Expression et caractérisation de la phosphate alcaline de l'Euryarchaeote Hyperthermophile Pyrococcus abyssi

Abstract : Alkaline phosphatases (E.C. 3.1.3.1.) are defined as aspecific phosphomonoesterases, optimally active under alkaline conditions. Despite these enzymes are considered as ubiquitous, no alkaline phosphatase have been studied from a hyperthermophilic archeaon. The first step of the present work was to isolate the first alkaline phosphatase from such an organism: Pyrococcus abyssi, an euryarchaeon discovered in a deep-sea hydrothermal vent which grows optimally at 96°C. The phoA gene was identified on P abyssi genome, cloned and the recombinant protein was expressed in Escherichia coli. This protein was purified, confirming phoA product was an alkaline phosphatase. Optimum temperature and pH were found to be 70°C and 11.0, respectively. The enzyme showed a high thermostability with half-lives at 100°C and 105°C of 18 hours and hours, respectively. Metal dependancy was investigated, revealing its metalloenzyme nature, which is a general property of alkaline phosphatases. The second part of this project consisted in determining the quaternary structure of P. abyssi alkaline phosphatase. Several experiments, showed that the homodimer is the only active form. This quaternary structure, two subunits of 52 kDa each, appeared to be dependant of pH, buffer composition and enzyme metal content. The last part of this thesis deals with the expressipn of recombinant P. abyssi alkaline phosphatase. Investigation was performed about the expression of the enzyme by the Escherichia coli system using the T7 promoter regulation, with or without the co-expression of tRNA, related to rare codons. Recombinant P. abyssi alkaline phosphatase was also performed using the methylotrophic yeast Pichia pastoris by secretion, in a 1.2 liters working volume fermentor.
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Sébastien Zappa. Expression et caractérisation de la phosphate alcaline de l'Euryarchaeote Hyperthermophile Pyrococcus abyssi. Alimentation et Nutrition. Institut National Polytechnique de Lorraine, 2002. Français. ⟨NNT : 2002INPL081N⟩. ⟨tel-01749731⟩

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