THE LONG TERM OBJECTIVE OF THE JOINT PROJECT IS TO WORK OUT ECONOMIC METHODS FOR THE CONTINUOUS ENZYMATIC SYNTHESIS OF HIGH-VALUE FINE CHEMICALS BY USING REDOX REACTION REQUIRING NAD(H), NADP(H) OR ARTIFICIAL DYES AS COFACTOR.
THE DEVELOPMENT OF THE FOUR DIFFERENT SYSTEMS STUDIED REQUIRES THE DESIGN OF NEW MEMBRANE-REACTORS BOTH IN AQUEOUS OR ORGANIC ENVIRONMENT.
THE PRODUCTION OF THE VALUABLE CHEMICALS IS EXPECTED :
1. 12-KETOCHENODEOXYCHOLIC AND 12-KETOURSODEOXYCHOLIC ACID WHICH ARE EXPENSIVE PRECURSORS FOR THE CHEMICAL SYNTHESIS OF DRUGS TO TREAT GALLSTONE ALIMENT.
2. GLUCONIC ACID AND MANNITOL FOR AGRO-FOOD INDUSTRY.
3. L-ASCORBIC ACID (VITAMIN C) FOR PHARMACEUTICAL INDUSTRY.
4. NEUTRAL STEROIDS AND LONG CHAIN ALCOHOLS FOR PHARMACEUTICAL AND AGROFOOD INDUSTRY.
Several hydroxysteroid dehydrogenases and alcohol dehydrogenases from T. brockii have been used for the synthesis of 12-ketochenodeoxycholic acid, 12-ketoursodeoxycholic acid and intermediates of broxaterol and muscarines. The reactions have been carried out in batch reactors with simultaneous nicotinamide adenine dinucleotide phosphate (NADP) or reduced NADP regeneration.
The work has included:
further extention and strengthening of the usefulness and versatility of dehydrogenases in organic synthesis;
demonstration that hydroxysteroid dehydrogenases are also effective catalysts with some unnatural substrates;
development and optimization of enzymatic methods for NADP or reduced NADP regeneration.
Microbial nicotinamide adenine dinucleotide (phosphate) (reduced) (NAD(P)(H)) dependent (dep) dehydrogenases (DH) are of great potential for the synthesis of fine chemicals (chiral synthons, steroids, carbohydrates, optically pure compounds (labelled) or pharmaceutical precursors). To exploit their catalytic potential 2 main criteria must be fulfilled: optimized bioprocessing methods and downstream processing methods (production, purification, adaptation to large scale); appropriate reactor technology for economic use of enzymes and coenzymes considering enzyme stability and coenzyme regeneration.
Research into the methodology has given thefollowing results:
development of a simplified synthesis of N6-(2-aminoethyl)-nicotinamide adenine dinucleotide, N6-(2-aminoethyl)-nicotinamide adenine dinucleotide phosphate and, as spin off, N6-(2-aminoethyl)-flavin adenine dinucleotide (mild conditions, yield 15 to 40%;
nicotinamide adenine dinucleotide phosphate (reduced)-dep 12 alpha-hydroxy steroid-DH (Clostridium group P) gave a cheap process for production (0.9 E6 U/900 l) and total purification (130 U/mg, yield 53%), enzyme characterization and laboratory scale synthesis of 12-ketochenodeoxycholic acid;
nicotinamide adenine dinucleotide (reduced)-dep L-phenylatanine-DH (Sporosarcina urae) gave a bioprocess optimization (2E5 U/50 l), downstream processing by 2-step affinity chromatography (100 U/mg, yield 70%) and full characterization and synthesis of 2-15 NH2-phenylalanine and 2-15 NH2-tyrosine are in progress;
with nicotinamide adenine dinucleotide phosphate (reduced)-dep glucose-DH (Cryptococcus uniguttulatus) native nicotinamide adenine dinucleotide phosphate and N6-functionalized nicotinamide adenine dinucleotide phosphate (low molecular and high molecular) are equally accepted by glucose-DH;
nicotinamide adenine dinucleotide phosphate (reduced)-dep sec alcohol-DH (Thermoanaerobium brockii) on a laboratory scale gives the synthesis of S-(+)-sulcatol, (4R-2H)-reduced nicotinamide adenine dinucleotide phosphate and R-3-bromo-5-(l-hydroxy-2-bromoethyl) isoxazole (broxaterol precursor);
nicotinamide adenine dinucleotide (reduced)-dep mannitol-DH (Saccharomyces cerevisiae) gave medium optimization (2.95E4 U/220 l) purification (36 U/mg) and characterization nicotinamide adenine dinucleotide (phosphate) (reduced) scale simultaneous mannitol and gluconic acid production;
retention of nicotinamide adenine dinucleotide (phosphate) (reduced) by a negatively charged urea formaldehyde (UF) membrane (M cut off 2000) with retention greater than 99% and 99.9% under process conditions.
USE OF STEROID DEHYDROGENASES TO CONTINUOUSLY TRANSFORM IN A CONTINUOUS-FLOW MEMBRANE REACTOR, BILE ACIDS AND NEUTRAL STEROIDS USING ENLARGED NADP MOLECULES.
THE FOLLOWING REACTIONS WILL BE STUDIED :
1. OXIDATION OF CHOLIC ACID TO 12-KETOCHENODEOXYCHOLIC ACID,
2. REDUCTION OF DEHYDROCHOLIC ACID TO 12-K-KETOCHENODEOXYCHOLIC ACID OR 12-KETOURSODEOOXYCHOLIC ACID,
3. INSOMERIZATION OF CHENODEOXYCHOLIC ACID TO URSODEOXYCHOLIC ACID,
4. REDUCTION OF 20 KETOSTEROIIDS TO 20 B-HYDROXYSTEROIDS COUPLED TO TE OXIDATION OF 3 B, OR 17 B OR 3 X-HYDROXYSTEROIDS.
GLUMATE DEHYDROGENASE, FORMATE DEHYDROGENASE, GLUCOSE DEHYDROGENASE AND B OR 3X-HYDROXYSTEROIDS DEHYDROGENASES WILL BE USED TO REGENERATE NADP, NAD AND NADP (H).
Funding SchemeCSC - Cost-sharing contracts