DEVELOPMENT OF NEW ENZYME REACTORS FOR CONTINUOUS PRODUCTIONS OF WATER SOLUBLE AND WATER INSOLUBLE PRODUCTS REQUIRING REGENERATION OF REDOX COFACTORS.
THE FOLLOWING CHEMICALS WILL BE PRODUCED :
1. L-ALANINE, L-METHIONINE,
2. LONG CHAIN ALDEHYDES,
3. LACTIC ACID FROM MALIC ACID,
4. HIGH-ADDED-VALUE LIPIDS.
The development of many industrial enzymatic processes is dependent upon the discovery of thermostable enzymes (ie those extracted from extremophiles) or methodologies for activity stabilization (ie immobilization) in the presence of organic solvents, with coenzyme regeneration.
Research was carried out in order to contribute to solving the general problem of enzyme stability and coenzyme regeneration in aqueous or mixed solvent and water solutions, in enzyme reactors and in liquid membranes.
The potentiality of some extremophilic organisms' cells and enzymes which are thermostable and resistant to common protein denaturing agents and to organic solvents was studied. These enzymes were purified and produced by genetic engineering.
A new alcohol dehydrogenase (ADH), a beta-galactosidase and a malic enzyme were purified from the extremophile Sulfolobus solfataricus. These thermostable and solvent resistant enzymes were used for the synthesis of long chain aldehydes, chiral compounds, L-amino acids and for malic acid conversion and lactose hydrolysis.
2 gene banks were constructed and the beta-galactosidase gene was isolated from the extremophile, cloned, sequenced and expressed in Escherichia coli. New types of solid gas bioreactors were developed and have been used with coenzyme regeneration for the synthesis of aldehydes or transesterification reactions catalyzed by lipases, whose activities and regioselectivities were investigated in different systems (eg microemulsions, etc) with and without immobilization. L-amino acid and aldehyde productions were investigated in liquid membranes with coenzyme regeneration.
Extremophile enzymes appear to be different with little or no homology with the corresponding ones from mesophiles; they are thermostable and solvent resistant proteins whose structures can be used as models for producing proteins by protein engineering.
BIOCONVERSION OF HYDROPHILIC AND HYDROPHOBIC COMPOUNDS BY THERMOSTABLE ENZYMES ISOLATED FROM SULFOLOBUS SOLFATARICUS.
THE FOLLOWING BIOCONVERSIONS WILL BE DEVELOPED :
1. DECARBOXYLATION OF L-MALATE TO PYRUVATE (WHICH IS THE LIMITING STEP OF MALOLACTIC FERMENTATION) USING MACROMOLECULARIZED NAD+ OR NADP+ IN A MEMBRANE REACTOR.
2. PRODUCTION OF ALDEHYDES FROM LONG CHAIN ALCOHOLS BY IMMOBILIZED ALCOHOL DEHYDROGENASE ABLE TO WORK IN ORGANIC PHASE.
3. SCREENING FOR A THERMOSTABLE AND SOLVENT-RESISTANT LIPASE.
4. INVESTIGATION ON HOW DNA REPLICATES AT TIGH TEMPERATURE AND OF THE USE OF THERMOSTABLE DNA POLYMERASE FOR DNA MANIPULATION IN VITRO.
5. CONSTRUCTION OF A GENE BANK FROM THERMOSTABLE SULFOLOBUS SOLFATARICUS AND ITS USE FOR CLONING OF GALACTOSIDASE.
Funding SchemeCSC - Cost-sharing contracts