Effect of pH on the Biotransformation of (R)-1-(4-bromo-phenyl)-ethanol by using Aspergillus niger as Biocatalyst
Molecular chirality is a fundamental phenomenon that plays an important role in biological processes. Currently, much attention has been focused on the production of chiral alcohols. Chiral alcohols such as (R)-1-(4-bromo-phenyl)-ethanol is important as a useful starting material used in pharmaceutical industries for drug synthesis. There are many factors that influenced the bioconversion and some of the crucial ones include the pH of media, biotransformation time, substrate concentration and agitation speed. All of these factors significantly affect the percentage of enantiomeric excess as well as the percentage conversion of the chiral alcohol. In this work, biocatalytic production of (R)-1-(4-bromo-phenyl)-ethanol was achieved via asymmetric reduction of 1-(4-bromo-phenyl)-ethanone using the shake-flask method at the reaction conditions of 30° C, 1 mmol of substrate, agitation speed of 150 rpm and at various pHs. Since Aspergillus niger can easily grow and produce enzyme that is suitable in converting the substrate, therefore, it was used as a biocatalyst in the reaction. Based on the results obtained, pH 7 gave the highest enantiomeric excess of 99.9% and conversion of 94.7% for the reduction time of 48 h.
September 06, 2010; Accepted: September 06, 2010;
Published: October 19, 2010
Biotransformation process has been increasingly utilized in order to replace
the conventional chemical processes and to facilitate the formation of new products
in many areas such as pharmaceutical and agrochemical to mention a few (Goretti
et al., 2009). The technique is now established as a useful tool
in the production of intermediates in fine chemical processes (Valadez-Blanco
and Livingston, 2009). One of the most successful and popular applications
in biotransformation is the asymmetric reduction of substituent of acetophenone
to a chiral phenylethanol using the whole-cell biocatalyst. According to Xiao
et al. (2005) biotransformation using growing cells is still a preferable
method for the synthesis of most of the cofactor-dependent products industrially.
Valadez-Blanco and Livingston (2009) reported that the
enantioselective reduction of ketones in organic synthesis plays a major role
in the production of chiral intermediates. The reaction of acetophenone to chiral
phenylethanol has also been widely studied as a model reaction for ketone bioreductions
in order to produce a specific chiral alcohol.
Chirality is a key factor in the efficiency of many enantiomers (Zilbeyaz
and Kurbanoglu, 2008). Enantiomerically pure secondary chiral alcohols such
as (R)-1-(4-bromo-phenyl)-ethanol as well as a chiral alcohol with additional
functional group serve as an important chiral building block and a useful starting
materials for the synthesis of various biologically active compound especially
in pharmaceutical, chemical and agrochemicals industries (Shimizu
et al., 1998; Zilbeyaz et al., 2010).
In fact, the demand for chiral alcohols has recently boosted in accordance to
the need for optically active drugs (Kurbanoglu et al.,
2007). These chiral alcohols have also been utilized as ligands for various
metals for a number of asymmetric reactions (Moon Kim and
Jin Kyoon, 1999).
This study is aimed to achieve in particular the highest products enantiomeric excess as well as the conversion by verifying the pH of reaction medium from 6 to 9. The model reaction is the asymmetric reduction of 1-(4-bromo-phenyl)-ethaone by Aspergillus niger producing (R)-1-(4-bromo-phenyl)-ethanol.
BIOCATALYST FOR THE SYNTHESIS OF CHIRAL ALCOHOL
Whole-cells microorganisms are normally preferred in many biocatalytic applications.
Jurcek et al. (2008) reported that there are
increasingly important of using microorganisms for relatively easy introduction
of chiral centers into the new molecules.
This has been based on the fact that microorganisms are able to transform
a great variety of organic molecules, both natural and synthetic, into the requested
chiral products with high enantioselectivity (Jurcek et
al., 2008). The enantiopure alcohols which are significantly employed
as chiral building blocks for fine chemicals can be prepared through the biocatalytic
methods using the whole-cell microorganisms as biocatalyst (Drepper
et al., 2006).
Figure 1 shows the schematic process of reduction of 1-(4-bromo-phenyl)-ethanone to (R) 1-(4-Bromo-phenyl)-ethanol using Aspergillus niger as a biocatalyst at a specified condition.
Moreover, Zilbeyaz and Kurbanoglu (2008) found that
the whole-cell of A. niger fungus is an effective biocatalyst for this
enantioselective bioreduction to obtain the corresponding (R)-alcohol.
Therefore, as the biocatalytic reaction are carried out by the whole fungal
cells, it was observed that the ketone was reduced to its corresponding alcohol,
which means that the alcohol dehydrogenases is present in the enzymatic system
of the microorganisms (Keppler et. al., 2005).
The usage of A.niger is significantly effective since the application
of whole-cell is rather simple, generally cheaper and highly advantageous for
practical synthesis of chiral alcohols (Shimizu et al.,
1998). In addition, the usage of whole microbial cells is preferable for
carrying out the desired reduction process since they do not require the addition
of NADPH/NADP cofactors for regeneration. This is due to the fact that they
contain multiple dehydrogenases and all the enzymes as well as cofactors are
well protected within their natural cellular environment (Mandal
et al., 2004). As a result, the enzyme becomes more stable and thus,
extending the life of a biocatalyst (Zilbeyaz and Kurbanoglu,
2008). Likewise, it properly acts under mild reaction conditions, biodegradable
and environmentally friendly and has a remarkable predominance due to their
high enantioselectivity (Xiao et al., 2005).
Therefore, an increasing important application of microorganism for relatively
easy introduction of chiral centers into the new molecules has been mainly based
on the fact that microorganisms are able to transform a great variety of organic
molecules, both natural and synthetic, into the desired chiral products with
COMPARISON BETWEEN BIOLOGICAL AND CHEMICAL REACTIONS
According to Nakamura et al. (2003) biocatalysis
has a unique characteristic when compared to the chemical catalyst. Some features
that distinguish biocatalyst from chemical catalyst are listed as follow:
||Catalyst preparation: Some of the biocatalysts for
asymmetric reduction, such as the isolated-enzyme and whole-cells are commercially
available and ready to use
||Selectivity: Very high enantio-, regio- and chemo-selectivities
can be achieved due to the strict recognition of the substrate by enzymes.
For example, in the reduction of ethyl propyl ketone, biological based reaction
has achieved high enantioselectivities (98%). In contrast, chemical catalyst
can perform highly enantioselective reduction when two adjacent groups of
the carbonyl carbon of the ketones are significantly different
||Safety of the reaction: Biocatalytic reductions are generally safe.
The reaction conditions are mild, the solvent is usually water and harzardous
reagents are not necessary. For instance, ethanol and glucose were used
as hydrogen sources instead of explosive hydrogen gas. This is because hydrogen
sources are necessary to perform a reduction reaction
||Natural catalysts: Biocatalysts, i.e., microorganisms, plants,
animal cells, or their isolated enzymes, are reproducible and can be easily
decomposed in the environment after use
Nowadays, there is a considerable interest in finding the most efficient routes
to produce chiral alcohols. The compound can be synthesized in enantiomerically
pure form from prochiral ketones either biologically, using a biocatalytic system,
or chemically via stereoselective reduction, using either a catalytic system
or a stoichiometric amount of reducing agent (Hage et
al., 2001). Both the fungus (Mellerius tremellosus) and the metal
based catalysts (iridium) are suitable systems for aryl ketone reduction but
there is a slight different on the percentage of Enantiomeric Excess (% e.e)
and product yield/conversion gained. The results are summarized as in Table
||Alcohol production from biocatalyzed and metal-catalyzed asymmetric
reductions of aryl ketones
|Nd: Not defined, Rac: Racemic, ee: Enantiomeric excess
According to Table 1, the comparison of results showed that the corresponding chiral alcohols could be obtained with moderate to high enantioselectivities (e.es up to 98%). In addition, it also shows that biocatalytic approach using the fungus is the most suitable for the enantioselective reduction of aryl ketones.
MATERIALS AND METHODS
Culture medium and biotransformation: The method is a modification from
the study of Zilbeyaz and Kurbanoglu (2008). The media
fermentation was prepared with addition of glucose 20 g L-1, yeast
extract 3 g L-1 and normal peptone 4 g L-1 in the 1 L
of Erlenmeyer flask. Then, before it was sterilized at 121°C for 15 min,
the culture medium was set at pH 7 using 1 N HCl and 1 N NaOH. The pH of the
culture medium was then adjusted to 6.0, 6.5, 7.5, 8.0, 8.5 and 9.0 with the
same molar of HCl and NaOH. The pH of the media was kept constant throughout
80 h biotransformation time. All cultures were grown in 250 mL of the Erlenmeyer
flasks containing 150 mL of culture medium. The conidia from 5 days old cultures
that were already prepared were used for inoculation. The conidial suspension
was prepared in a sterilized 200 mL distilled water by gently scratching the
conidia from an agar plate with a sterile wire loop and then vigorously shaken
to break the conidial clumps. About 15 mL of conidial suspension was then added
into each flask. The organism was maintained at the same weight for all flasks,
i.e., 5 g L-1. Flasks were incubated in an orbital shaker (Labtech
LSI-3016 A) at 30°C and 150 rpm. Only after 48 h of fermentation time, 1-(4-bromo-phenyl)-ethanone
(1 mmol) was added directly to each culture medium and then the incubation was
continued at 30°C and 150 rpm for 60 h reduction time (Zilbeyaz
and Kurbanoglu, 2008).
Purification of product: After 4 h of incubation, the first sample from each pH was withdrawn for purification and analysis. The samples were withdrawn in every 4 h as the reduction progressed. The mycelium was separated by filtration and the filtrate was saturated with sodium chloride and then extracted with ethyl acetate. The mycelia were also extracted using the same solvent. The extracts were combined and then dried over Na2SO4 to remove any moisture content.
Analytical method: The consideration of analytical methodologies needed
for the determination of chiral compounds in pharmaceutical and biological samples
is a key component to the successful compound of chiral drugs (Williams
et al., 1998). For this process, 2 mL of the extracted product which
was that previously purified was centrifuged in order to separate the two phase
of product sample using microcentrifuge (Profuge 6K) at 6600 rpm for 10 min.
The upper layer of the product which is organic solvent was withdrawn using
the syringe and was put into 10 mL of beaker. After that, the product sample
was filtered using the 0.45 μm nylon membrane filter (Double Rings Filter
Paper, 101) before it was put into the bottle sample. Then the samples were
injected into the HPLC. HPLC with Regis Reversible HPLC OD Column (4.6x250 mm,
10 um) purchased from Fisher Scientific using the eluent of n-hexane-i-Propanol,
90:10, with flow rate of 1.0 mL min-1 and detections was performed
at 254 nm at 1 mL min-1 of flow rate. All experiments were replicated
twice and averaged values were presented in this study. The optical purity was
expressed by the e.e value as given by:
Meanwhile, the conversion rate was determined from the ratio of reacted substrate concentration, ([S0]-[S1]) to its initial substrate concentration ([S0]) given by:
RESULTS AND DISCUSSION
pH profiles in the range between 6.0-9.0 were examined for the synthesis of
(R)-1-(4-bromo-phenyl)-ethanol. The initial substrate concentration for
this experiment was 1 mmol and the initial pH was set at 7. These ranges of
pH were analyzed for the optimization of microbial transformation process so
that it can be utilized for further analysis. It is well known that pH plays
a crucial role in any enzymatic reaction. As can be recognized, variation in
pH will alter the ionic state of substrate and the enzymes involved in this
reaction thus, leading to the change in enzymatic activities and enantioselectivity
(Lou et al., 2004).
||Enantiomeric excess profile on different pH. Initial substrate
concentration 1 mM, agitation 150 rpm, temperature 30°C
Figure 2 shows the profile of the enantiomeric excess performance
for differential pH.
Based on Fig. 2, it is apparent that the highest enantiomeric
excess was achieved at pH 7. The percentage of enantiomeric excess maintained
around 98 to 99%. The best reduction time to carry out the synthesis was found
at 48 h reduction time with 99.9% enantiomeric excess. After 48 h reduction
time, it decreased slightly to 98.3% at 80 h reduction time. Meanwhile, for
pH 6.0, it has a weak biotransformation which gave the lowest percentage of
enantiomeric excess (92.2%) at 80 h reduction time. The lower enantioselectivity
at lower pH could be due to the activity of the enzyme and normally the relationship
between the bacterial growth rate and pH is approximately parabolic where when
it close to optimum pH value, the growth rate changes little with changes in
extracellular pH but it declines more rapidly close to the acidic and alkaline
growth limits (Krist et al., 1998). Moreover,
from the figure, all the profiles showed an increment of enantiomeric excess
from 4 h to 48 h reduction times but then decrease slightly until 80 h reduction
time except for pH 6 which gradually decreases after 48 h reduction process.
These results strongly suggested that the variation of the enantioselectivity
with pH was directly related to the OH group which is chiral alcohol that produced
from reduction of ketone (Wang and Shi, 1998).
Figure 3 shows the comparison of product conversion at different
pH. The graph shows that the product conversion for all the pHs increased
with time. The product conversion gradually increases as the pH increases.
||Conversion profile on for different ph along 80 h incubation
time. Initial substrate concentration 1 mM, agitation 150 rpm, temperature
|| Results of (%) enantiomeric excess and (%) conversion
|Initial substrate concentration 1 mmol, 150 rpm, 30°C
at reduction time of 48 h
The effect was more apparent with the lower pH. This can be proven when the
product conversion gradually decreased when pH was adjusted to 6. Based on the
figure, pH 7 gives a good conversion with the highest conversion of 99.3%. Meanwhile,
it gives the lowest product conversion at pH 6 as well as pH 6.5 for the period
of 80 h. This is because the activity of the enzyme that produces by wild-type
of A. niger was slow in basic condition. Besides, since the enzymes are
protein, they are very sensitive to the changes of pHs and the changes
of pHs might affect the shape of an enzyme as well as change the shape
or charge properties of the substrate. In addition, generally the activity of
enzyme was completely loss at the lowest and highest p (Krist
et al., 1998). All these factors will affect the reaction of this
reduction as well as the product conversion.
Table 2 shows the results of the percentage of enantiomeric excess and conversion for different pHs at 48 h biotransformation time.
According to Table 1, it clearly shows that the pH significantly
influenced the enzymatic enantioselectivity as well as slightly affected the
conversion of 1-(4-bromo-phenyl)-ethanone to form chiral alcohol. The conversion
gradually increased when the pH of the growth medium was increased from pH 6.0
to pH 7. The highest conversion was achieved at 94.7% for pH 7 and it gradually
decreased from pH 7.5 to pH 9. Meanwhile the maximum e.e reached 99.9% at pH
7 and drastically decreased to 96.98% at pH 9. Meanwhile, the results give the
lowest enantiomeric excess and conversion was achieved at 96.36 and 65.6%, respectively
for pH 6. This data was collected at 48 h reduction time. Therefore, based on
the graph above, it can be proven that the optimum pH for reduction activity
was found to be at pH 7 since it gave the highest e.e as well as highest conversion.
Besides, it can be concluded that Aspergillus niger has a weak growth
as well as biotransformation at pH 6. This is due to the same reason as stated
previously in the enantiomeric excess discussion. This is also thought to be
due to the homeostatic mechanisms that control the intracellular pH (Krist
et al., 1998). In addition, according to the Table
2, comparison of the results show that the corresponding chiral alcohols
could be obtained with moderate to high enantioselectivities (e.e up to 100%).
(R)-1-(4-bromo-phenyl)-ethanol can be effectively produced by the fermentation
of the whole-cell Aspergillus niger fungus. In this study, the conversion
and product e.e were remarkably improves by adjusting the seven different pH
levels that can be seen through out the reduction time. Hence, the apparent
activity of different enzymes inside the cell maybe regulated through the adjustment
of the operation conditions. Among the controllable operating factors such as
temperature and concentration of substrate adjustment, pH is the most important
factor (Chen et al., 2002). Based on the results
obtained, it can be concluded that pH 7 gave the highest e.e which is 99.9%
and highest conversion which is 94.7% at 48 h reduction time. This optimum condition
can be utilized for further experiment in the future such as for different substrate
concentration and different biotransformation time in order to produce the highest
percentage of enantioselectivity and product concentration.
Authors would like to thank MOSTI for providing the Science Fund (305/ PJKIMIA/6013204) to carry out this project. F.Z. Abas would also like to thank USM for providing the Fellowship Scholarship under the Research University Scheme.
Chen, J., K.P. Wang, J.Y. Houng and S.L. Lee, 2002. Design of the pH profile for asymmetric bioreduction of ethyl 4-chloro-3-oxobutyrate on the basis of a data-driven method. Biotechnol. Prog., 18: 1414-1422.
Drepper, T., T. Eggert, W. Hummel, C. Leggewie and M. Pohl et al., 2006. Novel biocatalysts for white biotechnology. Biotechnol. J., 1: 777-786.
Goretti, M., C. Ponzoni, E. Caselli, E. Marchigiani and M.R. Cramarossa et al., 2009. Biotransformation of electron-poor alkenes by yeasts: Asymmetric reduction of (4S)-(+)-carvone by yeast enoate reductases. Enzyme Microbial. Technol., 45: 463-468.
Hage, A., D.G.I. Petra, J.A. Field, D. Schipper and J.B.P.A. Wijnberg et al., 2001. Asymmetric reduction of ketones via whole cell bioconversions and transfer hydrogenation: complementary approaches. Tetrahedron Asymmetry, 12: 1025-1034.
Jurcek, O., M. Wimmerova and Z. Wimmer, 2008. Selected chiral alcohols: Enzymic resolution and reduction of convenient substrates. Coordination Chem. Rev., 252: 767-781.
Keppler, A.F., A.L.M. Porto, I.H. Schoenlein-Crusius, J.V. Comasseto and L.H. Andrade, 2005. Enzymatic evaluation of different Aspergillus strains by biotransformation of cyclic ketones. Enzyme Microbial. Technol., 36: 967-975.
Krist, K.A., T. Ross and T.A. McMeekin, 1998. Final optical density and growth rate; effects of temperature and NaCl differ from acidity. Int. J. Food Microbiol., 43: 195-203.
Kurbanoglu, E.B., K. Zilbeyaz, N.I. Kurbanoglu and H. Kilic, 2007. Enantioselective reduction of substituted acetophenones by Aspergillus niger. Tetrahedron Asymmetry, 18: 1159-1162.
Lou, W.Y., M.H. Zong, Y.Y. Zhang and H. Wu, 2004. Efficient synthesis of optically active organosilyl alcohol via asymmetric reduction of acyl silane with immobilized yeast. Enzyme Microbial. Technol., 35: 190-196.
Mandal, D., A. Ahmad, M. Islam Khan and R. Kumar, 2004. Enantioselective bioreduction of acetophenone and its analogous by the fungus Trichothecium sp. J. Mol. Catalysis B Enzymatic, 27: 61-63.
Moon Kim, B. and P. Jin Kyoon, 1999. A short synthesis of a chiral alcohol as a new chiral auxiliary for asymmetric reactions. Bull. Korean Chem. Soc., 20: 744-745.
Nakamura, K., R. Yamanaka, T. Matsuda and T. Harada, 2003. Recent developments in asymmetric reduction of ketones with biocatalysts. Tetrahedron Asymmetry, 14: 2659-2681.
Direct Link |
Shimizu, S., M. Kataoka and K. Kita, 1998. Chiral alcohol synthesis with yeast carbonyl reductases. J. Mol. Catalysis B Enzymatic, 5: 321-325.
Valadez-Blanco, R. and A.G. Livingston, 2009. Enantioselective whole-cell biotransformation of acetophenone to S-phenylethanol by Rhodotorula glutinis: Part I. Product formation kinetics and feeding strategies in aqueous media. Biochem. Eng. J., 46: 44-53.
Wang, Z.X. and Y. Shi, 1998. A pH study on the chiral ketone catalyzed asymmetric epoxidation of hydroxyalkenes. J. Organic Chem., 63: 3099-3104.
Williams, R.C., C.M. Riley, K.W. Sigvardson, J. Fortunak and P. Ma et al., 1998. Pharmaceutical development and specification of stereoisomers. J. Pharmaceutical Biomed. Analysis, 17: 917-924.
Xiao, M.T., Y.Y. Huang, X.A. Shi and Y.H. Guo, 2005. Bioreduction of phenylglyoxylic acid to R-(-)-mandelic acid by Saccharomyces cerevisiae FD11b. Enzyme Microbial. Technol., 37: 589-596.
Zilbeyaz, K. and E.B. Kurbanoglu, 2008. Production of (R)-1-(4-Bromo-phenyl)-ethanol by locally isolated Aspergillus niger using ram horn peptone. Bioresour. Technol., 99: 1549-1552.
Zilbeyaz, K., M. Taskin, E.B. Kurbanoglu, N.I. Kurbanoglu and H. Kilic, 2010. Production of (R)-1-phenylethanols through bioreduction of acetophenones by a new fungus isolate Trichothecium roseum. Chirality, 22: 543-547.
CrossRef | PubMed | Direct Link |