Silanized maghemite for cross-linked enzyme aggregates of recombinant xylanase from Trichoderma reesei

https://doi.org/10.1016/j.molcatb.2016.07.006Get rights and content

Highlights

  • Development of Xyl-CLEA-silanized maghemite.

  • Xyl-CLEA-silanized maghemite improved activity recovery of xylanase.

  • Presence of silanized maghemite enhanced thermal stability of enzyme.

  • Reusability of Xyl-CLEA-silanized maghemite was increased from Xyl-CLEA.

Abstract

Numerous state-of-the-art technologies and new types of carriers have been developed recently to improve enzyme immobilization. Cross-linked enzyme aggregate (CLEA) technology is a lucrative prospect, as several robust biocatalysts have been generated using this simple method of carrier-free immobilization with the possibility of using semipurified enzyme. However, the low lysine content in the enzyme remains a challenge for effective crosslinking. In this work, maghemite (γ- Fe2O3), a recently sought after nanoparticle, was silanized with (3-aminopropyl) triethoxysilane (APTES) for use in the preparation of cross-linked enzyme aggregates of recombinant xylanase from Trichoderma reesei (Xyl-CLEA-silanized maghemite). Prior screening revealed ethanol to be the best precipitant, and a 0.2: 1 (v:v) glutaraldehyde to enzyme ratio was essential to form active CLEAs. The Xyl-CLEA-silanized maghemite succeeded in increasing the activity recovery 1.66- and 1.5-fold compared to conventional Xyl-CLEAs and Xyl-CLEA-BSA, respectively. The silanization of maghemite with APTES was proven feasible when a 0.0075:1 (v:v) maghemite to enzyme ratio was able to achieve a 78% activity recovery of the xylanase aggregates, whereas the non-silanized maghemite only achieved a 47% activity recovery. At an elevated temperature of 60 °C, Xyl-CLEA-silanized maghemite retained approximately 50% of its initial activity, compared to the free enzyme, for which the activity recovery had plummeted to 20%. Conversely, Xyl-CLEA suffered a total loss of activity at this temperature, whereas Xyl-CLEA-BSA retained only a 6% activity. Xyl-CLEA-silanized maghemite also successfully retained more than 50% of its activity after up to 6 cycles, whereas Xyl-CLEA retained approximately 10% of its initial activity after only 5 cycles. The surface morphology, particle size and loading of silanized maghemite were confirmed by field emission scanning electron microscopy (FESEM) and Fourier transform infrared (FTIR) spectroscopy. The combination of the effective surface modification, high enzyme activity recovery, improved stability and enhanced reusability of Xyl-CLEA-silanized maghemite presents an attractive process for xylanase immobilization and provides a promising catalyst for the biomass industry.

Introduction

The past two decades have shown tremendous development in the design of cross-linked enzyme aggregates. This carrier-free immobilization strategy has attracted increasing attention due to its simplicity in preparation and robustness in industrial applications [1], [2]. CLEAs hold several prominent advantages, including a highly concentrated catalytic activity, high stability against extreme operating conditions, low production cost due to the exclusion of carriers, ease of synthesis, facile recovery and reusability, as well as the fact that no extensive purification of enzymes is needed [2], [3]. The synthesis includes two main procedures, which are the precipitation of enzymes by aggregating agents such as salts, water-miscible organic solvents or non-ionic polymers, followed by the subsequent crosslinking of the precipitated enzymes by a bifunctional reagent such as glutaraldehyde [3], [4]. Although it is a straightforward immobilization strategy, the preparation of CLEAs remains challenging for enzymes with few lysine residues. Ineffective crosslinking occurs in enzymes with low amine content, resulting in CLEAs with low mechanical stability and thus enabling the release of enzyme molecules into the reaction media [4], [5]. Common solutions proposed are the introduction of a proteic feeder such as bovine serum albumin [5], [6], polylysine [7] or polyionic polymers containing abundant amine groups such as polyethylenimine (PEI) [8], [9] to increase the number of amino groups and facilitate intermolecular crosslinking, which increases the stability of the final CLEAs. In other cases, the particle size of the CLEAs, which is usually small (below 10 μm), or it being too soft greatly hinders the process of recovery [1]. These CLEAs are not mechanically resistant and may require physical support to increase the rigidity for some industrial applications [10].

Although various modifications have been made to further stabilize the CLEAs, the rapid development of nanostructured materials has stimulated strong interest in using magnetic nanoparticles to improve the quality of the immobilized enzyme. Effective surface functionalization provides these magnetic nanoparticles with recognition ability, enables controlled interaction between the magnetic cores and targeted biological species, and offers better aqueous dispersion and biocompatibility [11]. Enhanced stability is possible for repeated usage in continuous bioseparations and enables greater control over the catalytic process [1]. The CLEAs can be quickly and efficiently recovered by using an external magnetic field and can be recycled for iterative uses [12], [13], [14]. The removal of magnetic nanoparticles from a solution by using magnetic fields is selective and applicable to separations that would be otherwise impossible or unfeasible by other separation methods[14]. Previous studies on magnetic CLEAs commonly employed magnetite as a magnetic support, with the amino group (3-aminopropyl)triethoxysilane (APTES) being the most favoured functional group for bonding various bioactive molecules to the nanoparticles [12], [13], [15].

Although magnetite has been extensively used, Kang et al. [16], in their study of human lung cancer A549, discovered that maghemite nanoparticles (γ-Fe2O3) had a greater binding specificity compared to magnetite nanoparticles (Fe3O4). Maghemite is possibly a better adsorbent than magnetite due to its larger active surface area [17]. In addition, due to its spinel structure with two magnetically nonequivalent interpenetrating sublattices, maghemite exhibits a strong magnetic behaviour that has been used practically in various biomedical and biological applications including magnetic resonance imaging (MRI) contrast enhancement, biomagnetic separations and magnetic drug targeting. These wide applications of maghemite nanoparticles originate from their nontoxicity, biocompatibility, biodegradability, low particle dimension, large surface area and suitable magnetic properties [18]. To fully exploit the advantages of maghemite, it is essential to investigate its potential in stabilizing the cross-linked enzyme aggregates. This work, to our knowledge, is the first report of CLEA preparation by introducing silanized maghemite nanoparticles into the enzyme solution to produce a mechanically stable biocatalyst for the effective hydrolysis of hemicellulosic material. The present work reports an improvement in recovered activity and stability and in reusability, compared to conventional CLEAs, both with and without the addition of BSA. FESEM and FTIR analyses were also carried out to further characterize these insoluble biocatalysts.

Section snippets

Materials

Recombinant xylanase from Trichoderma reesei ATCC 53850 was expressed in Pichia pastoris and produced as described elsewhere [19]. The crude enzyme extract was partially purified using Minimate (Pall). Beechwood xylan, 3,5-dinitrosalycyclic acid, glutaraldehyde, bovine serum albumin and poly-l-lysine were purchased from Sigma Aldrich. All other chemicals were of analytical grade and purchased from Merck and Fisher. The initial activity of the xylanase after the partial purification step was

Effect of precipitant

The optimum precipitation and anticipated crosslinker concentration are two of the most important parameters affecting cross-linked enzyme aggregates. To determine the best precipitant for this recombinant xylanase, nine commonly used protein-precipitating agents were tested, including organic solvents (acetone, acetonitrile, tert-butanol, ethanol, n-propanol, isopropanol), polyethylene glycol (PEG 6000, PEG 8000) and ammonium sulfate (Fig. 1). Although the initial precipitation step had

Conclusions

A new approach in developing CLEAs has been explored, incorporating maghemite in the preparation of cross-linked xylanase aggregates. A significant increase in the catalytic recovery was observed for Xyl-CLEA-silanized maghemite compared to Xyl-CLEA and Xyl-CLEA-BSA. Combining the good properties of CLEAs with the striking characteristics of silanized maghemite nanoparticles has succeeded in enhancing the xylanase stability as well as enabling more cycles of reusability, which are crucial

Acknowledgements

The authors would like to express their heartiest gratitude to the Ministry of Science and Technology Malaysia for the financial support (Project No: 02-01-02-SF0886; Vote no: 4B093) and to the Ministry of Higher Education Malaysia, Universiti Teknologi Malaysia and Universiti Malaysia Pahang.

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