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CONTINUOUS SYNTHESIS OF FINE CHEMICALS BY COFACTOR DEPENDENT ENZYMES WITH SIMULTANEOUS COFACTOR REGENERATION

Objective


To produce nicotinamide adenine dinucleotide phosphate (NADP) dependent 12 alpha-hydroxysteroid dehydrogenase (12 alpha-HSDH), the first inexpensive anaerobic bioprocess was developed by replacing the usual brain heart infusion medium (150 $/kg) by yeast autolysate (7 $/kg) giving approximately 90% reduction in medium cost. By a convenient 2-step affinity chromatography procedure using inexpensive affinity ligands, immobilised on solid supports, pure and concentrated 12 alpha-HSDH was isolated directly from crude cell extract (yield 53%).
Both operations are easily adaptable to large scale processing to produce 12 alpha-HSDH which is of potential interest for the analysis of conjugated and unconjugated bile acids in medicine.

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.
IT IS INTENDED TO DEVELOP SIMPLIFIED METHODS FOR THE SYNTHESIS OF MACROMOLOCULAR DERIVATIVE OF NAD(P) AND FAD. WITH EMPHASIS ON POLYETHYLENE GLYCOL-NAD(P), (CO)ENZYME ACTIVITIES (KM AND VMAX) WILL BE STUDIES FOR THE ENZYMES 3"ALPHA"-, 7"ALPHA"-, 7"BETA"-, AND 12"ALPHA"- HYDROXY STEROID DEHYDROGENASE, MANNITOL DEHYDROGENASE, GLUCOSE DEHYDROGENASE, GLUTAMATE DEHYDROGENASE, FORMATE DEHYDROGENASE AND THERMOSTABLE ALCOHOL DEHYDROGENASE AND L-PHENYLALANINE DEHYDROGENASE. BASED ON THE RESULTS, A REACTOR CONCEPT WILL CHOSEN FOR THE ENZYMES OF MOST IMPORTANCE FOR THE ENZYMATIC SYNTHESIS AND COENZYME REGENERATION.

Funding Scheme

CSC - Cost-sharing contracts

Coordinator

Gesellschaft für Biotechnologische Forschung mbH
Address
Mascheroder Weg 1
38124 Braunschweig
Germany

Participants (2)

Consiglio Nazionale delle Ricerche (CNR)
Italy
Address
Via Mario Bianco 9
20131 Milano
FRAUNHOFER-GESELLSCHAFT ZUR FOERDERUNG DER ANGEWANDTEN FORSCHUNG E.V.
Germany
Address
Leorodstrasse 54
80636 Muenchen