Immobilized protease on the magnetic nanoparticles used for the hydrolysis of rapeseed meals

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Abstract

(3-aminopropl) triethoxysilaneand modified magnetic nanoparticles with the average diameter of 25.4 nm were synthesized in water-phase co-precipitation method. And then these nanoparticles were covalently coupled with alkaline protease as enzyme carrier by using 1,4-phenylene diisothlocyanate as coupling agent. Experiments showed that the immobilized protease can keep the catalytic bioactivity, which can reach to 47.8% when casein was served as substrate. Results showed that the catalytic activity of immobilized protease on these magnetic nanoparticles could retain 98.63±2.37% after 60 days. And it is more stable than the free protease during the shelf-life test. The enzyme reaction conditions such as optimum reaction temperature and pH are the same as free protease. Furthermore, mix-and-separate experiments showed that the immobilized protease could be recycled through the magnetic nanoparticles after the biocatalysis process. When the rapeseed meals were used as substrate, the degree of hydrolysis of immobilized alkaline protease achieved 9.86%, while it was 10.41% for the free protease. The macromolecular proteins of rapeseed meals were hydrolyzed by immobilized protease into small molecules such as polypeptides or amino acids. Thus, a novel efficient and economic way for the recycling of enzymes in the application of continuous production of active peptides was provided based on these magnetic nanoparticles.

Introduction

Enzymes are natural biocatalyst with very high catalytic efficiency and selectivity which are widely used in many areas such as food processing and medical science. However, the stability of these enzymes is feeble, and there are still challenges in the recycling of the valuable enzymes, which limit their applications in industrial production. Not until recently, the immobilized enzyme technologies overcome these difficulties by immobilizing enzymes onto carriers through physical or chemical methods. Immobilized enzymes maintain natural catalytic activities and can be easily separated from reaction systems for recycling use. Consequently, immobilized enzyme technologies provide a feasible way for continuous production [1]. A large number of research reports showed that immobilized enzymes have better performance than free enzymes in many respects [2], [3], [4].

Various methods have been used to immobilize enzymes, such as adsorption-crosslinking [5], encapsulation [6], covalent coupling [7] and so on. Among these methods, covalent coupling attracts wide attention due to the stability of combination between carriers and enzymes. The carriers designed for the enzymes can be polymers [8], inorganic materials [9] or composites [10]. As magnetic nanoparticles can be separated from the reaction systems by applying external magnetic field [11] and therefore these nanoparticles have been widely used for biological and medical researches [12], for example, separation of stem cells [13], immobilization of lipase [14]. Such magnetic carriers are generally of core-shell structure. And the shells can be modified with various functional groups (such as hydroxyl, amino, sulfhydryl and so on) by copolymerization or chemical surface modification. And the enzymes, antibodies, nucleic acids are then covalent coupled to the magnetic nanoparticles through organic or inorganic surface modified shells.

Meanwhile, the rapeseed protein is a kind of vegetable protein, which has a nutritive value no less than that of the animal protein. Rapeseed peptide, degraded from rapeseed meals or rapeseed proteins, is a type of small-molecule peptide with solubility, emulsifying properties and water holding capacities which are better than rapeseed protein. In addition, rapeseed peptide has several excellent nutritional physiology properties such as fast absorption and high absorption rate [15]. Thus, it is an important economic resource for the high value rapeseed peptide from rapeseed meals. The enzyme method to obtain peptides by hydrolyzing protein attracts considerable attention because of its mild reaction conditions, safety, controllability, and the capability of large-scale production of specific peptides [16]. Alkaline protease is a kind of proteolytic enzyme produced by fermenting bacillus subtilis 2709, and its catalytic site is serine [17]. Alkaline protease exhibits outstanding capability to degrade protein, and has many free amino acids which serve as the active sites for covalent conjugation.

In this study, amino silane coated magnetic nanoparticles were synthesized in water phase, then alkaline proteases were immobilized onto the surface of these magnetic nanoparticles carriers with 1,4-phenylene diisothlocyanate as coupling agent. The resultant immobilized protease was used for biocatalysis for different kinds of proteins in rapeseed meals, and it can be separated easily by applying external magnetic field. This work provides a promising way to recycle the enzymes and also provides a feasible way to produce active peptides continuously by mix-and-separate.

Section snippets

Material and reagents

FeCl3·6H2O, FeSO4·7H2O, aqueous solution (25%) and N,N-dimethylformamide (DMF) were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). (3-aminopropl) triethoxysilaneand (APTES, 98%), 1,4-phenylene diisothlocyanate (PDC, 98%) were obtained from Alfa Aesar (Ward Hill, USA). Fluorescamine (99%) was provided by Sigma-Aldrich Fine Chemicals (St. Louis, USA). All reagents were of analytical grade.

Casein was purchased from Huashun Chemical Reagent Co. Ltd. (Wuhan, China), which was

Characterization of the composite nanoparticles

The X-ray diffraction (XRD) pattern was measured first to confirm the crystal structure of the bare Fe3O4 particles (Fig. 1). The main diffraction peaks of the sample were located at 2θ=30.4159°, 35.8136°, 43.3978°, 53.8213°, 57.3240° and 62.9344°, corresponding to (2 2 0), (3 1 1), (4 0 0), (4 2 2), (5 1 1) and (4 4 0) crystal face of inverse spinel Fe3O4 with a face-centered cubic phase, which are consistent with the characteristic diffraction peaks of normative Fe3O4 [21]. The XRD pattern indicated the

Conclusions

In this work, immobilized protease was prepared by immobilizing alkaline protease onto the surface of amino silane coated magnetic nanoparticles. All the results demonstrated that the immobilized protease presented good catalytic activity, and can be separated easily by applying external magnetic field, providing a feasible way to produce active peptides continuously.

Acknowledgements

The work was supported by the National High Technology Research and Development Program of China (863 Program: 2007AA10Z328, 2007AA100703), the National Natural Science Foundation of China (Grant no. 30670553). We also thank Analytical and Testing Center (HUST) for the help of measurement. This work was also supported by the Graduate Innovation Fund of Huazhong University of Science and Technology (HF05222007170).

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