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Continuous enzymatic production of 5′-nucleotides using free nuclease P1 in ultrafiltration membrane reactor
Journal of Membrane Science 345 (2009) 217–222
Contents lists available at ScienceDirect
Journal of Membrane Science journal homepage: www.elsevier.com/locate/memsci
Continuous enzymatic production of 5 -nucleotides using free nuclease P1 in ultrafiltration membrane reactor Lu-E Shi a , Guo-Qing Ying b,∗ , Zhen-Xing Tang b , Jian-Shu Chen b , Wen-Yue Xiong b , Hong Wang b a b
College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, Zhejiang 310036, China College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, China
a r t i c l e
i n f o
Article history: Received 28 May 2009 Received in revised form 11 August 2009 Accepted 1 September 2009 Available online 6 September 2009 Keywords: Enzyme membrane reactor Nucleotides Preparation
a b s t r a c t In this paper, production of 5 -nucleotides using a free enzyme membrane reactor was presented. The enzyme membrane reactor consists in a coupling of a membrane separation process with an enzymatic reaction. Retention of substrate (RNA) and catalyst (nuclease P1) by the ultrafiltration membranes was observed. The best enzyme membrane reactor configuration was achieved with a polyethersulfone membrane of 30 kDa molecular weight cut-off, since the substrate and biocatalyst were retained with no loss of enzyme activity. Enzyme stability and optimum temperature were investigated. Other optimized operating conditions were also obtained: working temperature 65 ◦ C, flow rate 2.0 mL/min (transmembrane pressure 0.050 MPa), and reactor volume 100 mL. The results demonstrated that enzymatic production of 5 -nucleotides from RNA was efficiently conducted by employing an ultrafiltration membrane reactor. © 2009 Elsevier B.V. All rights reserved.
1. Introduction 5 -Nucleotides have been widely used in pharmaceutical and food industries [1,2]. They can be used to synthesize the antivirus and anticancer drugs as the intermediate. The nucleotide derivates have important uses in the treatment of the illness of human central nervous system and circulatory system. The interest in nucleotides has grown during the last few years due to the applications in antivirus and anticancer treatment. 5 -Nucleotides can be produced directly by fermentation using microorganisms, chemical method, or enzymatic method. The enzymatic method has incomparable advantages than others, such as low complexity and high yields, which make it to be the most widely used method in industry [3]. Many industrial enzymatic hydrolysis reactions are performed in batch reactors. However, such processes suffer from disadvantages such as low efficiency due to start up and shut down procedures, high labor costs or batch to batch variations. Another main process limitation is the need to inactivate and separate enzymes at the end of each step, entailing high processing costs [4]. Ultrafiltration enzymatic membrane reactors greatly reduce or eliminate the bottlenecks associated with bath reactor hydrolysis [5]. Enzyme membrane bioreactor has been developed with the prosperity of biochemical engineering and membrane separation techniques. The enzyme membrane reactor which integrates
∗ Corresponding author. E-mail address: [email protected] (G.-Q. Ying). 0376-7388/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.memsci.2009.09.001
enzymatic hydrolysis, products separation and catalyst recovery simultaneously in one device has attracted more and more attention during the last 10 years. Membrane processing is a relatively new unit operation for nucleotides production. As a practical use of the theory integrated bio-reaction separation process, enzyme membrane reactor has its overwhelming virtues. Now it has been widely used in food industry, biochemical engineering, pharmaceutics and environmental process [6–10]. Here enzyme membrane reactor system was employed to increase the yield by reducing inhibition due to product accumulation. Nuclease P1 and unconverted RNA are retained within the bioreactor system by membrane with a specific molecular cutoff size, whereas synthesized nucleotides are removed from the bioreactor by ultrafiltration through membrane. Substrate is continuously fed to the reactor in order to compensate for permeate flux and maintain constant reaction volume in the reactor. Additional advantages of the continuous recycling membrane reactor are its high efficiency, low labor costs, possibility of full automation of the apparatus. Preparation of 5 -nucleotides by enzymatic hydrolysis of RNA using free enzyme membrane bioreactor was studied in this paper. Operational parameters were studied too. 2. Materials Collector-magnetic heating blender (DF-II): Medical Instrument Factory, Jintan, Jiangsu Province, China; Nitrogen bottles (YQD-6A): ShangHai Regulator Factory Co., Ltd; enzyme membrane reactor and polyethersulfone (PES) membrane: Chinese Academy of sci-
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ences, ShangHai Branch, China; all other reagents were of analytical grade. 2.1. Enzyme production and isolation For nuclease P1 production, the bacteria was grown at 30 ◦ C in a medium (pH 7.0) consisting of (w/v): sucrose 3.0%, potassium dihydrogen phosphate 0.10%, ferrous sulfate 0.010%, sodium nitrate 0.30%, magnesium sulfate 0.050%, potassium chloride 0.050%, and potato extract 20%. This 24 h grown mother culture (10 mL) was used to inoculate 50 mL of production medium containing (w/v): glucose 6.0%, peptone 0.20%, groundnut meal 0.50%, zinc sulfate 0.030%, calcium carbonate 0.040%, and potassium dihydrogen phosphate 0.10%. The pH of the medium was adjusted to 5.4 with HCl. Erlenmeyer flasks (500 mL) containing 50 mL of medium were incubated at 28 ◦ C in an orbital shaker at 240 rpm for 49 h. The 5 -phosphodiesterase solution was harvested by centrifugation at 4000 rpm at 4 ◦ C for 10 min, and the supernatant thus obtained was used as the crude enzyme preparation. The enzyme was purified to homogeneity according to SDS-PAGE by thermal deactivation, ultrafiltration, (NH4 )2 SO4 precipitation, phenyl sepharose chromatography, ion-exchange chromatography and gel filtration. 2.2. Enzyme activity assay Enzyme activity was measured in terms of the acid-soluble nucleotides amount produced by RNA hydrolysis catalyzed by nuclease P1. Enzyme solution (0.10 mL) was incubated with substrate solution (1.0% RNA, 0.125 M acetate buffer, pH 5.4, 3.0 mM Zn2+ ) at 69 ◦ C for 15 min. The reaction was stopped by the addition of 2.0 mL ice-cold nucleic acid precipitator (0.25% ammonium molybdate dissolved in 2.5% perchloric acid). The mixtures were settled at ice-bath for more than 10 min. The precipitated RNA was removed by centrifugation at 4000 rpm at 4 ◦ C for 10 min. The supernatant fluid was diluted 50-fold with distilled water. The absorbance at 260 nm of the diluted solution was read with a blank incubation without enzyme. The activity (U) of enzyme was calculated according to the following formula:
2.3. Determination of nucleotides 2.3.1. Spectrophotometer 1.0 mL precipitator (0.25% ammonium molybdate dissolved in 2.5% perchloric acid) was added into 1.0 mL sample. The precipitated RNA was removed by centrifugation at 4000 rpm at 4 ◦ C for 10 min. The supernatant fluid was diluted 100-fold with distilled water, absorbance being measured at 260 nm. Nucleotides concentration was calculated according to the following formula: C=
OD260 × 200 40
C: 5 -nucleotides concentration (mg/ml); 200: diluted solution multiples; 40: concentration of 1.0 mg/mL nucleotide extinction coefficient. 2.3.2. HPLC method Column: Shimadzu WAX-1 anion exchange column, Ø4.0 mm × 50 mm. The mobile phase—20 mmol/L (pH 7.0) phosphate buffer:480 mmol/L (pH 6.8) phosphate buffer = 1:1, flow rate 1.0 mL/min, 260 nm UV detection, room temperature. 2.4. Continuous stirred tank reactor (CSTR) A convective 300 mL glass stirred tank reactor including a flat ultrafiltration membrane (30 kDa cut-off) having an internal diameter of 8 cm was used for the continuous production of 5 -nucleotides (Fig. 1). Mixing was ensured by magnetic stirrer (180–300 rpm). The reactor was operated at constant temperature using water bath. Temperature and magnetic stirring were controlled by collector-magnetic heating blender as shown in Fig. 1(3). The substrate solution (pH 5.4) containing 1.0% RNA and 3.0 mM Zn2+ was fed through a filter with a peristaltic pump at a constant flow rate as required for the assay. The reaction was started by the injection of nuclease P1 (200 U) into the reactor. Pressure was maintained by means of a valve and pressure gauge. Samples were periodically taken at the reactor outlet. 2.5. Cleaning of membrane
4.0×50 = OD260 × a × 133.3 0.1 × 15
Before and after each run, the reactor was cleaned according to the following procedure:
a: A dilution factor of the enzyme before enzyme activity assay. One unit of enzyme activity was defined as the amount of enzyme that produced an increase in the optical density of 1.0 in 1 min at 260 nm.
(1) The membrane was rinsed with running water at room temperature for 10 min. (2) The system was circulated with a sodium hydroxide solution (0.25 M) at 60 ◦ C for 15 min. This was followed by rinsing with
enzyme activity (units/ml) = OD260 ×
Fig. 1. Schematic diagram of enzymatic membrane bioreactor. (1) Substrate reservoir; (2) pump; (3) collector-magnetic heating blender; (4) enzyme reactor; (5) products collector; (6) nitrogen bottle; (7) pressure gauge.
L.-E. Shi et al. / Journal of Membrane Science 345 (2009) 217–222
Fig. 2. Membrane flux curve of different MWCO.
running water at room temperature for 10 min. This stage was usually repeated. (3) The system was circulated with a nitric acid solution (0.10%) at 60 ◦ C for 15 min. Finally the system was rinsed with demineralized water for 30 min. 2.6. Selection of membrane molecular weight cut-off In order to assure that the membrane molecular weight cut-off (MWCO) is large enough to allow the hydrolysis product to pass through the membrane module, nucleotides’ molecular weight should be referenced. On the other hand, MWCO must be small enough to ensure that no enzyme and substrate leaks through the membrane module. 3. Results and discussion 3.1. Characterization of membrane 3.1.1. Membrane flux The substrate permeability of new membrane module was measured to test the initial membrane flux at 65 ◦ C. Fig. 2 indicates that polyethersulfone (PES) membrane flux is quite different with different molecular weight cut-off. Membrane flux was reduced with the extension of operating time. Membrane flux dropped by two main factors: first, concentration polarization, second was the result of macromolecular material absorption fouling. When enzyme and substrate cycled through membrane, they accumulated on the membrane surface at the high pressure side of membrane, which led to concentration gradient and concentration polarization. As shown in Fig. 2, after an initial flux decline, a quasi-steady-state was reached. Membrane with higher molecular weight, its membrane flux declined less than that with lower molecular weight. Membrane whose molecular weight was 30 kDa, after running 300 min almost had no membrane flux decline phenomenon, which suggests that fouling phenomenon is negligible with suitable molecular weight membrane.
219
Fig. 3. Substrate concentration of retentate and permeate through membrane (30,000 kDa).
for RNA hydrolysis must be able to retain enzyme as well as RNA. Molecular weight of the enzyme is about 42 kDa and that of the substrate is more than 100 kDa. Therefore, the membrane pores have to be well defined. 10 mg/mL RNA solution and 1.0 mg/mL nuclease P1 solution was circulated in the enzyme membrane reactor for 5 h in two separate experiments at 65 ◦ C. As seen in Figs. 3 and 4, there were no substrate RNA and nuclease P1 in permeate side. It could be concluded that the membrane had good retention to substrate and enzyme, though there existed a little loss of substrate RNA and nuclease P1 in the retentate side. Maybe some of them were attached on the reactor wall or on the top of membrane. A complete retention of the enzyme and substrate within the reactor is the prerequisite for continuous operation of enzyme membrane reactor. This offers the great advantage of extensive and continuous use of enzymes. The same curve could be observed using membrane molecular weight of 6, 10, 20 kDa (data not shown). But when membrane molecular weight was 50 kDa, there was some leakage of enzyme and substrate (data not shown). So membrane with molecular weight of 6, 10, 20, and 30 kDa are all suitable to retain enzyme and substrate.
3.2. Evaluation of ultrafiltration membrane 3.2.1. RNA and enzyme retention To find a suitable membrane is a fundamental task in the process carried out in the membrane bioreactor. A membrane to be suitable
Fig. 4. Enzyme concentration of retentate and permeate through membrane (30,000 kDa).
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Fig. 6. Effect of temperature on membrane flux under different pressure. Fig. 5. Effect of membrane molecular weight on nucleotides production.
3.2.2. Nucleotides permeation Nucleotides produced during the enzyme hydrolytic reaction should permeate through the membrane in order to avoid nucleotides accumulation and inhibition phenomena. Thus, the membrane was tested in model experiments. Various initial concentrations (5, 10, and 20 mg/mL) nucleotides were circulated at a flow rate of 1.5 mL/min in the membrane reactor at 65 ◦ C. Nucleotides concentration was determined as a function of time. The data (data not shown) have proven that nucleotides permeated very easily across the membrane (molecular weight of 6, 10, 20, and 30 kDa) and the equilibrium concentration could be reached after 3 h. 3.2.3. Size of membrane molecular weight cut-off The steady-state for RNA hydrolysis to nucleotides was reached about after 3 h reaction with membrane molecular weight from 6 to 50 kDa (Fig. 5). From Fig. 5 we can see that membrane molecular weight did not significantly affect the yield. But the nucleotide product was relatively low with a membrane of 50 kDa. This is because the withholding molecular weight is too large and the reaction time is too short, and it is difficult to control speed. At the same time, using membrane with molecular weight of 50 kDa, part of nucleic acids and nuclease P1 infiltrate through the reactor, which also leads to partial low production concentration. In a word, membrane with 50 kDa molecular weight is not suitable for the preparation of nucleotides. Among all the membrane used, higher levels of nucleotides could be obtained with membrane of 30 kDa. And membrane with higher molecular weight is easier to reach a certain speed with smaller pressure, which required less nitrogen. So, production cost could be reduced using higher molecular weight membrane. Membrane with 30 kDa molecular weight was selected for the following experiment.
ture was beneficial in that it allowed a high permeate flux through membrane. So just take into account the membrane flux, higher temperature is more suitable. 3.3.2. Study of enzyme stability and optimum temperature Enzyme was incubated at different temperatures for 12 h and then cooled at ice-bath. The residual enzyme activity was determined at pH 5.4, 69 ◦ C under standard assay conditions. The maximum activity of the enzyme has been taken as 100%. The effect of enzyme denaturation with time at 40, 50, 60, 65, and 70 ◦ C are shown in Fig. 7. Temperature has a significant effect on enzyme stability. As seen in Fig. 8, the optimum temperature for hydrolysis of nucleotides is 69 ◦ C. But enzyme was more stable with lower temperature. The loss of enzyme activity was greatly reduced at 65 ◦ C. When enzyme incubated at 70 ◦ C for 300 min, 33% enzyme activity was lost, but at 65 ◦ C, only about 3% enzyme activity was lost. Therefore, a temperature of 65 ◦ C is more attractive. In industrial processes, finding a working temperature that gives high enzyme activity with good stability represents a compromise between lower process costs and higher productivities. Fig. 9 shows the effect of temperature on nucleotides production. As seen in Fig. 9 temperature had a greater influence on the reaction. When temperature increased, it can accelerate reaction rate, which led to produce more and more nucleotides. But when the temperature reached 70 ◦ C, nucleotides was less than
3.3. Working temperature 3.3.1. Effect of temperature on membrane flux The best temperature for enzymatic hydrolysis of nucleic acid is 65 ◦ C. High temperature will affect the enzyme activity, so only within the scope of 20–65 ◦ C was inspected the temperature impact of membrane flux. The mixtures of substrate and enzyme were adopted in this experiment. The experimental results are shown in Fig. 6. As seen in Fig. 6, membrane flux was increased with the increase of temperature. Membrane with bigger initial membrane flux was increased more as temperature increased. A high tempera-
Fig. 7. Thermal stability of nuclease P1.
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Fig. 8. Optimum temperature of nuclease P1.
that at 65 ◦ C. Generally the impact of temperature on reaction performances in two areas, on the one hand for the chemical reaction, the temperature rises every 10 ◦ C, the reaction rate can be doubled. Therefore, the enzyme decomposition will help improve the RNA hydrolysis rate with the increase of temperature; on the other hand temperature is too high, would undermine the structure of protein, a protein heat inactivation phenomenon, making the active site of the spatial structure changes, influence the integration of RNA and enzyme activity site, thereby inhibiting the enzyme’s catalytic reaction. These two factors were affecting the enzyme activity and effectivity. Thermal stability and hydrolysis rate should be comprehensively considered. Considering all the factors studied above, 65 ◦ C as working temperature is more suitable.
Fig. 10. Effect of pressure on membrane flux of 30,000 kDa.
the substrate and enzyme do not have enough reaction time. But later, the difference was smaller and smaller, especially among 0.6, 1.2, 1.6, and 2.0 mL/min. But the membrane reactor productivity (nucleotides concentration multiply flow rate) was increased with the increase of flow rate. So, 2.0 mL/min was the optimum flow rate. 3.5. Residence time
The enhancement of transmembrane pressure in the range of 0.03–0.07 MPa increased the membrane flux (Fig. 10). So even there existed concentration polarization and membrane fouling which had effect on membrane flux, constant flow rate can be reached by adjusting the transmembrane pressure. Fig. 11 shows the flow rate effect on nucleotides production. As shown in Fig. 11, at first with the increase of flow rate, the nucleotides in permeation side was reduced (within 150 min). This is because with high flow rate,
Residence time was calculated as a total volume of the reactor divided by the flow rate [11,12] (t = V/J, V – reactor volume, J – membrane flux), when membrane flux is fixed, t is in proportion to V. In this experiment, membrane flux was kept at 2.0 mL/min. As can be seen in Fig. 12, when flow rate was certain, nucleotides content was increased gradually with the increase of residence time. It is the same as literature reported [13]. The increase in the nucleotides concentration with the increase of residence time is a consequence of total time that substrates keep reacting in the reactor. The residence time can be considered as the average time that substrates keep reacting in the reactor and, as a consequence, if residence time increases substrate conversion also increase. The results clearly show that, at low residence time, products concentration decreased, and this effect was particularly significant for small residence time.
Fig. 9. Effect of temperature on nucleotides production.
Fig. 11. Effect of membrane flux on nucleotides production.
3.4. Effect of flow rate
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from RNA was efficiently conducted by employing an ultrafiltration membrane reactor. References
Fig. 12. Effect of enzyme membrane reactor volume on nucleotides production.
4. Conclusions In this paper, production of 5 -nucleotides from RNA was successfully obtained by a free enzyme membrane reactor. The best enzyme membrane reactor configuration was achieved with a polyethersulfone membrane of 30 kDa molecular weight cut-off, since the substrate and biocatalyst was retained with no loss of enzyme activity. The optimized operating conditions were also obtained: working temperature 65 ◦ C, flow rate 2.0 mL/min (transmembrane pressure 0.050 MPa), reactor volume 100 mL. The results demonstrated that enzymatic production of 5 -nucleotides
[1] A. Kuninaka, Nucleotides and related compounds, in: H.J. Rehm, G. Reed (Eds.), Biotechnology, vol. 6, Wiley-VCH, Germany, 1996, pp. 561–612. [2] R. Gerald, W. Tilak, Yeast derived products, in: Yeast Technology, AVI, New York, 1991, pp. 369–412. [3] M.D. Benaiges, J. Lopez-Santin, C. Sola, Production of 5 -ribonucleotides by enzymatic hydrolysis of RNA, Enzyme Microb. Technol. 12 (1990) 86–89. [4] D. Paolucci-Jeanjean, M.P. Belleville, G.M. Rios, A comprehensive study of the loss of enzyme activity in a continuous membrane reactor-application to starch hydrolysis, J. Chem. Technol. Biotechnol. 76 (2001) 273–278. [5] A. Guadix, F. Camacho, E.M. Guadix, Production of whey protein hydrolysates with reduced allergenicity in a stable membrane reactor, J. Food Eng. 72 (4) (2006) 398–405. [6] E.J. Tomotani, M. Vitolo, Production of high-fructose syrup using immobilized invertase in a membrane reactor, J. Food Eng. 80 (2) (2007) 662–667. [7] F.J. Hernández, A.P. de los Ríos, D. Gómez, M. Rubio, G. Villora, A new recirculating enzymatic membrane reactor for ester synthesis in ionic liquid/supercritical carbon dioxide biphasic systems, Appl. Catal. B: Environ. 67 (1–2) (2006) 121–126. [8] T.M. LaPara, C.G. Klatt, R. Chen, Adaptations in bacterial catabolic enzyme activity and community structure in membrane-coupled bioreactors fed simple synthetic wastewater, J. Biotechnol. 121 (3) (2006) 368–380. [9] W.S. Long, A.H. Kamaruddin, S. Bhatia, Enzyme kinetics of kinetic resolution of racemic ibuprofen ester using enzymatic membrane reactor, Chem. Eng. Sci. 60 (18) (2005) 4957–4970. [10] R. Kapel, A. Chabeau, J. Lesage, G. Riviere, R. Ravallec-Ple, D. Lecouturier, M. Wartelle, D. Guillochon, P. Dhulster, Production, in continuous enzymatic membrane reactor, of an anti-hypertensive hydrolysate from an industrial alfalfa white protein concentrate exhibiting ACE inhibitory and opioid activities, Food Chem. 98 (1) (2006) 120–126. [11] D. Darnoko, M. Cheryan, W.E. Artz, Saccharification of cassava starch in an ultrafiltration reactor, Enzyme Microb. Technol. 11 (1989) 154–159. [12] A.G. Przywecka, L. Slominska, Saccharification of potato starch in an ultrafiltration reactor, J. Food Eng. 79 (2007) 539–545. [13] L. Giorno, E. Drioli, G. Carvoli, A. Cassano, L. Donato, Study of an enzyme membrane reactor with immobilized fumarase for production of l-malic acid, Biotechnol. Bioeng. 72 (1) (2001) 77–84.
Contents lists available at ScienceDirect
Journal of Membrane Science journal homepage: www.elsevier.com/locate/memsci
Continuous enzymatic production of 5 -nucleotides using free nuclease P1 in ultrafiltration membrane reactor Lu-E Shi a , Guo-Qing Ying b,∗ , Zhen-Xing Tang b , Jian-Shu Chen b , Wen-Yue Xiong b , Hong Wang b a b
College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, Zhejiang 310036, China College of Pharmaceutical Science, Zhejiang University of Technology, Hangzhou, Zhejiang 310014, China
a r t i c l e
i n f o
Article history: Received 28 May 2009 Received in revised form 11 August 2009 Accepted 1 September 2009 Available online 6 September 2009 Keywords: Enzyme membrane reactor Nucleotides Preparation
a b s t r a c t In this paper, production of 5 -nucleotides using a free enzyme membrane reactor was presented. The enzyme membrane reactor consists in a coupling of a membrane separation process with an enzymatic reaction. Retention of substrate (RNA) and catalyst (nuclease P1) by the ultrafiltration membranes was observed. The best enzyme membrane reactor configuration was achieved with a polyethersulfone membrane of 30 kDa molecular weight cut-off, since the substrate and biocatalyst were retained with no loss of enzyme activity. Enzyme stability and optimum temperature were investigated. Other optimized operating conditions were also obtained: working temperature 65 ◦ C, flow rate 2.0 mL/min (transmembrane pressure 0.050 MPa), and reactor volume 100 mL. The results demonstrated that enzymatic production of 5 -nucleotides from RNA was efficiently conducted by employing an ultrafiltration membrane reactor. © 2009 Elsevier B.V. All rights reserved.
1. Introduction 5 -Nucleotides have been widely used in pharmaceutical and food industries [1,2]. They can be used to synthesize the antivirus and anticancer drugs as the intermediate. The nucleotide derivates have important uses in the treatment of the illness of human central nervous system and circulatory system. The interest in nucleotides has grown during the last few years due to the applications in antivirus and anticancer treatment. 5 -Nucleotides can be produced directly by fermentation using microorganisms, chemical method, or enzymatic method. The enzymatic method has incomparable advantages than others, such as low complexity and high yields, which make it to be the most widely used method in industry [3]. Many industrial enzymatic hydrolysis reactions are performed in batch reactors. However, such processes suffer from disadvantages such as low efficiency due to start up and shut down procedures, high labor costs or batch to batch variations. Another main process limitation is the need to inactivate and separate enzymes at the end of each step, entailing high processing costs [4]. Ultrafiltration enzymatic membrane reactors greatly reduce or eliminate the bottlenecks associated with bath reactor hydrolysis [5]. Enzyme membrane bioreactor has been developed with the prosperity of biochemical engineering and membrane separation techniques. The enzyme membrane reactor which integrates
∗ Corresponding author. E-mail address: [email protected] (G.-Q. Ying). 0376-7388/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.memsci.2009.09.001
enzymatic hydrolysis, products separation and catalyst recovery simultaneously in one device has attracted more and more attention during the last 10 years. Membrane processing is a relatively new unit operation for nucleotides production. As a practical use of the theory integrated bio-reaction separation process, enzyme membrane reactor has its overwhelming virtues. Now it has been widely used in food industry, biochemical engineering, pharmaceutics and environmental process [6–10]. Here enzyme membrane reactor system was employed to increase the yield by reducing inhibition due to product accumulation. Nuclease P1 and unconverted RNA are retained within the bioreactor system by membrane with a specific molecular cutoff size, whereas synthesized nucleotides are removed from the bioreactor by ultrafiltration through membrane. Substrate is continuously fed to the reactor in order to compensate for permeate flux and maintain constant reaction volume in the reactor. Additional advantages of the continuous recycling membrane reactor are its high efficiency, low labor costs, possibility of full automation of the apparatus. Preparation of 5 -nucleotides by enzymatic hydrolysis of RNA using free enzyme membrane bioreactor was studied in this paper. Operational parameters were studied too. 2. Materials Collector-magnetic heating blender (DF-II): Medical Instrument Factory, Jintan, Jiangsu Province, China; Nitrogen bottles (YQD-6A): ShangHai Regulator Factory Co., Ltd; enzyme membrane reactor and polyethersulfone (PES) membrane: Chinese Academy of sci-
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ences, ShangHai Branch, China; all other reagents were of analytical grade. 2.1. Enzyme production and isolation For nuclease P1 production, the bacteria was grown at 30 ◦ C in a medium (pH 7.0) consisting of (w/v): sucrose 3.0%, potassium dihydrogen phosphate 0.10%, ferrous sulfate 0.010%, sodium nitrate 0.30%, magnesium sulfate 0.050%, potassium chloride 0.050%, and potato extract 20%. This 24 h grown mother culture (10 mL) was used to inoculate 50 mL of production medium containing (w/v): glucose 6.0%, peptone 0.20%, groundnut meal 0.50%, zinc sulfate 0.030%, calcium carbonate 0.040%, and potassium dihydrogen phosphate 0.10%. The pH of the medium was adjusted to 5.4 with HCl. Erlenmeyer flasks (500 mL) containing 50 mL of medium were incubated at 28 ◦ C in an orbital shaker at 240 rpm for 49 h. The 5 -phosphodiesterase solution was harvested by centrifugation at 4000 rpm at 4 ◦ C for 10 min, and the supernatant thus obtained was used as the crude enzyme preparation. The enzyme was purified to homogeneity according to SDS-PAGE by thermal deactivation, ultrafiltration, (NH4 )2 SO4 precipitation, phenyl sepharose chromatography, ion-exchange chromatography and gel filtration. 2.2. Enzyme activity assay Enzyme activity was measured in terms of the acid-soluble nucleotides amount produced by RNA hydrolysis catalyzed by nuclease P1. Enzyme solution (0.10 mL) was incubated with substrate solution (1.0% RNA, 0.125 M acetate buffer, pH 5.4, 3.0 mM Zn2+ ) at 69 ◦ C for 15 min. The reaction was stopped by the addition of 2.0 mL ice-cold nucleic acid precipitator (0.25% ammonium molybdate dissolved in 2.5% perchloric acid). The mixtures were settled at ice-bath for more than 10 min. The precipitated RNA was removed by centrifugation at 4000 rpm at 4 ◦ C for 10 min. The supernatant fluid was diluted 50-fold with distilled water. The absorbance at 260 nm of the diluted solution was read with a blank incubation without enzyme. The activity (U) of enzyme was calculated according to the following formula:
2.3. Determination of nucleotides 2.3.1. Spectrophotometer 1.0 mL precipitator (0.25% ammonium molybdate dissolved in 2.5% perchloric acid) was added into 1.0 mL sample. The precipitated RNA was removed by centrifugation at 4000 rpm at 4 ◦ C for 10 min. The supernatant fluid was diluted 100-fold with distilled water, absorbance being measured at 260 nm. Nucleotides concentration was calculated according to the following formula: C=
OD260 × 200 40
C: 5 -nucleotides concentration (mg/ml); 200: diluted solution multiples; 40: concentration of 1.0 mg/mL nucleotide extinction coefficient. 2.3.2. HPLC method Column: Shimadzu WAX-1 anion exchange column, Ø4.0 mm × 50 mm. The mobile phase—20 mmol/L (pH 7.0) phosphate buffer:480 mmol/L (pH 6.8) phosphate buffer = 1:1, flow rate 1.0 mL/min, 260 nm UV detection, room temperature. 2.4. Continuous stirred tank reactor (CSTR) A convective 300 mL glass stirred tank reactor including a flat ultrafiltration membrane (30 kDa cut-off) having an internal diameter of 8 cm was used for the continuous production of 5 -nucleotides (Fig. 1). Mixing was ensured by magnetic stirrer (180–300 rpm). The reactor was operated at constant temperature using water bath. Temperature and magnetic stirring were controlled by collector-magnetic heating blender as shown in Fig. 1(3). The substrate solution (pH 5.4) containing 1.0% RNA and 3.0 mM Zn2+ was fed through a filter with a peristaltic pump at a constant flow rate as required for the assay. The reaction was started by the injection of nuclease P1 (200 U) into the reactor. Pressure was maintained by means of a valve and pressure gauge. Samples were periodically taken at the reactor outlet. 2.5. Cleaning of membrane
4.0×50 = OD260 × a × 133.3 0.1 × 15
Before and after each run, the reactor was cleaned according to the following procedure:
a: A dilution factor of the enzyme before enzyme activity assay. One unit of enzyme activity was defined as the amount of enzyme that produced an increase in the optical density of 1.0 in 1 min at 260 nm.
(1) The membrane was rinsed with running water at room temperature for 10 min. (2) The system was circulated with a sodium hydroxide solution (0.25 M) at 60 ◦ C for 15 min. This was followed by rinsing with
enzyme activity (units/ml) = OD260 ×
Fig. 1. Schematic diagram of enzymatic membrane bioreactor. (1) Substrate reservoir; (2) pump; (3) collector-magnetic heating blender; (4) enzyme reactor; (5) products collector; (6) nitrogen bottle; (7) pressure gauge.
L.-E. Shi et al. / Journal of Membrane Science 345 (2009) 217–222
Fig. 2. Membrane flux curve of different MWCO.
running water at room temperature for 10 min. This stage was usually repeated. (3) The system was circulated with a nitric acid solution (0.10%) at 60 ◦ C for 15 min. Finally the system was rinsed with demineralized water for 30 min. 2.6. Selection of membrane molecular weight cut-off In order to assure that the membrane molecular weight cut-off (MWCO) is large enough to allow the hydrolysis product to pass through the membrane module, nucleotides’ molecular weight should be referenced. On the other hand, MWCO must be small enough to ensure that no enzyme and substrate leaks through the membrane module. 3. Results and discussion 3.1. Characterization of membrane 3.1.1. Membrane flux The substrate permeability of new membrane module was measured to test the initial membrane flux at 65 ◦ C. Fig. 2 indicates that polyethersulfone (PES) membrane flux is quite different with different molecular weight cut-off. Membrane flux was reduced with the extension of operating time. Membrane flux dropped by two main factors: first, concentration polarization, second was the result of macromolecular material absorption fouling. When enzyme and substrate cycled through membrane, they accumulated on the membrane surface at the high pressure side of membrane, which led to concentration gradient and concentration polarization. As shown in Fig. 2, after an initial flux decline, a quasi-steady-state was reached. Membrane with higher molecular weight, its membrane flux declined less than that with lower molecular weight. Membrane whose molecular weight was 30 kDa, after running 300 min almost had no membrane flux decline phenomenon, which suggests that fouling phenomenon is negligible with suitable molecular weight membrane.
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Fig. 3. Substrate concentration of retentate and permeate through membrane (30,000 kDa).
for RNA hydrolysis must be able to retain enzyme as well as RNA. Molecular weight of the enzyme is about 42 kDa and that of the substrate is more than 100 kDa. Therefore, the membrane pores have to be well defined. 10 mg/mL RNA solution and 1.0 mg/mL nuclease P1 solution was circulated in the enzyme membrane reactor for 5 h in two separate experiments at 65 ◦ C. As seen in Figs. 3 and 4, there were no substrate RNA and nuclease P1 in permeate side. It could be concluded that the membrane had good retention to substrate and enzyme, though there existed a little loss of substrate RNA and nuclease P1 in the retentate side. Maybe some of them were attached on the reactor wall or on the top of membrane. A complete retention of the enzyme and substrate within the reactor is the prerequisite for continuous operation of enzyme membrane reactor. This offers the great advantage of extensive and continuous use of enzymes. The same curve could be observed using membrane molecular weight of 6, 10, 20 kDa (data not shown). But when membrane molecular weight was 50 kDa, there was some leakage of enzyme and substrate (data not shown). So membrane with molecular weight of 6, 10, 20, and 30 kDa are all suitable to retain enzyme and substrate.
3.2. Evaluation of ultrafiltration membrane 3.2.1. RNA and enzyme retention To find a suitable membrane is a fundamental task in the process carried out in the membrane bioreactor. A membrane to be suitable
Fig. 4. Enzyme concentration of retentate and permeate through membrane (30,000 kDa).
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Fig. 6. Effect of temperature on membrane flux under different pressure. Fig. 5. Effect of membrane molecular weight on nucleotides production.
3.2.2. Nucleotides permeation Nucleotides produced during the enzyme hydrolytic reaction should permeate through the membrane in order to avoid nucleotides accumulation and inhibition phenomena. Thus, the membrane was tested in model experiments. Various initial concentrations (5, 10, and 20 mg/mL) nucleotides were circulated at a flow rate of 1.5 mL/min in the membrane reactor at 65 ◦ C. Nucleotides concentration was determined as a function of time. The data (data not shown) have proven that nucleotides permeated very easily across the membrane (molecular weight of 6, 10, 20, and 30 kDa) and the equilibrium concentration could be reached after 3 h. 3.2.3. Size of membrane molecular weight cut-off The steady-state for RNA hydrolysis to nucleotides was reached about after 3 h reaction with membrane molecular weight from 6 to 50 kDa (Fig. 5). From Fig. 5 we can see that membrane molecular weight did not significantly affect the yield. But the nucleotide product was relatively low with a membrane of 50 kDa. This is because the withholding molecular weight is too large and the reaction time is too short, and it is difficult to control speed. At the same time, using membrane with molecular weight of 50 kDa, part of nucleic acids and nuclease P1 infiltrate through the reactor, which also leads to partial low production concentration. In a word, membrane with 50 kDa molecular weight is not suitable for the preparation of nucleotides. Among all the membrane used, higher levels of nucleotides could be obtained with membrane of 30 kDa. And membrane with higher molecular weight is easier to reach a certain speed with smaller pressure, which required less nitrogen. So, production cost could be reduced using higher molecular weight membrane. Membrane with 30 kDa molecular weight was selected for the following experiment.
ture was beneficial in that it allowed a high permeate flux through membrane. So just take into account the membrane flux, higher temperature is more suitable. 3.3.2. Study of enzyme stability and optimum temperature Enzyme was incubated at different temperatures for 12 h and then cooled at ice-bath. The residual enzyme activity was determined at pH 5.4, 69 ◦ C under standard assay conditions. The maximum activity of the enzyme has been taken as 100%. The effect of enzyme denaturation with time at 40, 50, 60, 65, and 70 ◦ C are shown in Fig. 7. Temperature has a significant effect on enzyme stability. As seen in Fig. 8, the optimum temperature for hydrolysis of nucleotides is 69 ◦ C. But enzyme was more stable with lower temperature. The loss of enzyme activity was greatly reduced at 65 ◦ C. When enzyme incubated at 70 ◦ C for 300 min, 33% enzyme activity was lost, but at 65 ◦ C, only about 3% enzyme activity was lost. Therefore, a temperature of 65 ◦ C is more attractive. In industrial processes, finding a working temperature that gives high enzyme activity with good stability represents a compromise between lower process costs and higher productivities. Fig. 9 shows the effect of temperature on nucleotides production. As seen in Fig. 9 temperature had a greater influence on the reaction. When temperature increased, it can accelerate reaction rate, which led to produce more and more nucleotides. But when the temperature reached 70 ◦ C, nucleotides was less than
3.3. Working temperature 3.3.1. Effect of temperature on membrane flux The best temperature for enzymatic hydrolysis of nucleic acid is 65 ◦ C. High temperature will affect the enzyme activity, so only within the scope of 20–65 ◦ C was inspected the temperature impact of membrane flux. The mixtures of substrate and enzyme were adopted in this experiment. The experimental results are shown in Fig. 6. As seen in Fig. 6, membrane flux was increased with the increase of temperature. Membrane with bigger initial membrane flux was increased more as temperature increased. A high tempera-
Fig. 7. Thermal stability of nuclease P1.
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Fig. 8. Optimum temperature of nuclease P1.
that at 65 ◦ C. Generally the impact of temperature on reaction performances in two areas, on the one hand for the chemical reaction, the temperature rises every 10 ◦ C, the reaction rate can be doubled. Therefore, the enzyme decomposition will help improve the RNA hydrolysis rate with the increase of temperature; on the other hand temperature is too high, would undermine the structure of protein, a protein heat inactivation phenomenon, making the active site of the spatial structure changes, influence the integration of RNA and enzyme activity site, thereby inhibiting the enzyme’s catalytic reaction. These two factors were affecting the enzyme activity and effectivity. Thermal stability and hydrolysis rate should be comprehensively considered. Considering all the factors studied above, 65 ◦ C as working temperature is more suitable.
Fig. 10. Effect of pressure on membrane flux of 30,000 kDa.
the substrate and enzyme do not have enough reaction time. But later, the difference was smaller and smaller, especially among 0.6, 1.2, 1.6, and 2.0 mL/min. But the membrane reactor productivity (nucleotides concentration multiply flow rate) was increased with the increase of flow rate. So, 2.0 mL/min was the optimum flow rate. 3.5. Residence time
The enhancement of transmembrane pressure in the range of 0.03–0.07 MPa increased the membrane flux (Fig. 10). So even there existed concentration polarization and membrane fouling which had effect on membrane flux, constant flow rate can be reached by adjusting the transmembrane pressure. Fig. 11 shows the flow rate effect on nucleotides production. As shown in Fig. 11, at first with the increase of flow rate, the nucleotides in permeation side was reduced (within 150 min). This is because with high flow rate,
Residence time was calculated as a total volume of the reactor divided by the flow rate [11,12] (t = V/J, V – reactor volume, J – membrane flux), when membrane flux is fixed, t is in proportion to V. In this experiment, membrane flux was kept at 2.0 mL/min. As can be seen in Fig. 12, when flow rate was certain, nucleotides content was increased gradually with the increase of residence time. It is the same as literature reported [13]. The increase in the nucleotides concentration with the increase of residence time is a consequence of total time that substrates keep reacting in the reactor. The residence time can be considered as the average time that substrates keep reacting in the reactor and, as a consequence, if residence time increases substrate conversion also increase. The results clearly show that, at low residence time, products concentration decreased, and this effect was particularly significant for small residence time.
Fig. 9. Effect of temperature on nucleotides production.
Fig. 11. Effect of membrane flux on nucleotides production.
3.4. Effect of flow rate
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from RNA was efficiently conducted by employing an ultrafiltration membrane reactor. References
Fig. 12. Effect of enzyme membrane reactor volume on nucleotides production.
4. Conclusions In this paper, production of 5 -nucleotides from RNA was successfully obtained by a free enzyme membrane reactor. The best enzyme membrane reactor configuration was achieved with a polyethersulfone membrane of 30 kDa molecular weight cut-off, since the substrate and biocatalyst was retained with no loss of enzyme activity. The optimized operating conditions were also obtained: working temperature 65 ◦ C, flow rate 2.0 mL/min (transmembrane pressure 0.050 MPa), reactor volume 100 mL. The results demonstrated that enzymatic production of 5 -nucleotides
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