| Abstract |
The majority of Earth’s environments are characterized by extreme biological conditions, which
facilitates their characterization as inhospitable by humans. Despite this, life has adapted to these harsh
habitats, as many organisms have been found to colonize and thrive in them, characterized as
extremophiles. The most widespread in this group are psychrophiles, meaning organisms that have
adapted to life in cold environments. This doesn’t come as a surprise, as most of Earth’s oceans and
around one fifth of Earth’s soils are permanently at temperatures below 5° C or covered by permafrost,
respectively. Psychrophilic organisms possess a special biotechnological interest, in terms of their
biocatalysts. Cold-adapted enzymes are, in general, characterized by higher catalytic activity at low
temperatures and heat- lability compared with their mesophilic counterparts. Both of this characteristics
are considered advantageous in many biotechnological applications.
In a previous study at MINOTECH biotechnology, a bacterial collection of 262 strains, collected from
the Antarctic Ocean, was screened for the expression of alkaline phosphatases activity, based on a
chromogenic-based high-throughput assay. The genome of nine strains with high and temperaturedepended
expression of alkaline phosphatase activity were sequenced and, based on sequence
similarity and the preservation of the active site residues with other characterized alkaline
phosphatases, the gene APT110α from the strain TAP110α was isolated. The corresponding gene
encoding for a putative psychrophilic alkaline phosphatase was cloned in E.coli for further
characterization.
In this thesis, the above research initiative was continued, with the biochemical and biophysical
characterization of TAP110α alkaline phosphatase, together with the rational redesigning of its
properties. In detail, a full purification scheme is standardized and presented for the isolation of
TAP110α. Furthermore, the kinetic parameters for the enzyme were calculated, while crystallization of
the protein resulted in the acquisition of diffraction data, in order to elucidate its three-dimensional
structure. Additionally, circular dichroism spectrometry was used to assess the thermal stability and
secondary structure of the enzyme. Lastly, based on the resulting information, amino acid substitutions
were designed and performed, in order to enhance both the catalytic activity and the heat- lability of
the enzyme. Similar characterization steps were followed for the resulting mutants.
Based on the results of these experiments, TAP110α alkaline phosphatase shows a striking
thermostability, resembling that of mesophilic homologous. Furthermore, the designed mutations
D179H and D179E result to a more heat-labile enzyme, while the mutant D179E shows also an increase
in activity. Characterization of all the designed mutants may further improve the TAP110α alkaline
phosphatase. Lastly, elucidation of its three-dimensional structure will give insights of its temperature
adaptation, and provide means for a more insightful and targeted redesigning of its properties.
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Characterization and putative biotechnological applications of a psychrophilic alkaline phosphatase from the bacterial strain TAB110a
