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Expression, purification and functional characterization of the recombinant HydA1 hydrogenase from Chlamydomonas reinhardtii

Hydrogenases are a class of enzymes responsible for hydrogen gas production in many microorganisms. Their study is considered of great interest for biotechnological applications to support a fossil fuel-free world. To this end, the construction of artificial devices that are able to use solar energy to produce hydrogen (the so-called “nano-leaf”) has been proposed. Because of its relatively simple structure and high specific activity, Chlamydomonas reinhardtii HydA1 hydrogenase (CrHydA1) is an enzyme of outstanding interest for this purpose. Thus, the aim of this study is to produce and characterize CrHydA1.
Since CrHydA1 is quickly inactivated by oxygen, this study required the development of protocols for CrHydA1 production, manipulation and storage in strictly anoxic conditions. The recombinant expression system in Escherichia coli developed at the National Renewable Energy Laboratory (NREL), USA, was used to produce and purify CrHydA1 to homogeneity with a specific activity of about 60 μmol H2 • min-1 • mg protein-1 and a yield of 0.4 mg protein per litre of culture. Produced samples were stored at -20°C for months without any loss of activity. The recombinant expression system was further characterized in terms of recombinant proteins expression levels; various modifications of the purification protocol were evaluated.
CrHydA1 hydrogen oxidation activity was characterized at different pH and temperature. Besides the specific information on hydrogen oxidation activity, these experiments demonstrated that CrHydA1 is not only very stable in alkaline conditions, up to pH 11, but also very stable in terms of temperature, up to 60°C. Moreover, 50% activity was conserved after 10 hours incubation at 50°C. This intrinsic thermo stability of CrHydA1 is an important factor in its biotechnological application in the construction of the “nano-leaf” due to the fact that when exposed to solar light, this device will reach high temperatures and the availability of thermo stable components will be essential.
In the absence of a crystal structure for CrHydA1, a computer model, based on homology, was produced and evaluated. Also, for the very first time, the CrHydA1 protein structure was experimentally studied by circular dichroism spectroscopy and the far UV spectrum led to the prediction of the secondary structure content that is in good accordance with that predicted with the model.
Preliminary trials of CrHydA1 immobilization on different electrodes were carried out. The obtained results suggest the possibility of immobilizing the enzyme on TiO2 or carbon based electrodes.
Finally, the data presented in this work on the production, purity and stability of CrHydA1 together with preliminary immobilization results, confirm the feasibility of using this enzyme in the construction of artificial “nano-leaf” device.

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1 1. Introduction The shortage of fossil fuels, that today are the main energy source on Earth, is proposing new challenges to science to find new, renewable and cost effective energy sources. Solar energy is the main candidate, being virtually endless, clean, abundant and free for everybody, and hydrogen gas (H2) is considered the best candidate as an energy carrier (Sartbaeva 2008) where solar energy can be stored, even if its production using directly or indirectly solar energy is not simple (Lewis 2006). Over the last years, the use of different microorganisms that are able to produce this valuable fuel has been studied with increasing interest (Rupprecht 2006) because the production of hydrogen through biological techniques (the so-called bio-hydrogen) is considered a cheap and environmental friendly solution. Thus, the study of hydrogen production in biology has led to many interesting opportunity for future technologies that today are to be improved with multidisciplinary approaches. 1.1 Hydrogen in life sciences Hydrogen gas has an important role in life sciences as testified by the great number of microorganisms that can metabolize it. Hydrogen metabolism was identified in many prokaryotic species belonging to Bacteria, but also in Archaea and in some Eukaryotes. Hydrogen metabolism is usually related to energetic issues in the cell and might have started its evolution very early, when the Earth’s atmosphere was still hydrogen-rich and oxygen-free (Vignais 2007). Then, its importance has been reduced by the oxygen based metabolism, which today has the most important role in higher plants and animals; nevertheless, today hydrogen metabolism still has an important role for many microorganisms that have conserved the ability to use it. Hydrogen gas is a substrate for the growth of many microorganisms that are able to activate and oxidize it, extracting from H2 the energy necessary for their life. Examples are methanogenic bacteria, sulphate reducers (e.g. Desulfovibrio), Fe3+ reducers (e.g. Geobacter), and denitrifying bacteria (Vignais 2007). Hydrogen can be produced by other microorganisms as a sink for unnecessary energy. This is the case of many fermentative bacteria such as those belonging to the genus Clostridium (Thauer 1977, Vignais 2007), of some photosynthetic purple non-sulphur (PNS) bacteria such as Rhodobacter sphaeroides (Koku 2002) and Rhodospirillum rubrum (Melnicki 2008) and many cyanobacteria (Dutta 2005). Between the Eukaryotes, hydrogen

Laurea liv.II (specialistica)

Facoltà: Scienze Matematiche, Fisiche e Naturali

Autore: Simone Morra Contatta »

Composta da 106 pagine.

 

Questa tesi ha raggiunto 72 click dal 20/04/2010.

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