Department of Physics
University of Rome, La Sapienza
PhD Research Project - XXIII Cycle
Infrared Spectroscopic, Thermodynamic, and AFM Investigations of Immobilized Enzymes
in Nanostructured Polymers
Candidate: Gihan S. Kamel
Supervisors: Prof. Stefano Lupi - Prof. Federico Bordi
Enzymes are biomolecules that catalyze (i.e. increase of the rate) chemical reactions. Almost all
enzymes are proteins. In enzymatic reactions, the molecules at the beginning of the process are called
substrates, and the enzyme converts them into different molecules, the products. Almost all processes in a
biological cell need enzymes to occur at significant rates. Since enzymes are selective for their substrates and
speed up only a few reactions from among many possibilities, the set of enzymes made in a cell determines
which metabolic pathways occur in that cell.
Suitable supports for technical applications should maintain a high level of enzyme activity, while
preventing a possible leaching out during the reaction. Macroscopic, planar devices find limited applications,
due to the maximum concentration of functional enzyme that can be immobilized within the designated
surface area of the device. On the contrary, particles of nanoscopic size are very well-suited for the
immobilization of enzymes as they provide large surface areas. As additional advantages, nanoscale materials
offer lower mass transfer resistance and less fouling phenomena during bioprocess development. Moreover,
the immobilization of the biomolecules on colloidal particles can be easily obtained by adsorption from
solution. Proteins at the colloidal particle surface are capable of self-assembling, thus opening the possibility
of nanoscale surface patterning, particularly attractive for potential applications that include novel diagnostic
or therapeutic agents on the cell size scale.
Within this framework, the present project will focus on developing nanostructures conjugated to
particular enzymes, microbial lipases and esterases. The development of pharmaceutical products based on
this mechanism requires the tuning of reliable methods for the controlled production of acylated oligosaccharides. In this sense, the enzymatic immobilization could be a useful tool to modulate enzyme
selectivity and specificity. It is clear that, particularly in the case of lipases, a deep understanding of
interactions of the enzyme with the surface onto which it is immobilized and, more in general, with its "nanoenvironment" at the supportsolvent interface, is a major requirement for the advancement of technological
applications.
Different techniques will be used to characterize the physico-chemical properties of lipase immobilized on to
the solid surfaces of the different nanostructures.
(A) IR-SPECTROSCOPY CHARACTERIZATION:
Infrared spectroscopy has proved to be a powerful tool for the study of biological molecules and the
application of this technique to biological problems is continually expanding, particularly with the advent of
increasingly sophisticated sampling techniques such as infrared imaging. Biological systems, including lipids,
proteins, peptides, biomembranes, nucleic acids, animal tissues, microbial cells, plants and clinical samples,
have all been successfully studied by using infrared spectroscopy. The strong difference between absorption
bands that are characteristic for different protein conformations can also be observed in biologically relevant
peptides and proteins. Based on these relations between secondary structure and IR absorption frequencies,
the structural components of proteins may be determined.
The most significant advances in infrared spectroscopy, however, have come about as a result of the
introduction of Fourier-transform spectrometers. Bruker Optics, IFS 66v/s interferometer equipped with
Globar IR source, KBr beam splitter and MCT (mercury cadmium telluride) detector will be used for the
samples characterization. Mid-infrared region is to be selected for the performance of the measurements.
Nanostrucrured polymer and enzyme under study were synthesized by the group of Cleofe Palocci,
Biotechnology laboratory, Chemistry Department, La Sapienza.
(B) THERMODYNAMIC STUDIES CHARACTERIZATION:
Monolayers at the air-water interface have been investigated. However, the intensity of research in
the field increased tremendously with the onset of the current trends to nanotechnology, molecular
electronics, organic electronics and bio-engineering because it was felt that the technology could be useful on
the way there, allowing the manipulation of many materials at the molecular level. Langmuiur-Blodgett
technology allows the deposition onto solid substrates of well packed molecular monolayers and multilayers
with a perfectly controlled monolayer substructure.
Biodegradable polymers such as PLA, may often be utilised as drug delivery systems because their
degradation products are metabolized in the human body.
Interfacial properties of spread monolayers of poly(lactic-acid), PLA, are to be studied at the airwater interface by measuring surface pressure-mean molecular area (area per monomer) isotherms. Also,
Langmuir balance is to be applied to study how the interfacial behaviour of the biopolymer in the compound
mixed system will be changed under different circumstances.
The interfacial properties of these monolayers will be also studied using IR-spectroscopic tools as a
complemetray study. As a first step, we will investigate the spectroscopic properties of the monolayers
deposited on solid (IR transparent) substrates. The second step will be based on the simultaneous
measurement of the IR spectroscopic properties for the mixed system at the interface. This task will be
accomplished through a development of an experimental set-up in which the Langmuir-Blodgett technique
will be coupled to the Michelson interferometer.
(C) ATOMIC FORCE MICROSCOPY (AFM) CHARACTERIZATION:
AFM is a powerful technique also for studying supported lipid films. Its potentialities come from the
capacity of imaging lipid films at high lateral and vertical resolution in a liquid environment, the ability of
investigating local mechanical properties and interaction force and the possibility of using the AFM tip as a
nanotool to locally modify the films. Samples are usually prepared by using a Langmuir–Blodgett trough
which allows us to transfer lipid films from the air–water interface to a solid support.
In this project, AFM technique will be used to investigate the mechanism of interaction between the
lipase and the poly(lactic-acid). Also, to study the stucture and conformation of biological materials, in order
to correlate them to their functionnality.
Our knowledge about the physicochemical properties of proteins coupled to different synthetic surfaces is
still limited. However, in developing of enzyme-based devices a deep understanding of the protein/surface
interaction and of the possible changes induced in the protein conformation is needed. Immobilization onto
the surface of the nanostructures may lead to a deformation of the enzyme that, due to the relatively fragile
and varied nature of proteins, frequently leads to a marked loss of biological activity or complete inactivation.
In contrast, attachment of enzyme to solid supports may result in enhanced enzyme activity in organic
solvents as compared with that of native enzyme in the same reaction media.
References:
1- Smith AD (Ed) et al. (1997)) Oxford Dictionary of Biochemistry and Molecular Biology Oxford University
Press.
2- Garrett RH, Grisham CM. (1999) Biochemistry, Second Edition Saunders College Publishing. 426-427.
3- Langmuir (2004), 20, 10639-10647.
4- Palocci et al. (2007) Biomacromolecules.
5- Corvis, Y., Walcarius, A., Rink, R., Mrabet, N. T., and Rogalska, E. (2005). Analytical Chemistry, 10.1021.
6- Wang, P., Dai, S., Waezsada, S. D., Tsao, A. Y., and Davison, B. H. (2001).Biotech. Bioeng., 74(3):249–255.
7- O. Albrecht / Colloids and Surfaces A: Physicochem. Eng. Aspects 284–285 (2006) 175–179.
8- E. Kiss et al. / Journal of Colloid and Interface Science 325 (2008) 337–345