Production of (R)-mandelic acid by immobilized cells of Saccharomyces cerevisiae on chitosan carrier

Process Biochemistry 42 (2007) 1465–1469 www.elsevier.com/locate/procbio

Short communication

Production of (R)-mandelic acid by immobilized cells of Saccharomyces cerevisiae on chitosan carrier Gui-Yin Li a,b, Ke-Long Huang a,*, Yu-Ren Jiang a, Ping Ding a a

College of Chemistry and Chemical Engineering, Central South University, Hunan, Changsha 410083, PR China b Hunan Vocational College of Science and Technology, Hunan, Changsha 410004, PR China Received 18 December 2006; received in revised form 20 June 2007; accepted 20 June 2007

Abstract The asymmetric microbial reduction of phenylglyoxylic acid (PGA) to (R)-mandelic acid ((R)-MA) with immobilized Saccharomyces cerevisiae cells on globular chitosan was studied. The immobilization conditions and characterization of the immobilized cells were carried out. Chitosan–acetic acid solution was injected into a mixture of 20% NaOH and 30% CH3OH aqueous solution to obtain globular chitosan, and then the globular chitosan was treated with 1% solution of glutaraldehyde to immobilize yeast cells, which were used to synthesize (R)-MA. The optimum conditions were identified as the substrate concentration of 10 mmol L1, pH of 6.5 and reaction temperature of 30 8C with the yield of 62% for (R)-MA and the enantiomeric excess (e.e.) of 98% for (R)-MA. The immobilized cells showed good operation and storage stability. # 2007 Elsevier Ltd. All rights reserved. Keywords: Saccharomyces cerevisiae; Globular chitosan; Immobilized cells; Asymmetric reduction; Phenylglyoxylic acid; (R)-Mandelic acid

1. Introduction Enantiomers of mandelic acid (MA) are valuable chemicals that have been utilized extensively for synthetic purposes as well as stereo-chemical investigations [1], and (R)-mandelic acid ((R)-MA) is used as an intermediate for the manufacture of semi-synthetic penicillins and cephalosporins and also for the synthesis of various other pharmaceuticals [2]. Many methods were reported to prepare (R)-MA such as diastereomeric crystallization, asymmetric synthesis and kinetic resolution [3–7]. However, the conventional chemical synthetic procedure produce the racemic mixtures of (R)- and (S)-isomers, and the maximum theoretical yield of the desired product (R)-MA is only 50% through further resolution methods. Biotransformation is one of the most important fields in the biological technology due to its high enantioselectivity, mild reaction conditions and environmental compatibility. Biotransformation has been applied widely in the asymmetric production of many chiral drugs and their intermediates [8]. In biotransformation, whole cells are used more than isolated enzymes because coenzyme regeneration is required for

* Corresponding author. Tel.: +86 731 8879850; fax: +86 731 8879850. E-mail address: [email protected] (K.-L. Huang). 1359-5113/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2007.06.015

sustained catalytic activity. It is well known that Saccharomyces cerevisiae (baker’s yeast) showed good capacity as a redox biocatalyst in a variety of stereoselective reductions and in the regeneration of coenzyme NAD(P)H in vivo continuously [9,10]. Asymmetric reduction of prochiral carbonyl compounds by yeast cells was an attractive method to synthesize enantiomeric pure alcohols [11]. The highly selective biotransformation of phenylglyoxylic acid (PGA) to (R)-MA can be performed by S. cerevisiae as catalyst [12,13]. However, a frequently occurring problem in biocatalytic processes with free cells is the dilute and complex products, requires concentration and purification for the recovery of the desired product. Immobilized cells may be re-used, reduce cell wastes, minimize loss of product and facilitate product recovery, in particular when in situ product removal is performed. Many support materials for yeast cells immobilization have been reported including calcium alginate [14], k-carrageenan gel [15], polyacrylamide [16], g-alumina [17] and acrylamide/ sodium acrylate copolymers [18]. As one of the most abundant renewable polysaccharides prepared from chitin through chemical N-deacetylation, chitosan has attracted much attention for its good biocompatibility, easiness to use and low price [19]. On account of the presence of the amino groups in the chitosan molecule providing a binding site for proteins, chitosan can be used as a

1466

G.-Y. Li et al. / Process Biochemistry 42 (2007) 1465–1469

solid support for the preparation of immobilized enzymes or cells. There are many reports about immobilized enzymes by using chitosan as a carrier [20–22]. However, few studies on immobilized cells using chitosan as a carrier were reported [23,24]. The aim of this paper was to produce (R)-MA by the biocatalysis of immobilized S. cerevisiae cells on globular chitosan carrier using PGA as the substrate in batch mode. The effects of substrate concentration, pH and reaction temperature on yield and e.e. of the (R)-MA were systematically studied. 2. Materials and methods 2.1. Strain and chemicals Strains (S. cerevisiae, strain no. 3) used in this work were obtained from our culture collection. Phenylglyoxylic acid (PGA, >95%), (R)-mandelic acid ((R)MA) and (S)-mandelic acid ((S)-MA) were purchased from Sigma Co. Hydroxy-b-cyclodextrin was supplied by Fluka Co. Chitosan (CTS, MW: 4.9  105, degree of deacetylation: 95%) was procured from Dalian Xindie Chitin Co. Ltd. Twenty-five percentage of glutaraldehyde solution was purchased from Hunan Normal University Chemical Company. Other chemical reagents were analytical grade reagents without further purification. All solutions were prepared with distilled water.

2.2. Media and culture conditions Culture medium for cell growth (g L1): glucose 50, peptone 3.0, yeast extract 2.5, K2HPO4 1.0, MgSO47H2O 0.5, NaCl 0.5, Fe2(SO4)3 0.01, ZnSO4 0.01, pH 6.5. Culture medium for bioreduction (g L1): glucose 50, peptone 3.0, (NH4)2SO4 3.0, K2HPO4 1.0, MgSO47H2O 0.5, NaCl 0.5, Fe2(SO4)3 0.01, ZnSO4 0.01, pH 6.5. PGA was added into the medium in the designed concentrations (5.0, 10.0, 15.0, 20.0, 25.0 mmol L1).

2.3. Permeabilization of S. cerevisiae S. cerevisiae was cultivated in Rough bottles at 30 8C for 24 h in culture medium for cell growth. The cell suspensions were collected in sterile centrifuge tubes, and centrifuged at 6687.5  g for 15 min at 4 8C. The obtained cake was washed several times with 0.9% sterile physiological saline and stored at 4 8C.

2.4. Preparation of globular chitosan A 2% (w/v) chitosan–acetate solution was obtained by adding 1 g chitosan in 50 ml 1% aqueous acetic acid while stirring at 80 8C, autoclaved at 121 8C for 15 min to prepare a chitosan–acetate sterile solution, Then the chitosan–acetic acid solution was injected into a mixture of 20% NaOH and 30% CH3OH aqueous solution, and filtrated to obtain globular chitosan. One gram of globular chitosan was treated with 25 mL 1% aqueous glutaraldehyde solution in the presence of 2.5 mL acetic acid as catalyst and stirred at room temperature for 6 h. The globular chitosan–glutaraldehyde complex was washed several times with distilled water until the washings were free of glutaraldehyde.

2.5. Immobilization of S. cerevisiae Permeabilized yeast cells (0.2 g wet weight) were suspended to 6.0 mL of 0.9% physiological saline and the suspensions were added to chitosan–glutaraldehyde complex according to the mass ratio of yeast cell suspensions (Y) to chitosan–glutaraldehyde complex (CG) of 0.5 and allowed to stand for 18 h at 30 8C with occasional stirring. The supernatant was removed by centrifugation

at 4012.5  g for 15 min at 4 8C and chitosan cell complex washed with distilled water until the washings were free of cells. The obtained immobilized cells were then collected and stored at 4 8C.

2.6. Bioconversion of PGA to (R)-MA with immobilized cells Immobilized S. cerevisiae cells were immersed in fresh bioreduction medium and fermented at 30 8C for designed time under gentle rotary shaking. PGA was added into the medium in the designed concentrations (5.0, 10.0, 15.0, 20.0, 25.0 mmol L1). In the course of the reduction, liquid samples were taken and analysed for PGA, (S)-MA, (R)-MA concentration and the e.e. of (R)-MA. All samples were analysed in triplicate. After a certain time of bioconversion, the cells were removed by centrifugation at 4012.5  g for 15 min at 4 8C. Then the supernatant was adjusted to pH 1.0 with concentrated hydrochloric acid, and extracted with ethyl acetate. The ethyl acetate phase was collected, dried over anhydrous Na2SO4 and concentrated under reduced pressure to obtain a crude crystal of (R)-MA. Recrystallization of the crude crystal from ethyl acetate gave a white crystal powder of (R)-MA. mp: 120–122 8C (literature [25], 118–121 8C). Each resulting crystal was dissolved in distilled water at a concentration of 1.0% (w/v). Optical rotation of the product was determined with a WZZ-15 Automatic Polarimeter (Shanghai Physical Optics Apparatus Company) ½a20 D¼  150 (ca. 1.0, H2O) (literatures [26]): ½a20 ¼ 154 (ca. 1.0, H O). 2 D

2.7. Analytical methods For the determination of dry weight, the cells were washed twice with distilled water and separated by centrifugation after each cycle. The dry weight was determined by drying the wet cells at 105 8C, after 3 h, the cell weight dried to a constant weight. The concentration of PGA, (R)-MA, (S)-MA were determined with a CE system equipped with a photodiode array detector (P/ACETM MDQ, Beckman Coulter Co., USA) containing a 61 cm  75 mm (i.d.) uncoated fused silica capillary. The CE conditions were 100 mmol L1 Tris–potassium phosphate buffer (pH 7.6) containing 150 g L1 hydroxypropyl-b-cyclodextrin (chiral selector), and at detection of 24 kV and 214 nm. The retention time for PGA, (R)-MA and (S)-MA was 12.46, 17.69 and 18.15 min, respectively. PGA, (R)MA and (S)-MAwere identified by comparing the retention times with authentic standards in the electropherograms and also checked with the blank test solutions. The peak area of each component was obtained by the average of three times detection of certain concentration. The detection concentration of each component varied from 0.05 to 1.00 mmol L1. The linearity of the plot of peak area (Y) versus concentration (X, mmol L1) for each component in CE was investigated. The regression equations for these components were given as follows: Y ¼ 14774:50 þ 398951:6X;

PGA :

R ¼ 0:99956 ð0:05  1:00 mmol L1 Þ ðRÞ-MA :

Y ¼ 2896:51 þ 384388:39X;

R ¼ 0:9983 ð0:05  1:00 mmol L1 Þ ðSÞ-MA :

Y ¼ 1011:382 þ 367297:34X;

R ¼ 0:9992 ð0:05  1:00 mmol L1 Þ The conversion rate (c) was determined from the ratio of reacted substrate concentration ([S0]  [S]) to its initial substrate concentration ([S0]): c¼

½S0   ½S  100% ½S0 

In this equation [S0] is the initial PGA concentration, [S] the PGA concentration calculated from the regression equation of PGA, and [S0]  [S] is the reacted PGA concentration. The product yield (Y) was determined from the ratio of the product (R)-MA concentration ([(R)-MA]) to PGA initial substrate concentration ([S0]): Y¼

½ðRÞ-MA  100% ½S0 

G.-Y. Li et al. / Process Biochemistry 42 (2007) 1465–1469

1467

The enantiomeric excess (e.e.) of (R)-MA was calculated as follows:

e:e: ¼

½ðRÞ-MA  ½ðSÞ-MA  100% ½ðRÞ-MA þ ½ðSÞ-MA

[(R)-MA] and [(S)-MA] are the concentrations of (R)-MA and (S)-MA, respectively.

3. Results and discussion 3.1. Comparison of the asymmetric reduction with free and immobilized yeast cells The immobilized cells have a different environment from free cells, while the environment affects their ability of asymmetric reduction. Fig. 1 shows the reaction time course of the asymmetric reduction of phenylglyoxylic acid catalyzed by immobilized and free S. cerevisiae cells. Both the free S. cerevisiae cells and immobilized cells have the ability to convert PGA to (R)-MA, but there is a difference between the conversion rate of PGA and the e.e. of (R)-MA. The gradient of concentration of substrate in the reaction medium is different from the gradient of concentration inside the chitosan beads. After 24 h reaction, 57% of substrate was consumed using free cells whereas 68% substrate was transformed by immobilized yeast cells. The enantioselectivity is improved since the immobilization shield plays an important role by creating a barrier to substrate partition, and the enantiomeric excess of the product (R)-MA is 97% e.e. It is well known that baker’s yeast possesses several alcohol dehydrogenases with varying substrate selectivity and even opposing enantioselectivity. When the cells are immobilized, the selectivity of these reactions is better than that observed from free cells.

Fig. 1. Reaction time course of the asymmetric reduction of phenylglyoxylic acid catalyzed by immobilized and free Saccharomyces cerevisiae cells (pH 6.8, t = 30 8C, 10 mmol L1 phenylglyoxylic acid).

Fig. 2. FT-IR spectra for chitosan, globular chitosan and immobilized cell matrices.

3.2. Characterization of immobilized yeast cells 3.2.1. Infrared characterization of CTS, globular chitosan and immobilized cell matrices IR spectra is used to characterize the functional groups of the immobilized cell matrices. The IR spectra of CTS, globular chitosan and immobilized cell matrices recorded on a Hitachi 270-50 IR spectrophotometer using KBr discs is shown in Fig. 2. As known from Fig. 2, in curve a, characteristically strong and broad overlapping bands at 3440 cm1 are observed, corresponding to the stretching vibration of N–H and O–H. The very strong peaks at 1095, 1070 and 1076 cm1 (Fig. 2a–c) are the typical stretching vibration of the C–O bond [27]. Compared with curve a, the presence of a new pronounced sharp amide carbonyl band at 1654 cm1 of globular chitosan (Fig. 2b) indicates that chitosan react with glutaraldehyde to form Schiff base, and the appearance of the new peak at 2871 cm1 is assigned to the Femi vibration of C–H of aldehyde on the side chains. Compared with curve b, the disappearance of the peak 2871 cm1 in curve c indicates that yeast cells have already immobilized on globular chitosan carrier. 3.2.2. Effect of pH on the asymmetric reduction The pH profile in the range of 5.0–7.5 was examined for the product of (R)-MA by immobilized cells in Fig. 3. The efficient resolution of PGA was observed when the pH was held between 6.0 and 6.5 by the immobilized cells whereas the free cells showed an optimum pH of 6.5 [13]. The optimal pH for immobilized cells shifted less to the acidic side. This may be explained by electrostatic potential theory. Chitosan is a positively charged carrier, thus the electrostatic potential within the immobilized cells is higher than that in the bulk solution The concentration of [H+] within the immobilized enzyme will be lower than that in the bulk solution which causes the pH– activity curve to shift to the acid [22]. The optimal pH for immobilized cells was 6.5 with the yield of (R)-MA 62% and the e.e. of (R)-MA 98%.

1468

G.-Y. Li et al. / Process Biochemistry 42 (2007) 1465–1469

Fig. 3. Effect of pH on the reaction (reaction conditions: 2.5 g immobilized cells, T = 24 h, t = 30 8C, PGA concentration: 10 mmol L1, reaction medium volume: 50 mL).

3.2.3. Effect of temperature on the asymmetric reduction The temperature profile in the range of 20–45 8C was examined for the product of (R)-MA by immobilized cells in Fig. 4. As shown in Fig. 4, the immobilized cells showed better biocatalytic activities in a broader range of temperature from 25 to 35 8C, and an optimum temperature was 35 8C for the efficient of resolution of PGA. Higher temperatures resulted in more detrimental to PGA resolution than lower temperatures. However, the enanotioselectivity of the reduction was not significantly influenced in the reaction temperature from 25 to 40 8C. The optimum temperature range was 25–35 8C for the yield of (R)-MA and e.e. of the desired product (R)-MA. 3.2.4. Effect of PGA concentrations on the asymmetric reduction In order to find the optimum concentration of substrate PGA, several levels of substrate PGA concentrations from 5.0 to

Fig. 5. Effect of substrate concentration on the reaction (reaction conditions: 2.5 g immobilized cells, t = 30 8C, T = 24 h, pH 6.5, reaction medium volume: 50 mL).

25.0 mmol L1 were presented to determine their effects on the yield of (R)-MA and e.e. of (R)-MA. As shown in Fig. 5, the optimum concentrations of PGA was 10 mmol L1 with the yield of (R)-MA 62% and the e.e. of (R)-MA 98%. At the PGA concentrations above 15 mmol L1, substrate PGA possessed an apparently negative influence on the reduction activity of yeast cells, and substrate inhibition of bioconversion occurred. In many cases, the stereochemical results obtained from microbial reactions are affected by the substrate concentrations. At the same concentration of biomass, the yield and optical purity of desired product are affected notably by the substrate concentration. In general, decreasing the substrate concentration can enhance the yield and the enantioselectivity of the product. 3.2.5. Operation stability of immobilized yeast cells To investigate the operation stability of immobilized cells, several repetitive use of immobilized cells to convert PGA to (R)-MA were operated. After each cycle the beads were separated by filtration and washed with distilled water three times, the immobilized cells were then collected and immersed in fresh bioreduction medium with 10 mmol L1 PGA and fermented at 30 8C for 24 h under gentle rotary shaking. Within six batches, the yield of (R)-MA was 62%, 60%, 57%, 54%, 50% and 48%, respectively, though the stereospecific degree of asymmetric reduction of the immobilized cells decreases lightly. Thus, the immobilized cells demonstrated better operation stability.

Fig. 4. Effect of temperature on the reaction (reaction conditions: 2.5 g immobilized cells, T = 24 h, pH 6.5, PGA concentration: 10 mmol L1, reaction medium volume: 50 mL).

3.2.6. Storage stability of immobilized yeast cells The wet immobilized cells were stored at 4 8C for 20 days, and the yield was measured every 3 days. The yield of (R)-MA decreased from 62% to 47% after 20 days. The results indicated that the immobilized cell had good storage stability.

G.-Y. Li et al. / Process Biochemistry 42 (2007) 1465–1469

4. Conclusions (R)-MA is possibly produced by the fermentation of free or immobilized S. cerevisiae cells. The use of immobilized whole cells in industrial processes has attracted considerable attention due to the advantages such as an increase of yield and cellular stability and a decrease of procedure expenses due to the ease for cell recovery and reutilization over traditional processes. Immobilization is the restriction of cell mobility within a defined space. Immobilization provides high cell concentrations and cell reuse. The immobilized S. cerevisiae cells with chitosan as carrier using glutaraldehyde as cross-linking reagent showed significant advantages over free cells. The optimum substrate concentration, pH and reaction temperature were found to be 10 mmol L1, 6.5 and 30 8C, respectively, in bioconversion of PGA using the immobilized cells. Under the optimum conditions the yield and the enantiomeric excess of (R)-MA were as high as 62% and 98% e.e., respectively. Hence there is a great potential for the use of immobilized S. cerevisiae cells for the biosynthesis of industrial (R)-MA. The immobilized method could be useful model for other biochemical reactions with yeast cells. Acknowledgments This work was supported by research grants from the National Science Foundation of China (Project No. 20376085). References [1] Challener CA, editor. Chiral intermediates. London: Ashgate Publishing; 2001. p. 502. [2] Fulenmeier A, Quitt P, Volgler K, Lanz P. 6-Acyl derivatives of aminopenicillanic acid. U.S. Patent 3,957,758 (1976). [3] Ganapati D, Yadav P, Sivakumar. Enzyme-catalysed optical resolution of mandelic acid via methyl-mandelate in non-aqueous media. Biochem Eng J 2004;19:101–7. [4] Blacker AJ, Houson IN. Preparation of mandelic acid derivatives.WO Patent 02,066,410 (August 29, 2002). [5] Strauss UT, Faber K. Deracemization of (RS)-mandelic acid using a lipase–mandelate racemase two-enzyme system. Tetrahedron: Asymmetic 1999;10:4079–81. [6] Silvia R, Amaya SVU, Massimo P, Aur T, Jose MG, Roberto FL, et al. Influence of the enzyme derivative preparation and substrate structure on the enantioselectivity of penicillin G acylase. Enzyme Microb Technol 2002;31:88–93. [7] Kim BY, Hwang KC, Song HS, Chung N, Bang WG. Optical resolution of RS-mandelic acid by Pseudomonas sp. Biotechnol Lett 2000;22:1871–5.

1469

[8] Patel RN. Biocatalytic synthesis of intermediates for the synthesis of chiral drug substances. Curr Opin Biotechnol 2001;12:587–604. [9] Scheulze B, Wubbolts MG. Biocatalysis for industrial production of fine chemicals. Curr Opin Biotechnol 1999;10:609–15. [10] Lou WY, Zong MH, Fan XD. Asymmetic synthesis of ()-1-trimethylsilyl-ethanol with immobilized Saccharomyces cerecisiae cells in water/ organic solvent biphasic system. Chinese J Chem Eng 2003;11(2):136–40. [11] Athanasiou N, Smallridge AJ, Trewhella MA. Baker’s yeast mediated reduction of b-keto esters and b-keto amides in an organic solvent system. J Mol Catal B: Enzyme 2001;11:893–6. [12] Xiao MT, Huang YY, Meng C, Guo YH. Kinetics of asymmetric reduction of phenylglyoxylic acid to R-()-mandelic acid by Saccharomyces cerevisiae FDllb. Chinese J Chem Eng 2006;14(1):73–80. [13] Xiao MT, Huang YY, Shi XA, Guo YH. Bioreduction of phenylglyoxylic acid to R-()-mandelic acid by Saccharomyces cerevisiae FD11b. Enzyme Microb Technol 2005;37:589–96. [14] Evelyn MB, Ifoeng CJ, Straathof AJ, Jaap AJ, Joseph JH. Immobilization affects the rate and enantioselectivity of 3-oxo ester reduction by baker’s yeast. Enzyme Microb Tech 2002;31:656–64. [15] Luong JH. Cell immobilization in k-carrageenan for ethanol production. Biotechnol Bioeng 1985;27:1652–61. [16] Mandwal AK, Tripathi CKM, Trivedi PD, Joshi AK, Agarwal SC, Bihari V. Production of L-phenylacetyl carbinol by immobilized cells of Saccharomyces cerevisiae. Biotechnol Lett 2004;26:217–21. [17] Isono Y, Araya G, Hoshind A. Immobilization of Saccharomyces cerevisiae on g-alumina particles using spray-dryer. Process Biochem 1995;30:743–6. ¨ ztop HN, O ¨ ztop AY, Karadag E, Isikver Y, Saraydin D. Immobilization [18] O of Saccharomyces cerevisiae on to acrylamide–sodium acrylate hydrogels for production of ethyl alcohol. Enzyme Microb Technol 2003;32: 114–9. [19] Ding P, Huang KL, Li GY. Kinetics of adsorption of Zn(II) ion on chitosan derivatives. Int J Biol Macromol 2006;39:222–7. [20] Wang G, Xu JJ, Ye LH. Highly sensitive sensors based on the immobilization of tyrosinase in chitosan. Bioelectrochemistry 2002;57:33–8. [21] Juang RS, Wu FC, Tseng RL. Use of chemically modified chitosan beads for sorption and enzyme immobilization. Adv Environ Res 2002;6:171–6. [22] Zeng J, Zheng LY. Studies on Penicillium sp. ZDZ1 chitosanase immobilized on chitin by cross-linking reaction. Process Biochem 2002;38: 531–5. [23] Valentino MK, Gaston P. Stability of chitosan gel as entrapment matrix of viable Scenedesmus bicellularis cells immobilized on screens for tertiary treatment of wastewater. Bioresour Technol 1996;56:147–55. [24] Krastanov A, Blazheva D, Yanakieva I, Kratchanova M. Conversion of sucrose into palatinose in a batch and continuous processes by immobilized Serratia plymuthica cells. Enzyme Microb Technol 2006;39: 1306–12. [25] Heike L, Andreas SM. A contribution to the mandelic acid phase diagram. Thermochim Acta 2004;415:55–61. [26] Mori T, Furui M, Nakamichi K, Takahashi E. Process for producing Dmandelic acid from benzoylformic acid. United States Patent 5,441,888 (August 15, 1995). [27] Tan XC, Tian YX, Cai PX. Glucose biosensor based on glucose oxidase immobilized in sol–gel chitosan/silica hybrid composite film on Prussian blue modified glass carbon electrode. Anal Bioanal Chem 2005;381: 500–7.