Synthesis of enantiomerically pure compounds by biocatalysis

Abstract

Biocatalytic resolution has drawn much attention to obtain enantiomerically enriched compounds by exploiting the selectivity of enzymes for one form of the enantiomers of a racemic molecule. The development of efficient enzyme systems and processes has been an important research objective in this field. In this thesis, I carried out development of novel cell surface displayed systems for the production of enatiomerically pure compounds. First, recombinant Escherichia coli displaying lipase on the cell surface was examined as a whole cell biocatalyst for enantioselective resolution of racemic compounds. The Pseudomonas fluorescens lipase was displayed on the cell surface of E. coli by fusing the lipase gene to the Salmonella typhimurium outer membrane protein C (OmpC) gene by Cterminal deletion-fusion strategy. When racemic ethyl 3-hydroxybutyrate, methyl mandelate and cis-3-acetoxy-4-phenylazetidin-2-one were used as substrates, (R)-3-hydroxybutyric acid, (S)-mandelic acid and (3S, 4R)-cis-3-hydroxy-4-phenylazetidin-2-one were successfully obtained with the enantiomeric excess of greater than 99%. The cell surface displayed lipase was found to be maintained without much loss of activity and selectivity during 10 repeated reactions over 120 hours. Second, a novel cell surface display system was developed employing FadL as an anchoring motif, which is an outer membrane protein involved in long-chain fatty acid transport in Escherichia coli. A thermostable Bacillus sp. TG43 lipase (44.5 kDa) could be successfully displayed on the cell surface of E. coli in an active form by C-terminal deletion-fusion of lipase at the ninth external loop of FadL. Cell surface displayed lipase was quite stable even at 60℃ and 70℃, and retained over 90% of the full activity after incubation at 50℃ for a week. As a potential application, cell surface displayed lipase was used as a whole cell catalyst for kinetic resolution of racemic methyl mandelate. In 36 h of reaction, (S)-mandelic...