THE PROBLEM OF STABILITY OF PROTEINS AND ENZYMES IS CENTRAL IN MODERN BIOTECHNOLOGY AND OFTEN CONSTITUTES A MAJOR LIMITATION FOR THE SUCCESSFUL APPLICATION OF ENZYMES. UNDERSTANDING THE MOLECULAR BASIS OF THE UNUSUAL STABILITY OF THERMOPHILIC ENZYMES WILL OPEN THE WAY FOR THE STABILISATION OF ENZYMES OF MESOPHILIC ORIGIN. THERMOPHILIC ENZYMES ITSELF CAN BE OF GREAT VALUE AND DO OFFER SEVERAL SPECIFIC ADVANTAGES FOR BIOTECHNOLOGICAL APPLICATIONS. PRELIMINARY OBSERVATIONS INDICATE THAT THERMOPHILIC ENZYMES ARE RATHER STABLE IN ORGANIC SOLVENTS, AN ASPECT THAT CAN HAVE IMPORTANT CONSEQUENCES IN THE FIELD OF BIOTECHNOLOGY. A SPECIFIC AND IMPORTANT APPLICATION OF THIS COULD BE FOR THE ENZYMATIC SYNTHESIS OF PEPTIDES USING PROTEASES, SINCE PROTEOLYSIS IS REVERSED UNDER THOSE CONDITIONS.
Thermophilic enzymes are usually much more resistant to heat and most common protein denaturants than their counterparts from mesophilic sources and enzymes offer several specific advantages for biotechnological applications.
Research was carried out in order to characterize the functional, conformational and stability properties of enzymes isolated from thermophilic bacteria, in particular, from Thermotoga maritima, Sulfolobus solfataricus and Bacillus thermoproteolyticus. Relatively large scale fermentations, including optimization of the growth conditions of archaebacteria, were performed in order to isolate enzymes in suitable quantity. Many techniques (circular dichroism, fluorescence, nuclear magnetic resonance (NMR), calorimetry, ultracentrifugation, hydrogen deuterium exchange) were used to analyse the folding, association and stability of the newly isolated enzymes. A study was made of the independent folding of protein domains of thermolysin, with the view to establish the minimum size of a polypeptide chain able to fold into a stable globular structure.
Lactate dehydrogenase (LDH), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and amylase were isolated from T maritima and their functional, association and stability properties investigated. LDH exhibits long term stability up to 85 C and GAPDH shows extreme stability, (transition point at 107 and 109 C for the apoenzymes and holoenzymes, respectively). Conformational studies were also carried out on alcohol dehydrogenase and beta-galactosidase from Sulfolobus. The molecular mechanism of protein degradation by proteolyic enzymes was investigated using thermolysin as a model, establishing that exposed and flexible loops are the vulnerable sites. The role of protein domains in folding and stability of proteins was examined by studying the conformational association and stability properties of C-terminal fragments of thermolysin.
IT IS PROPOSED TO STUDY THE FUNCTIONAL, STRUCTURAL AND FOLDING PROPERTIES OF ENZYMES FROM THERMOPHILES, EXTREME THERMOPHILES AND ARCHAEBACTERIA. A SELECTED SMALL NUMBER OF ENZYMES WILL BE ISOLATED FROM SULFOLOBUS SOLFATARICUS, BACILLUS STEAROTHERMOPHILUS AND THERMUS AQUATICUS. FUNCTIONAL AND STRUCTURAL PROPERTIES AS WELL AS FOLDING AND ASSEMBLY FEATURES WILL BE STUDIED.
DETAILED STUDIES WILL BE CARRIED OUT ON THE STRUCTURE-FUNCTION RELATIONSHIPS, AS WELL AS FOLDING AND STABILITY PROPERTIES, OF THE METALLOPROTEASE THERMOLYSIN, WHICH CAN BE CONSIDERED AS A MODEL THERMOPHILIC AND STABLE PROTEIN. SINCE THERMOLYSIN IS A TWO DOMAIN PROTEIN, THE CONFORMATIONAL AND STABILITY PROPERTIES OF FRAGMENTS ENCOMPASSING DOMAINS AND SUBDOMAINS IN THE INTACT PROTEIN WILL BE INVESTIGATED, WITH THE VIEW TO CONTRIBUTE TO A BETTER UNDERSTANDING THE HIERARCHIC STRUCTURE AND FOLDING OF GLOBULAR PROTEINS.
SINCE THERMOPHILIC ENZYMES ARE RATHER STABLE TO AQUEOUS ORGANIC SOLVENTS AND UNDER THESE SOLVENT CONDITIONS PROTEASES CATALYZE PEPTIDE BOND FORMATION, THE ENZYME-ASSISTED SYNTHESIS OF PEPTIDES WILL BE INVESTIGATED, USING ALSO PEPTIDE FRAGMENTS DERIVED FROM THERMOLYSIN.
Fields of science
- natural sciencesbiological sciencesmicrobiologybacteriology
- natural scienceschemical sciencesanalytical chemistrycalorimetry
- natural scienceschemical sciencesorganic chemistryalcohols
- natural sciencesbiological sciencesbiochemistrybiomoleculesproteinsenzymes
- engineering and technologyindustrial biotechnologybioprocessing technologiesfermentation