Skip to main content

FOLDING ASSEMBLY STABILITY AND GENETIC MODIFICATION OF PENICILLIN ACYCLASE AND ITS PRECURSOR

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 SUBUNITS A AND B.

THIS PROCESSING WHICH IS CARRIED OUT BY PROTEASES DURING RELEASE OF THE SUBUNITS INTO THE PERIPLASMIC SPACE IS NOT FULLY UNDERSTOOD TODAY. IT IS IMPORTANT TO ELUCIDATE THIS PROCESS IN ORDER TO IMPROVE THE SECRETION OF 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.
STUDY OF THE PROTEOLYTIC PROCESSING OF THE BACTERIAL PENICILLIN ACYLASE PRECURSOR DURING SECRETION THROUGH THE CELL MEMBRANE IN THE PERIPLASMIC SPACE.

IN PARTICULAR :
1. A PROCEDURE WILL BE ESTABLISHED TO PREPARE PENICILLIN ACYLASE PRECURSOR PROTEIN IN LARGE AMOUNT. THE GENETICALLY ENGINEERED FORMS WITH AND WITHOUT LEADER PEPTIDE WILL BE ISOLATED.
2. THE PURIFIED PRECURSORS WILL BE USED FOR ANALYSIS OF THE PROTEASES INVOLVED IN PROCESSING AND THEIR CHARACTERIZATION.
3. PURIFICATION OF THESE PROTEASES AND SETTING-UP OF AN IN VITRO PROCESSING SYSTEM.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

LUDWIG-MAXIMILIANS UNIVERSITY OF MUNICH
Address
Maria-ward-strasse 1A
80638 Muenchen
Germany

Participants (2)

Boehringer Mannheim GmbH
Germany
Address

Penzberg
UNIVERSITY OF NEWCASTLE UPON TYNE
United Kingdom
Address
6 Kensington Terrace
NE1 7RU Newcastle Upon Tyne