Supervisors info:
Γαλανοπούλου Ντία Αναπλ. Καθηγήτρια , Κωνσταντίνος Βοργιάς Καθηγητής , Δημήτριος Χατζηνικολάου Επίκ. Καθηγητής
Summary:
The TIM-barrel fold is abundant in various enzyme families catalyzing
completely different biochemical reactions, many of which with biotechnological
interest. The remarkable versatility of this motif is further highlighted
because it is adopted by enzymes that come from organisms who live in a wide
range of conditions (temperature, acidity, salinity, pressure). These enzyme
families have been studied extensively in biochemical, structural and
thermodynamic level. Chitinase 40 (Chi40) is a thermophilic chitinase, the
enzymatic domain of which was found to adopt the TIM-barrel fold. An important
and unique characteristic of Chi40 is the reversibility in its fold after
thermal denaturation. This characteristic is very important and enables us to
study the mechanism and structural factors that might be responsible for this
phenomenon. The aim of this dissertation thesis is to establish a high
throughput technology in order to study a large number of random mutations in
the catalytic domain of the enzyme. Our target is to identify the
folding-refolding mechanism in atomic level. First, we designed an effective
system for Chi40 gene expression and active protein production. The wild type
Chi40 gene has a high percentage of GC that prohibits gene-protein engineering
experiments. This problem was solved by designing and synthesizing a synthetic
gene of Chi40 with optimized characteristics, which makes easier further
genetic modifications and better expression of the enzyme in the bacterial
system of E. coli. We further studied the kinetics of synthetic Chi40
production testing different expression conditions such as (a) growth media (b)
modes of induction (c) combination of the above conditions in different E. coli
systems. As the expression levels differed dramatically in each combination, we
focused on finding the best conditions for the expression of secreted Chi40. We
also applied an activity assay using a photometric method, in which pNP-(NAG)2
was the synthetic substrate. Using the total secreted Chi40, we managed to
measure protein denaturation using Differential Scanning Fluorimetry (DSF). In
addition, we purified the protein by a combination of two chromatographic
techniques, hydrophobic and ion exchange chromatography. The identification of
the enzyme was held by mass spectrometry (MS), while native-PAGE
electrophoresis and Dynamic Light Scattering (DLS) experiments prove that the
enzyme appear in a monomeric state without forming aggregates. In addition,
thermostability measurements by Circular Dichroism (CD) experiments showed that
Chi40 has a Τm of 60.9oC in neutral pH. Our results show that thermal
denaturation is a quick and efficient method for the study of the mechanism of
reversible denaturation of Chi40 in the case of many random mutations produced
during this study. Our target is to apply these data to various enzymes of
biotechnological interest so as to improve their stability during
biotechnological applications.
Keywords:
Chitinolytic enzymes, Chitinase 40, Structural motif (α/β)8, TIM barrel
