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Enzymatic synthesis of Cephalexin in recyclable aqueous two-phase systems composed by two pH responsive polymers
Accepted Manuscript Title: Enzymatic synthesis of Cephalexin in recyclable aqueous two-phase systems composed by two pH responsive polymers Author: Shutao Li Xuejun Cao PII: DOI: Reference:
S1369-703X(14)00154-5 http://dx.doi.org/doi:10.1016/j.bej.2014.06.002 BEJ 5960
To appear in:
Biochemical Engineering Journal
Received date: Revised date: Accepted date:
28-3-2014 25-5-2014 2-6-2014
Please cite this article as: S. Li, X. Cao, Enzymatic synthesis of Cephalexin in recyclable aqueous two-phase systems composed by two pH responsive polymers, Biochemical Engineering Journal (2014), http://dx.doi.org/10.1016/j.bej.2014.06.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Enzymatic synthesis of Cephalexin in recyclable aqueous
ip t
two-phase systems composed by two pH responsive polymers
Shutao Li, Xuejun Cao*
cr
State Key Laboratory of Bioreactor Engineering, Department of Bioengineering,
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East China University of Science and Technology; 130 Meilong Road, Shanghai
an
200237, China; Email: [email protected]
ABSTRACT
was
studied,
using
M
The synthesis of Cephalexin in novel recyclable aqueous two-phase systems immobilized
penicillin
acylase
as
biocatalyst,
te
d
7-amino-3-desacetoxy cephalosporanic acid (7-ADCA) as nucleophile and phenyl glycine methyl ester (PGME) as acyl donor. The aqueous two-phase system (ATPS)
Ac ce p
was composed by two pH-response copolymers PADBA and PMDB, which could be recycled efficiently. In the ATPS, high partition coefficient (2.57) for Cephalexin and low partition coefficient (0.21) for 7-ADCA were obtained. Then the synthesis of Cephalexin in ATPS and conventional one-phase system was compared. Higher Cephalexin yield was obtained in ATPS. In addition, synthesis in ATPS was improved at higher substrates concentrations, obtaining yield of 98.2% with 50 mM 7-ADCA and 150 mM PGME at pH 5.8, 20 °C. Even reducing acyl donor to nucleophile ratio to 2, the yield can still above 90%. PADBA/PMDB recycling aqueous two-phase system is suitable for synthesis of Cephalexin.
Page 1 of 33
Keywords: Aqueous Two Phase; Immobilised enzymes; Biocatalysis; Product inhibition; Cephalexin; pH-sensitive polymer 1. Introduction
ip t
Cephalexin is a semi-synthetic cephalosporin antibiotics. Traditionally, it is
cr
chemically synthesized by a multi-step process, with high cost and pollution of
us
environment. At present, enzymatic synthesis of Cephalexin is attracting attention owing to economic and environmental benefits compared to conventional chemical
an
processes. However, there are still some problems need to be resolved [1-5]. The requirement of a significant excess of acyl donor phenyl glycine methyl ester
M
(PGME) causes high cost and waste. Moreover, product accumulation may inhibit the enzyme efficiency in aqueous phase.
te
d
ATPS has low interfacial tension and high water content in two phases which are suitable for bioconversions. On the other hand, suitable ATPS can partition the
Ac ce p
products into the phase not containing the enzyme so that the inhibition of product can be removed. Wei et al. [6] synthesized Cephalexin in ATPS composed of PEG 400 and magnesium sulfate, with a high product yield. Aguirre et al. [7] reported an aqueous two-phase system composed of PEG 400 and ammonium sulfate, which was used for the bioconversion of Cephalexin. However, traditional ATPS cannot be recycled to result in high cost and environmental pollution. Recently, scientists have attempted to synthesize new phase-forming copolymers that can be recycled by changing pH, temperature, ionic strength and so on [8, 9]. In our laboratory, Cao’s group has been working on the exploration of novel
Page 2 of 33
recycling ATPS and their applications in biotechnology fields. For example, Miao et al. [10]prepared a recyclable thermo-sensitive copolymer PNB to form ATPS with PADB (isoelectric point 4.0), which was used to transform penicillin G to 6-APA by
ip t
penicillin G acylase. Li et al. [11] improved the pH-response copolymer PADB
cr
(isoelectric point 4.0) to PADB (isoelectric point 2.6) and formed ATPS with PNB,
us
which was used to transform Ceph-G to 7-ADCA by penicillin G acylase. Yan et al. [12] in our lab prepared novel recyclable ATPS composed of two pH-response
an
polymers (PADB (isoelectric point 2.9) and PADBA) to partition demeclocycline. All their studies obtained good experimental results.
M
In this study, we report a new recyclable ATPS composed by two pH-response copolymers PMDB and PADBA. Both the two copolymers were synthesized in our
te
d
laboratory and could be recycled by adjusting pH. The novel recyclable ATPS was used as reaction media for the synthesis of Cephalexin to improve yield and to
Ac ce p
reduce the cost of production without pollution of environment. 2. Material and Methods 2.1 Materials
2-Dimethylamino ethyl methacrylate (DMAEMA) was obtained from
Wanduofu Fine Chemicals Co., Ltd. (Shandong, China). Methacrylic acid (MAA), butyl methacrylate (BMA), acrylic acid (AA), butyl methacrylate (BMA), allyl alcohol (Aal) and two initiators ammonium persulfate (APS) and sodium bisulfite (NaHSO3)were purchased from Ling Feng Chemical Co. (Shanghai, China). 7-amino-3-desacetoxycephalosporanic acid (7-ADCA) was obtained from
Page 3 of 33
Beijing Redwood Fine Chemical Co., Ltd. Phenyl glycine methyl ester (PGME) was purchased from Yangzhou Ao Xin Chemical Factory (Jiangsu, China). Cephalexin was obtained from North China Pharmaceutical Factory (Hebei, China).
Lai Ge biological technology Co., Ltd.
2.2.1 Preparation of copolymers PADBA and PMDB
us
2.2 Preparation of PADBA/PMDB aqueous two-phase systems
cr
ip t
Immobilized penicillin acylase (IPGA) with 90 IUH/g was obtained from Hu Nan Fu
an
Copolymer PADBA was synthesized as illustrated in Fig.1 (a). 0.1235 g APS and 0.1235 g NaHSO3 as initiators were added into 118ml water containing 5 ml AA, 4
M
ml DMAEMA, 0.5 ml BMA and 1.284 ml Aal. The copolymerization reaction was carried out for 24 h at 55 °C under the protection of N2. After the reaction finished,
te
d
the product was washed by ethanol and then dried in a vacuum oven to constant mass [12]. The yield of PADBA was defined as the ratio of the experimental value to
Ac ce p
theoretical value.
Copolymer PMDB was synthesized as illustrated in Fig.1 (b). 0.1235 g APS and
0.1235 g NaHSO3 as initiators were added into 118ml water containing 5.1 ml MAA,
0.53 ml DMAEMA and 0.5 ml BMA. The copolymerization reaction was carried out for 24 h at 55 °C under the protection of N2. After the reaction finished, the product was dissolved in 3 M NaOH solution and filtrated to remove the undissolved impurities. Then the pH of the solution was adjusted to 3.1 by adding 3 M HCl. The precipitate was collected, and then dried in the vacuum to constant mass [13]. The yield of PMDB was defined as the ratio of the experimental value to theoretical value.
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2.2.2 Phase-forming test and phase diagram Dissolve PADBA and PMDB in 170 mM NaOH solution, respectively. Different concentrations of PADBA were used to test the possibility of forming ATPS with
ip t
different concentrations of PMDB. Stood mixed systems at room temperature (25 °C)
cr
until a distinct interphase was observed, then took out aliquots from the top and
us
bottom phase to measure the PADBA and PMDB concentration in both phases. The concentration of copolymers was determined by UV spectrophotometer as the
an
following method [12]: Two samples were prepared from the top phase and the bottom phase, respectively. The absorbance at 207 nm was the summation of PMDB
M
and PADBA. The concentration of PADBA was determined at 415 nm after reacting with 2% (w/w) potassium permanganate for 6 h. Then the concentration of PADBA was
te
d
used to calculate the absorbance of PADBA at 207 nm with the standard curve. The absorbance of PMDB was calculated by subtracting the absorbance of PADBA from the
Ac ce p
summation. Then the concentration of PMDB was calculated by the standard curve of
PMDB at 207 nm.
2.2.3 Recycling of copolymers The pH-response copolymers can be precipitated from solution by adjusting pH
to its isoelectric point (pI) by adding HCl. The precipitate could be separated by centrifugation and dried in a vacuum oven to constant mass. The recovery of copolymers can be calculated by the percentage of the dry weight of recovered polymer to that of the initial polymer weight. The pI was determined as the pH of the solution when the zeta potential is zero.
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Different pH values of the polymer solution (20 ppm) were adjusted and then the zeta potentials were measured using Zetasizer Nano ZEN3600 (Malvern). 2.3 Bioconversion reaction of Cephalexin in ATPS
ip t
2.3.1 Analytic methods
cr
The concentrations of Cephalexin, PGME and 7-ADCA in the reaction mixtures
us
were determined by high performance liquid chromatography (HPLC) with a reversed-phase column C18 (150 mm×4.6 mm, 5 μm) from Dikma. The mobile phase
an
was consisted of sodium phosphate buffer (20 mM, pH 6.0) and methanol with a volume ratio of 30:70. The flow rate was set to be 1.0 ml/min. The injected volume
M
was 20μL. The detection wavelengths were 220 nm and 260 nm. The retention times of the components were 7.6 min for PGME (220 nm), 1.2 min for 7-ADCA (260 nm)
calibration curves.
te
d
and 5.8 min for Cephalexin (260 nm). The concentrations were calculated using
Ac ce p
2.3.2 Partition of 7-ADCA, PGME and Cephalexin in ATPS ATPS formed by mixing 7% (w/v) PADBA and 4% (w/v) PMDB with same volume
was used for the partition of 7-ADCA, PGME and Cephalexin. pH and temperature of ATPS were adjusted to 5.8 and 20 °C, respectively. Different salts including LiCl, KCl, NaCl, Gu-HCl, NH4Cl, KBr, Na2SO4, (NH4)2SO4 with concentrations from 10 mM to 80 mM were added into systems to affect partition coefficients, respectively. After aqueous two-phase systems reached equilibrium, the samples were taken out from top and bottom phase by a syringe. Their concentrations were determined by HPLC. The partition coefficient is defined as the ratio of concentration of a
Page 6 of 33
substance in the top phase to that in the bottom phase. To investigate the relationship between the electrostatic potential difference and the concentration of salts, the electrostatic potential difference between two phases
ip t
was measured after aqueous two-phase systems reached equilibrium using interphase
cr
potentiometer JIE09 (Powereach).
us
2.3.3 Synthesis of Cephalexin in ATPS
Syntheses of Cephalexin with immobilized penicillin acylase proceed according
an
to the reaction (Fig.2). Synthesis was performed under kinetic control. In the study, 15 ml ATPS formed by mixing same volume of 7% (w/v) PADBA and 4% (w/v) PMDB
M
were put into 25 ml graduated glass tube to carry out the reaction, at the same pH and temperature as the determination of partition coefficients. One salt referred to
te
d
the partition coefficients was added as desired amount. Substrates concentrations were 30 mM and 90 mM for 7-ADCA and PGME, respectively. Amount of loaded
Ac ce p
enzyme was 3.38 IUH/ml. Control synthesis was run in parallel where a
homogeneous aqueous system (phosphate buffer 0.1 M) was used as reaction medium under the same experimental conditions. For the synthesis of Cephalexin in the ATPS, the effect of increasing the substrate concentration and reducing the excess acyl donor were then studied. The range of 7-ADCA concentration in the whole system was from 30 mM to 70 mM and nucleophile to acyl donor molar ratio was from 1:1 to1:3. Samples were taken at specified intervals and were properly diluted prior to be assayed by HPLC. The yield of Cephalexin was molar ratio of product (Cephalexin) to limiting substrate (7-ADCA). In ATPS, the parameter was
Page 7 of 33
determined considering the amounts of product in both phases. 3. Results and discussions 3.1 Yield and recovery of copolymers
ip t
The isoelectric point (pI) is defined as the pH at which the net charge on a
cr
macromolecule is zero[14]. Fig.3 is the zeta potentials of both polymers at different
us
pH. pI of PADBA is 4.01 and PMDB is 3.09. The pI of PADBA is dependent on the ratio of AA to DMAEMA, the pI of PMDB is dependent on the ratio of MAA to
an
DMAEMA.
pH of the solution was adjusted to the pI of copolymer by adding HCl and
M
precipitate could be separated by centrifugation and dried in a vacuum oven. The recycled copolymer could be used to form ATPS again. Table1 is the yield and
te
d
recovery of PADBA and PMDB, Each data was average value of three parallel experimental points. The yield of PADBA is 73.1% and PMDB is 70.7%. The recovery
Ac ce p
of PADBA is 97.4% and PMDB is 97.2%. The recoveries are quite high and stable.
3.2 Phase diagram
Presented on Fig.4 is the phase diagram of PADBA/PMDB ATPS at room
temperature (25 °C). Bimodal curve divides a region of component compositions capable of forming two immiscible aqueous phases (region above the bimodal curve) from those which forms one phase (region below the bimodal curve). T, B and M represent the compositions of two polymers in top phase, the compositions in bottom phase and the total composition of the copolymers in the ATPS. According to the diagram, PADBA is mainly enriched in top phase while PMDB is mainly in bottom
Page 8 of 33
phase. The volume ratio of phase could be calculated according to length ratio of line MB and MT. In the experiment, the concentration of PADBA and PMDB was 3.5% (w/w) and 2% (w/w) in total phase systems which will give a volume ratio of about 2:1
3.3 Synthesis of Cephalexin
us
3.3.1 Partition of 7-ADCA, PGME and Cephalexin in ATPS
cr
ip t
when the two phases are in equilibrium.
The partition coefficients of 7-ADCA, PGME and Cephalexin in ATPS were
an
determined in this experiment. The partitioning behavior of a substance in ATPS may be affected by several factors including physicochemical properties of the polymers
M
(structure, molecular weight) and salt ions type etc. In the ATPS, partition coefficient of Cephalexin, 7-ADCA and PGME were 1.25, 0.2 and 1.0 without any salt (Fig.5
te
d
(a)). After adding salts, the maximum partition coefficient of Cephalexin reached 2.57 (Fig.5 (b)) in the presence of 20 mM NaCl, While at the NaCl concentration,
Ac ce p
the partition coefficients of 7-ADCA and PGME was 0.21 and 0.94 (Fig.5 (c) and (d)), respectively. In the reactions of Cephalexin synthesis, higher product yield is to be expected since 7-ADCA was biased in the PMDB-rich phase where the reaction occurs, while Cephalexin could enrich in the PADBA-rich phase where its hydrolysis
could be avoided.
Cephalexin and 7-ADCA have a structural difference in the side chain, Cephalexin has more hydrophobicity. In the ATPS, Cephalexin biased to the PADBA-rich phase while 7-ADCA biased to the PMDB-rich phase without adding any salts, it was believed that hydrophobicity interaction between them and the
Page 9 of 33
environment was the reason of the phenomenon. After adding inorganic salts, the salts will partition unequally between the phases. This unequal distribution, along with the requirement of electroneutrality leads to an electrostatic potential difference
ip t
between two phases (Donna potential). Fig.6 is the relationship between the
cr
electrostatic potential difference and the concentration of salts. According to the
us
Fig.5 (b) and Fig.6, the electrostatic potential difference is positively associated with the partition coefficient of Cephalexin. The adding of NaCl increases the
an
electrostatic potential difference between the two phases in the range of 10~30 mM NaCl concentration, and then produce an obvious repulsion to drive Cephalexin and
M
7-ADCA into the PADBA-rich phase. However, compared with the hydrophobicity interaction between 7-ADCA and the environment, the change of electrostatic
te
d
potential difference is so small that it has little influence on the partition of 7-ADCA. With the increasing of concentration of NaCl, the electrostatic potential difference
Ac ce p
dropped. This may attribute to high ionic strength. 3.3.2 Synthesis of Cephalexin in ATPS and single aqueous phase Cephalexin synthesis was performed in the ATPS and 0.1 M phosphate buffer
used as control. In the ATPS, 20 mM NaCl was added. Experiments were done in triplicate and reported results are the average of these triplicates. Results are illustrated in Fig.7 that records the time course of Cephalexin synthesis in the ATPS and in single aqueous phase. The reaction was quick during the first 1 h and then becomes stable as Cephalexin accumulated. In the single aqueous phase, the yield of Cephalexin
Page 10 of 33
reached to maximal yield with 73.58% at 3.5 h, and then began to drop significantly. In the ATPS, the viscosity of the copolymers resulted in longer time for the yield to reach maximum. The yield of Cephalexin reached to maximal yield with 97.3% at 5
ip t
h, and then declined insignificantly with time.
cr
The yield of Cephalexin was the equilibrium of three different catalytic
us
activities of the same penicillin acylase: a) synthesis of Cephalexin; b) hydrolysis of Cephalexin previously synthesized; c) hydrolysis of the activated acyl donor[15, 16].
an
Hydrolysis of Cephalexin can be promoted when Cephalexin accumulated around penicillin acylase. Furthermore, Cephalexin is a stronger inhibitor to penicillin
M
acylase. With the increase in concentration of Cephalexin, the activity of penicillin acylase was inhibited at a higher degree. Consequently, a transient maximum yield
te
d
of Cephalexin in the single aqueous phase was observed in Fig.7. However, the ATPS provided a reaction medium in which Cephalexin hydrolysis was avoided.
Ac ce p
This was clearly attributed to the partition of the Cephalexin to the PADBA-rich phase where its hydrolysis was strongly inhibited. Here, with bioconversion reaction went on, nearly 84% Cephalexin moved to the PADBA-rich phase at the presence of 20 mM NaCl. In this way, inhibition of penicillin acylase was removed to a large extent and the reaction equilibrium was shifted to the favorable synthesis direction. Additionally, penicillin acylase is likely to be in a more protective environment than in single aqueous medium[1, 17]. 3.3.3 Effect of substrates concentration on synthesis of Cephalexin in ATPS The effect of the substrate concentration on product yield was investigated in
Page 11 of 33
the ATPS with 20 mM NaCl. The details were shown in Fig.8, 98.2% product yield was obtained in the ATPS at 50 mM 7-ADCA at 6 h. With the increasing of substrates concentration, the yield reached maximum need longer time and the
ip t
maximum yield decreased.
cr
The effect of the substrate concentration on product yield is attributed to the
us
solubility of 7-ADCA. At 70 mM 7-ADCA and 210 mM PGME, the 7-ADCA is initially partly insoluble in the ATPS and mass transfer limitations are likely to be
that more precipitate may be produced.
an
significant. On the other hand, the by-product phenyl glycine is quite insoluble so
M
3.3.4 Effect of acyl donor to nucleophile ratio on synthesis of Cephalexin in ATPS Excess PGME has proven to be necessary to attain high yield of Cephalexin,
te
d
mainly because penicillin acylase can also act as an esterase and hydrolyze PGME to the side product. It’s a drawback of the synthesis of Cephalexin. The effect of lower
Ac ce p
molar ratios of PGME to 7-ADCA on product yield was investigated. In the experiment, 30 mM was chosen as the concentration of 7-ADCA due to the reaction equilibrium time, Results were presented in Fig.9. Results at a ratio of 2.5 were similar to 3, and the maximum yield can reach 95.4%. Even reducing the ratio to 2, the maximum yield can still keep above 90%. This may attribute to the partition of 7-ADCA. In the ATPS, the 7-ADCA and immobilized penicillin acylase were biased in the bottom phase, while the PGME was distributed evenly. That’s to say, under the same conditions of substrates concentration, there were more 7-ADCA around the penicillin acylase and inhibit the hydrolysis of PGME.
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4. Conclusion In this study, a new recyclable pH-pH aqueous two-phase system was applied to synthetic reaction of Cephalexin. This ATPS was suitable for the reaction due to the
ip t
removing inhibition of product. Even at lower acyl donor to nucleophile ratio, yield
cr
of Cephalexin still keeps very high. It is believed that synthesis reaction in the
us
recycling ATPS is potential application value in future bio-separation and
an
biochemical industry, not limited to synthesis of Cephalexin.
Acknowledgment
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The authors very appreciate financial support from National Natural Science Foundation of China (No. 21376078) and National Special Fund for State Key
Ac ce p
te
d
Laboratory of Bioreactor Engineering (2060204).
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References [1] C. Aguirre, M. Toledo, V. Medina, A. Illanes, Effect of cosolvent and pH on the kinetically controlled synthesis of cephalexin with immobilised penicillin
ip t
acylase, Process Biochemistry, 38 (2002) 351-360.
cr
[2] X. CAO, J. ZHU, D. WEI, B.-K. HUR, Biosynthesis of cephalexin in
us
PEG400-ammonium sulfate and PEG400-magnesium sulfate aqueous two-phase systems, Journal of microbiology and biotechnology, 14 (2004)
an
62-67.
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M
controlled synthesis of ampicillin with immobilized penicillin acylase in organic media, Applied biochemistry and biotechnology, 97 (2002) 165-179.
te
d
[4] A. Illanes, Z. Cabrera, L. Wilson, C. Aguirre, Synthesis of cephalexin in ethylene glycol with glyoxyl-agarose immobilised penicillin acylase: temperature and
Ac ce p
pH optimisation, Process Biochemistry, 39 (2003) 111-117.
[5] A. Illanes, L. Wilson, O. Corrotea, L. Tavernini, F. Zamorano, C. Aguirre, Synthesis of cephalexin with immobilized penicillin acylase at very high substrate concentrations in fully aqueous medium, Journal of Molecular Catalysis B: Enzymatic, 47 (2007) 72-78.
[6] D.-Z. Wei, J.-H. Zhu, X.-J. Cao, Enzymatic synthesis of cephalexin in aqueous two-phase systems, Biochemical engineering journal, 11 (2002) 95-99. [7] C. Aguirre, I. Concha, J. Vergara, R. Riveros, A. Illanes, Partition and substrate concentration effect in the enzymatic synthesis of cephalexin in aqueous
Page 14 of 33
two-phase systems, Process Biochemistry, 45 (2010) 1163-1167. [8] G. Chen, A.S. Hoffman, Graft copolymers that exhibit temperature-induced phase transitions over a wide range of pH, Nature, 373 (1995) 49-52.
cr
complexation, Macromolecules, 18 (1985) 83-85.
ip t
[9] J. Ricka, T. Tanaka, Phase transition in ionic gels induced by copper
us
[10] S. Miao, J. Chen, X. Cao, Preparation of a novel thermo-sensitive copolymer forming recyclable aqueous two-phase systems and its application in
an
bioconversion of Penicillin G, Separation and Purification Technology, 75 (2010) 156-164.
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[11] H. Li, X. Cao, Bioconversion of cephalosporin-G to 7-ADCA in a pH-thermo
te
(2011) 1753-1758.
d
sensitive recycling aqueous two-phase systems, Process Biochemistry, 46
[12] B. Yan, X. Cao, Preparation of aqueous two-phase systems composed of two
Ac ce p
pH-response polymers and liquid–liquid extraction of demeclocycline, Journal of Chromatography A, 1245 (2012) 39-45.
[13] W. Liang, X. Cao, Preparation of a pH-sensitive polyacrylate amphiphilic copolymer and its application in cellulase immobilization, Bioresource Technology, 116 (2012) 140-146.
[14] R. Chang, Physical chemistry for the chemical and biological sciences, University Science Books, 2000. [15] R. Fernandez ‐ Lafuente, G. JM, Dynamic reaction design of enzymic biotransformations in organic media: equilibrium‐controlled synthesis of
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antibiotics by penicillin G acylase, Biotechnology and applied biochemistry, 24 (1996) 139-145. [16] R. Fernandez-Lafuente, C. Rosell, J. Guisan, The presence of methanol exerts a
ip t
strong and complex modulation of the synthesis of different antibiotics by
cr
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[17] C.M. Rosell, M. Terreni, R. Fernandez-Lafuente, J.M. Guisan, A criterion for
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the selection of monophasic solvents for enzymatic synthesis, Enzyme and
Ac ce p
te
d
M
Microbial Technology, 23 (1998) 64-69.
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Figure captions
Fig.1 (a). Synthesis of pH-response copolymer PADBA.
ip t
x, y, z, m stands for monomer ratio of AA, DMAEMA, BMA, Aal, respectively.
us
Fig.1 (b). Synthesis of pH-response copolymer PMDB.
cr
The functional monomers mole ratio is AA: DM: BMA: Aal = 23:8:1:6.
x, y, z, m stands for monomer ratio of AA, DMAEMA, BMA, respectively. The
an
functional monomers mole ratio is MAA: DM: BMA = 19:1:1.
Fig.2 Reaction scheme for Immobilized penicillin acylase-catalyzed synthesis of
M
Cephalexin. (1) PGME, (2) 7-ADCA), (3) Cephalexin, (4) Phenylglycine (PG). Fig.3 Zeta potential of two polymers at different pH
temperature (25 °C)
te
d
Fig.4 The phase diagram of PADBA/PMDB aqueous two-phase systems at room
Ac ce p
Fig.5 Effect of salts on partition coefficient: (a) no salts (b) Cephalexin; (c) 7-ADCA; (d) PGME.
Different salts with concentrations from 10 mM to 80 mM with 10 mM step
were added into systems. The partition coefficient was measured at 20 °C and pH 5.8 by HPLC.
Fig.6 Effect of salts on the electrostatic potential difference The concentrations of salts were from 10 mM to 80 mM with 10 mM step. The electrostatic potential difference was measured at 20 °C and pH 5.8. Fig.7 Synthesis of Cephalexin in PADBA/PMDB systems with 20 mM NaCl and
Page 17 of 33
single aqueous phase. Reaction condition: 30 mM 7-ADCA, 90 mM PGME, pH 5.8, 20 °C and 3.38 IUH/ml.
ip t
The pH, temperature and amount of loaded enzyme have been optimized in the
cr
single aqueous phase at previous works.
us
Fig.8 Effect of substrates concentration on synthesis of Cephalexin in PADBA/PMDB systems with 20 mM NaCl
an
Reaction condition: pH 5.8, 20 °C and 3.38 IUH/ml. The range of 7-ADCA concentration in the whole system was from 30 mM to 70 mM.
M
Fig.9 Effect of acyl donor to nucleophile ratio on synthesis of Cephalexin in PADBA/PMDB systems with 20 mM NaCl.
te
d
Reaction condition: 30 mM 7-ADCA, pH 5.8, 20 °C and 3.38 IUH/ml. The
Ac ce p
range of nucleophile to acyl donor molar ratio was from 1:1 to1:3
Page 18 of 33
Yield
Recovery
PADBA
73.1%
97.4%
PMDB
70.7%
97.2%
Ac ce p
te
d
M
an
us
cr
ip t
Table 1 The yield and recovery of PADBA and PMDB
Page 19 of 33
Ac ce p
te
d
M
an
us
cr
ip t
Highlights A recyclable ATPS composed by two pH‐response copolymers was prepared Substrates K7‐ADCA and KPGME biased in bottom phase, and product KCephalexin biased in top phase Synthesis of Cephalexin in ATPS has been conducted with 98.2% yield Ratio of acyl donor to nucleophile decreased from 3 to 2
Page 20 of 33
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Figure.1 (a)
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Figure.1 (b)
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Figure.2
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Figure.3
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Figure.4
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Figure.5 (a)
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Figure.5 (b)
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Figure.5 (c)
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Figure.5 (d)
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Figure.6
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Figure.7
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Figure.8
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Figure.9
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S1369-703X(14)00154-5 http://dx.doi.org/doi:10.1016/j.bej.2014.06.002 BEJ 5960
To appear in:
Biochemical Engineering Journal
Received date: Revised date: Accepted date:
28-3-2014 25-5-2014 2-6-2014
Please cite this article as: S. Li, X. Cao, Enzymatic synthesis of Cephalexin in recyclable aqueous two-phase systems composed by two pH responsive polymers, Biochemical Engineering Journal (2014), http://dx.doi.org/10.1016/j.bej.2014.06.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Enzymatic synthesis of Cephalexin in recyclable aqueous
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two-phase systems composed by two pH responsive polymers
Shutao Li, Xuejun Cao*
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State Key Laboratory of Bioreactor Engineering, Department of Bioengineering,
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East China University of Science and Technology; 130 Meilong Road, Shanghai
an
200237, China; Email: [email protected]
ABSTRACT
was
studied,
using
M
The synthesis of Cephalexin in novel recyclable aqueous two-phase systems immobilized
penicillin
acylase
as
biocatalyst,
te
d
7-amino-3-desacetoxy cephalosporanic acid (7-ADCA) as nucleophile and phenyl glycine methyl ester (PGME) as acyl donor. The aqueous two-phase system (ATPS)
Ac ce p
was composed by two pH-response copolymers PADBA and PMDB, which could be recycled efficiently. In the ATPS, high partition coefficient (2.57) for Cephalexin and low partition coefficient (0.21) for 7-ADCA were obtained. Then the synthesis of Cephalexin in ATPS and conventional one-phase system was compared. Higher Cephalexin yield was obtained in ATPS. In addition, synthesis in ATPS was improved at higher substrates concentrations, obtaining yield of 98.2% with 50 mM 7-ADCA and 150 mM PGME at pH 5.8, 20 °C. Even reducing acyl donor to nucleophile ratio to 2, the yield can still above 90%. PADBA/PMDB recycling aqueous two-phase system is suitable for synthesis of Cephalexin.
Page 1 of 33
Keywords: Aqueous Two Phase; Immobilised enzymes; Biocatalysis; Product inhibition; Cephalexin; pH-sensitive polymer 1. Introduction
ip t
Cephalexin is a semi-synthetic cephalosporin antibiotics. Traditionally, it is
cr
chemically synthesized by a multi-step process, with high cost and pollution of
us
environment. At present, enzymatic synthesis of Cephalexin is attracting attention owing to economic and environmental benefits compared to conventional chemical
an
processes. However, there are still some problems need to be resolved [1-5]. The requirement of a significant excess of acyl donor phenyl glycine methyl ester
M
(PGME) causes high cost and waste. Moreover, product accumulation may inhibit the enzyme efficiency in aqueous phase.
te
d
ATPS has low interfacial tension and high water content in two phases which are suitable for bioconversions. On the other hand, suitable ATPS can partition the
Ac ce p
products into the phase not containing the enzyme so that the inhibition of product can be removed. Wei et al. [6] synthesized Cephalexin in ATPS composed of PEG 400 and magnesium sulfate, with a high product yield. Aguirre et al. [7] reported an aqueous two-phase system composed of PEG 400 and ammonium sulfate, which was used for the bioconversion of Cephalexin. However, traditional ATPS cannot be recycled to result in high cost and environmental pollution. Recently, scientists have attempted to synthesize new phase-forming copolymers that can be recycled by changing pH, temperature, ionic strength and so on [8, 9]. In our laboratory, Cao’s group has been working on the exploration of novel
Page 2 of 33
recycling ATPS and their applications in biotechnology fields. For example, Miao et al. [10]prepared a recyclable thermo-sensitive copolymer PNB to form ATPS with PADB (isoelectric point 4.0), which was used to transform penicillin G to 6-APA by
ip t
penicillin G acylase. Li et al. [11] improved the pH-response copolymer PADB
cr
(isoelectric point 4.0) to PADB (isoelectric point 2.6) and formed ATPS with PNB,
us
which was used to transform Ceph-G to 7-ADCA by penicillin G acylase. Yan et al. [12] in our lab prepared novel recyclable ATPS composed of two pH-response
an
polymers (PADB (isoelectric point 2.9) and PADBA) to partition demeclocycline. All their studies obtained good experimental results.
M
In this study, we report a new recyclable ATPS composed by two pH-response copolymers PMDB and PADBA. Both the two copolymers were synthesized in our
te
d
laboratory and could be recycled by adjusting pH. The novel recyclable ATPS was used as reaction media for the synthesis of Cephalexin to improve yield and to
Ac ce p
reduce the cost of production without pollution of environment. 2. Material and Methods 2.1 Materials
2-Dimethylamino ethyl methacrylate (DMAEMA) was obtained from
Wanduofu Fine Chemicals Co., Ltd. (Shandong, China). Methacrylic acid (MAA), butyl methacrylate (BMA), acrylic acid (AA), butyl methacrylate (BMA), allyl alcohol (Aal) and two initiators ammonium persulfate (APS) and sodium bisulfite (NaHSO3)were purchased from Ling Feng Chemical Co. (Shanghai, China). 7-amino-3-desacetoxycephalosporanic acid (7-ADCA) was obtained from
Page 3 of 33
Beijing Redwood Fine Chemical Co., Ltd. Phenyl glycine methyl ester (PGME) was purchased from Yangzhou Ao Xin Chemical Factory (Jiangsu, China). Cephalexin was obtained from North China Pharmaceutical Factory (Hebei, China).
Lai Ge biological technology Co., Ltd.
2.2.1 Preparation of copolymers PADBA and PMDB
us
2.2 Preparation of PADBA/PMDB aqueous two-phase systems
cr
ip t
Immobilized penicillin acylase (IPGA) with 90 IUH/g was obtained from Hu Nan Fu
an
Copolymer PADBA was synthesized as illustrated in Fig.1 (a). 0.1235 g APS and 0.1235 g NaHSO3 as initiators were added into 118ml water containing 5 ml AA, 4
M
ml DMAEMA, 0.5 ml BMA and 1.284 ml Aal. The copolymerization reaction was carried out for 24 h at 55 °C under the protection of N2. After the reaction finished,
te
d
the product was washed by ethanol and then dried in a vacuum oven to constant mass [12]. The yield of PADBA was defined as the ratio of the experimental value to
Ac ce p
theoretical value.
Copolymer PMDB was synthesized as illustrated in Fig.1 (b). 0.1235 g APS and
0.1235 g NaHSO3 as initiators were added into 118ml water containing 5.1 ml MAA,
0.53 ml DMAEMA and 0.5 ml BMA. The copolymerization reaction was carried out for 24 h at 55 °C under the protection of N2. After the reaction finished, the product was dissolved in 3 M NaOH solution and filtrated to remove the undissolved impurities. Then the pH of the solution was adjusted to 3.1 by adding 3 M HCl. The precipitate was collected, and then dried in the vacuum to constant mass [13]. The yield of PMDB was defined as the ratio of the experimental value to theoretical value.
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2.2.2 Phase-forming test and phase diagram Dissolve PADBA and PMDB in 170 mM NaOH solution, respectively. Different concentrations of PADBA were used to test the possibility of forming ATPS with
ip t
different concentrations of PMDB. Stood mixed systems at room temperature (25 °C)
cr
until a distinct interphase was observed, then took out aliquots from the top and
us
bottom phase to measure the PADBA and PMDB concentration in both phases. The concentration of copolymers was determined by UV spectrophotometer as the
an
following method [12]: Two samples were prepared from the top phase and the bottom phase, respectively. The absorbance at 207 nm was the summation of PMDB
M
and PADBA. The concentration of PADBA was determined at 415 nm after reacting with 2% (w/w) potassium permanganate for 6 h. Then the concentration of PADBA was
te
d
used to calculate the absorbance of PADBA at 207 nm with the standard curve. The absorbance of PMDB was calculated by subtracting the absorbance of PADBA from the
Ac ce p
summation. Then the concentration of PMDB was calculated by the standard curve of
PMDB at 207 nm.
2.2.3 Recycling of copolymers The pH-response copolymers can be precipitated from solution by adjusting pH
to its isoelectric point (pI) by adding HCl. The precipitate could be separated by centrifugation and dried in a vacuum oven to constant mass. The recovery of copolymers can be calculated by the percentage of the dry weight of recovered polymer to that of the initial polymer weight. The pI was determined as the pH of the solution when the zeta potential is zero.
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Different pH values of the polymer solution (20 ppm) were adjusted and then the zeta potentials were measured using Zetasizer Nano ZEN3600 (Malvern). 2.3 Bioconversion reaction of Cephalexin in ATPS
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2.3.1 Analytic methods
cr
The concentrations of Cephalexin, PGME and 7-ADCA in the reaction mixtures
us
were determined by high performance liquid chromatography (HPLC) with a reversed-phase column C18 (150 mm×4.6 mm, 5 μm) from Dikma. The mobile phase
an
was consisted of sodium phosphate buffer (20 mM, pH 6.0) and methanol with a volume ratio of 30:70. The flow rate was set to be 1.0 ml/min. The injected volume
M
was 20μL. The detection wavelengths were 220 nm and 260 nm. The retention times of the components were 7.6 min for PGME (220 nm), 1.2 min for 7-ADCA (260 nm)
calibration curves.
te
d
and 5.8 min for Cephalexin (260 nm). The concentrations were calculated using
Ac ce p
2.3.2 Partition of 7-ADCA, PGME and Cephalexin in ATPS ATPS formed by mixing 7% (w/v) PADBA and 4% (w/v) PMDB with same volume
was used for the partition of 7-ADCA, PGME and Cephalexin. pH and temperature of ATPS were adjusted to 5.8 and 20 °C, respectively. Different salts including LiCl, KCl, NaCl, Gu-HCl, NH4Cl, KBr, Na2SO4, (NH4)2SO4 with concentrations from 10 mM to 80 mM were added into systems to affect partition coefficients, respectively. After aqueous two-phase systems reached equilibrium, the samples were taken out from top and bottom phase by a syringe. Their concentrations were determined by HPLC. The partition coefficient is defined as the ratio of concentration of a
Page 6 of 33
substance in the top phase to that in the bottom phase. To investigate the relationship between the electrostatic potential difference and the concentration of salts, the electrostatic potential difference between two phases
ip t
was measured after aqueous two-phase systems reached equilibrium using interphase
cr
potentiometer JIE09 (Powereach).
us
2.3.3 Synthesis of Cephalexin in ATPS
Syntheses of Cephalexin with immobilized penicillin acylase proceed according
an
to the reaction (Fig.2). Synthesis was performed under kinetic control. In the study, 15 ml ATPS formed by mixing same volume of 7% (w/v) PADBA and 4% (w/v) PMDB
M
were put into 25 ml graduated glass tube to carry out the reaction, at the same pH and temperature as the determination of partition coefficients. One salt referred to
te
d
the partition coefficients was added as desired amount. Substrates concentrations were 30 mM and 90 mM for 7-ADCA and PGME, respectively. Amount of loaded
Ac ce p
enzyme was 3.38 IUH/ml. Control synthesis was run in parallel where a
homogeneous aqueous system (phosphate buffer 0.1 M) was used as reaction medium under the same experimental conditions. For the synthesis of Cephalexin in the ATPS, the effect of increasing the substrate concentration and reducing the excess acyl donor were then studied. The range of 7-ADCA concentration in the whole system was from 30 mM to 70 mM and nucleophile to acyl donor molar ratio was from 1:1 to1:3. Samples were taken at specified intervals and were properly diluted prior to be assayed by HPLC. The yield of Cephalexin was molar ratio of product (Cephalexin) to limiting substrate (7-ADCA). In ATPS, the parameter was
Page 7 of 33
determined considering the amounts of product in both phases. 3. Results and discussions 3.1 Yield and recovery of copolymers
ip t
The isoelectric point (pI) is defined as the pH at which the net charge on a
cr
macromolecule is zero[14]. Fig.3 is the zeta potentials of both polymers at different
us
pH. pI of PADBA is 4.01 and PMDB is 3.09. The pI of PADBA is dependent on the ratio of AA to DMAEMA, the pI of PMDB is dependent on the ratio of MAA to
an
DMAEMA.
pH of the solution was adjusted to the pI of copolymer by adding HCl and
M
precipitate could be separated by centrifugation and dried in a vacuum oven. The recycled copolymer could be used to form ATPS again. Table1 is the yield and
te
d
recovery of PADBA and PMDB, Each data was average value of three parallel experimental points. The yield of PADBA is 73.1% and PMDB is 70.7%. The recovery
Ac ce p
of PADBA is 97.4% and PMDB is 97.2%. The recoveries are quite high and stable.
3.2 Phase diagram
Presented on Fig.4 is the phase diagram of PADBA/PMDB ATPS at room
temperature (25 °C). Bimodal curve divides a region of component compositions capable of forming two immiscible aqueous phases (region above the bimodal curve) from those which forms one phase (region below the bimodal curve). T, B and M represent the compositions of two polymers in top phase, the compositions in bottom phase and the total composition of the copolymers in the ATPS. According to the diagram, PADBA is mainly enriched in top phase while PMDB is mainly in bottom
Page 8 of 33
phase. The volume ratio of phase could be calculated according to length ratio of line MB and MT. In the experiment, the concentration of PADBA and PMDB was 3.5% (w/w) and 2% (w/w) in total phase systems which will give a volume ratio of about 2:1
3.3 Synthesis of Cephalexin
us
3.3.1 Partition of 7-ADCA, PGME and Cephalexin in ATPS
cr
ip t
when the two phases are in equilibrium.
The partition coefficients of 7-ADCA, PGME and Cephalexin in ATPS were
an
determined in this experiment. The partitioning behavior of a substance in ATPS may be affected by several factors including physicochemical properties of the polymers
M
(structure, molecular weight) and salt ions type etc. In the ATPS, partition coefficient of Cephalexin, 7-ADCA and PGME were 1.25, 0.2 and 1.0 without any salt (Fig.5
te
d
(a)). After adding salts, the maximum partition coefficient of Cephalexin reached 2.57 (Fig.5 (b)) in the presence of 20 mM NaCl, While at the NaCl concentration,
Ac ce p
the partition coefficients of 7-ADCA and PGME was 0.21 and 0.94 (Fig.5 (c) and (d)), respectively. In the reactions of Cephalexin synthesis, higher product yield is to be expected since 7-ADCA was biased in the PMDB-rich phase where the reaction occurs, while Cephalexin could enrich in the PADBA-rich phase where its hydrolysis
could be avoided.
Cephalexin and 7-ADCA have a structural difference in the side chain, Cephalexin has more hydrophobicity. In the ATPS, Cephalexin biased to the PADBA-rich phase while 7-ADCA biased to the PMDB-rich phase without adding any salts, it was believed that hydrophobicity interaction between them and the
Page 9 of 33
environment was the reason of the phenomenon. After adding inorganic salts, the salts will partition unequally between the phases. This unequal distribution, along with the requirement of electroneutrality leads to an electrostatic potential difference
ip t
between two phases (Donna potential). Fig.6 is the relationship between the
cr
electrostatic potential difference and the concentration of salts. According to the
us
Fig.5 (b) and Fig.6, the electrostatic potential difference is positively associated with the partition coefficient of Cephalexin. The adding of NaCl increases the
an
electrostatic potential difference between the two phases in the range of 10~30 mM NaCl concentration, and then produce an obvious repulsion to drive Cephalexin and
M
7-ADCA into the PADBA-rich phase. However, compared with the hydrophobicity interaction between 7-ADCA and the environment, the change of electrostatic
te
d
potential difference is so small that it has little influence on the partition of 7-ADCA. With the increasing of concentration of NaCl, the electrostatic potential difference
Ac ce p
dropped. This may attribute to high ionic strength. 3.3.2 Synthesis of Cephalexin in ATPS and single aqueous phase Cephalexin synthesis was performed in the ATPS and 0.1 M phosphate buffer
used as control. In the ATPS, 20 mM NaCl was added. Experiments were done in triplicate and reported results are the average of these triplicates. Results are illustrated in Fig.7 that records the time course of Cephalexin synthesis in the ATPS and in single aqueous phase. The reaction was quick during the first 1 h and then becomes stable as Cephalexin accumulated. In the single aqueous phase, the yield of Cephalexin
Page 10 of 33
reached to maximal yield with 73.58% at 3.5 h, and then began to drop significantly. In the ATPS, the viscosity of the copolymers resulted in longer time for the yield to reach maximum. The yield of Cephalexin reached to maximal yield with 97.3% at 5
ip t
h, and then declined insignificantly with time.
cr
The yield of Cephalexin was the equilibrium of three different catalytic
us
activities of the same penicillin acylase: a) synthesis of Cephalexin; b) hydrolysis of Cephalexin previously synthesized; c) hydrolysis of the activated acyl donor[15, 16].
an
Hydrolysis of Cephalexin can be promoted when Cephalexin accumulated around penicillin acylase. Furthermore, Cephalexin is a stronger inhibitor to penicillin
M
acylase. With the increase in concentration of Cephalexin, the activity of penicillin acylase was inhibited at a higher degree. Consequently, a transient maximum yield
te
d
of Cephalexin in the single aqueous phase was observed in Fig.7. However, the ATPS provided a reaction medium in which Cephalexin hydrolysis was avoided.
Ac ce p
This was clearly attributed to the partition of the Cephalexin to the PADBA-rich phase where its hydrolysis was strongly inhibited. Here, with bioconversion reaction went on, nearly 84% Cephalexin moved to the PADBA-rich phase at the presence of 20 mM NaCl. In this way, inhibition of penicillin acylase was removed to a large extent and the reaction equilibrium was shifted to the favorable synthesis direction. Additionally, penicillin acylase is likely to be in a more protective environment than in single aqueous medium[1, 17]. 3.3.3 Effect of substrates concentration on synthesis of Cephalexin in ATPS The effect of the substrate concentration on product yield was investigated in
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the ATPS with 20 mM NaCl. The details were shown in Fig.8, 98.2% product yield was obtained in the ATPS at 50 mM 7-ADCA at 6 h. With the increasing of substrates concentration, the yield reached maximum need longer time and the
ip t
maximum yield decreased.
cr
The effect of the substrate concentration on product yield is attributed to the
us
solubility of 7-ADCA. At 70 mM 7-ADCA and 210 mM PGME, the 7-ADCA is initially partly insoluble in the ATPS and mass transfer limitations are likely to be
that more precipitate may be produced.
an
significant. On the other hand, the by-product phenyl glycine is quite insoluble so
M
3.3.4 Effect of acyl donor to nucleophile ratio on synthesis of Cephalexin in ATPS Excess PGME has proven to be necessary to attain high yield of Cephalexin,
te
d
mainly because penicillin acylase can also act as an esterase and hydrolyze PGME to the side product. It’s a drawback of the synthesis of Cephalexin. The effect of lower
Ac ce p
molar ratios of PGME to 7-ADCA on product yield was investigated. In the experiment, 30 mM was chosen as the concentration of 7-ADCA due to the reaction equilibrium time, Results were presented in Fig.9. Results at a ratio of 2.5 were similar to 3, and the maximum yield can reach 95.4%. Even reducing the ratio to 2, the maximum yield can still keep above 90%. This may attribute to the partition of 7-ADCA. In the ATPS, the 7-ADCA and immobilized penicillin acylase were biased in the bottom phase, while the PGME was distributed evenly. That’s to say, under the same conditions of substrates concentration, there were more 7-ADCA around the penicillin acylase and inhibit the hydrolysis of PGME.
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4. Conclusion In this study, a new recyclable pH-pH aqueous two-phase system was applied to synthetic reaction of Cephalexin. This ATPS was suitable for the reaction due to the
ip t
removing inhibition of product. Even at lower acyl donor to nucleophile ratio, yield
cr
of Cephalexin still keeps very high. It is believed that synthesis reaction in the
us
recycling ATPS is potential application value in future bio-separation and
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biochemical industry, not limited to synthesis of Cephalexin.
Acknowledgment
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The authors very appreciate financial support from National Natural Science Foundation of China (No. 21376078) and National Special Fund for State Key
Ac ce p
te
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Laboratory of Bioreactor Engineering (2060204).
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References [1] C. Aguirre, M. Toledo, V. Medina, A. Illanes, Effect of cosolvent and pH on the kinetically controlled synthesis of cephalexin with immobilised penicillin
ip t
acylase, Process Biochemistry, 38 (2002) 351-360.
cr
[2] X. CAO, J. ZHU, D. WEI, B.-K. HUR, Biosynthesis of cephalexin in
us
PEG400-ammonium sulfate and PEG400-magnesium sulfate aqueous two-phase systems, Journal of microbiology and biotechnology, 14 (2004)
an
62-67.
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M
controlled synthesis of ampicillin with immobilized penicillin acylase in organic media, Applied biochemistry and biotechnology, 97 (2002) 165-179.
te
d
[4] A. Illanes, Z. Cabrera, L. Wilson, C. Aguirre, Synthesis of cephalexin in ethylene glycol with glyoxyl-agarose immobilised penicillin acylase: temperature and
Ac ce p
pH optimisation, Process Biochemistry, 39 (2003) 111-117.
[5] A. Illanes, L. Wilson, O. Corrotea, L. Tavernini, F. Zamorano, C. Aguirre, Synthesis of cephalexin with immobilized penicillin acylase at very high substrate concentrations in fully aqueous medium, Journal of Molecular Catalysis B: Enzymatic, 47 (2007) 72-78.
[6] D.-Z. Wei, J.-H. Zhu, X.-J. Cao, Enzymatic synthesis of cephalexin in aqueous two-phase systems, Biochemical engineering journal, 11 (2002) 95-99. [7] C. Aguirre, I. Concha, J. Vergara, R. Riveros, A. Illanes, Partition and substrate concentration effect in the enzymatic synthesis of cephalexin in aqueous
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two-phase systems, Process Biochemistry, 45 (2010) 1163-1167. [8] G. Chen, A.S. Hoffman, Graft copolymers that exhibit temperature-induced phase transitions over a wide range of pH, Nature, 373 (1995) 49-52.
cr
complexation, Macromolecules, 18 (1985) 83-85.
ip t
[9] J. Ricka, T. Tanaka, Phase transition in ionic gels induced by copper
us
[10] S. Miao, J. Chen, X. Cao, Preparation of a novel thermo-sensitive copolymer forming recyclable aqueous two-phase systems and its application in
an
bioconversion of Penicillin G, Separation and Purification Technology, 75 (2010) 156-164.
M
[11] H. Li, X. Cao, Bioconversion of cephalosporin-G to 7-ADCA in a pH-thermo
te
(2011) 1753-1758.
d
sensitive recycling aqueous two-phase systems, Process Biochemistry, 46
[12] B. Yan, X. Cao, Preparation of aqueous two-phase systems composed of two
Ac ce p
pH-response polymers and liquid–liquid extraction of demeclocycline, Journal of Chromatography A, 1245 (2012) 39-45.
[13] W. Liang, X. Cao, Preparation of a pH-sensitive polyacrylate amphiphilic copolymer and its application in cellulase immobilization, Bioresource Technology, 116 (2012) 140-146.
[14] R. Chang, Physical chemistry for the chemical and biological sciences, University Science Books, 2000. [15] R. Fernandez ‐ Lafuente, G. JM, Dynamic reaction design of enzymic biotransformations in organic media: equilibrium‐controlled synthesis of
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antibiotics by penicillin G acylase, Biotechnology and applied biochemistry, 24 (1996) 139-145. [16] R. Fernandez-Lafuente, C. Rosell, J. Guisan, The presence of methanol exerts a
ip t
strong and complex modulation of the synthesis of different antibiotics by
cr
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[17] C.M. Rosell, M. Terreni, R. Fernandez-Lafuente, J.M. Guisan, A criterion for
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the selection of monophasic solvents for enzymatic synthesis, Enzyme and
Ac ce p
te
d
M
Microbial Technology, 23 (1998) 64-69.
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Figure captions
Fig.1 (a). Synthesis of pH-response copolymer PADBA.
ip t
x, y, z, m stands for monomer ratio of AA, DMAEMA, BMA, Aal, respectively.
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Fig.1 (b). Synthesis of pH-response copolymer PMDB.
cr
The functional monomers mole ratio is AA: DM: BMA: Aal = 23:8:1:6.
x, y, z, m stands for monomer ratio of AA, DMAEMA, BMA, respectively. The
an
functional monomers mole ratio is MAA: DM: BMA = 19:1:1.
Fig.2 Reaction scheme for Immobilized penicillin acylase-catalyzed synthesis of
M
Cephalexin. (1) PGME, (2) 7-ADCA), (3) Cephalexin, (4) Phenylglycine (PG). Fig.3 Zeta potential of two polymers at different pH
temperature (25 °C)
te
d
Fig.4 The phase diagram of PADBA/PMDB aqueous two-phase systems at room
Ac ce p
Fig.5 Effect of salts on partition coefficient: (a) no salts (b) Cephalexin; (c) 7-ADCA; (d) PGME.
Different salts with concentrations from 10 mM to 80 mM with 10 mM step
were added into systems. The partition coefficient was measured at 20 °C and pH 5.8 by HPLC.
Fig.6 Effect of salts on the electrostatic potential difference The concentrations of salts were from 10 mM to 80 mM with 10 mM step. The electrostatic potential difference was measured at 20 °C and pH 5.8. Fig.7 Synthesis of Cephalexin in PADBA/PMDB systems with 20 mM NaCl and
Page 17 of 33
single aqueous phase. Reaction condition: 30 mM 7-ADCA, 90 mM PGME, pH 5.8, 20 °C and 3.38 IUH/ml.
ip t
The pH, temperature and amount of loaded enzyme have been optimized in the
cr
single aqueous phase at previous works.
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Fig.8 Effect of substrates concentration on synthesis of Cephalexin in PADBA/PMDB systems with 20 mM NaCl
an
Reaction condition: pH 5.8, 20 °C and 3.38 IUH/ml. The range of 7-ADCA concentration in the whole system was from 30 mM to 70 mM.
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Fig.9 Effect of acyl donor to nucleophile ratio on synthesis of Cephalexin in PADBA/PMDB systems with 20 mM NaCl.
te
d
Reaction condition: 30 mM 7-ADCA, pH 5.8, 20 °C and 3.38 IUH/ml. The
Ac ce p
range of nucleophile to acyl donor molar ratio was from 1:1 to1:3
Page 18 of 33
Yield
Recovery
PADBA
73.1%
97.4%
PMDB
70.7%
97.2%
Ac ce p
te
d
M
an
us
cr
ip t
Table 1 The yield and recovery of PADBA and PMDB
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Ac ce p
te
d
M
an
us
cr
ip t
Highlights A recyclable ATPS composed by two pH‐response copolymers was prepared Substrates K7‐ADCA and KPGME biased in bottom phase, and product KCephalexin biased in top phase Synthesis of Cephalexin in ATPS has been conducted with 98.2% yield Ratio of acyl donor to nucleophile decreased from 3 to 2
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Figure.1 (a)
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Figure.1 (b)
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Figure.4
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Figure.5 (a)
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Figure.5 (b)
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Figure.8
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