsorption of CS onto the magnetic Fe3O4 nanoparticles by electrostatic attraction. The addition of TPP might cross-link the adsorbed CS with positively charged amino

Conclusion

Chitosan coated magnetic Fe3O4–CSN was prepared successfully, and rAXE was covalently immobilized on the surface as layer by layer. The rAXE was covalently immobilized to Fe3O4–CSN using the simple dialdehyde cross linking agent glutaraldehyde. Central composite design of response surface methodology was used to determine the optimum immobilization conditions of enzyme to the Fe3O4–CSN surface, using X, Y and Z as three independent variables. Thus, the reaction conditions for immobilization of

Acknowledgements

This research was supported by the Technology Development Program for Agriculture and Forestry, under the Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea. This research was also supported in part by Korea Research Council of Fundamental Science & Technology (Joint Degree and Research Center for Biorefinery). We would like to thank the Research Institute of Bioindustry at Chonbuk National University for kindly providing the facilities for which to conduct this research.

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