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ETUDE DE LA STRUCTURE ET DE LA FONCTION DE L'ALPHA-AMYLASE D'ORGE

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Alpha-amylases are starch degrading enzymes with important biotechnical applications. So far a structural model is available for a mammalian alpha-amylases and 2 microbial alpha-amylases. Dependent on the enzyme, different products are obtained. It is therefore relevant to determine a structure for a cereal alpha-amylase which characteristically produces larger oligodextrins. A multidisciplinary strategy was thus applied to improve the insight at a molecular level into structure/function relationship and structure/stability relationship of the dominant barley malt alpha-amylase 2 and its interaction with the endogenous inhibitor barley amylase/subtilisin inhibitor (BASI).

In order to study the functional roles of the individual domains the barley alpha-amylase 1 and alpha-amylase 2 were subjected to limited proteolysis. A single bond was cleaved, however, it was located in 1 of the loops of the alpha/beta barrel and not in the region between the barrel and the carboxyl-terminal domain. This form had about 40% of the activity of the intact enzyme and the isolated fragments corresponding to residues 1-294 and 295-403 were purified for the high pI isozyme and found to be inactive. In the case of porcine pancreatic alpha-amylase parallel studies indicated cleavage in a different loop from the barrel domain. Thus the structural integrity of the enzyme does not lead to cleavage between domains indicating that they may both be required for function.

In order to examine the functional role of specific amino acid sidechains a yeast expression system was established for complementary deoxyribonucleic acids (cDNA) encoding a member of either isozyme family although reasonable levels of protein was produced only in the case of the low pI isozyme. By change of expression plasmid a similar expression level has been obtained also for the other isozyme. 4 products obtained for the low pI isozyme in the initial system and large scale production has allowed the purification of these individual forms for chemical and functional characterisation. In this way carboxyl terminal processing was concluded to occur in the germinating plant seed and the yeast expression system was found to give rise to disulphide formation between free sulphur hydrogen groups in the recombinant proteins and glutathione. The carboxyl terminal processing was demonstrated also with isolated protoplasts of aleurone cells to be performed by 1 or serval carboxypeptidase(s).

The structure of the first alpha-amylase from a higher plant, the barley malt high pI isozyme form alpha-2-2 has been determined and a model constructed at 3 angstroms resolution. Techniques have been developed to synthesize nonhydrolyzable oligosaccharide analogues suitable for photoaffinity labelling of endo-alpha-glucanases. A complementary deoxyribonucleic acid yeast expression system for production of high yields of higher plant proteins has also been developed. It was discovered that carboxy terminal processing of the amylase took place in the aleurone cells catalyzed by 1 or more carboxypeptidases from malt and resulting in several forms of the protein.

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Université d'Aix-Marseille III (Université de Droit d'Économie et des Sciences)
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13397 Marseille
Frankreich

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