Cells of Candida utilis for in vitro (R)-phenylacetylcarbinol production in an aqueous/octanol two-phase reactor

Abstract

(R)-Phenylacetylcarbinol (PAC), a pharmaceutical precursor, was produced from benzaldehyde and pyruvate by pyruvate decarboxylase (PDC) of Candida utilis in an aqueous/organic two-phase emulsion reactor. When the partially purified enzyme in this previously established in vitro process was replaced with C. utilis cells and the temperature was increased from 4 to 21 degrees C, a screen of several 1-alcohols (C4-C9) confirmed the suitability of 1-octanol as the organic phase. Benzyl alcohol, the major by-product in the commercial in vivo conversion of benzaldehyde and sugar to PAC by Saccharomyces cerevisiae, was not formed. With a phase volume ratio of 1:1 and 5.6 g C. utilis l-1 (PDC activity 2.5 U ml-1), PAC levels of 103 g l-1 in the octanol phase and 12.8 g l-1 in the aqueous phase were produced in 15 h at 21 degrees C. In comparison to our previously published process with partially purified PDC in an aqueous/octanol emulsion at 4 degrees C, PAC was produced at a 4-times increased specific rate (1.54 versus 0.39 mg U-1 h-1) with simplified catalyst production and reduced cooling cost. Compared to traditional in vivo whole cell PAC production, the yield on benzaldehyde was 26% higher, the product concentration increased 3.9-fold (or 6.9-fold based on the organic phase), the productivity improved 3.1-fold (3.9 g l-1 h-1) and the catalyst was 6.9-fold more efficient (PAC/dry cell mass 10.3 g g-1).

Comparative studies on enzyme preparations and role of cell components for (R)‐phenylacetylcarbinol production in a two‐phase biotransformation

“…Whole cells of C. utilis are efficient catalysts for PAC production in an aqueous/octanol system (Rosche et al, 2005b) and the present study gives more insights into possible factors behind their improved performance compared to partially purified preparations, that is, higher stability as well as higher productivity at low enzyme concentration. The study identifies phospholipids as a cell component that enhanced PAC production and shows that a synthetic surfactant, AOT, improved both PAC production and PDC stability when using a partially purified PDC preparation.…”

“…the by-product benzyl alcohol was not formed and high PAC concentrations were achieved. With a 1:1 phase volume ratio at 218C, the PAC concentrations after 15 h were 103 g/L in the octanol phase and 13 g/L in the aqueous phase (Rosche et al, 2005b). This result corresponded to a four-times increased specific productivity (1.54 vs. 0.39 mg PAC/U/h) compared to the previously published process with partially purified PDC at 48C (Rosche et al, 2002b;Sandford et al, 2005).…”

“…Biotransformations were carried out in 4 or 20 mL teflon sealed screw cap glass vials at 218C as described by Rosche et al (2005b) and detailed conditions for each study are given in the captions of the corresponding figures. The reaction mixture had a 1:1 phase volume ratio and was magnetically stirred which resulted in an emulsion of octanol droplets in the water phase.…”

“…To minimize PDC deactivation, an enzymatic aqueous/ organic two-phase reactor was developed where major concentrations of substrate benzaldehyde and product PAC are partitioned into the organic (octanol) phase while PDC and pyruvate are located in the aqueous phase (Rosche et al, 2002b(Rosche et al, , 2005bSandford et al, 2005). When C. utilis PDC was added in the form of resting cells (whole cell PDC) in this rapidly stirred emulsion system, cell viability, and metabolic activity were lost immediately (Rosche et al, 2005b).…”

“…When C. utilis PDC was added in the form of resting cells (whole cell PDC) in this rapidly stirred emulsion system, cell viability, and metabolic activity were lost immediately (Rosche et al, 2005b). However, this catalyst with characteristics between isolated enzymes and living cells maintained the advantages of the enzymatic system, viz.…”