Heterologous expression and characterization of 1,2-dioxigenase of gentisate from Pseudarthrobacter phenanthrenivorans Sphe3 (Master thesis)

Λάππα, Ειρήνη


Polycyclic aromatic hydrocarbons are a large group of chemicals with two or more aromatic rings. Their physicochemical properties affect both humans and the environment. This is why the scientific community is increasingly interested in exploring the methods of degradation of these substances. The bacterium Pseudarthrobacter phenanthrenivorans Sphe3 was isolated from a region of Epirus contaminated with creosote. It is capable of degrading polycyclic aromatic hydrocarbons, such as phenanthrene. Gentisic acid is an intermediate metabolite, key in the aerobic bacterial degradation of aromatic compounds. In the present study, an 1112 bp gene encoding gentisate dioxygenase was cloned into the plasmid vector pET29c and overexpressed in Escherichia coli BL21 bacterial cells. Thereafter, the enzyme was purified by Ni2+-NTA affinity column and its biochemical characteristics were studied. Gentisate dioxoxygenase follows Michaelis-Menten kinetics. The values of Km and kcat/Km for gentisic acid are 24.9 μM and 28.75 s-1 M-1 103, respectively. The activity of the enzyme is optimal at 50°C and pH 7.5-8. The activity is increased after incubation of the enzyme in 0.2 mM Fe2+/L-ascorbate for 30 min. O-phenanthroline has a strong inhibitory effect, as well as Cu2+, Ni2+ to a lesser influence.
Institution and School/Department of submitter: Πανεπιστήμιο Ιωαννίνων. Σχολή Επιστημών Υγείας. Τμήμα Ιατρικής
Subject classification: Υδρογονάνθρακες
Keywords: Πολυκυκλικοί αρωματικοί υδρογονάνθρακες,Βιοαποδόμηση,Βακτήρια,Γενετικό οξύ,Polycyclic aromatic hydrocarbons,Biodegradation,Bacteria,Gentisic acid
URI: http://olympias.lib.uoi.gr/jspui/handle/123456789/29426
Appears in Collections:Διατριβές Μεταπτυχιακής Έρευνας (Masters)

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