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Production of N-carbamoyl-d -hydroxyphenylglycine by d -hydantoinase activity of a recombinant Escherichia coli
Process Biochemistry 35 (1999) 285 – 290 www.elsevier.com/locate/procbio
Production of N-carbamoyl-D-hydroxyphenylglycine by D-hydantoinase activity of a recombinant Escherichia coli Yi-Chuan Chen a, Bang-Ding Yin a, Sung-Chyr Lin a,*, Wen-Hwei Hsu b a
Department of Chemical Engineering, National Chung Hsing Uni6ersity, Taichung 402, Taiwan b Institute of Molecular Biology, National Chung Hsing Uni6ersity, Taichung 402, Taiwan Received 7 April 1999; received in revised form 11 May 1999; accepted 16 May 1999
Abstract Recombinant Escherichia coli BL21 (DE3) harbouring plasmid pET36 encoding D-hydantoinase from Pseudomonas putida were used as biocatalyst for the production of N-carbamoyl-D-hydroxyphenylglycine from DL-p-hydroxyphenylhydantoin. The optimum D-hydantoinase activity was observed at 40°C and pH 8.5. At a substrate concentration of 300 mg/l, an initial reaction rate of 24.52 mM/h g cells was obtained and 91% of the substrate was converted into product after a 24-h reaction. Recombinant cells were immobilized within calcium alginate beads with diameters ranging from 2 to 3 mm. The specific activity of the immobilized cells increased with cell loads, probably due to reduced mass transfer resistance. The immobilized cells also exhibited an optimal pH of 8.5. However, under the conditions described above, the initial reaction rate with the immobilized cells as the biocatalysts was reduced by 87% to 3.19 mM/h g cells, probably due to the formation of cell aggregates inaccessible to substrate. Thermostability and reusability of D-hydantoinase were increased upon immobilization. The initial reaction rate with immobilized cells was increased with temperature at least up to 60°C. More than 95% of the D-hydantoinase activity was recovered after three cycles for the immobilized cells, compared to 22% for the free cell systems. © 1999 Elsevier Science Ltd. All rights reserved. Keywords: Calcium alginate;
D-Hydantoinase; D-p-Hydroxyphenylglycine;
1. Introduction D-Amino acids are important precursors for the production of many semi-synthetic antibiotics, such as amoxicillin, cefadroxil, cefatrizinen, cefaparole, and cefaperazon. Among b-lactam antibiotics, amoxicillin is one of the most widely used broad-spectrum antibiotics because of limited bacterial resistance. Amoxicillin is produced by the reaction of 6-amino penicillanic acid (6-APA), derived from the catalytic degradation of penicillin with penicillin acylase, and D-p-hydroxyphenylglycine (D-p-HPG) [1 – 3]. D-p-HPG can be produced from DL-5-substituted hydantoins via chemical processes or biological processes. Although the chemical processes for the synthesis of D-p-HPG are well established [4], the application of a chemical process for industrial production of D-p-
* Corresponding author. Tel.: +886-4-2854724, ext. 104; fax: + 886-4-2854734. E-mail address: [email protected] (S.-C. Lin)
Escherichia coli; Immobilization
HPG has been challenged due to its low product yield, low product purity, and the use of a large quantity of organic solvent. A chemo-enzymic process for the production of D-p-HPG from DL-5-substituted hydantoins has been proposed by Yamada and co-workers [4–6]. In this process, DL-5-substituted hydantoin is asymmetrically hydrolyzed to N-carbamoyl-D-amino acid by D-specific hydantoinase and this compound is further converted chemically to the corresponding D-amino acid under acidic conditions. Alternatively, N-carbamoyl-D-amino acids can be enzymically cleaved to D-p-HPG with carbamoylase (EC 3.5.1.6) [7–10]. Compared to the chemical processes, the enzymic process has the advantages of high product yield, high optical purity and low environmental impacts, and, therefore, has attracted extensive interest in D-hydantoinases and carbamoylase. D-Hydantoinases are identical to dihydropyrimidinases (EC 3.5.2.2) that catalyze the hydrolytic ring opening of dihydropyrimidines to N-carbamoyl-b-
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amino acids and of DL-5-substituted hydantoins to the corresponding N-carbamoyl-D-amino acids [15]. D-Hydantoinases are widely distribute among bacteria, including Agrobacterium sp. [7,11 – 13], Arthrobacterium sp. [9,10], Bacillus sp. [14,15], Corynebacterium sp. [16], and Pseudomonas sp. [5,16 – 22]. However, the D-hydantoinase activity obtained in these wild-type strains is relatively low. It is, therefore, desirable to enhance the production of D-hydantoinase via genetic engineering. A recombinant E. coli-harbouring plasmid containing Dhydantoinase gene from P. putida has recently been constructed [23,24]. Under the control of T5lac promoter and lactose induction, the recombinant E. coli exhibited a D-hydantoinase activity about 20-fold higher than that of the gene donor strain [24]. In this study, the use of the recombinant E. coli cells exhibiting D-hydantoinase activity as biocatalysts for the production of N-carbamoyl-D-hydroxyphenylglycine from DL-p-hydroxyphenylhydantoin was systematically investigated. 2. Materials and methods
both free cells and immobilized cells as biocatalysts were investigated. To study the effect of temperature on the D-hydantoinase activities of the biocatalysts, predetermined amount of biocatalysts, 3 ml of cell concentrate for the free-cell systems, and 3 g of calcium alginate beads for the immobilized systems, were respectively added into 50 mM Tris–HCl buffer, pH 8.5, containing D-p-hydroxyphenyl hydantoin (D-p-HPH, Tokyo Chemicals, Tokyo, Japan) at a concentration of 300 mg/l to a total reaction volume of 25 ml [23]. The reaction mixtures were then incubated at temperatures ranging from 28 to 70°C with shaking for 1 h. To investigate the effect of pH on the D-hydantoinase activities of the biocatalysts, fixed amount of biocatalysts were added into predetermined amount of 50 mM Tris–HCl buffers containing D-pHPH (300 mg/l) at pH ranging from 7.0 to 9.0 to a total reaction volume of 25 ml. The reactions were conducted at 35°C with shaking for 1 h and then terminated by adding 0.2 ml of trichlorous acid (TCA, Wako Pure Chemical, Osaka, Japan). Biocatalysts were subsequently removed from the reaction mixtures by centrifugation. The cell-free supernatant was stored at − 20°C for further analysis.
2.1. Microorganism and growth conditions
2.4. Acti6ity assay
E. coli BL21(DE3) harbouring pET 36 encoding D-hydantoinase gene from P. putida was grown in 500 ml basal medium containing 100 mg/ml ampicillin in a 2-l Erlenmeyer flask at 37°C [23]. The expression of D-hydantoinase was induced by adding lactose into the culture to a final concentration of 5 mM when the optical density of the culture at 600 nm reached 1.0. The induced culture was harvested 3 h after the induction by centrifugation at 10 000× g for 10 min. The harvested pellets were subsequently resuspended in 85 ml of a mixture of basal medium and glycerol (50:50, v/v) and stored at −20°C.
2.2. Whole cell immobilization To immobilize the recombinant cells, 100 ml of the stored culture described above were washed with 50 mM Tris –HCl buffer, pH 8.5, and then resuspended in 1 ml 50 mM Tris–HCl buffer, pH 8.5. The cell paste was subsequently suspended in 100 ml of 50 mM, pH 8.5, Tris –HCl buffer containing 3% (w/v) sterilized low viscosity sodium alginate (Sigma). The mixture was then dropped into 500 ml of 8% calcium chloride solution through a 23-gauge syringe with moderate stirring and further incubated at 4°C with agitation for 3 h [25,26]. Spherical calcium alginate beads with diameters around 2 – 3 mm were obtained.
2.3. Biocatalyst reactions The effects of temperature and pH on the rate of N-carbamoyl-D-hydroxyphenylglycine production with
The D-hydantoinase activities of free cells and immobilized cells were analyzed by measuring the amount of N-carbamoyl-D-HPG produced from the enzyme reactions with high-performance liquid chromatography (HPLC) with a Jasco HPLC system (Tokyo, Japan) equipped with a C18 column (LiChroCART, 5 mm, 4.6×250 mm, Merck, NJ, USA). The solvent system consisted of mobile phase A (0.01%, v/v, phosphorous acid; TEDIA, Fairfield, OH) and mobile phase B (acetonitrile; TEDIA; [23,27]). For each assay, 20 ml of the cell-free sample was injected and eluted with 100% mobile phase A for 3 min followed by a gradient elution of 0–5% mobile phase B within 15 min at a flow rate of 0.5 ml/min at 35°C. The absorbance of the eluent was monitored with an UV detector at 220 nm. One unit of D-hydantoinase activity was defined as the amount of enzyme capable of converting 1 mmol of D-p-HPH to N-carbamoyl-D-HPG within 1 min under the reaction conditions.
3. Results and discussion
3.1. Production of N-carbamoyl-D -HPG with recombinant E. coli To access the D-hydantoinase activity of the recombinant E. coli cells, 3 ml of cell concentrate were washed with 50 mM Tris–HCl buffer, pH 8.5, and subsequently resuspended in 25 ml of 50 mM Tris–HCl
Y.-C. Chen et al. / Process Biochemistry 35 (1999) 285–290
buffer, pH 8.5, containing D-p-HPH at a concentration of 300 mg/l. The reaction mixture was incubated at 35°C with moderate agitation. Samples were taken periodically for quantitative analysis. The time course of the reaction is shown in Fig. 1. The reaction rate remained constant for the first 3 h and then gradually decreased. A conversion factor, defined as {[initial substrate concentration] −[residual substrate concentration]}/[initial substrate concentration]×100%, of 91% was achieved after a 24-h reaction, which is significantly higher than the level, 75.5%, reported for resting cells of Pseudomonas desmolyticum under similar reaction conditions [26]. A reaction time of 1 h was applied to all subsequent experiments, unless specified. Before accessing the optimal conditions for N-carbamoyl-D-HPG production, the effect of substrate concentration on reaction rate was investigated. The initial rates of enzyme reactions under the condition described above with substrate concentrations ranging from 50 to 500 mg/l were determined (Fig. 2). The reaction rate increased linearly with substrate concentration over the concentration range studied. Substrate at concentration above 500 mg/l was not included in this study because of the low water solubility of D-p-HPH. A substrate concentration of 300 mg/l was used for all subsequent experiments.
3.2. Whole cell immobilization The recombinant E. coli cells were immobilized by gel entrapment with calcium alginate. The effect of cell loads, the amount of cells immobilized within unit mass of calcium alginate determined by measuring the weight of lyophilized cells recovered from biocatalysts dissolved with 0.1 M sodium phosphate buffer, pH 7.0 [28], on reaction rate was investigated. Immobilized
Fig. 1. Reaction profile of D-p-hydroxyphenyl hydantoin hydrolysis with recombinant E. coli cells. Concentrations of substrate ( ) and product ( ) and conversion factors (), defined as [initial substrate concentration −residual substrate concentration]/[initial substrate concentration] ×100%, were determined over the course of the reaction. The reactions were conducted at pH 8.5 and 40°C.
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Fig. 2. Effect of initial substrate concentration on the rate of D-p-hydroxyphenyl hydantoin hydrolysis. The reactions were conducted at pH 8.5 and 40°C.
biocatalysts with cell loads ranging from 0.54 to 5.57 mg DCW/g bead were used. For each experiment, 1 g of beads was added into 30 ml of reaction mixture and incubated at pH 8.5 and 40°C. The time courses of N-carbamoyl-D-HPG production are shown in Fig. 3. It was observed that the rates of product formation increased with cell load. The specific activities, defined as the number of D-hydantoinase activity units per gram of DCW, of the four immobilized biocatalysts are shown in Table 1. Surprisingly, the specific activity of the immobilized biocatalysts increased with the cell load. For example, when the cell load of the immobilized cells was increased from 0.542 to 5.577 mg DCW/ g bead, the specific activity was increased from 0.35 to 2.33 IU/g DCW. One possible explanation for this phenomenon is that the porosity of the calcium alginate beads increases with cell load. The increase in porosity reduces mass transfer resistance and makes the immobilized cells more accessible to substrate for reaction.
Fig. 3. Effect of cell load on the D-hydantoinase activity of the immobilized E. coli cells. Cell loads of 0.54 ( ), 1.66 (), 2.77 (") and 5.58 ( ) mg DCW/g bead were used throughout the study.
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Table 1 Cell loads and specific activities of the immobilized recombinant E. coli with D-hydantoinase activity Immobilized systems
Cell load (mg DCW/g bead)
Specific activity (IU/g DCW)
Biocatalyst Biocatalyst Biocatalyst Biocatalyst
0. 542 1.657 2.773 5.577
0.353 0.772 1.602 2.333
A B C D
Conversion factors achieved at different periods with the four immobilized biocatalysts are shown in Table 2. The conversion factors obtained with the immobilized biocatalysts were much lower than those obtained with the free cell systems. The possible mechanisms for the reduction in D-hydantoinase activity upon immobilization are addressed in next session. A cell load of 2.77 mg DCW/g bead was used for all subsequent reactions involving immobilized biocatalysts systems.
3.3. En6ironmental effects on enzyme acti6ity To evaluate the optimal conditions for the production of N-carbamoyl-D-hydroxyphenylglycine using free-cell and immobilized systems, the effects of environmental factors such as pH and temperature were investigated. For both free-cell and immobilized systems hydantoinase activity increased with pH up to a maximum of pH 8.5 and declined thereafter (Fig. 4). However, the hydantoinase activity of the immobilized systems was significantly lower than that of the free-cell system. For example, at pH 8.5 the rate of N-carbamoyl-D-hydroxyphenylglycine production was 2.7 mM/h g DCW for the immobilized system, compared to 20.1 mM/h g DCW for the free-cell system. The exact mechanism for the reduction of D-hydantoinase activity of the recombinant cells upon immobilization is still unclear. Since the conditions used for cell entrapment with calcium alginate are relatively moderate, the large reduction in hydantoinase activity cannot be attributed solely to enzyme inactivation during the process of immobilization. The formation of cell aggregates
Table 2 Conversion of D-HPH into N-carbamoyl-D-HPG with immobilized E. coli with D-hydantoinase activity Conversion (%)
Biocatalyst A
Biocatalyst B
Biocatalyst C
Biocatalyst D
5.5 h 9h 13 h 24 h
0.11 90.01 0.21 9 0.02 0.46 9 0.08 3.45 9 0.02
0.58 90.00 1.4590.05 2.49 90.14 5.179 0.13
2.409 0.08 4.88 90.08 7.50 90.18 12.359 0.19
8.4490.48 14.229 0.07 19.089 0.16 25.769 0.25
Fig. 4. Effect of pH on the D-hydantoinase activities of the free ( ) and immobilized ( ) E. coli cells.
with cores inaccessible to the substrate within the calcium alginate beads might also cause the reduction of enzyme activity. From a process engineer’s point of view, it is desirable to conduct the bioconversion at elevated temperature due to the low water solubility of the substrate (D,L-HPH). At high temperature, the solubility of the substrate, and thus the reaction rate, will be increased. Furthermore, the level of microbial contamination can also be reduced by conducting the bioconversion at higher temperature. The effect of temperature on enzyme activity for both free cells and immobilized cells is shown in Fig. 5. In the free-cell systems, optimal hydantoinase activity was observed at 40°C, and further increase in reaction temperature led to a sharp reduction in reaction rate, possibly due to enzyme denaturation. The response of immobilized cells to temperature changes was sharply different from that of free cells. With immobilized cells as biocatalysts, the reaction rate increased with temperature over the range of temperature studied. For example, when the temperature was increased from 30 to 60°C, the reaction rate was increased by more than 100% from 2.27 to 4.76 mM/h g DCW. Apparently, the thermostability of D-hydantoinase in the recombinant cells was significantly increased upon immobilization. However, the reaction rate achieved at 60°C with immobilized biocatalysts was still much lower than that achieved at 35°C with the free cell systems, due to the inherited low activity of the immobilized systems.
Y.-C. Chen et al. / Process Biochemistry 35 (1999) 285–290
Although it is possible to conduct the enzyme reaction with immobilized biocatalysts at temperatures above 60°C, kinetic data obtained at high reaction temperature were not shown in this paper due to difficulties encountered in HPLC analysis. At elevated temperatures, interfering peaks overlapping with the product peak were observed on the HPLC chromatograms. The presence of these peaks makes the accurate quantification of product concentration very difficult. Results of HPLC analysis indicated that these interfering peaks might correspond to the thermal degradation products of the substrate, by-products of the bioconversion, and/or other compounds released due to the disintegration of the biocatalysts (data not shown). To conduct enzyme reaction at higher temperatures, it is, therefore, necessary to use matrixes that are more heat-resistant than calcium alginate.
3.4. Recycling of biocatalysts One of the major advantages of immobilized enzyme systems is the ease of recycling of the biocatalysts. The reusabilities of free cells and immobilized cells were compared. For both systems, fixed amount of biocatalysts was added into the 50 mM Tris – HCl buffer, pH 8.5, containing substrate at a concentration of 300 mg/l and incubated at 40 and/or 60°C with moderate agitation for 1 h. After each cycle, the biocatalysts were recovered by centrifugation and rinsed with fresh 50 mM Tris–HCl buffer, pH 8.5. The recovered biocatalysts were then used for subsequent reactions. The
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Fig. 6. Recycling experiment for free whole cell enzyme. Residual activity of the biocatalysts was defined as the [reaction rate of each run]/[reaction rate of the first run]×100%.
effect of recycling on the activity of the biocatalysts was shown in Fig. 6. Residual activity of the biocatalysts was defined as the [reaction rate of each run]/[reaction rate of the first run]×100%. Sharp decline in D-hydantoinase activity was observed for the free-cell systems. The mechanism for the sharp decline in D-hydantoinase activity during the first two cycles is still unclear. On the contrary, the reusability of the biocatalysts was significantly enhanced upon immobilization. For the immobilized systems at 40°C, the D-hydantoinase activity of the biocatalysts was essentially unchanged after two cycles. Even after six cycles, a residual activity of 69% was recovered for the immobilized systems. The effect of recycling on the activity of the immobilized cells at 60°C was also studied. Compared to the immobilized systems at 40°C, the stability of the immobilized cells was lower at 60°C. Nevertheless, the reusability of immobilized cells at 60°C was still higher than that of the free cell systems at 40°C. The decline in residual activity of the immobilized systems could be attributed to the denaturation of D-hydantoinase and the disintegration of the alginate beads. Both the level of D-hydantoinase denaturation and the degree of gel disintegration were more critical at high temperature.
4. Conclusions
Fig. 5. Effect of temperature on the D-hydantoinase activities of the free ( ) and immobilized ( ) E. coli cells.
The recombinant E. coli strain used in this study exhibited higher D-hydantoinase activity over the donor strain of the D-hydantoinase gene and other Pseudomonas sp. The E. coli cells exhibited an optimal D-hydantoinase activity at pH 8.5 and 40°C. However, on an industrial scale, it may be desirable to conduct the bioconversion at pH above 8.5, because under alkaline conditions the concentration of D-HPH can be increased significantly due to the increase in overall
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substrate solubility and the racemic conversion of L-HPH into D-HPH. [29]. While the optimal pH remained at 8.5 for the immobilized E. coli cells, the optimal temperature for D-hydantoinase activity of the E. coli cells was increased by at least 20 – 60°C upon immobilization. The reusability of the recombinant E. coli was also increased significantly upon immobilization. However, the specific D-hydantoinase activity of the immobilized cells was significantly lower than that of the free cells, probably due to diffusion limitation. It is, therefore, desirable to use immobilized enzyme systems or develop other matrices with lower mass transfer resistance and higher physical strength for cell entrapment. The use of permeabilized recombinant E. coli exhibiting lower mass transfer resistance for the production of N-carbamoylD-hydroxyphenylglycine is currently under investigation. Acknowledgements This work was supported by a grant, DOH88-HR-622, from the National Health Research Institute, Taiwan. References [1] Lapointe G, Viau S, Leblanc D, Robert N, Morin A. Cloning, sequencing, expression of the D-hydantoinase gene from Pseudomonas putida and other microorganisms. Appl Environ Microbiol 1994;60:888 – 95. [2] Syldatk C, Laufer A, Muller R, Hoke H. Production of optically pure d- and l-a-amino acids by bioconversion of d,l-5-monosubstituted hydantoin derivatives. In: Fiechter A, editor. Advances in Biocehmical Engineering/Biotechnology, vol. 41. Berlin: Springer, 1990:30 – 75. [3] Louwrier A, Knowlest CJ. The aim of industrial enzymic amoxycillin production: characterization of a novel carbamoylase enzyme in the form of a crude, cell-free extract. Biotechnol Appl Biochem 1997;25:143–9. [4] Yamada H, Takahashi S, Kii Y, Kumagai H. Distribution of hydantoin hydrolyzing activity in microorganisms. J Ferment Technol 1978;56:484–91. [5] Takahashi S, Kii Y, Kumagai H, Yamada H. Purification, crystallization and properties of hydantoinase from Pseudomonas striata. J Ferment Technol 1978;56:492–8. [6] Shimizu S, Shimada H, Takahashi S, Ohashi T, Yamada H. Synthesis of N-carbamoyl-D-2-thienylglycine and D-2-thienglycine by microbial hydantoinase. Agric Biol Chem 1980;44:2233 – 4. [7] Oliveri R, Fascetti E, Angelini L, Degen L. Microbial transformation of racemic hydantoins to D-amino acids. Biotechnol Bioeng 1981;23:2173–83. [8] Yokozeki K, Nakamori S, Eguchi C, Yamada K, Mitsugi K. Screening of microorganisms producing D-p-hydroxyphenylglycine from DL-5-(p-hydroxyphenyl) hydantoin. Agric Biol Chem 1987;51:355 – 62. [9] Runser S, Ohleyer E. Properties of the hydantoinase from Agrobacterium sp. IP I-671. Biotechnol Lett 1990;12:259– 64. [10] Runser S, Chinski N, Ohleyer E. D-p-Hydroxyphenylglycine production from D,L-5-p-hydroxyphenylhydantoin by Arthrobacterium sp. Appl Microbiol Biotechnol 1990;33:382–8.
[11] Moller A, Syldatk C, Schulze M, Wagner F. Stereo- and substrate-specificity of a hydantoinase and D-N-carbomyl amino acid amidohydrolase of Arthrobacter crystalloietes AM 2. Enzyme Microb Technol 1988;10:618 – 25. [12] Runser S, Meyer P. Purification and biochemical characterization of the hydantoin hydrolyzing enzyme from Agrobacterium species ahydantoinase with no 5,6-dihydropyrimidine amidohydrolase activity. Eur J Biochem 1993;213:1315– 24. [13] Meyer P, Runser S. Efficient production of the industrial biocatalysts hydantoinase and N-carbamoyl amino acid amidohydrolase: novel non-metabolizable inducers. Microbiol Biotechnol 1993;109:67 – 74. [14] Mukohara Y, Ishikawa T, Watabe K, Nakamura H. A thermostable hydantoinase of Bacillus stearothermophilus NS1122A: cloning, sequencing, and high expression of the enzyme gene, and some properties of the expressed enzyme. Biosci Biotechnol Biochem 1994;58:1621 – 6. [15] Lee SG, Lee DC, Hong SP, Sung MH. Thermostable D-hydantoinase from thermophilic Bacillus stearothermophilus SD-1: characteristics of purified enzyme. Appl Microbiol Biotechnol 1995;43:270 – 6. [16] Morin A, Leblanc A, Paleczek A, Hummel W, Kula MR. Comparison of seven microbial D-hydantoinase. J Biotechnol 1990;16:37 – 48. [17] Morin A, Hummel W, Kula MR. Production of hydantoinase from Pseudomonas fluorescens strain DSM 84. Appl Microbiol Biotechnol 1986;25:91 – 6. [18] Morin A, Hummel W, Kula MR. Rapid detection of microbial hydantoinase on solid medium. Biotechnol Lett 1986;8:573–6. [19] Morin A, Hummel E, Kula ER. Enrichment and selection of hydantoinase-producing microorganisms. J Gen Microbiol 1987;133:1201– 7. [20] Chavalier P, Roy D, Morin A. Hydantoinase activity of immobilized nongrowing Pseudomonas putida cells. Appl Microbiol Biotechnol 1989;30:482 – 6. [21] Kim DM, Kim HS. Enzymatic synthesis of D-p-hydroxyphenylglycine from DL-p-hydroxyphenylhydantoin. Enzyme Microb Technol 1993;15:530 – 4. [22] Gokhale DV, Bastawde KB, Patil SG, Kalkote UR, Joshi RR, Joshi RA, Ravindranathan T, Gaikwad BG, Jogdand VV, Nene D. Chemoenzymatic synthesis of D(− )phenylglycine using hydantoinase of Pseudomonas desmolyticum resting cells. Enzyme Microb Technol 1996;18:353 – 7. [23] Jih YL. Overexpression of Pseudomonas putida D-hydantoinase gene in Escherichia coli and Pseudomonas putida. MS thesis, National Chung Hsing University, Taichung, Taiwan, 1995. [24] Chien HR, Jih YL, Yang WY, Hsu WH. Identification of the open reading frame for the Pseudomonas putida D-hydantoinase gene and expression of the gene in Escherichia coli. Biochim Biophys Acta 1998;1395:68 – 77. [25] Fraser JE, Bickerstaff GF. Entrapment in calcium alginate. In: Bickerstaff GF, editor. Methods in Biotechnology, vol. 1:. Totowa, NJ: Humana Press, 1997:61 – 6. [26] Das N, Kayastha AM, Malhotra OP. Immobilization of ureas from pigeonpea (Cajanus cajan L.) in polyacrylamide gels and calcium alginate beads. Biotechnol Appl Biochem 1998;27:25–9. [27] Chien HCR, Hsu WH. Rapid and sensitive detection of D-hydantoinase producing microorganisms by using microtiter plate assay. Bioetchol Tech 1996;10:879 – 82. [28] Chibata I, Tosa T, Sato T. Methods of cell immobilization. In: Demain AL, Solomon NA, editors. Manual of Industrial Microbiology and Biotechnology, American Society for Microbiology, Washington, DC, 1986:217 – 29. [29] Dinelli D, Marconi E, Cecere F, Galli G, Morisi F. 2 A new method for the production of optically active amino acids. Enzyme Eng 1978;3:477 – 81.
Production of N-carbamoyl-D-hydroxyphenylglycine by D-hydantoinase activity of a recombinant Escherichia coli Yi-Chuan Chen a, Bang-Ding Yin a, Sung-Chyr Lin a,*, Wen-Hwei Hsu b a
Department of Chemical Engineering, National Chung Hsing Uni6ersity, Taichung 402, Taiwan b Institute of Molecular Biology, National Chung Hsing Uni6ersity, Taichung 402, Taiwan Received 7 April 1999; received in revised form 11 May 1999; accepted 16 May 1999
Abstract Recombinant Escherichia coli BL21 (DE3) harbouring plasmid pET36 encoding D-hydantoinase from Pseudomonas putida were used as biocatalyst for the production of N-carbamoyl-D-hydroxyphenylglycine from DL-p-hydroxyphenylhydantoin. The optimum D-hydantoinase activity was observed at 40°C and pH 8.5. At a substrate concentration of 300 mg/l, an initial reaction rate of 24.52 mM/h g cells was obtained and 91% of the substrate was converted into product after a 24-h reaction. Recombinant cells were immobilized within calcium alginate beads with diameters ranging from 2 to 3 mm. The specific activity of the immobilized cells increased with cell loads, probably due to reduced mass transfer resistance. The immobilized cells also exhibited an optimal pH of 8.5. However, under the conditions described above, the initial reaction rate with the immobilized cells as the biocatalysts was reduced by 87% to 3.19 mM/h g cells, probably due to the formation of cell aggregates inaccessible to substrate. Thermostability and reusability of D-hydantoinase were increased upon immobilization. The initial reaction rate with immobilized cells was increased with temperature at least up to 60°C. More than 95% of the D-hydantoinase activity was recovered after three cycles for the immobilized cells, compared to 22% for the free cell systems. © 1999 Elsevier Science Ltd. All rights reserved. Keywords: Calcium alginate;
D-Hydantoinase; D-p-Hydroxyphenylglycine;
1. Introduction D-Amino acids are important precursors for the production of many semi-synthetic antibiotics, such as amoxicillin, cefadroxil, cefatrizinen, cefaparole, and cefaperazon. Among b-lactam antibiotics, amoxicillin is one of the most widely used broad-spectrum antibiotics because of limited bacterial resistance. Amoxicillin is produced by the reaction of 6-amino penicillanic acid (6-APA), derived from the catalytic degradation of penicillin with penicillin acylase, and D-p-hydroxyphenylglycine (D-p-HPG) [1 – 3]. D-p-HPG can be produced from DL-5-substituted hydantoins via chemical processes or biological processes. Although the chemical processes for the synthesis of D-p-HPG are well established [4], the application of a chemical process for industrial production of D-p-
* Corresponding author. Tel.: +886-4-2854724, ext. 104; fax: + 886-4-2854734. E-mail address: [email protected] (S.-C. Lin)
Escherichia coli; Immobilization
HPG has been challenged due to its low product yield, low product purity, and the use of a large quantity of organic solvent. A chemo-enzymic process for the production of D-p-HPG from DL-5-substituted hydantoins has been proposed by Yamada and co-workers [4–6]. In this process, DL-5-substituted hydantoin is asymmetrically hydrolyzed to N-carbamoyl-D-amino acid by D-specific hydantoinase and this compound is further converted chemically to the corresponding D-amino acid under acidic conditions. Alternatively, N-carbamoyl-D-amino acids can be enzymically cleaved to D-p-HPG with carbamoylase (EC 3.5.1.6) [7–10]. Compared to the chemical processes, the enzymic process has the advantages of high product yield, high optical purity and low environmental impacts, and, therefore, has attracted extensive interest in D-hydantoinases and carbamoylase. D-Hydantoinases are identical to dihydropyrimidinases (EC 3.5.2.2) that catalyze the hydrolytic ring opening of dihydropyrimidines to N-carbamoyl-b-
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amino acids and of DL-5-substituted hydantoins to the corresponding N-carbamoyl-D-amino acids [15]. D-Hydantoinases are widely distribute among bacteria, including Agrobacterium sp. [7,11 – 13], Arthrobacterium sp. [9,10], Bacillus sp. [14,15], Corynebacterium sp. [16], and Pseudomonas sp. [5,16 – 22]. However, the D-hydantoinase activity obtained in these wild-type strains is relatively low. It is, therefore, desirable to enhance the production of D-hydantoinase via genetic engineering. A recombinant E. coli-harbouring plasmid containing Dhydantoinase gene from P. putida has recently been constructed [23,24]. Under the control of T5lac promoter and lactose induction, the recombinant E. coli exhibited a D-hydantoinase activity about 20-fold higher than that of the gene donor strain [24]. In this study, the use of the recombinant E. coli cells exhibiting D-hydantoinase activity as biocatalysts for the production of N-carbamoyl-D-hydroxyphenylglycine from DL-p-hydroxyphenylhydantoin was systematically investigated. 2. Materials and methods
both free cells and immobilized cells as biocatalysts were investigated. To study the effect of temperature on the D-hydantoinase activities of the biocatalysts, predetermined amount of biocatalysts, 3 ml of cell concentrate for the free-cell systems, and 3 g of calcium alginate beads for the immobilized systems, were respectively added into 50 mM Tris–HCl buffer, pH 8.5, containing D-p-hydroxyphenyl hydantoin (D-p-HPH, Tokyo Chemicals, Tokyo, Japan) at a concentration of 300 mg/l to a total reaction volume of 25 ml [23]. The reaction mixtures were then incubated at temperatures ranging from 28 to 70°C with shaking for 1 h. To investigate the effect of pH on the D-hydantoinase activities of the biocatalysts, fixed amount of biocatalysts were added into predetermined amount of 50 mM Tris–HCl buffers containing D-pHPH (300 mg/l) at pH ranging from 7.0 to 9.0 to a total reaction volume of 25 ml. The reactions were conducted at 35°C with shaking for 1 h and then terminated by adding 0.2 ml of trichlorous acid (TCA, Wako Pure Chemical, Osaka, Japan). Biocatalysts were subsequently removed from the reaction mixtures by centrifugation. The cell-free supernatant was stored at − 20°C for further analysis.
2.1. Microorganism and growth conditions
2.4. Acti6ity assay
E. coli BL21(DE3) harbouring pET 36 encoding D-hydantoinase gene from P. putida was grown in 500 ml basal medium containing 100 mg/ml ampicillin in a 2-l Erlenmeyer flask at 37°C [23]. The expression of D-hydantoinase was induced by adding lactose into the culture to a final concentration of 5 mM when the optical density of the culture at 600 nm reached 1.0. The induced culture was harvested 3 h after the induction by centrifugation at 10 000× g for 10 min. The harvested pellets were subsequently resuspended in 85 ml of a mixture of basal medium and glycerol (50:50, v/v) and stored at −20°C.
2.2. Whole cell immobilization To immobilize the recombinant cells, 100 ml of the stored culture described above were washed with 50 mM Tris –HCl buffer, pH 8.5, and then resuspended in 1 ml 50 mM Tris–HCl buffer, pH 8.5. The cell paste was subsequently suspended in 100 ml of 50 mM, pH 8.5, Tris –HCl buffer containing 3% (w/v) sterilized low viscosity sodium alginate (Sigma). The mixture was then dropped into 500 ml of 8% calcium chloride solution through a 23-gauge syringe with moderate stirring and further incubated at 4°C with agitation for 3 h [25,26]. Spherical calcium alginate beads with diameters around 2 – 3 mm were obtained.
2.3. Biocatalyst reactions The effects of temperature and pH on the rate of N-carbamoyl-D-hydroxyphenylglycine production with
The D-hydantoinase activities of free cells and immobilized cells were analyzed by measuring the amount of N-carbamoyl-D-HPG produced from the enzyme reactions with high-performance liquid chromatography (HPLC) with a Jasco HPLC system (Tokyo, Japan) equipped with a C18 column (LiChroCART, 5 mm, 4.6×250 mm, Merck, NJ, USA). The solvent system consisted of mobile phase A (0.01%, v/v, phosphorous acid; TEDIA, Fairfield, OH) and mobile phase B (acetonitrile; TEDIA; [23,27]). For each assay, 20 ml of the cell-free sample was injected and eluted with 100% mobile phase A for 3 min followed by a gradient elution of 0–5% mobile phase B within 15 min at a flow rate of 0.5 ml/min at 35°C. The absorbance of the eluent was monitored with an UV detector at 220 nm. One unit of D-hydantoinase activity was defined as the amount of enzyme capable of converting 1 mmol of D-p-HPH to N-carbamoyl-D-HPG within 1 min under the reaction conditions.
3. Results and discussion
3.1. Production of N-carbamoyl-D -HPG with recombinant E. coli To access the D-hydantoinase activity of the recombinant E. coli cells, 3 ml of cell concentrate were washed with 50 mM Tris–HCl buffer, pH 8.5, and subsequently resuspended in 25 ml of 50 mM Tris–HCl
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buffer, pH 8.5, containing D-p-HPH at a concentration of 300 mg/l. The reaction mixture was incubated at 35°C with moderate agitation. Samples were taken periodically for quantitative analysis. The time course of the reaction is shown in Fig. 1. The reaction rate remained constant for the first 3 h and then gradually decreased. A conversion factor, defined as {[initial substrate concentration] −[residual substrate concentration]}/[initial substrate concentration]×100%, of 91% was achieved after a 24-h reaction, which is significantly higher than the level, 75.5%, reported for resting cells of Pseudomonas desmolyticum under similar reaction conditions [26]. A reaction time of 1 h was applied to all subsequent experiments, unless specified. Before accessing the optimal conditions for N-carbamoyl-D-HPG production, the effect of substrate concentration on reaction rate was investigated. The initial rates of enzyme reactions under the condition described above with substrate concentrations ranging from 50 to 500 mg/l were determined (Fig. 2). The reaction rate increased linearly with substrate concentration over the concentration range studied. Substrate at concentration above 500 mg/l was not included in this study because of the low water solubility of D-p-HPH. A substrate concentration of 300 mg/l was used for all subsequent experiments.
3.2. Whole cell immobilization The recombinant E. coli cells were immobilized by gel entrapment with calcium alginate. The effect of cell loads, the amount of cells immobilized within unit mass of calcium alginate determined by measuring the weight of lyophilized cells recovered from biocatalysts dissolved with 0.1 M sodium phosphate buffer, pH 7.0 [28], on reaction rate was investigated. Immobilized
Fig. 1. Reaction profile of D-p-hydroxyphenyl hydantoin hydrolysis with recombinant E. coli cells. Concentrations of substrate ( ) and product ( ) and conversion factors (), defined as [initial substrate concentration −residual substrate concentration]/[initial substrate concentration] ×100%, were determined over the course of the reaction. The reactions were conducted at pH 8.5 and 40°C.
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Fig. 2. Effect of initial substrate concentration on the rate of D-p-hydroxyphenyl hydantoin hydrolysis. The reactions were conducted at pH 8.5 and 40°C.
biocatalysts with cell loads ranging from 0.54 to 5.57 mg DCW/g bead were used. For each experiment, 1 g of beads was added into 30 ml of reaction mixture and incubated at pH 8.5 and 40°C. The time courses of N-carbamoyl-D-HPG production are shown in Fig. 3. It was observed that the rates of product formation increased with cell load. The specific activities, defined as the number of D-hydantoinase activity units per gram of DCW, of the four immobilized biocatalysts are shown in Table 1. Surprisingly, the specific activity of the immobilized biocatalysts increased with the cell load. For example, when the cell load of the immobilized cells was increased from 0.542 to 5.577 mg DCW/ g bead, the specific activity was increased from 0.35 to 2.33 IU/g DCW. One possible explanation for this phenomenon is that the porosity of the calcium alginate beads increases with cell load. The increase in porosity reduces mass transfer resistance and makes the immobilized cells more accessible to substrate for reaction.
Fig. 3. Effect of cell load on the D-hydantoinase activity of the immobilized E. coli cells. Cell loads of 0.54 ( ), 1.66 (), 2.77 (") and 5.58 ( ) mg DCW/g bead were used throughout the study.
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Table 1 Cell loads and specific activities of the immobilized recombinant E. coli with D-hydantoinase activity Immobilized systems
Cell load (mg DCW/g bead)
Specific activity (IU/g DCW)
Biocatalyst Biocatalyst Biocatalyst Biocatalyst
0. 542 1.657 2.773 5.577
0.353 0.772 1.602 2.333
A B C D
Conversion factors achieved at different periods with the four immobilized biocatalysts are shown in Table 2. The conversion factors obtained with the immobilized biocatalysts were much lower than those obtained with the free cell systems. The possible mechanisms for the reduction in D-hydantoinase activity upon immobilization are addressed in next session. A cell load of 2.77 mg DCW/g bead was used for all subsequent reactions involving immobilized biocatalysts systems.
3.3. En6ironmental effects on enzyme acti6ity To evaluate the optimal conditions for the production of N-carbamoyl-D-hydroxyphenylglycine using free-cell and immobilized systems, the effects of environmental factors such as pH and temperature were investigated. For both free-cell and immobilized systems hydantoinase activity increased with pH up to a maximum of pH 8.5 and declined thereafter (Fig. 4). However, the hydantoinase activity of the immobilized systems was significantly lower than that of the free-cell system. For example, at pH 8.5 the rate of N-carbamoyl-D-hydroxyphenylglycine production was 2.7 mM/h g DCW for the immobilized system, compared to 20.1 mM/h g DCW for the free-cell system. The exact mechanism for the reduction of D-hydantoinase activity of the recombinant cells upon immobilization is still unclear. Since the conditions used for cell entrapment with calcium alginate are relatively moderate, the large reduction in hydantoinase activity cannot be attributed solely to enzyme inactivation during the process of immobilization. The formation of cell aggregates
Table 2 Conversion of D-HPH into N-carbamoyl-D-HPG with immobilized E. coli with D-hydantoinase activity Conversion (%)
Biocatalyst A
Biocatalyst B
Biocatalyst C
Biocatalyst D
5.5 h 9h 13 h 24 h
0.11 90.01 0.21 9 0.02 0.46 9 0.08 3.45 9 0.02
0.58 90.00 1.4590.05 2.49 90.14 5.179 0.13
2.409 0.08 4.88 90.08 7.50 90.18 12.359 0.19
8.4490.48 14.229 0.07 19.089 0.16 25.769 0.25
Fig. 4. Effect of pH on the D-hydantoinase activities of the free ( ) and immobilized ( ) E. coli cells.
with cores inaccessible to the substrate within the calcium alginate beads might also cause the reduction of enzyme activity. From a process engineer’s point of view, it is desirable to conduct the bioconversion at elevated temperature due to the low water solubility of the substrate (D,L-HPH). At high temperature, the solubility of the substrate, and thus the reaction rate, will be increased. Furthermore, the level of microbial contamination can also be reduced by conducting the bioconversion at higher temperature. The effect of temperature on enzyme activity for both free cells and immobilized cells is shown in Fig. 5. In the free-cell systems, optimal hydantoinase activity was observed at 40°C, and further increase in reaction temperature led to a sharp reduction in reaction rate, possibly due to enzyme denaturation. The response of immobilized cells to temperature changes was sharply different from that of free cells. With immobilized cells as biocatalysts, the reaction rate increased with temperature over the range of temperature studied. For example, when the temperature was increased from 30 to 60°C, the reaction rate was increased by more than 100% from 2.27 to 4.76 mM/h g DCW. Apparently, the thermostability of D-hydantoinase in the recombinant cells was significantly increased upon immobilization. However, the reaction rate achieved at 60°C with immobilized biocatalysts was still much lower than that achieved at 35°C with the free cell systems, due to the inherited low activity of the immobilized systems.
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Although it is possible to conduct the enzyme reaction with immobilized biocatalysts at temperatures above 60°C, kinetic data obtained at high reaction temperature were not shown in this paper due to difficulties encountered in HPLC analysis. At elevated temperatures, interfering peaks overlapping with the product peak were observed on the HPLC chromatograms. The presence of these peaks makes the accurate quantification of product concentration very difficult. Results of HPLC analysis indicated that these interfering peaks might correspond to the thermal degradation products of the substrate, by-products of the bioconversion, and/or other compounds released due to the disintegration of the biocatalysts (data not shown). To conduct enzyme reaction at higher temperatures, it is, therefore, necessary to use matrixes that are more heat-resistant than calcium alginate.
3.4. Recycling of biocatalysts One of the major advantages of immobilized enzyme systems is the ease of recycling of the biocatalysts. The reusabilities of free cells and immobilized cells were compared. For both systems, fixed amount of biocatalysts was added into the 50 mM Tris – HCl buffer, pH 8.5, containing substrate at a concentration of 300 mg/l and incubated at 40 and/or 60°C with moderate agitation for 1 h. After each cycle, the biocatalysts were recovered by centrifugation and rinsed with fresh 50 mM Tris–HCl buffer, pH 8.5. The recovered biocatalysts were then used for subsequent reactions. The
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Fig. 6. Recycling experiment for free whole cell enzyme. Residual activity of the biocatalysts was defined as the [reaction rate of each run]/[reaction rate of the first run]×100%.
effect of recycling on the activity of the biocatalysts was shown in Fig. 6. Residual activity of the biocatalysts was defined as the [reaction rate of each run]/[reaction rate of the first run]×100%. Sharp decline in D-hydantoinase activity was observed for the free-cell systems. The mechanism for the sharp decline in D-hydantoinase activity during the first two cycles is still unclear. On the contrary, the reusability of the biocatalysts was significantly enhanced upon immobilization. For the immobilized systems at 40°C, the D-hydantoinase activity of the biocatalysts was essentially unchanged after two cycles. Even after six cycles, a residual activity of 69% was recovered for the immobilized systems. The effect of recycling on the activity of the immobilized cells at 60°C was also studied. Compared to the immobilized systems at 40°C, the stability of the immobilized cells was lower at 60°C. Nevertheless, the reusability of immobilized cells at 60°C was still higher than that of the free cell systems at 40°C. The decline in residual activity of the immobilized systems could be attributed to the denaturation of D-hydantoinase and the disintegration of the alginate beads. Both the level of D-hydantoinase denaturation and the degree of gel disintegration were more critical at high temperature.
4. Conclusions
Fig. 5. Effect of temperature on the D-hydantoinase activities of the free ( ) and immobilized ( ) E. coli cells.
The recombinant E. coli strain used in this study exhibited higher D-hydantoinase activity over the donor strain of the D-hydantoinase gene and other Pseudomonas sp. The E. coli cells exhibited an optimal D-hydantoinase activity at pH 8.5 and 40°C. However, on an industrial scale, it may be desirable to conduct the bioconversion at pH above 8.5, because under alkaline conditions the concentration of D-HPH can be increased significantly due to the increase in overall
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substrate solubility and the racemic conversion of L-HPH into D-HPH. [29]. While the optimal pH remained at 8.5 for the immobilized E. coli cells, the optimal temperature for D-hydantoinase activity of the E. coli cells was increased by at least 20 – 60°C upon immobilization. The reusability of the recombinant E. coli was also increased significantly upon immobilization. However, the specific D-hydantoinase activity of the immobilized cells was significantly lower than that of the free cells, probably due to diffusion limitation. It is, therefore, desirable to use immobilized enzyme systems or develop other matrices with lower mass transfer resistance and higher physical strength for cell entrapment. The use of permeabilized recombinant E. coli exhibiting lower mass transfer resistance for the production of N-carbamoylD-hydroxyphenylglycine is currently under investigation. Acknowledgements This work was supported by a grant, DOH88-HR-622, from the National Health Research Institute, Taiwan. References [1] Lapointe G, Viau S, Leblanc D, Robert N, Morin A. Cloning, sequencing, expression of the D-hydantoinase gene from Pseudomonas putida and other microorganisms. Appl Environ Microbiol 1994;60:888 – 95. [2] Syldatk C, Laufer A, Muller R, Hoke H. Production of optically pure d- and l-a-amino acids by bioconversion of d,l-5-monosubstituted hydantoin derivatives. In: Fiechter A, editor. Advances in Biocehmical Engineering/Biotechnology, vol. 41. Berlin: Springer, 1990:30 – 75. [3] Louwrier A, Knowlest CJ. The aim of industrial enzymic amoxycillin production: characterization of a novel carbamoylase enzyme in the form of a crude, cell-free extract. Biotechnol Appl Biochem 1997;25:143–9. [4] Yamada H, Takahashi S, Kii Y, Kumagai H. Distribution of hydantoin hydrolyzing activity in microorganisms. J Ferment Technol 1978;56:484–91. [5] Takahashi S, Kii Y, Kumagai H, Yamada H. Purification, crystallization and properties of hydantoinase from Pseudomonas striata. J Ferment Technol 1978;56:492–8. [6] Shimizu S, Shimada H, Takahashi S, Ohashi T, Yamada H. Synthesis of N-carbamoyl-D-2-thienylglycine and D-2-thienglycine by microbial hydantoinase. Agric Biol Chem 1980;44:2233 – 4. [7] Oliveri R, Fascetti E, Angelini L, Degen L. Microbial transformation of racemic hydantoins to D-amino acids. Biotechnol Bioeng 1981;23:2173–83. [8] Yokozeki K, Nakamori S, Eguchi C, Yamada K, Mitsugi K. Screening of microorganisms producing D-p-hydroxyphenylglycine from DL-5-(p-hydroxyphenyl) hydantoin. Agric Biol Chem 1987;51:355 – 62. [9] Runser S, Ohleyer E. Properties of the hydantoinase from Agrobacterium sp. IP I-671. Biotechnol Lett 1990;12:259– 64. [10] Runser S, Chinski N, Ohleyer E. D-p-Hydroxyphenylglycine production from D,L-5-p-hydroxyphenylhydantoin by Arthrobacterium sp. Appl Microbiol Biotechnol 1990;33:382–8.
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