(R)-phenylacetylcarbinol or R-PAC is a chiral precursor for the synthesis of pharmaceuticals ephedrine and pseudoephedrine. PAC is produced through biotransformation of pyruvate and benzaldehyde catalyzed by pyruvate decarboxylase (PDC) enzyme. The present research project aims at characterizing a two-phase aqueous/organic process for enzymatic PAC production. In a comparative study of several selected yeast PDCs, the highest PAC formation was achieved in systems with relatively high benzaldehyde concentrations when using C. utilis PDC. C. tropicalis PDC was associated with the lowest by-product acetoin formation although it also produced lower PAC concentrations. C. utilis PDC was therefore selected as the biocatalyst for the development of the two-phase PAC production. From an enzyme stability study it was established that PDC deactivation rates in the twophase aqueous/octanol-benzaldehyde system were affected by: (1) soluble octanol and benzaldehyde in the aqueous phase, (2) agitation rate, (3) aqueous/organic interfacial area, and (4) initial enzyme concentration. PDC deactivation was less severe in the slowly stirred phase-separated system (low interfacial area) compared to the rapidly stirred emulsion system (high interfacial area), however the latter system was presumably associated with a faster rate of organic-aqueous benzaldehyde transfer. To find a balance between maintaining enzyme stability while enhancing PAC productivity, a two-phase system was designed to reduce the interfacial contact by decreasing the organic to aqueous phase volume ratio. Lowering the ratio from 1:1 to 0.43:1 resulted in increased overall PAC production at 4??C and 20??C (2.5 M MOPS, partially purified PDC) with a higher concentration at the higher temperature. The PAC was highly concentrated in the organic phase with 212 g/L at 0.43:1 in comparison to 111 g/L at 1:1 ratio at 20??C. The potential of further two-phase process simplification was evaluated by reducing the expensive MOPS concentration to 20 mM (pH controlled at 7.0) and employment of whole cell PDC. It was found that 20??C was the optimum temperature for PAC production in such a system, however under these conditions lowering the phase ratio resulted in decreased overall PAC production. Two-phase PAC production was relatively low in 20 mM MOPS compared to biotransformations in 2.5 M MOPS. Addition of 2.5 M dipropylene glycol (DPG) into the aqueous phase with 20 mM MOPS at 0.25:1 ratio and 20??C improved the production with organic phase containing 95 g/L PAC. Although the productivity was lower, the system may have the benefit of a reduction in production cost.
| Identifer | oai:union.ndltd.org:ADTP/233927 |
| Date | January 2006 |
| Creators | Gunawan, Cindy, 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 Cindy Gunawan, http://unsworks.unsw.edu.au/copyright |
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