Objective
EVENTHOUGH THIS SEARCH IS FAR FROM BEING CONSIDERED AS AN APPROPRIATE MODEL FOR PROTEIN DESIGN (NO STRUCTURAL STUDY IS CARRIED OUT) IT TACKLES AN IMPORTANT STEP IN PROTEIN SECRETION AND FOLDING, NAMELY THE SPLITING OF A PRECURSOR PROTEIN INTO ACTIVE SUBMITS A AND B.
THIS PROCESSING WHICH IS CARRIED OUT BY PROTEASES DURING RELEASE OF THE SUBMIT INTO THE PERIPLASMIC SPACE IS NOT FULLY UNDERSTOOD TODAY. IT IS IMPORTANT TO ELUCIDATE THIS PROCESS IN ORDER TO IMPROVE THE SECRETION 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.
MECHANISM OF FOLDING OF PENICILLIN ACYLASE AND PRODUCTION OF 7-AMINO-CEPHALOSPORIC ACID BY ENZYMES PRODUCED BY GENETIC ENGINEERING.
IN PARTICULAR :
1. THE GENE(S) OF INTEREST WILL BE CLONED IN DIFFERENT HOSTS IN ORDER TO PRODUCE LARGE QUANTITIES FOR THE FOLDING STUDIES.
2. THE MATURE AND PRECURSOR PENICILLIN ACYLASE WILL BE ISOLATED AND PURIFIES.
3. SITE DIRECTED MUTAGENIS WILL BE CARRIED OUT AT DNA LEVEL.
4. THE ALTERED GENE(S) WILL BE CLONED AND SEQUENCED.
5. THE ALTERED GENE(S) WILL BE EXPRESSED AND THE GENE PRODUCT WILL BE ANALYZED.
Fields of science (EuroSciVoc)
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
CORDIS classifies projects with EuroSciVoc, a multilingual taxonomy of fields of science, through a semi-automatic process based on NLP techniques. See: The European Science Vocabulary.
- natural sciences biological sciences microbiology bacteriology
- natural sciences biological sciences genetics DNA
- medical and health sciences medical biotechnology genetic engineering
- medical and health sciences basic medicine pharmacology and pharmacy pharmaceutical drugs antibiotics
- natural sciences biological sciences biochemistry biomolecules proteins enzymes
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Coordinator
8132 TUTZING
Germany
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