Preparation, Characterization and Optimization of Chitosan Nanoparticles as Carrier for Immobilization of Thermophilic Recombinant Esterase

Abstract

Immobilization of biologically important molecules on myriad nano-sized materials has attracted great attention. Through this study, thermophilic esterase enzyme was obtained using recombinant DNA technology and purified applying one-step His-Select HF nickel affinity gel. The synthesis of chitosan was achieved from chitin by deacetylation process and degree of deacetylation was calculated as 89% by elemental analysis. Chitosan nanoparticles were prepared based on the ionic gelation of chitosan with tripolyphosphate anions. The physicochemical properties of the chitosan and chitosan nanoparticles were determined by several methods including SEM (Scanning Electron Microscopy), FT-IR (Fourier Transform Infrared Spectroscopy) and DLS (Dynamic Light Scattering). The morphology of chitosan nanoparticles was spherical and the nanospheres' average diameter was 75.3 nm. The purified recombinant esterase was immobilized efficiently by physical adsorption onto chitosan nanoparticles and effects of various immobilization conditions were investigated in details to develope highly cost-effective esterase as a biocatalyst to be utilized in biotechnological purposes. The optimal conditions of immobilization were determined as follows; 1.0 mg/mL of recombinant esterase was immobilized on 1.5 mg chitosan nanoparticles for 30 min at 60• C, pH 7.0 under 100 rpm stirring speed. Under optimized conditions, immobilized recombinant esterase activity yield was 88.5%. The physicochemical characterization of enzyme immobilized chitosan nanoparticles was analyzed by SEM, FT-IR and AFM (Atomic Force Microscopy).

Physicochemical Characterization of Nanobiocomposites

“…The strength of these two peaks' intensity also decreased dramatically. These changes in the FTIR spectrum confirmed the immobilization of esterase on chitosan NPs [30].…”

“…The presence of an electronegative group around the nucleus will result in a higher resonant frequency in general. In [30].…”

Thermoalkalophilic recombinant esterase entrapment in chitosan/calcium/alginate‐blended beads and its characterization

“…(2011). 35 The degree of deacetylation of chitosan was investigated by a LECO-CHNS-932 elemental analyzer (Monchengladbach, Germany) and it was calculated as about 87.3%. Also, molecular weight of chitosan was investigated by a viscosimetric method at the room temperature 36 and it was found in the range of middle molecular weight category.…”

Polymer Materials in Environmental Chemistry

“…SEM has been widely used for decades in the study and analysis of biopolymer systems to gain information pertaining to the structure, morphology, size, shape, surface modifications, wear and tear, etc. SEM studies have played pivotal roles in the analysis of biopolymers [115][116][117], biopolymer nanoparticles [118,119], biopolymer assisted nanoparticles [21][22][23], biopolymer nanocomposites [120][121][122][123][124], and biopolymer fibers [125][126][127][128]. SEM images can be helpful in observing and analyzing different particle structures varying from fibers to microparticles and nanocrystals [129,130]; SEM provides an effective way to analyze these particles and their surfaces, and a medium to measure the particle size and diameter as well [81] (Figure 5).…”