(<i>R</i>)‐phenylacetylcarbinol production in aqueous/organic two‐phase systems using partially purified pyruvate decarboxylase from <i>Candida utilis</i>

Sandford, Vanessa; Breuer, Michael; Hauer, Bernhard; Rogers, Peter L.; Rosche, Bettina

doi:10.1002/bit.20513

Cited by 36 publications

(32 citation statements)

References 26 publications

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“…Progress in enzymatic PAC production (Shin & Rogers 1996, Bringer-Meyer & Sahm 1988, Iwan et al 2001, Leksawasdi et al 2003, Rosche et al 2003a has led to the development of an aqueous/octanol two-phase emulsion reactor operated at 4°C with 80 g PAC l )1 produced in 49 h using an initial activity of 4.25 U ml )1 partially purified PDC and a 1:1 phase ratio (Rosche et al 2002b, Sandford et al 2005). In the present study Candida utilis PDC is added in the form of resting cells as a less expensive catalyst and the temperature is raised from 4 to 21°C in order to increase rates and to cut cooling cost.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2005

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(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).

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Section: Introductionmentioning

confidence: 99%

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

et al. 2005

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“…Typical R-PAC levels reported in the literature for its fermentative production are 10-15 g/L (Shin and Rogers, 1995;Tripathi et al, 1997). A novel organic-aqueous phase enzyme based R-PAC production method using partially purified PDC from C. utilis has been developed by our group with R-PAC levels of more than 100 g/L in the organic phase (Rosche et al, 2002b;Sandford et al, 2005).…”

Section: Introductionmentioning

confidence: 99%

pH shift enhancement ofCandida utilis pyruvate decarboxylase production

Chen

Breuer

Hauer

et al. 2005

Biotechnol. Bioeng.

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Pyruvate decarboxylase (PDC) catalyses the synthesis of asymmetric carbinols, e.g., chiral precursors for pharmaceuticals such as ephedrine and pseudoephedrine. The production of PDC by Candida utilis in a minimal medium was improved by manipulating the pH during fermentation in a 5 L bioreactor. At an aeration rate of 0.1 vvm with a stirrer speed of 300 rpm at constant pH 6, a specific PDC activity of 141 U/g dry cell weight (DCW) was achieved (average of two fermentations +/-13%). By allowing the yeast to acidify the growth medium from pH 6 to 2.9, the final specific PDC activity increased by a factor of 2.7 to 385 U/g DCW (average from 4 fermentations +/-16%). The effect of this pH drift on PDC production was confirmed by another experiment with a manual shift of pH from 6 to 3 by addition of 5 M sulfuric acid. The final PDC activity was 392 U/g DCW (average from two fermentations +/-5%). However, experiments with constant pH of 6, 5, 4, or 3 resulted in average specific activities of only 102 to 141 U/g DCW, suggesting that a transitional pH change rather than the absolute pH value was responsible for the increased specific PDC activity.

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“…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).…”

Section: Introductionmentioning

confidence: 67%

“…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).…”

Section: Introductionmentioning

confidence: 99%

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

Satianegara

Rogers²,

Rosche³

2006

Biotech & Bioengineering

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Whole cell pyruvate decarboxylase (PDC) from Candida utilis enhanced the enzymatic production of (R)-phenylacetylcarbinol (PAC) in an aqueous/octanol biotransformation compared to the partially purified PDC especially for a lower range of initial activities (0.3-2.5 U/mL). With an initial activity of 1.1 U/mL and at a 1:1 phase volume ratio, whole cell PDC achieved a maximum specific PAC production of 42 mg/U (2.8 g/L/h) in comparison to 13 mg/U (0.9 g/L/h) for partially purified PDC. The enhanced performance of whole cell PDC was associated with high stability towards the substrate benzaldehyde. The strong PDC inactivation by benzaldehyde was minimal even when whole cells were broken as long as cell debris was not removed from the broken cells. Biotransformations with various cellular components added to partially purified PDC revealed that membrane components especially 2 mg/mL phosphatidylcholine enhanced PAC concentrations. The role of surfactants was further confirmed from the results with synthetic surfactant sodium bis(2-ethyl-1-hexyl)sulfosuccinate (AOT). It was apparent that the membrane components in whole cells were sufficient for optimal PAC production and no further surfactant addition is required for optimal performance.

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(R)‐phenylacetylcarbinol production in aqueous/organic two‐phase systems using partially purified pyruvate decarboxylase from Candida utilis

Cited by 36 publications

References 26 publications

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

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

pH shift enhancement ofCandida utilis pyruvate decarboxylase production

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

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