Photonic crystals in biology

Poster Session, Tuesday, June 15
Theme A1 - B702
Immobilization and Characterization of Thermophilic Recombinant Esterase on Chitosan Nanoparticles
1 ,Taylan Turan 1 and 1 *
zmir Institute of Technology, Faculty of Science, Department of Chemistry, Gülbahce Köyü Kampüsü, Urla, Izmir, 35430, Turkey
1
Abstract— Immobilization of biologically important molecules on myriad nano-sized materials has attracted great attention. Through this
study, homemade esterase enzyme was obtained using recombinant DNA technology. Enzyme over expression in Escherichia coli and
purification were carried out successfully. At the next step, chitosan was synthesized from chitin by deacetylation process and degree of
deacetylation was calculated as 89% by elemental analysis. Characterization of chitosan was studied by applying FT-IR (Fourier Transform
Infrared Spectroscopy). After preparation of chitosan and chitosan/esterase enzyme nano-particles, their surface morphologies and structures
were examined by AFM (Atomic Force Microscopy) and SEM (Scanning Electron Microscopy). In order to investigate the characterization of
esterase enzyme immobilized on chitosan nano-particles, the experimental studies in terms of activity, substrate specificity, the effect of
temperature and pH on the activity, optimum temperature and pH values, the effect of variety of metals, inhibitors and detergents on activity and
thermal stability are being under examination. The characterization data of enzyme immobilized chitosan nano-particles will be compared with
free enzyme for their potential uses as biocatalysts in variety of biotechnological applications.
Recently the majority of industrial enzymes produced by
micro-organisms are being utilized widely and their utilization
on very diverse area of industry is increasing rapidly in the
world day by day. Especially, enzymes from thermophiles
have many advantages for industrial applications because they
are very thermo stabile and thermo active under high
temperatures. In that study, we have used recombinant
thermophilic esterase enzyme from Balçova Geothermal
region [1]. Esterase enzymes are special interest in a variety of
biotechnological applications because of their many useful
properties.
Immobilization of enzymes is very important not only for
reuse of them but also for use them more efficient. As
compared to free enzymes in solution immobilized enzymes
are more robust and more resistant to environmental changes.
Therefore, many kinds of immobilization techniques have
been improved for many years on various supports and by
different methods. One of the most used biopolymer as
immobilization carriers, chitosan offer a unique set of
characteristics: biocompatibility, biodegradability to harmless
products, nontoxicity, physiological inertness, antibacterial
properties, heavy metal ions chelation, gel forming properties
and hydrophilicity, and remarkab le affin ity to proteins [2].
Recently, researchers have indicated that nano-sized materials
can be used for immobilization of enzymes. To the best of our
knowledge, studies of enzyme immobilization on Chitosan
nano-particles have been rarely reported. In this study,
chitosan nano-particles were prepared by ionic gelation
methodology. Thermophilic recombinant esterase was initially
over-expressed in E.coli and then purified by one-step affinity
chromatography with high yield and good purity.
Thermophilic recombinant esterase was immobilized on
chitosan nano-particles and conditions for the immobilization
and characterization of the immobilized enzyme were studied
systematically. Figure 1 shows AFM images of
Chitosan/Recombinant-Esterase Nano-particles. Figure 2
shows SEM images of chitosan nano-particles and
chitosan/Recombinant-Esterase nano-particles.
a) b)
Figure 2. SEM images of a) Chitosan nano-particles, b)
Chiosan/Esterase nano-particles.
As a result of our studies, it is expected that the immobilized
enzy me will exh ibit remarkab ly improved stability properties
to various parameters, such as temperature, reuse and storage
time. Thus nano-particles will be suitable for an immobilized
enzyme carrier.
*Corresponding author: gulsahsanli@iyte.edu.tr
[1] Tekedar, H., 2009. Molecular Cloning, Overexpression and
Characterization of Thermostable Esterase and Lipase from
Thermophilic Bacillus sp., Master’s Thesis, IYTE.
[2] Krajewska, B., 2004. Application of chitin- and chitosan-based
materials for enzyme immobilizations: a review, Enzyme and
Microbial Technology, 35: 126-139.
[3] Tang, Z. X., Qian, J. Q., Shi, L. E., 2007. Preparation of Chitosan
Nanop articles as carrier for immobilzed enzyme, Appl. Biochem. and
Biotech., 136: 77-96.
Figure 1. AFM images of Chitosan /Recombinant-Esterase Nanoparticles
6th Nanoscience and Nanotechnology Conference, zmir, 2010 293