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Production of d -amino acid precursors with permeabilized recombinant Escherichia coli with d -hydantoinase activity
Process Biochemistry 35 (2000) 915 – 921 www.elsevier.com/locate/procbio
Production of D-amino acid precursors with permeabilized recombinant Escherichia coli with D-hydantoinase activity Bang-Ding Yin a, Yi-Chuan Chen 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 18 August 1999; received in revised form 18 October 1999; accepted 27 November 1999
Abstract Recombinant Escherichia coli cells expressing D-hydantoinase were used as the biocatalysts for the production of N-carbamoylfrom DL-p-hydroxyphenylhydantoin. Although high concentrations of DMSO could lead to enzyme denaturation, in the presence of 1.5% DMSO, the rate of product formation increased by more than 80% due to enhanced permeability of the cell membrane and increased substrate concentration. Reduced mass transfer resistance, achieved by the permeabilization of cell membrane with CTAB and glutaraldehyde, led to a 60% increase in the rate of production. However, in addition to causing a shift of optimal pH toward lower pH, permeabilization of the cell membrane resulted in reduced enzyme stability toward thermal and organic denaturation. Nevertheless, the stability of the D-hydantoinase of the recombinant cells toward pH, temperature and organic solvents can be significantly enhanced by immobilization. © 2000 Elsevier Science Ltd. All rights reserved.
D-hydroxyphenylglycine
Keywords: Escherichia coli; Immobilization; Permeabilization;
D-hydantoinase;
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]. A chemo-enzymatic process for the production of D-p-HPG from DL-5-substituted hydantoins has been proposed by Yamada and coworkers [4 – 6]. In this process, DL-5-substituted hydantoins were asymmetrically hydrolyzed to N-carbamoyl-D-amino acids by Dspecific hydantoinase, and these compounds were
* Corresponding author. Tel.: +886-4-2854724 ext. 104; fax: + 886-4-2854734. E-mail address: [email protected] (S.-C. Lin)
Calcium alginate
further chemically converted to the corresponding Damino acid under acidic conditions. Alternatively, Ncarbamoyl-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. A recombinant Escherichia coli containing a plasmid expressing a D-hydantoinase gene from P. putida has recently been constructed [11,12]. The recombinant E. coli exhibited a D-hydantoinase activity about 20-fold higher than that of the gene donor strain [12]. In a previous study, the use of recombinant E. coli cells exhibiting D-hydantoinase activity as biocatalysts for the production of N-carbamoyl-D-hydroxyphenylglycine (N-carbamoyl-HPG) from DL-p-hydroxwas systematically yphenylhydantoin (D-HPH) investigated [13]. Although it has been shown that the recombinant E.coli cells exhibits high D-hydantoinase activity and enhanced thermal stability and reusability
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upon immobilization, the rate of D-carbamoyl-HPG production is low, due to low substrate solubility and high substrate mass-transfer resistance. Although the D-hydantoinase activity of the recombinant E. coli is relatively high compared to the donor strain, the rate of D-carbamoyl-HPG production is limited by the low water solubility of the substrate (9–13 mg/ml), DL-HPH [14]. It is, therefore, desirable to develop reaction systems that are capable of enhancing reaction rate by providing higher concentration of substrate. DMSO is an excellent solvent for the substrate. The solubility of DL-HPH in DMSO is estimated at about 250 mg/ml. Kim and Kim [15] have investigated the effect of organic solvents on the production of D-HPG with bacterial strain KBEL 101 expressing D-hydantoinase and reported that in the presence of 5% DMSO the relative D-hydantoinase activity of the cells is increased by twofold. To reduce the mass transfer resistance of substrate in the production of 6-APA with recombinant E. coli expressing penicillin acylase, SivaRaman and coworkers [16] reported the successful permeabilization of E. coli cell membrane and cell wall with N-cetyl-N,N,N-trimethylammonium bromide (CTAB). We, therefore, conducted a systematic investigation on the effect of DMSO on the enzymic activity of recombinant E. coli and the effect of membrane permeability on the rate of D-carbomyl-HPG production.
2. Materials and methods
2.1. Microorganism and growth conditions E. coli BL21(DE3) harbouring pET 36 encoding Dhydantoinase 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 [11,13]. 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 by centrifugation at 10 000×g for 10 min 5 h after induction. 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 One hundred mililitres of the stored culture described above was washed with 50 mM Tris –HCl buffer pH 8.5 and resuspended in 1 ml 50 mM Tris– HCl buffer pH 8.5. The cell paste was then 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 [13,17,18].
2.3. Permeablization of cells The method developed by Prabhune et al. for the permeabilization of cell was adopted [16]. Cells suspended in 50 mM potassium phosphate buffer, pH 7.8 containing 0.1% (w/v) CTAB (Sigma) were incubated at 4°C for 30 min with moderate agitation. The CTAB-treated cells, recovered by centrifugation, were then resuspended in 50 mM potassium phosphate buffer, pH 7.8 containing 1% (w/v) glutaraldehyde and incubated at room temperature for 1 h. The cells were subsequently recovered by centrifugation and rinsed with 50 mM potassium phosphate buffer, pH 7.8.
2.4. Biocatalyst reactions Predetermined amounts of the biocatalysts, 3 ml of cell concentrate for the free-cell systems and 3 g of calcium alginate beads for the immobilized systems, were added to 50 mM Tris–HCl buffer at pH values ranging from 5.5 to 8.5 to a total reaction volume of 25 ml [13]. Unless specified otherwise, the concentration of DL-p-HPH (Tokyo Chemicals, Tokyo, Japan) was 750 mg/l. In some experiments 0.5–10% (v/v) of DMSO was added to the reaction mixtures to investigate the effect of DMSO on enzyme activity. The reaction mixtures were then incubated for 1 h at temperatures ranging from 30 to 70°C. The reactions were 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.
2.5. Acti6ity assay The D-hydantoinase activities of biocatalysts were determined by measuring the amount of N-carbamoyl-D-HPG produced from the enzyme reactions with high- performance liquid chromatography (HPLC) using a Jasco HPLC system (Tokyo, Japan) equipped with a C18 column (LiChroCART, 5 mm, 4.6×250 mm, Merck, NJ). Gradient elutions with a mobile phase system consisting of 0.01% (v/v) phosphorous acid (TEDIA) acetonitrile (TEDIA) at 35°C were used [13,19]. The absorbance of the eluent was monitored with an UV detector at 220 nm.
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3. Results and discussion
3.1. Effect of DMSO on
D -hydantoinase
acti6ity
The effect of DMSO concentration on the D-hydantoinase activity of the recombinant E. coli was investigated by comparing the rates of D-carbamoyl-HPG production in the presence of DMSO at concentrations ranging from 0 to 10% (v/v) with a substrate concentration of 350 mg/l, Fig. 1. It was observed that in the presence of 0.5% DMSO the specific reaction rate, defined as the rate of D-carbamoyl-HPG production per gram of biocatalysts (dry cell weight), was increased by more than 10% from 17.0 to 18.9 mM/h.gDCW. The increase in reaction rate is believed to be a result of enhanced substrate availability to the enzyme. In the presence of DMSO the structural integrity of the cell membrane is disturbed, and this led to enhanced substrate permeability and thus higher reaction rate. This result is contradictory to that reported by Kim and Kim [15] who have found that the effect of DMSO on cell permeability to be negligible. The difference might result from differences in membrane structure between bacterial strain KBEL 101 and the recombinant E.coli. In the presence of 2% DMSO the specific reaction rates declined to 17.0 mM/h.gDCW, equal to the value obtained in the absence of DMSO. The slight decrease in specific reaction rate might have resulted from the denaturation of enzyme. In addition to enhancement in permeability of the cell membrane, DMSO at elevated concentration might alter the conformation of protein
Fig. 1. The effect of DMSO concentration on the D-hydantoinase activity of () untreated recombinant cells, ( ) permeabilized cells, and () immobilized permeabilized cells. Experiments were conducted at pH 7.0 and a temperature of 35°C with a substrate concentraion of 300 mg/l. Activity of the bioctalaysts was accessed by determining the initial specific reaction rate, the rate of production formation per gram of dry cell weight or beads within the first hour.
Fig. 2. Optimal DMSO concentration for the solubilization of substrate and the production of D-carbamoyl-HPG with whole cell biocatalysts exhibiting D-hydantoinase. The reaction mixtures were prepared by mixing aqueous buffer containing saturated D-HPH and DMSO containing D-HPH at a concentration of 250 mg/ml at various ratios. The reactions were conducted at pH 7.0 and a temperature of 35°C.
molecules and thus reduce the activity of the enzyme. The increase in specific reaction rate observed at low DMSO concentration due to enhanced substrate availability was probably balanced by the decline in specific reaction rate at higher DMSO concentration due to enzyme denaturation. At DMSO concentrations above 2%, the denaturation of enzyme became the dominating factor affecting the overall activity of the biocatalysts and led to a significant decline in D-hydantoniase activity. For example, in the presence 10% DMSO the specific reaction rate dropped by more than 50% to 6.9 mM/h.gDCW (Fig. 1). The stability of D-hydantoinase activity of the recombinant E. coli toward DMSO was apparently lower than that of KBEL 101 which exhibits maximal activity in the presence of 5% DMSO [15]. In addition to membrane permeability and enzyme stability, DMSO can also affect the rate of N-carbamoyl-HPG formation by increasing the solubility and thus the concentration of substrate in the reaction mixture. Therefore, even though the optimal DMSO concentration for D-hydantoinase activity of the recombinant E. coli was 0.5%, the optimal DMSO concentration for the production of N-carbamoyl-D-HPG may be higher. To determine the optimal DMSO concentration for N-carbamoyl-D-HPG production, the rates of product formation were compared for reaction mixtures containing different concentrations of DMSO and thus substrate, prepared by mixing aqueous buffer containing saturated D-HPH and DMSO containing 250 mg/
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ml D-HPH at various ratios, Fig. 2. The optimal specific reaction rate, 41.9 mM/h.gDCW, was observed in the presence of 1.5% DMSO. This value was at least 70% higher than that obtained in the absence of DMSO. At higher DMSO concentration, the advantage of higher substrate concentration was offset by the decline in enzyme activity due to denaturation by DMSO.
3.2. En6ironmental effects on
D -hydantoinase
acti6ity
The effect of pH on the D-hydantoinase activity of the recombinant E. coli is shown in Fig. 3. The optimal pH in reaction systems containing 0.5% DMSO shifted from 8.5 to 7.5 [13]. At pHs ranging from 7.0 to 8.0, the specific reaction rates were above 30 mM/h.gDCW. When the pH was increased to 8.5, the specific reaction rate declined sharply to 30.0 mM/h.gDCW. Since it has been reported that the pKa of a Tris buffer system is not affected by the presence of organic solvent [20], the shift in optimal pH could have resulted from the decrease in water activity and/or the change in the cellular environment where the enzyme located. The effect of temperature on the D-hydantoinase activity of the recombinant E. coli is shown in Fig. 4. The thermal stability of the recombinant E. coli was not significantly affected by DMSO. As in DMSO-free systems [13], the maximal reaction rate in the presence of 0.5% DMSO was observed at 40°C. When the reaction temperature was increased from 35 to 40°C, the specific reaction rate was increased from 29.8 to 38.2 mM/h.gDCW. Further increase in reaction temperature resulted in sharp decline in specific reaction rate, probably due to the thermal denaturation of D-hydantoinase.
Fig. 3. The effect of pH on the D-hydantoinase activity of () untreated recombinant cells, ( ) permeabilized cells, and () immobilized permeabilized cells. The reactions were conducted at a temperature of 35°C with a substrate concentration of 750 mg/l in the presence of 0.5% DMSO.
Fig. 4. The effect of temperature on the D-hydantoinase activity of ()untreated recombinant cells, ( ) permeabilized cells, and () immobilized permeabilized cells. The reactions were conducted at pH 7.0 with a substrate concentration of 750 mg/l in the presence of 0.5% DMSO.
3.3. Effect of permeabilization on acti6ity
D -hydantoinase
One of the major disadvantages of using immobilized whole cells as biocatalysts has been the diffusional limitation of the substrate. SivaRaman and coworkers [16] have reported the use of CTAB and glutaraldehyde for enhancing the permeability of E. coli cells with penicillin acylase activity. Since the permeabilization of cell membrane with CTAB might lead to the leakage of active enzymes from cells, glutaraldehyde was used for the fixation of enzymes by chemical crosslinking. The effect of permeabilization with CTAB and glutaraldehyde on the activity of the recombinant E. coli with D-hydantoinase activity was investigated. The kinetic profiles of D-carbamoyl-HPG production with permeabilized cells and untreated cells were compared (Fig. 5). It was observed that the specific reaction rate was increased by more than 60% from 23.4 to 38.0 mM/h.gDCW with the permeabilized cells. The increase in specific reaction rate can be attributed to the reduced substrate mass transfer resistance across cell membrane. However, it should be pointed out that crosslinking with glutaraldehyde cannot only effectively fix enzyme molecules within cell membrane but also lead to extensive denaturation of enzymes. The observed increase in specific reaction rate was in fact a combined result of enhanced mass transfer and reduced enzyme activity. Therefore, it is proposed that if non-denaturing or more gentle processes for enzyme fixation can be developed, the effect of permeabilization with CTAB on rate of D-carbamoyl-HPG will be even more significant.
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min in Fig. 6a, was significantly reduced in the reaction product obtained with permeabilized cells (Fig. 6b). The permeabilization of cells with CTAB effectively kills the cells and thus eliminates the production of contaminating extracellular metabolites.
3.4. En6ironmental effects on of permeabilized cells
Fig. 5. The rates of product formation with untreated cells () and permeabilized cells ( ). The reactions were conducted at pH 7.0 and at a temperature of 35°C with a substrate concentration of 750 mg/l in the presence of 0.5% DMSO.
In addition to enhanced reaction rate, the use of permabilized cells as biocatalysts has the advantage of reduced cell viability. One of the problems in employing whole cells as biocatalysts is the formation of extracellular metabolites that may lead to product contamination. The HPLC chromatograms of reaction products with permeabilized cells and untreated cells are shown in Fig. 6. The concentration of byproducts, extracellular metabolites, secreted by the recombinant cells during the course of the reaction, eluted between 17 and 22
D -hydantoinase
acti6ity
The effect of pH on the D-hydantoinase activity of the permeabilized E. coli cells is also shown in Fig. 3. Under the same reaction condition, the optimal pH for the permeabilized cells shifted from 7.5 to 6.5. The specific reaction rate for the permeabilized cells at pH 6.5, 45.9 mM/h.gDCW, was more than 35% higher than that for the untreated cells at its optimal pH, 7.5, at the same temperature. The instability of the permeabilized cells at high pH might have resulted from the loss of metabolites and thus the change in cellular environment upon permeabilization. The effect of temperature on the D-hydantoinase activity of the permeabilized E. coli cells is shown in Fig. 4. The highest specific reaction rate, 44.9 mM/ h.gDCW, was observed at 35°C, 10°C lower than that obtained with the untreated cells under the same reaction condition. Apparently, the combined effects of permeabilization with CTAB and enzyme fixation with glutaraldehyde leads to the reduction of overall thermal stability of the recombinant cells. Since it has been widely observed that crosslinking or immobilisation of enzymes per se in general will not adversely affect, if not enhance, the thermal stability of enzyme. The re-
Fig. 6. HPLC of the reaction productions obtained after 15 h reaction with untreated cells (a) and with permeabilized cells (b). The reactions were conducted at pH 7.0 and at a temperature of 35°C with a substrate concentration of 300 mg/l. The peaks eluted at 15.1 and 23.5 min are D-carbamoyl-HPG and HPH, respectively.
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duction in thermostability of D-hydantoinase may be due to other factors. The permeabilization of cells apparently can lead to the loss of cellular metabolites and other small molecules, such as carbohydrates, which collectively may be responsible for maintaining the stability of enzymes [20]. It is therefore considered that that the reduction in overall thermal stability of the permeabilized cells is a result of altered cellular environment due to permeabiliztion rather than crosslinking. The effect of DMSO concentration on the D-hydantoinase activity of the permeabilized E. coli cells is shown in Fig. 1. The D-hydantoinase activity of the permeabilized E. coli cells decreased with DMSO concentration. Even in the presence of DMSO concentration as low as 0.5%, the specific reaction rate was reduced by nearly 20%. This result may seem contradictory to that obtained with the untreated cells which exhibit maximal activity in the presence of 0.5% DMSO (Fig. 1). As described above the increase in D-hydantoinase activity for the untreated cells in the presence of 0.5% DMSO resulted from the increase in membrane permeability. For the permeabilized cells membrane, however, permeability is no longer a limiting factor for the enzyme reaction and, therefore, the presence of DMSO becomes a significant denaturing factor under in the destabilized cellular environment.
3.5. Effect of immobilization on of permeabilized cells
D -hydantoinase
acti6ity
The effects of environmental factors on the activity of the permeabilized cells upon immobilization by matrix entrapment with calcium alginate were studied. The effects of pH and temperature on the activity of the immobilized permeabilized cellls were shown in Fig. 3 and Fig. 4, respectively. Although the optimal pH remained at 6.5, the enzyme activity was relatively unaffected by pH in the range between pH 6.0 and 7.0, compared to the free permeabilized cells, also shown in Fig. 3. The optimal temperature of the permeabilized cells was increased by 20°C upon immobilization. The specific reaction rate with the immobilized cells increased with temperature up to 55°C at which the specific reaction rate reached its optima, Fig. 4. However, it should be pointed out that the specific reaction rate was sharply reduced upon immobilization probably due to low cell load and/or added mass transfer resistance [13]. The effect of DMSO concentration on the activity of the immobilized permeabilized cells was shown in Fig. 1. The D-hydantoinase activity of the permeabilized cells seems to be less sensitive toward the presence of DMSO. At DMSO concentration as high as 10%, a decline of less than 15% in specific reaction rate activity was observed, compared to a decline of more than 50% in the presence of only 5% DMSO for the
free permeabilized cells, also shown in Fig. 1. The stability of the D-hydantoinase of the permeabilized cells toward organic solvents was significantly increased upon immobilization.
4. Conclusions From a process engineer’s point of view, the use of isolated enzymes for the production of speciality chemicals and pharmaceutical intermediates are generally favoured over the use of whole cell biocatalysts partly because of the high mass transfer resistance and the secretion of contaminating metabolites associated with the use of whole cell biocatalysts. The use of recombinant E. coli expressing D-hydantoinase for the production of D-amino acid precursor in a previous study was further complicated by the low water solubility of substrates. In this study the use of co-solvents such as DMSO to enhance the rate of D-amino acid precursor production with whole cell D-hydantoinase has been established. It was also demonstrated that the use of permeabilized cells as biocatalysts can enhance the production rate by reducing mass transfer resistance and can improve product purity by eliminating the secretion of metabolites. However, the permeabilization of cells also lead to reduced enzyme stability toward organic and thermal denaturation and shift in optimal pH toward lower pH. The shift of optimal pH toward lower pH is particularly detrimental to conversion yield, because on an industrial scale it is preferable to conduct the production of D-carbamoyl-HPG from DLHPH under alkaline conditions at which the concentration of D-HPH can be increased significantly due to the increase in overall substrate solubility and the racemic conversion of L-HPH into D-HPH. [21]. Although the enzyme stability of the whole cell biocatalysts can be significantly enhanced by immobilization it is necessary to develop alternative approaches, which are not as denaturing as glutaraldeyde treatment, for the fixation of enzymes within the permeabilized cells.
Acknowledgements This work was in part supported by a grant, DOH88HR-622, from the National Health Research Institute, Taiwan.
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B.-D. Yin et al. / Process Biochemistry 35 (2000) 915–921 [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 Psedumonas strita. 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. Agri 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 micororganisms 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] Jih Y.L. Overexpression of Pseudomonas putida D-hydantoinase gene in Escherichia coli and Pseudomnas putida. MS
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.
Production of D-amino acid precursors with permeabilized recombinant Escherichia coli with D-hydantoinase activity Bang-Ding Yin a, Yi-Chuan Chen 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 18 August 1999; received in revised form 18 October 1999; accepted 27 November 1999
Abstract Recombinant Escherichia coli cells expressing D-hydantoinase were used as the biocatalysts for the production of N-carbamoylfrom DL-p-hydroxyphenylhydantoin. Although high concentrations of DMSO could lead to enzyme denaturation, in the presence of 1.5% DMSO, the rate of product formation increased by more than 80% due to enhanced permeability of the cell membrane and increased substrate concentration. Reduced mass transfer resistance, achieved by the permeabilization of cell membrane with CTAB and glutaraldehyde, led to a 60% increase in the rate of production. However, in addition to causing a shift of optimal pH toward lower pH, permeabilization of the cell membrane resulted in reduced enzyme stability toward thermal and organic denaturation. Nevertheless, the stability of the D-hydantoinase of the recombinant cells toward pH, temperature and organic solvents can be significantly enhanced by immobilization. © 2000 Elsevier Science Ltd. All rights reserved.
D-hydroxyphenylglycine
Keywords: Escherichia coli; Immobilization; Permeabilization;
D-hydantoinase;
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]. A chemo-enzymatic process for the production of D-p-HPG from DL-5-substituted hydantoins has been proposed by Yamada and coworkers [4 – 6]. In this process, DL-5-substituted hydantoins were asymmetrically hydrolyzed to N-carbamoyl-D-amino acids by Dspecific hydantoinase, and these compounds were
* Corresponding author. Tel.: +886-4-2854724 ext. 104; fax: + 886-4-2854734. E-mail address: [email protected] (S.-C. Lin)
Calcium alginate
further chemically converted to the corresponding Damino acid under acidic conditions. Alternatively, Ncarbamoyl-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. A recombinant Escherichia coli containing a plasmid expressing a D-hydantoinase gene from P. putida has recently been constructed [11,12]. The recombinant E. coli exhibited a D-hydantoinase activity about 20-fold higher than that of the gene donor strain [12]. In a previous study, the use of recombinant E. coli cells exhibiting D-hydantoinase activity as biocatalysts for the production of N-carbamoyl-D-hydroxyphenylglycine (N-carbamoyl-HPG) from DL-p-hydroxwas systematically yphenylhydantoin (D-HPH) investigated [13]. Although it has been shown that the recombinant E.coli cells exhibits high D-hydantoinase activity and enhanced thermal stability and reusability
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upon immobilization, the rate of D-carbamoyl-HPG production is low, due to low substrate solubility and high substrate mass-transfer resistance. Although the D-hydantoinase activity of the recombinant E. coli is relatively high compared to the donor strain, the rate of D-carbamoyl-HPG production is limited by the low water solubility of the substrate (9–13 mg/ml), DL-HPH [14]. It is, therefore, desirable to develop reaction systems that are capable of enhancing reaction rate by providing higher concentration of substrate. DMSO is an excellent solvent for the substrate. The solubility of DL-HPH in DMSO is estimated at about 250 mg/ml. Kim and Kim [15] have investigated the effect of organic solvents on the production of D-HPG with bacterial strain KBEL 101 expressing D-hydantoinase and reported that in the presence of 5% DMSO the relative D-hydantoinase activity of the cells is increased by twofold. To reduce the mass transfer resistance of substrate in the production of 6-APA with recombinant E. coli expressing penicillin acylase, SivaRaman and coworkers [16] reported the successful permeabilization of E. coli cell membrane and cell wall with N-cetyl-N,N,N-trimethylammonium bromide (CTAB). We, therefore, conducted a systematic investigation on the effect of DMSO on the enzymic activity of recombinant E. coli and the effect of membrane permeability on the rate of D-carbomyl-HPG production.
2. Materials and methods
2.1. Microorganism and growth conditions E. coli BL21(DE3) harbouring pET 36 encoding Dhydantoinase 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 [11,13]. 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 by centrifugation at 10 000×g for 10 min 5 h after induction. 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 One hundred mililitres of the stored culture described above was washed with 50 mM Tris –HCl buffer pH 8.5 and resuspended in 1 ml 50 mM Tris– HCl buffer pH 8.5. The cell paste was then 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 [13,17,18].
2.3. Permeablization of cells The method developed by Prabhune et al. for the permeabilization of cell was adopted [16]. Cells suspended in 50 mM potassium phosphate buffer, pH 7.8 containing 0.1% (w/v) CTAB (Sigma) were incubated at 4°C for 30 min with moderate agitation. The CTAB-treated cells, recovered by centrifugation, were then resuspended in 50 mM potassium phosphate buffer, pH 7.8 containing 1% (w/v) glutaraldehyde and incubated at room temperature for 1 h. The cells were subsequently recovered by centrifugation and rinsed with 50 mM potassium phosphate buffer, pH 7.8.
2.4. Biocatalyst reactions Predetermined amounts of the biocatalysts, 3 ml of cell concentrate for the free-cell systems and 3 g of calcium alginate beads for the immobilized systems, were added to 50 mM Tris–HCl buffer at pH values ranging from 5.5 to 8.5 to a total reaction volume of 25 ml [13]. Unless specified otherwise, the concentration of DL-p-HPH (Tokyo Chemicals, Tokyo, Japan) was 750 mg/l. In some experiments 0.5–10% (v/v) of DMSO was added to the reaction mixtures to investigate the effect of DMSO on enzyme activity. The reaction mixtures were then incubated for 1 h at temperatures ranging from 30 to 70°C. The reactions were 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.
2.5. Acti6ity assay The D-hydantoinase activities of biocatalysts were determined by measuring the amount of N-carbamoyl-D-HPG produced from the enzyme reactions with high- performance liquid chromatography (HPLC) using a Jasco HPLC system (Tokyo, Japan) equipped with a C18 column (LiChroCART, 5 mm, 4.6×250 mm, Merck, NJ). Gradient elutions with a mobile phase system consisting of 0.01% (v/v) phosphorous acid (TEDIA) acetonitrile (TEDIA) at 35°C were used [13,19]. The absorbance of the eluent was monitored with an UV detector at 220 nm.
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3. Results and discussion
3.1. Effect of DMSO on
D -hydantoinase
acti6ity
The effect of DMSO concentration on the D-hydantoinase activity of the recombinant E. coli was investigated by comparing the rates of D-carbamoyl-HPG production in the presence of DMSO at concentrations ranging from 0 to 10% (v/v) with a substrate concentration of 350 mg/l, Fig. 1. It was observed that in the presence of 0.5% DMSO the specific reaction rate, defined as the rate of D-carbamoyl-HPG production per gram of biocatalysts (dry cell weight), was increased by more than 10% from 17.0 to 18.9 mM/h.gDCW. The increase in reaction rate is believed to be a result of enhanced substrate availability to the enzyme. In the presence of DMSO the structural integrity of the cell membrane is disturbed, and this led to enhanced substrate permeability and thus higher reaction rate. This result is contradictory to that reported by Kim and Kim [15] who have found that the effect of DMSO on cell permeability to be negligible. The difference might result from differences in membrane structure between bacterial strain KBEL 101 and the recombinant E.coli. In the presence of 2% DMSO the specific reaction rates declined to 17.0 mM/h.gDCW, equal to the value obtained in the absence of DMSO. The slight decrease in specific reaction rate might have resulted from the denaturation of enzyme. In addition to enhancement in permeability of the cell membrane, DMSO at elevated concentration might alter the conformation of protein
Fig. 1. The effect of DMSO concentration on the D-hydantoinase activity of () untreated recombinant cells, ( ) permeabilized cells, and () immobilized permeabilized cells. Experiments were conducted at pH 7.0 and a temperature of 35°C with a substrate concentraion of 300 mg/l. Activity of the bioctalaysts was accessed by determining the initial specific reaction rate, the rate of production formation per gram of dry cell weight or beads within the first hour.
Fig. 2. Optimal DMSO concentration for the solubilization of substrate and the production of D-carbamoyl-HPG with whole cell biocatalysts exhibiting D-hydantoinase. The reaction mixtures were prepared by mixing aqueous buffer containing saturated D-HPH and DMSO containing D-HPH at a concentration of 250 mg/ml at various ratios. The reactions were conducted at pH 7.0 and a temperature of 35°C.
molecules and thus reduce the activity of the enzyme. The increase in specific reaction rate observed at low DMSO concentration due to enhanced substrate availability was probably balanced by the decline in specific reaction rate at higher DMSO concentration due to enzyme denaturation. At DMSO concentrations above 2%, the denaturation of enzyme became the dominating factor affecting the overall activity of the biocatalysts and led to a significant decline in D-hydantoniase activity. For example, in the presence 10% DMSO the specific reaction rate dropped by more than 50% to 6.9 mM/h.gDCW (Fig. 1). The stability of D-hydantoinase activity of the recombinant E. coli toward DMSO was apparently lower than that of KBEL 101 which exhibits maximal activity in the presence of 5% DMSO [15]. In addition to membrane permeability and enzyme stability, DMSO can also affect the rate of N-carbamoyl-HPG formation by increasing the solubility and thus the concentration of substrate in the reaction mixture. Therefore, even though the optimal DMSO concentration for D-hydantoinase activity of the recombinant E. coli was 0.5%, the optimal DMSO concentration for the production of N-carbamoyl-D-HPG may be higher. To determine the optimal DMSO concentration for N-carbamoyl-D-HPG production, the rates of product formation were compared for reaction mixtures containing different concentrations of DMSO and thus substrate, prepared by mixing aqueous buffer containing saturated D-HPH and DMSO containing 250 mg/
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ml D-HPH at various ratios, Fig. 2. The optimal specific reaction rate, 41.9 mM/h.gDCW, was observed in the presence of 1.5% DMSO. This value was at least 70% higher than that obtained in the absence of DMSO. At higher DMSO concentration, the advantage of higher substrate concentration was offset by the decline in enzyme activity due to denaturation by DMSO.
3.2. En6ironmental effects on
D -hydantoinase
acti6ity
The effect of pH on the D-hydantoinase activity of the recombinant E. coli is shown in Fig. 3. The optimal pH in reaction systems containing 0.5% DMSO shifted from 8.5 to 7.5 [13]. At pHs ranging from 7.0 to 8.0, the specific reaction rates were above 30 mM/h.gDCW. When the pH was increased to 8.5, the specific reaction rate declined sharply to 30.0 mM/h.gDCW. Since it has been reported that the pKa of a Tris buffer system is not affected by the presence of organic solvent [20], the shift in optimal pH could have resulted from the decrease in water activity and/or the change in the cellular environment where the enzyme located. The effect of temperature on the D-hydantoinase activity of the recombinant E. coli is shown in Fig. 4. The thermal stability of the recombinant E. coli was not significantly affected by DMSO. As in DMSO-free systems [13], the maximal reaction rate in the presence of 0.5% DMSO was observed at 40°C. When the reaction temperature was increased from 35 to 40°C, the specific reaction rate was increased from 29.8 to 38.2 mM/h.gDCW. Further increase in reaction temperature resulted in sharp decline in specific reaction rate, probably due to the thermal denaturation of D-hydantoinase.
Fig. 3. The effect of pH on the D-hydantoinase activity of () untreated recombinant cells, ( ) permeabilized cells, and () immobilized permeabilized cells. The reactions were conducted at a temperature of 35°C with a substrate concentration of 750 mg/l in the presence of 0.5% DMSO.
Fig. 4. The effect of temperature on the D-hydantoinase activity of ()untreated recombinant cells, ( ) permeabilized cells, and () immobilized permeabilized cells. The reactions were conducted at pH 7.0 with a substrate concentration of 750 mg/l in the presence of 0.5% DMSO.
3.3. Effect of permeabilization on acti6ity
D -hydantoinase
One of the major disadvantages of using immobilized whole cells as biocatalysts has been the diffusional limitation of the substrate. SivaRaman and coworkers [16] have reported the use of CTAB and glutaraldehyde for enhancing the permeability of E. coli cells with penicillin acylase activity. Since the permeabilization of cell membrane with CTAB might lead to the leakage of active enzymes from cells, glutaraldehyde was used for the fixation of enzymes by chemical crosslinking. The effect of permeabilization with CTAB and glutaraldehyde on the activity of the recombinant E. coli with D-hydantoinase activity was investigated. The kinetic profiles of D-carbamoyl-HPG production with permeabilized cells and untreated cells were compared (Fig. 5). It was observed that the specific reaction rate was increased by more than 60% from 23.4 to 38.0 mM/h.gDCW with the permeabilized cells. The increase in specific reaction rate can be attributed to the reduced substrate mass transfer resistance across cell membrane. However, it should be pointed out that crosslinking with glutaraldehyde cannot only effectively fix enzyme molecules within cell membrane but also lead to extensive denaturation of enzymes. The observed increase in specific reaction rate was in fact a combined result of enhanced mass transfer and reduced enzyme activity. Therefore, it is proposed that if non-denaturing or more gentle processes for enzyme fixation can be developed, the effect of permeabilization with CTAB on rate of D-carbamoyl-HPG will be even more significant.
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min in Fig. 6a, was significantly reduced in the reaction product obtained with permeabilized cells (Fig. 6b). The permeabilization of cells with CTAB effectively kills the cells and thus eliminates the production of contaminating extracellular metabolites.
3.4. En6ironmental effects on of permeabilized cells
Fig. 5. The rates of product formation with untreated cells () and permeabilized cells ( ). The reactions were conducted at pH 7.0 and at a temperature of 35°C with a substrate concentration of 750 mg/l in the presence of 0.5% DMSO.
In addition to enhanced reaction rate, the use of permabilized cells as biocatalysts has the advantage of reduced cell viability. One of the problems in employing whole cells as biocatalysts is the formation of extracellular metabolites that may lead to product contamination. The HPLC chromatograms of reaction products with permeabilized cells and untreated cells are shown in Fig. 6. The concentration of byproducts, extracellular metabolites, secreted by the recombinant cells during the course of the reaction, eluted between 17 and 22
D -hydantoinase
acti6ity
The effect of pH on the D-hydantoinase activity of the permeabilized E. coli cells is also shown in Fig. 3. Under the same reaction condition, the optimal pH for the permeabilized cells shifted from 7.5 to 6.5. The specific reaction rate for the permeabilized cells at pH 6.5, 45.9 mM/h.gDCW, was more than 35% higher than that for the untreated cells at its optimal pH, 7.5, at the same temperature. The instability of the permeabilized cells at high pH might have resulted from the loss of metabolites and thus the change in cellular environment upon permeabilization. The effect of temperature on the D-hydantoinase activity of the permeabilized E. coli cells is shown in Fig. 4. The highest specific reaction rate, 44.9 mM/ h.gDCW, was observed at 35°C, 10°C lower than that obtained with the untreated cells under the same reaction condition. Apparently, the combined effects of permeabilization with CTAB and enzyme fixation with glutaraldehyde leads to the reduction of overall thermal stability of the recombinant cells. Since it has been widely observed that crosslinking or immobilisation of enzymes per se in general will not adversely affect, if not enhance, the thermal stability of enzyme. The re-
Fig. 6. HPLC of the reaction productions obtained after 15 h reaction with untreated cells (a) and with permeabilized cells (b). The reactions were conducted at pH 7.0 and at a temperature of 35°C with a substrate concentration of 300 mg/l. The peaks eluted at 15.1 and 23.5 min are D-carbamoyl-HPG and HPH, respectively.
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duction in thermostability of D-hydantoinase may be due to other factors. The permeabilization of cells apparently can lead to the loss of cellular metabolites and other small molecules, such as carbohydrates, which collectively may be responsible for maintaining the stability of enzymes [20]. It is therefore considered that that the reduction in overall thermal stability of the permeabilized cells is a result of altered cellular environment due to permeabiliztion rather than crosslinking. The effect of DMSO concentration on the D-hydantoinase activity of the permeabilized E. coli cells is shown in Fig. 1. The D-hydantoinase activity of the permeabilized E. coli cells decreased with DMSO concentration. Even in the presence of DMSO concentration as low as 0.5%, the specific reaction rate was reduced by nearly 20%. This result may seem contradictory to that obtained with the untreated cells which exhibit maximal activity in the presence of 0.5% DMSO (Fig. 1). As described above the increase in D-hydantoinase activity for the untreated cells in the presence of 0.5% DMSO resulted from the increase in membrane permeability. For the permeabilized cells membrane, however, permeability is no longer a limiting factor for the enzyme reaction and, therefore, the presence of DMSO becomes a significant denaturing factor under in the destabilized cellular environment.
3.5. Effect of immobilization on of permeabilized cells
D -hydantoinase
acti6ity
The effects of environmental factors on the activity of the permeabilized cells upon immobilization by matrix entrapment with calcium alginate were studied. The effects of pH and temperature on the activity of the immobilized permeabilized cellls were shown in Fig. 3 and Fig. 4, respectively. Although the optimal pH remained at 6.5, the enzyme activity was relatively unaffected by pH in the range between pH 6.0 and 7.0, compared to the free permeabilized cells, also shown in Fig. 3. The optimal temperature of the permeabilized cells was increased by 20°C upon immobilization. The specific reaction rate with the immobilized cells increased with temperature up to 55°C at which the specific reaction rate reached its optima, Fig. 4. However, it should be pointed out that the specific reaction rate was sharply reduced upon immobilization probably due to low cell load and/or added mass transfer resistance [13]. The effect of DMSO concentration on the activity of the immobilized permeabilized cells was shown in Fig. 1. The D-hydantoinase activity of the permeabilized cells seems to be less sensitive toward the presence of DMSO. At DMSO concentration as high as 10%, a decline of less than 15% in specific reaction rate activity was observed, compared to a decline of more than 50% in the presence of only 5% DMSO for the
free permeabilized cells, also shown in Fig. 1. The stability of the D-hydantoinase of the permeabilized cells toward organic solvents was significantly increased upon immobilization.
4. Conclusions From a process engineer’s point of view, the use of isolated enzymes for the production of speciality chemicals and pharmaceutical intermediates are generally favoured over the use of whole cell biocatalysts partly because of the high mass transfer resistance and the secretion of contaminating metabolites associated with the use of whole cell biocatalysts. The use of recombinant E. coli expressing D-hydantoinase for the production of D-amino acid precursor in a previous study was further complicated by the low water solubility of substrates. In this study the use of co-solvents such as DMSO to enhance the rate of D-amino acid precursor production with whole cell D-hydantoinase has been established. It was also demonstrated that the use of permeabilized cells as biocatalysts can enhance the production rate by reducing mass transfer resistance and can improve product purity by eliminating the secretion of metabolites. However, the permeabilization of cells also lead to reduced enzyme stability toward organic and thermal denaturation and shift in optimal pH toward lower pH. The shift of optimal pH toward lower pH is particularly detrimental to conversion yield, because on an industrial scale it is preferable to conduct the production of D-carbamoyl-HPG from DLHPH under alkaline conditions at which the concentration of D-HPH can be increased significantly due to the increase in overall substrate solubility and the racemic conversion of L-HPH into D-HPH. [21]. Although the enzyme stability of the whole cell biocatalysts can be significantly enhanced by immobilization it is necessary to develop alternative approaches, which are not as denaturing as glutaraldeyde treatment, for the fixation of enzymes within the permeabilized cells.
Acknowledgements This work was in part supported by a grant, DOH88HR-622, from the National Health Research Institute, Taiwan.
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