Cost Effective Development Of Two-Phase Biotransformation For
(R)-Phenylacetylcarbinol (R-PAC) Production
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[pyruvate] (mM)
PAC, benzaldehyde, and benzoic acid concentrations were determined by HPLC with UV detection at 283
nm as described by Rosche et al. (2001). Concentrations of pyruvate and acetaldehyde were determined by an
enzymatic NADH coupled assay with lactate dehydrogenase and alcohol dehydrogenase, respectively (Rosche
et al. 2002). Acetoin was quantified by gas chromatography with a flame ionisation detector and PDC activity by
a carboligase assay as described previously (Rosche et al. 2002).
Benzaldehyde CO 2
Pyruvate
Pyruvate + H+
Products
From
R-PAC
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Acetaldehyde
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Acetoin
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CO 2
R-PAC
Biotransformation
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PAC production in 20 mM MOPS with pH control (using 3.6
M acetic acid to maintain pH at 7.0). Time profiles of relative
carboligase activity and chemical species in the (a) organic
and (b) aqueous phases at 4C and stirring speed of 255
rpm. The system contained (overall concentration) 10 mM
MOPS, 745 mM benzaldehyde, 715 mM pyruvate, 0.5 mM
TPP, 0.5 mM MgSO4, and 3.46 U carboligase ml-1. The initial
volume used in each phase was 75 ml
PAC production in 20 mM MOPS and 2.5 M DPG with pH
control (using 3.6 M acetic acid to maintain pH at 7.0). Time
profiles of relative carboligase activity and chemical
species in the (a) organic and (b) aqueous phases at 4C
and stirring speed of 255 rpm. The system contained
(overall concentration) 10 mM MOPS, 1.25 M DPG, 775 mM
benzaldehyde, 805 mM pyruvate, 0.5 mM TPP, 0.5 mM
MgSO4, and 4.40 U carboligase ml-1. The initial volumes
added were 75 ml for each phase, but upon equilibration the
ratio of aqueous to organic phase changed to 0.88:1.12
() pyruvate, () benzaldehyde, () PAC,
() enzyme activity, () benzoic acid,
() acetaldehyde and () acetoin
() pyruvate, () benzaldehyde, () PAC,
() enzyme activity, () benzoic acid,
() acetaldehyde and () acetoin.
5. References
(3)
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A 20 mM MOPS system with pH control resulted in a major decrease in final PAC concentration as well as in specific and
overall volumetric PAC productivities when compared to the same system with 2.5M MOPS. The yield on pyruvate decreased
due to greatly increased acetoin production (192 mM). This is likely to have resulted from the lower benzaldehyde partitioning
into the aqueous phase which would cause stronger competition of free acetaldehyde with benzaldehyde for the carboligase
reaction.
DPG was chosen as an additive in the present two-phase study since in contrast to glycerol, DPG addition enhanced the
benzaldehyde partitioning into 20 mM MOPS. From a comparison of the data for 2.5 M DPG and 20 mM MOPS with that of 2.5 M
MOPS. it is evident that: (1) final PAC concentrations in both systems were similar, although about a 25% decrease in specific
PAC productivity and overall volumetric productivity occurred with 20 mM MOPS and 2.5 M DPG (2) the concentrations of byproducts acetaldehyde and acetoin were of similar magnitude with final yields of PAC based on pyruvate utilized (YP/Pyr) virtually
the same.
In conclusion, 2.5 M MOPS or the addition of inexpensive DPG to the pH-controlled 20 mM MOPS system was important in
enhancing PAC productivity and yield on pyruvate and their roles were both that of increasing benzaldehyde partitioning into
the aqueous phase and enhancing PDC activity.
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(R)-Phenylacetylcarbinol
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4. Discussions & Conclusion
Laboratory scale biotransformations in the two-phase system were performed in a 500 ml Quickfit reactor
(dia. 7.3 cm, height 12.5 cm) immersed in a cooling water bath at 4ºC. The overhead stirrer (IKA, Model RW 20n)
with an R-1342 stainless steel impeller (stirrer dia. 5 cm, shaft dia. 0.8 cm) was set to 255 rpm, which was the
lowest possible agitation speed to maintain the phases as an emulsion. Addition of 3.6 M acetic acid to
maintain the pH level at 7.0 was performed with a high precision digital pH-stat controller (Radiometer, Model
PHM290) and a 50 ml autoburette (Radiometer, Model ABU901). Sixty ml of an aqueous phase biotransformation
buffer was mixed with 75 ml of an organic octanol phase for at least half an hour to equilibrate the
benzaldehyde concentrations in both phases. Fifteen ml of a concentrated PDC solution was then added to
initiate the biotransformation. The initial concentrations of substrates and chemical additive (if any) as well as
initial enzyme activity are given in the corresponding Figure captions. The profiles of enzyme activity,
substrates, product, and by-products concentrations in each phase were monitored at regular intervals over 81
h. Phases were immediately separated by centrifugation (19,000 g at 6ºC for 5 min) of a 1.5 ml emulsion sample.
Residual PDC carboligase activity was determined after removal of solutes by gel filtration as described by
Rosche et al. (2002). To another aliquot of the aqueous phase, 10% (w/v) trichloroacetic acid was added in order
to stop reactions and to precipitate protein for removal by centrifugation. Organic phases were extracted with a
100-fold volume of water for subsequent HPLC analysis of the extract.
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Time (h)
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Ephedra sp.
a natural source of
ephedrine
H+
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The aim of the present study is to replace the high buffer concentration in the
two phase system by pH-control through acid addition and to identify an
inexpensive additive which can maintain PDC stability and efficient PAC
production.
CO 2
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Enzymatic production of PAC in a benzaldehyde emulsion process with
partially purified Candida utilis PDC in phosphate buffer (40 mM) resulted in a
final PAC concentration of 28 g l-1 in 8 h (Shin & Rogers 1996). As the
biotransformation for PAC production is a proton consuming process the pH rose
and eventually resulted in the irreversible deactivation of PDC. High buffer
capacity (2.0-2.5 M MOPS, initial pH 6.5) was employed in small scale studies
without pH control to minimise the pH increase (Rosche et al. 2002). In a
benzaldehyde emulsion system (6C, initial pH of 6.5, 400 mM benzaldehyde and
600 mM pyruvate), Rhizopus javanicus and C. utilis PDC produced PAC
concentrations of 50.6 g l-1 in 29 h and 51.2 g l-1 in 21 h respectively (Rosche et al.
2003). In addition, MOPS was found to have a beneficial effect on enhancing PDC
stability as were a number of other solutes at high concentrations (e.g. 1 M KCl, 2
M glycerol and 0.75 M sorbitol) (Rosche et al. 2002). PAC production in the
benzaldehyde emulsion system was much less at 20 mM MOPS, however
supplementation with 2 M glycerol resulted in similar PAC production as with 2.5
M MOPS (Rosche et al. 2005). The development of an octanol/aqueous two-phase
system for PAC production in 2.5 M MOPS has resulted in increased final PAC
concentrations in the organic phase to more than 160 g PAC l-1 (Rosche et al.
2005).
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(b)
(R)-phenylacetylcarbinol (PAC) is a precursor for the production of the
anti-asthmatic and nasal decongestant compounds, ephedrine and
pseudoephedrine. Pyruvate decarboxylase (PDC), a biocatalyst found in several
ethanol producing microorganisms, converts the substrates pyruvate and
benzaldehyde to product, PAC and by-products, acetaldehyde and acetoin.
Protons are taken up in this reaction with a consequent rise in pH.
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1. Introduction
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[PAC], [benzaldehyde],
[acetoin], [acetaldehyde],
[benzoic acid] (mM) &
relative enzyme activity (%)
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[PAC], [benzaldehyde] (mM)
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[acetaldehyde], [acetoin],
[benzoic acid] (mM)
(a)
[acetaldehyde], [acetoin],
[benzoic acid] (mM)
Screening for a lower cost solute to replace the expensive 2.5 M MOPS identified dipropylene glycol (DPG)
as a suitable candidate. The addition of 2.5 M DPG to the reaction mixture containing 20 mM MOPS increased
benzaldehyde partitioning in the aqueous phase from a concentration of 33.9 to 54.2 mM. Subsequent
biotransformation with 20 mM MOPS and 2.5 M DPG resulted in a similar level of PAC production (151 g l-1 in the
organic phase and 17.2 g l-1 in the aqueous phase after 47 h) to that with 2.5 M MOPS (158 g l-1 in the organic
phase and 20.5 g l-1 in the aqueous phase after 34 h). However the specific PAC productivity was higher in the
latter system presumably due to the greater PDC protective effect of MOPS.
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Bettina
2
Rosche
3. Results
[PAC], [benzaldehyde] (mM)
An organic-aqueous two-phase process for production of PAC has been developed further through
optimal pH control and addition of a low cost solute to maintain PDC activity in the aqueous phase. The specific
rate of PAC production in the pH controlled system (pH = 7.0) with 2.5 M MOPS was 0.60 mg U-1 h-1 (reaction
completed at 34 h), a 1.6 times improvement over the same system without pH control (0.38 mg U-1 h-1 at 49 h).
An improved stability of PDC was evident at the end of biotransformation for the pH controlled system with 84%
residual carboligase activity, while 23% of enzyme activity remained in the absence of pH control. The positive
effect of maintaining a constant pH during two-phase biotransformation included also a higher yield of PAC for
pyruvate consumed of 0.82 mol mol-1. This compared to 0.73 mol mol-1 when the pH control was not applied due
to increased production of by- products acetaldehyde and acetoin in the latter case.
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Rogers ,
*Corresponding author: E-mail: noppol@hotmail.com
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2. Materials and Methods
Peter L.
[PAC], [benzaldehyde],
[acetoin], [acetaldehyde],
[benzoic acid] (mM) & relative
enzyme activity (%)
Abstract
1,*
Leksawasdi ,
Shin HS, Rogers PL (1996)
Biotechnology & Bioengineering,
49, 52.
Rosche B, Sandford V, Breuer M,
Hauer M, Rogers PL (2001) Applied
Microbiology and Biotechnology,
57, 309.
Rosche B, Leksawasdi N, Sandford
V, Breuer M, Hauer B, Rogers PL
(2002) Applied Microbiology and
Biotechnology, 60, 94.
Rosche B, Breuer M, Hauer B,
Rogers PL (2003) Biotechnology
Letters 25, 847.
Rosche B, Breuer M, Hauer B,
Rogers PL (2005) Journal of
Biotechnology 115, 91.
6. Keywords
Biotransformation, (R)-phenylacetylcarbinol, pyruvate decarboxylase,
enzyme catalysis, pH control, organic-aqueous two-phase system
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CH3
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of Food Engineering, Faculty of Agro-Industry, Chiang Mai University, Chiang Mai, Thailand
of Biotechnology and Biomolecular Sciences, University of New South Wales, Sydney, Australia