Overproduction and utilization of enzymes in synthetic organic chemistry

Abstract

The utility of papain in the synthesis of peptides containing D or unusual amino acids has been demonstrated. A novel one-step synthesis of tripeptides has been developed using papain. The amidase activity of a number of proteolytic enzymes has been selectively damaged, providing catalysts which are better suited for kinetically controlled peptide synthesis. Furylglycine derivatives were resolved with papain, providing both enantiomers in high optical purity. The enzymes phosphopentomutase and thymidine phosphorylase were overproduced in E. coli. Arabinose 5-phosphate was demonstrated to be a substrate for phosphopentomutase in a coupled enzyme system for the synthesis of purine nucleosides. Dideoxyribose 5-phosphate is not a substrate in the coupled system. The fda gene from Corynebacterium glutamicum was cloned and sequenced. The fda gene from E. coli was cloned and an E. coli strain which overproduced FDP adolase constructed. This enzyme was shown to exhibit exceptional operational stability in comparison with the rabbit muscle enzyme. Deoxyribose 5-phosphate aldolase was overproduced in E. coli. A convenient purification procedure was developed for the enzyme. The enzyme accepts acetaldehyde, propionaldehyde, acetone, and fluoroacetone as aldol donor substrates. The stereochemistry of the products obtained from the aldol condensation of acetaldehyde, acetone, or fluoroacetone with isobutyraldehyde is (S) in all cases. The enzyme accepts a broad range of simple aldehydes, as well as carbohydrates, as aldol acceptor substrates.