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FOLDING,ASSEMBLY,STABILITY AND GENETIC MODIFICATION OF PENICILLIN ACYLASE AND ITS PRECURSOR

Objectif

THIS PROJECT SETS OUT TO TACKLE AN IMPORTANT STEP IN PROTEIN SECRETION AND FOLDING; NAMELY THE SPLITTING OF A PRECURSOR PROTEIN INTO SUBUNITS A AND B THAT CONSTITUTE THE ACTIVE ENZYME.
THIS PROCESSING, WHICH IS CARRIED OUT BY PROTEASES DURING RELEASE OF THE SUBUNIT INTO THE PERIPLASMIC SPACE IS NOT YET FULLY UNDERSTOOD. IT IS IMPORTANT TO ELUCIDATE THIS PROCESS IN ORDER TO IMPROVE THE SECRETION OF PENICILLIN ACYLASE AND HOPEFULLY OF OTHER INDUSTRIAL ENZYMES PRODUCED BY GENETIC ENGINEERING.
Worldwide penicillin production in 1985 was 11000 tons, 40% of which was used for the manufacture of the important semisynthetic penicillin antibiotics. The biotechnological process today uses the enzyme penicillin acylase (PA) to achieve a purer intermediate with lower energy consumption. PA is produced from naturally occurring bacteria in which the enzyme occurs at relatively low levels. Its efficiency and the range of conditions under which it can be employed are limited. Improvements in PA production and in its stability and specificity are important to this highly competitive industry.

Improvements to PA should be attainable using recombinant deoxyribonucleic acid (DNA) technology to express the enzyme at higher levels, and by protein engineering techniques to modify the stability and specificity. Preliminary work has shown that the success of such approaches will depend upon understanding the fundamental processes of biosynthesis, assembly and activity of PA.

With respect to PA production, 2 fragments of PA have been produced at high levels by genetic engineering. Their assembly has been studied in depth, leading to 60% yield of functional enzyme. The novel process has potential for industrial production of enzyme. For PA processing in vivo a mechanism of processing from the precursor molecules has been elucidated. Essential conditions for the various steps are known and ground rules for modification of the enzyme by protein engineering have been established. With respect to stabilization, the principles of PA stability in terms of molecular structure are now known. Combinations of attachment to solid supports and chemical modification have led to a 50000-fold increase in stability.
The conclusions of this work constitute the essential scientific underpinning for future process development.
CHARACTERIZATION OF THE FOLDING PATHWAY OF AUTHENTIC AND RECOMBINANT PENICILLIN ACYLASE.

IN PARTICULAR :

1. THE PRECURSOR OF PENICILLIN ACYLASE WILL BE STUDIED USING UREA GRADIENT GEL ELECTROPHORESIS IN CONJUNCTION WITH IMMUNOPRECIPITATION AND IMMUNOBLOTTING AS A FUNCTION OF PH AND TEMPERATURE.
2. THE UNFOLDING AND ASSEMBLY OF PENICILLIN ACYLASE WILL BE CHARACTERIZED BY UREA GRADIENT GEL ELECTROPHORESIS; BY SPECTROSCOPIC AND HYDRODYNAMIC TECHNIQUES SUCH AS CIRCULAR DICHROISM, FLUORESCENCE SPECTROSCOPY, ULTRACENTRIFUGATION, HYDROGEN EXCHANGE AND IMMUNOLOGY.
3. THE CONFORMATIONAL DYNAMICS OF PENICILLIN ACYLASE AND ITS PRECURSOR WILL BE CHARACTERIZED BY FLUORESCENCE ANISOTROPIC DECAY.
4. THE STABILITY OF PENICILLIN ACYLASE AND ITS CHEMICAL AND GENETIC DERIVATES IN DIFFERENT SOLVENT ENVIRONMENTS WILL BE ASSESSED.

Thème(s)

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Appel à propositions

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Régime de financement

CSC - Cost-sharing contracts

Coordinateur

UNIVERSITY OF NEWCASTLE UPON TYNE
Contribution de l’UE
Aucune donnée
Adresse
RIDLEY BUILDING
NE1 7RU NEWCASTLE UPON TYNE
Royaume-Uni

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Participants (2)