Objective
THE PROBLEM OF STABILITY OF PROTEINS AND ENZYMES IS CENTRAL IN MODERN BIOTECHNOLOGY AND OFTEN CONSTITUES 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.
FURTHERMORE, THE RESULTS OF THIS STUDY COULD BE USEFUL TO LEARN MORE ABOUT THE ROLE OF METAL IONS IN METALLOENZYMES IN GENERAL. 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.
RESEARCH WILL BE CARRIED OUT IN ORDER TO CHARACTERIZE THE PHYSICO-CHEMICAL AND ENZYMOLOGICAL PROPERTIES OF PROTEINS FROM THERMOPHILES, THERMOACIDOPHILES AND HALOPHILES, IN PARTICULAR ARCHAEBACTERIA. THE FOUR MAIN LINES OF THE PROJECT ARE:
- THE GROWTH OF LARGE QUANTITIES OF EXTREME THERMOPHYLIC ORGANISMS LIKE METHANOCOCCUS THERMOLITHOTROPHICUS, THERMOTOGA AND SULFOLOBUS SOLFATARICUS, METHANOTRIX AND OTHERS;
- THE ISOLATION OF TYPICAL PROTEINS FROM THSES ORGANISMS FOR FURTHER CHARACTERISATION, IN PARTICULAR LACTATE DEHYDROGENASE AND AMYLASE;
- STUDY OF MOLECULAR WEIGHTS, QUARTENARY STRUCTURES, CONFORMATION AND DENATURATION-RENATURATION PATTERN AND INFLUENCE OF FOR EXAMPLE LIMITED PROTEOLYSIS ON THE LATTER;
- THE THERMODYNAMIC AND KINETIC ASPECTS OF THE EXPECTED SHIFT IN COLD INACTIVATION OF THESE PROTEINS WILL BE STUDIED AS WELL AS THE EFFECTS OF IMMOBILIZATION OF THE PROTEINS ON THIS BEHAVIOUR.
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 chemical sciences analytical chemistry calorimetry
- natural sciences chemical sciences organic chemistry alcohols
- natural sciences biological sciences biochemistry biomolecules proteins enzymes
- engineering and technology industrial biotechnology bioprocessing technologies fermentation
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Coordinator
93053 REGENSBURG
Germany
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