A reverse micelle strategy for fabricating magnetic lipase-immobilized nanoparticles with robust enzymatic activity

www.nature.com/scientificreports OPEN Received: 22 May 2017 Accepted: 8 August 2017 Published: xx xx xxxx A reverse micelle strategy for fabricating magnetic lipaseimmobilized nanoparticles with robust enzymatic activity Shixiong Yi1, Fangyin Dai1, Cunyi Zhao2 & Yang Si2 Enzyme-immobilized nanoparticles that are both catalysis effective and recyclable would have wide applications ranging from bioengineering and food industry to environmental fields; however, creating such materials has proven extremely challenging. Herein, we present a scalable methodology to create Candida rugosa lipase-immobilized magnetic nanoparticles (L-MNPs) by the combination of nonionic reverse micelle method and Fe3O4 nanoparticles. Our approach causes the naturally abundant and sustainable Candida rugose lipase to ordered-assemble into nanoparticles with high catalytic activity and durability. The resultant L-MNPs exhibit the integrated properties of high porosity, large surface area, fractal dimension, robust enzymatic activity, good durability, and high magnetic saturation (59 emu g−1), which can effectively catalyze pentyl valerate esterification and be easily separated by an external magnet in 60 second. The fabrication of such fascinating L-MNPs may provide new insights for developing functional enzyme-immobilized materials towards various applications. Enzymes are biocatalysts capable of accelerating a range of chemical transformations relevant to materials production, pharmaceutical development, and renewable energy, which are promising green and sustainable alternative to conventional synthetic strategies1–4. In order to adapt to organic reaction media and improve the recovery and reuse efficiency, usually hydrophilic enzymes need to be immobilized on or in specific carriers via physical adsorption or chemical binding5–7. Among the numerous supports investigated and applied for enzyme immobilization, nanomaterials, such as nanoparticles, nanotubes, graphene, and nanowires, combine the robust mechanical strength, high porosity, large surface area, lower mass transfer resistance, and high enzyme loading efficiency, which hold great promise as an exceptional nanoscale carrier for realizing enzyme immobilization8–12. However, conventional covalent immobilization of enzymes to nanocarrier was performed in aqueous phase, which was usually associated with a significant activity decrease because the active site might be blocked from substrate accessibility and multiple point-binding13, 14. The enzyme could also be denatured due to the random cross-linking between proteins and supports14, 15. Reverse micelles, as the forefront of microemulsion reaction medium, has attracted increasing interesting from the viewpoints of avoidance for the random cross-linking between proteins and supports16, 17. The reverse micelles are nano-sized spherical aggregates formed by certain surfactants in non-polar medium spontaneously, which solubilized small amounts of water in their interior so providing a stable aqueous microenvironment, the so-called “water-pool”, in non-aqueous medium18–20. The enzymes could be interfacial-activated for the oil-water interface and tended to localize at the spherical interface with the active sites orientated towards hydrophobic phase owing to a large associated hydrophobic region21. These organized enzymes would then form a self-immobilized particle in the presence of cross-linking agents. Several nanocarriers, including carbon nanotubes, copolymer particles, zeolite, and nanoclay, have recently been applied to enzyme immobilization using reverse micelles synthesis20–23. However, previous efforts mainly used these materials as non-functional nanosupports and focused excessively on the loading amount, ignoring the time-consuming and cost enzyme recovery procedures by separation or filtration, which presents major challenges in enzyme immobilization that must be addressed before their extensive practical applications. 1 State Key Laboratory of Silkworm Genome Biology & College of Biotechnology, Southwest University, Chongqing, 400715, P. R. China. 2Fiber and Polymer Science, University of California, Davis, CA, 95616, USA. Correspondence and requests for materials should be addressed to Y.S. (email: ) SCientifiC REPOrTS | 7: 9806 | DOI:10.1038/s41598-017-10453-4 1 www.nature.com/scientificreports/ Figure 1. (a) Schematic showing the synthetic steps of the GA-MNPs. (b) Synthesis of L-MNPs through the nonionic reverse micelle method. Herein, we demonstrate a scalable strategy for creating magnetic lipase-immobilized magnetic nanoparticles (L-MNPs) using a novel nonionic reverse micelle method and Fe3O4 nanoparticles. The premise of our design is that the Candida rugosa lipase are ordered incorporated into Fe3O4 particles with high catalytic efficiency and durability. The L-MNPs exhibited the integrated properties of high porosity, large surface area, robust enzymatic activity, good durability, and easy to magnetic separation, all originating from the synergistic effect of well-organized Candida rugose lipase and magnetic Fe3O4 nanoparticles. Results and Discussion Reverse Micelle Design: Optimizing Water Polarity. We designed the L-MNPs based on three criteria: (1) the lipase must assemble and bonding to MNPs with a stable covalent linking, (2) the lipase must be exhibit an ordered conformation with high enzymatic bioactivity, and (3) the L-MNPs should be easily collected and separated after reaction. The first two requirements were satisfied by a versatile and readily accessible nonionic reverse micelle method, which allowed quick, easy and reproducible preparation and high structural uniformity of the products. To satisfy the third criterion—we used Fe3O4 nanoparticles as magnetic nanocarriers for the ordered loading of lipase. The overall synthesis pathway was schematic showing in Fig. 1. The synthesis process began with the reverse micelles medium by optimizing the water polarity. The magnetic Fe3O4 nanoparticles (MNPs) were first modified with free amino groups on the surface (NH2-MNPs), then bonded with glutaric dialdehyde to form active aldehyde modified MNPs (GA-MNPs). Subsequently, the self-immobilization of lipase on GA-MNPs was carried out in an optimized reverse micelles medium, within vigorously stirring by an ultrasonic mixer. Finally, the lipase modified MNPs (L-MNPs) were facilely separated and collected by an external magnetic field. It was well known that the ionic systems such as sodium dioctylsulfosuccinate (AOT) reverse micelles would take remarkable influence on enzyme conformation. Usually enzymes showed lower activity compared to that in an aqueous system, which was attributed to the abnormally high polarity of interface regions of the water pool in an ionic surfactant reverse micelle24–28. Therefore, a nonionic reverse micelle system was introduced in this work, and the water polarity was optimized by adjusting the amount of solubilized. The po (...truncated)