Innovative approach to pharmaceutical biotechnology based on Layer-by-Layer technique
Author(s)
Guazzaroni, Melissa
Date Issued
March 16, 2012
Type
Doctoral Thesis
Abstract
In recent years, the use of enzymes as natural catalysts has received a great attention in the
development of organic synthesis, especially in the frame of green chemistry. In fact, due to the high
chemical and energy efficiency of enzymatic transformations, biocatalysis is one of the greenest
technologies that works perfectly with the emerging trend of bio-based sustainable feedstock.
Indeed, biocatalysts can prevent waste generation by performing catalytic processes with high
chemo-, stereo- and regio- selectivity under very mild reaction conditions of temperature, pH,
pressure and solvent, working predominantly in aqueous systems. These properties minimize the
problems of undesired side reactions and make the processes environmentally friendly. In addition to
the unquestionable advantages, there are some drawbacks that lead to a limitation of enzymes for
industrial applications: the high cost of isolation and purification of enzymes; the instability of their
structures once they are isolated from their natural environments; their sensitivity both to process
conditions other than the optimal ones, normally narrow-ranged, and to trace levels of substances
that can act as inhibitors. Also, unlike conventional heterogeneous chemical catalysts, most enzymes
operate dissolved in water in homogeneous catalysis systems, which is why they contaminate the
product and cannot be recovered in the active form from reaction mixtures for reuse. Several
methods have been proposed to overcome these limitations, one of the most successful being
enzyme immobilization. Immobilization is achieved by fixing enzymes to or within solid supports. By
mimicking the natural mode of occurrence in living cells, where enzymes for the most cases are
attached to cellular membranes, the systems stabilize the structure of enzymes, hence their activities.
Thus, as compared to free enzymes in solution, immobilized enzymes are more robust and more
resistant to environmental changes. I\/lore importantly, the heterogeneity of the immobilized enzyme
systems allows easy recovery of enzyme and product, multiple reuses of enzymes, continuous
operation of enzymatic processes, rapid termination of reactions and greater variety of bioreactor
designs. Nevertheless, compared with the free enzyme, the immobilized enzyme has usually its
activity lowered and the Michaelis-Menten constant increased. These alterations result from
structural changes introduced to the enzyme by the applied immobilization procedure and from the
creation of a microenvironment in which the enzyme works, different from the bulk solution. In spite
of these disadvantages, the creation of a microenvironment may allow to the enzyme to remain
active at different temperatures or pHs than would be predicted when immobilization do not occurs,
increasing the application possibilities.
The present PhD project will be focused on the development and characterization of novel
immobi ization systems of oxidative enzymes based on the Layer-by-Layer (LbL) method, and on their
applicat'on to pharmaceutical biotechnologies.
In part'cular, tyrosinase from Agaricus bisporus and laccase from Trametes versico/or were
immobi ized through two different procedures:
> Chemical immobilization, using the commercially available epoxy-resin Eupergit®C25OL as
support;
> Layer-by-Layer immobilization, based on the consecutive deposition of alternatively charged
polyelectrolytes onto a surface to form microcapsules. The polyelectrolyte films have the
ability to protect proteins from high-molecular-weight denaturing agents or bacteria and to
allow regulation of the permeability towards small substrates, which can enter the multilayer
and react with the catalytic site. Specifically, poly(sodium 4-styrenesulfonate) (PSS) was
chosen as negative layer and poly(allylamine hydrochloride) (PAH) as positive one. The
polyelectrolytes deposition took place on two different surfaces: one consisted of chemically
immobilized enzymes on Eupergit®C25OL and the other formed by enzymes supported on
particles of aluminum oxide (AIZO3).
Novel heterogeneous biocatalysts were first characterized for their kinetic properties and assayed for
their stability to changes in pH and temperature, and then they were used as catalysts for the
synthesis of high added-value molecules. In detail, tyrosinase-based biocatalysts were applied for the
synthesis of catechols that are molecules with significant pharmaceutical properties, including
antioxidant and antitumoral activities. The synthesis of catechols was conducted both in aqueous
than in biphasic medium, using dichloromethane and buffer as solvent. Laccase-based biocatalysts
were applied in the oxidation of alcohols to aldehyde in presence of molecular mediator. Both
enzymes, tyrosinase and laccase, performed reactions using dioxygen as the primary oxidant. Data
showed that the immobilization procedures increased the enzyme stability in the temperature and
pHs conditions assayed, being the LbL systems the most steady biocatalysts. Furthermore, although
heterogeneous enzymes were characterized by a slight decrease in catalytic efficiency (lower Vmax and
higher Km), they showed reactivity comparable to free enzyme when applied in the oxidation of
organic compounds, conducting reactions with high yields and conversions of substrate. Moreover,
immobilization allows easy recovery of the catalyst from the reaction mixture and its recycling for
more consecutive oxidation processes. For these numerous advantages, immobilization procedures
are suitable for possible industrial applications, representing an efficient alternative to expensive and
polluting chemical procedures for the preparation of these families of bioactive compounds.
Additional information
Dottorato di ricerca in Biotecnologie vegetali
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