Biocatalytic synthesis of ethyl (R)-2-hydroxy-4-phenylbutyrate with a newly isolated Rhodotorula mucilaginosa CCZU-G5 in an aqueous/organic biphasic system
© Wang et al.; licensee Springer. 2015
Received: 15 December 2014
Accepted: 21 January 2015
Published: 18 February 2015
Optically active ethyl (R)-2-hydroxy-4-phenylbutyrate [(R)-HPBE] is an important chiral building block for the synthesis of angiotensin-converting enzyme (ACE) inhibitors. It is reported that microbial or enzymatic reduction of ethyl 2-oxo-4-phenyl-butyrate (OPBE) is an attractive way to produce optically active (R)-HPBE.
The asymmetric reduction of OPBE to synthesize optically active (R)-HPBE with a newly isolated Rhodotorula mucilaginosa CCZU-G5 as catalyst was investigated in an aqueous/organic solvent biphasic system. R. mucilaginosa CCZU-G5 showed a good tolerance (the metabolic activity retention >80%) in the biphasic system composed of aqueous buffer and organic solvent with a log P value over 4.6. Isooctane was found to be the most suitable organic phase solvent. In the biphasic system, the volumetric phase ratio, OPBE concentration, cell concentration, reaction temperature, and buffer pH were optimized. Under the optimum conditions (volumetric phase ratio: 1/1, OPBE concentration: 100 mM, cell concentration: 0.075 g/mL, pH 7.5, 35°C), the final yield and the optical purity of (R)-HPBE reached 98.3% and >99.0% enantiomeric excess (ee), respectively, after 12 h of reaction.
All the results suggested that the OPBE-reducing enzymes in a newly isolated R. mucilaginosa cells possess highly stable and excellent stereoselectivity by establishing an aqueous/organic biphasic system.
Optically active ethyl (R)-2-hydroxy-4-phenylbutyrate [(R)-HPBE] is an important chiral building block for the synthesis of angiotensin-converting enzyme (ACE) inhibitors such as benazepril, enalapril, and lisinopril . In general, ACE inhibitors prevent the conversion of the precursor decapeptide angiotensin I to the powerful vasoconstrictor substance angiotensin II and have been demonstrated to be potent antihypertensive drugs . In recent years, various chemical and biological approaches for (R)-HPBE preparation have been reported, mainly in two ways: kinetic resolution and synthesis. However, chemical synthesis usually involves multiple steps and stringent reaction conditions , and resolution methods are limited by theoretical maximum yield of only 50% .
Microbial or enzymatic reduction of ethyl 2-oxo-4-phenyl-butyrate (OPBE) is an attractive way to produce optically active (R)-HPBE, since OPBE can be easily synthesized and is relatively cheap. Several biocatalysts have been used in the synthesis of (R)-HPBE, including the hydrolysis and transesterification catalyzed by lipase  and the reduction of OPBE catalyzed by isolated dehydrogenase  and whole cells [7,8]. Since the reduction reaction requires stoichiometric amounts of nicotinamide cofactors, whole cells rather than isolated enzymes were used preferentially to avoid enzyme purification and cofactor addition .
In the past decade, however, only a few microorganisms have been reported as efficient biocatalysts in the reduction of OPBE to (R)-HPBE. Chadha et al. reported that the enantioselective reduction of OPBE to (R)-HPBE could be achieved by using cell-free aqueous extracts of the callus of Daucus carota (wild carrot) with a high yield (90%) and enantiomeric excess (ee) (99%) . However, their process required a high cell/substrate ratio of 100:1, a large amount of cells, and a long reaction time of 10 days. Dao et al. and Lacerda et al. reported the successful reduction of OPBE with Pichia angusta and Saccharomyces cerevisiae, respectively, to give (R)-HPBE with a moderate enantioselectivity (81% ee) [7,11]. Recently, Chen et al. described the successful preparation of (R)-HPBE with favorable ee (99%) and yield (92%) by using Candida boidinii CIOC21 . However, the relatively low concentrations of the substrate (around 4.1 g/L) and product (around 3.8 g/L) in their process would restrict its application in large-scale production. In addition, Zhang et al. used Candida krusei SW2026 to produce (R)-HPBE from 20 g/L of OPBE with excellent ee (97.4%) and a moderate yield (82%) .
Recently, we have isolated a new yeast strain Rhodotorula mucilaginosa CCZU-G5 from vineyard soil samples and used it in preparing (R)-HPBE with high ee and yield. However, a severe substrate inhibition was observed when the tested OPBE concentration in an aqueous single-phase system was high due to the high hydrophobicity of the substrate and its toxicity to the cells, and the highest substrate concentration that the bacterium could transform was only 50 mM. The aqueous/organic solvent biphasic system is a good alternative to resolve the aforementioned problems occurred in the aqueous system. The organic solvent phase in the biphasic system acts as a substrate reservoir and prevents the cells in the aqueous phase from being damaged by high substrate concentration. This biphasic system has attracted great attention over the past few decades, and several successful examples have been reported [13-15].
In this study, resting cells of R. mucilaginosa CCZU-G5 were used as biocatalysts for asymmetric reduction of OPBE in an aqueous/organic solvent biphasic system. Various parameters such as substrate concentration, cell concentration, reaction temperature, and pH were investigated and optimized to improve the yield and optical purity (ee) of (R)-HPBE. Compared to the monophasic aqueous system, the asymmetric reduction of OPBE in the aqueous/organic solvent biphasic system gave excellent ee and a much higher yield due to reduced substrate and product inhibition. To our best knowledge, this is the first study using R. mucilaginosa cells for high-yield and high-purity production of (R)-HPBE.
(R)-HPBE was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA) OPBE was supplied by Wujin Fine Chemical Factory Co., Ltd. (Changzhou, Jiangsu, China). All other reagents and solvents were commercially available, and were of analytical grade purity.
Microbial strain and cultivation conditions
The yeast R. mucilaginosa CCZU-G5 was isolated from vineyard soil samples and preserved in China General Microbiological Cultures Collection Center (CGMCC 6328). It was grown in the following medium: glucose 20 g/L, yeast extract 10 g/L, peptone 15 g/L, (NH4)2SO4 1 g/L, MgSO4·7H2O 1 g/L, pH 7.0. The strain was incubated aerobically at 30°C and 180 rpm in 500-mL Erlenmeyer flasks with 70 mL sterilized medium. After 72 h of growth, the cells were harvested by centrifugation (8,000×g for 10 min) at 4°C, washed twice with 0.85% (w/v) NaCl, and then stored at 4°C for further use.
Bioconversion in monophasic aqueous system
The reduction of OPBE in the aqueous system was conducted in a 50-mL Erlenmeyer flask capped with a septum. Two grams of wet cells was suspended in 20.0 mL phosphate-buffered saline (PBS) (0.1 M, pH 7.0) with 1 g glucose and 0.4 mmol OPBE. The cell suspensions were subsequently incubated in a rotary incubator at 35°C and 180 rpm. The mixture was centrifuged to remove the cells at different time intervals, and the supernatant was extracted three times with ethyl acetate and dried over anhydrous Na2SO4 for gas chromatography (GC) analysis.
Tolerance assay of R. mucilaginosa CCZU-G5 in biphasic systems
Four grams of harvested cells was suspended in 100 mL PBS (0.1 M, pH 7.0) to give a final concentration of 0.04 g/mL. Ten milliliters of the cell suspension was added to 10 mL of each of the different organic solvents in a 50-mL Erlenmeyer flask capped with a septum. The cell suspension (0.04 g/mL) in PBS without any organic solvent was used as a control. All cell suspensions were subsequently incubated in a rotary incubator at 30°C and 180 rpm for 24 h. The cell suspensions were then centrifuged (8,000×g for 10 min) at 4°C. Then the cells were transferred to 10 mL of 20 g/L glucose and incubated at 30°C and 180 rpm. After 4 h, the suspensions were centrifuged, and the supernatants were analyzed using a spectrophotometer to determine the concentration of glucose through DNS method to obtain the amount of consumed glucose . The metabolic activity retention is defined as the ratio of the amount of glucose consumed by the cells pretreated in the biphasic system to that consumed by the cells pretreated in PBS.
Bioconversion in biphasic systems
The concentrations of OPBE and HPBE were determined with a gas chromatography. (GC-950, Shanghai Haixin Chromatographic Instrument Co., Ltd., Shanghai, China) equipped with a flame ionization detector and a SE-30 capillary column (30 m, i.d. 0.5 mm). The (R)-HPBE and (S)-HPBE were analyzed using an Agilent 1260 HPLC system (Sta. Clara, CA, USA) equipped with a Chiralcel OD-H column (4.6 mm × 250 mm, 5 μm, Diacel, Hyogo, Japan) using n-hexane:isopropanol (95:5, v/v) as eluent at a flow rate of 1.0 mL/min. The detection was performed at 225 nm.
Results and discussion
Tolerance of R. mucilaginosa CCZU-G5 in different biphasic systems
Tolerance and asymmetric reduction of OPBE to ( R )-HPBE with R. mucilaginosa CCZU-G5 in different organic/aqueous biphasic systems
Metabolic activity retention (%) a
Yield (%) b
ee (%) b
14.5 ± 0.6
11.3 ± 0.7
17.1 ± 0.7
14.3 ± 1.3
17.5 ± 0.6
36.1 ± 1.1
22.3 ± 0.7
44.1 ± 1.0
50.4 ± 1.3
53.1 ± 1.5
67.2 ± 0.9
69.3 ± 0.5
70.5 ± 0.8
89.3 ± 0.4
78.8 ± 0.7
90.3 ± 0.4
80.1 ± 0.6
98.1 ± 0.3
92.2 ± 0.4
63.1 ± 0.3
98.0 ± 0.4
52.8 ± 0.9
Selection of organic solvents
The asymmetric reduction of OPBE to (R)-HPBE with resting cells of R. mucilaginosa CCZU-G5 in an aqueous medium has been optimized in our previous work (20 mM OPBE, yield 76.3%, ee 99.2%, productivity 6.8 g/L/day). The aqueous systems supported a desirable ee value, but the highest substrate concentration that could be transformed with 0.1 g/mL cell was merely 50 mM. To enhance the substrate concentration and overcome the substrate-tolerance obstacle [14,19], the reaction was explored in the aqueous/organic biphasic system. With the addition of organic solvents, the solubility of the substrate could be enhanced. Besides, the hydrophilic microbial cells (in aqueous phase) could be separated from the hydrophobic substrate and the product in the organic phase. The selection of the organic solvent as substrate and product carrier in an aqueous/organic solvent biphasic system is based mainly on its biocompatibility towards the biocatalyst and adequate solubility of both substrate and product . Therefore, the influence of organic solvents on the catalytic activity and enantioselectivity was studied in 11 different biphasic systems with different log P values ranging from 0.68 to 5.6. In general, there was a positive correlation between the product yield and the log P value of organic solvent (Table 1), with the exceptions of n-hydride and n-decane, which have log P values of over 5 but gave a relatively low product yield. The maximal yield of 98.1% was achieved in the aqueous/isooctane biphasic system. The yield decreased significantly in the organic solvents with lower log P values such as butyl acetate and EtOAC, which gave a poor yield of 11.3%. The ee values of (R)-HPBE were higher than 99.0% when the log P values of the organic solvents were more than 3.2. Considering the yield and the ee value, the aqueous/isooctane biphasic system was selected for further study.
Effects of phase volume ratio
Effects of temperature
Effects of pH
Effects of cell concentration
Effects of substrate concentration
Comparison of the aqueous/organic solvent biphasic system with the monophasic aqueous system
Comparison between R. mucilaginosa CCZU-G5 and other reported microorganisms
Reaction time (h)
Yield (conversion) (%)
R. mucilaginosa CCZU-G5
Shi et al., 2009 
Candida krusei SW2026
Zhang et al., 2009 
Candida boidinii CIOC21
Chen et al., 2009 
0.14% in volume
Lacerda et al., 2006 
In this study, an aqueous/isooctane biphasic system was successfully established for asymmetric reduction of OPBE to (R)-HPBE with a newly isolated strain R. mucilaginosa CCZU-G5. Several factors such as volume ratio of the aqueous phase to the organic phase, reaction temperature, reaction pH, cell concentration, and substrate concentration significantly influenced the reaction rate and product yield. However, the optical purity of the product was not significantly affected and maintained at high levels of >99%. Under optimum reaction conditions (35°C, pH 7.5, 0.075 g/mL of cells, 100 mM OPBE, 1:1 of volume phase ratio), R. mucilaginosa CCZU-G5 exhibited excellent catalytic capability, giving product an excellent yield (98.3%) and ee (>99%).
This work was supported by National Natural Science Foundation of China (No. 21102011) and the Science and Technology Supporting Project of Changzhou (No. CE20145041).
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