The present study is part of a project to develop a high productivity enzymatic process for (R)-phenylacetylcarbinol (PAC), a precursor for the pharmaceuticals ephedrine and pseudoephedrine, with recent interest for a low cost and more stable biocatalyst pyruvate decarboxylase (PDC) preparation. PDC initially added in the form of Candida utilis cells, viz. whole cell PDC, showed higher stability towards substrate benzaldehyde and temperature in comparison to the partially purified preparation in an aqueous/benzaldehyde emulsion system. Increasing the temperature from 4?? to 21??C for PAC production with whole cell PDC resulted in similar final PAC levels of 39 and 43 g/L (258 and 289 mM) respectively from initial 300 mM benzaldehyde and 364 mM pyruvate. However, the overall volumetric productivity was enhanced by 2.8-fold. Enantiomeric excess values of 98 and 94% for R-PAC were obtained at 4?? and 21??C respectively and benzylalcohol (a potential by-product from benzaldehyde) was not formed. The potential of whole cell PDC was also evident in an aqueous/octanolbenzaldehyde emulsion system at 21??C with a 3-fold higher specific production compared to partially purified PDC. At 2.5 U/mL, PAC levels of 104 g/L in the organic phase and 16 g/L in the aqueous phase (60 g/L total reaction volume, 15 h) and a 99.1% enantiomeric excess for R-PAC were obtained with whole cell PDC. The study of cell membrane components provided a better understanding for the enhanced performance of whole cell PDC in comparison to partially purified PDC. It was apparent that surfactants, both biologically-occurring (e.g. phosphatidylcholine) and synthetically manufactured (e.g. bis(2-ethyl-1-hexyl)sulfosuccinate (AOT)), enhanced PDC stability and/or PAC production in the aqueous/octanol-benzaldehyde biotransformation system with the partially purified enzyme. Addition of 50 mM AOT to the biotransformation with partially purified PDC enhanced the enzyme half-life by 13-fold (19 h) and increased specific PAC production by 2-fold (36 mg/U). Chemical modification studies targeting the amino and carboxyl groups were carried out to achieve increased stability of partially purified PDC. However these were not successful and future work could be directed at PDC protein engineering as well as optimization and scale up of the two-phase process using whole cell PDC.
| Identifer | oai:union.ndltd.org:ADTP/234139 |
| Date | January 2006 |
| Creators | Satianegara, Gernalia, Biotechnology & Biomolecular Sciences, Faculty of Science, UNSW |
| Publisher | Awarded by:University of New South Wales. School of Biotechnology and Biomolecular Sciences |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | Copyright Gernalia Satianegara, http://unsworks.unsw.edu.au/copyright |
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