Cellulose (2011) 18:1563–1571 DOI 10.1007/s10570-011-9593-0
Immobilization of lipase onto cellulose ultrafine fiber membrane for oil hydrolysis in high performance bioreactor Peng-Cheng Chen • Xiao-Jun Huang • Fu Huang • Yang Ou • Ming-Rui Chen Zhi-Kang Xu
•
Received: 28 April 2011 / Accepted: 9 September 2011 / Published online: 18 September 2011 Ó Springer Science+Business Media B.V. 2011
Abstract Practical application of biphasic enzymeimmobilized membrane bioreactors (EMBR) requires efficient loading of the enzyme with retention of enzymatic activity. Here, we report a method to fabricate an ultrafine fiber membrane conjugated to lipase with high levels of enzyme loading and activity retention. A cellulose acetate (CA) non-woven ultrafine fiber membrane was prepared with 200 nm nominal fiber diameter by electrospinning, followed by alkaline hydrolysis to obtain regenerated cellulose (RC). The RC ultrafine fiber membrane was oxidized by exposure to NaIO4, simultaneously generating aldehyde groups to couple with pentaethylenehexamine (PEHA) as a spacer for lipase immobilization. A biphasic EMBR was assembled with the PEHAmodified and lipase-immobilized membranes. The effect of operation variables, namely aqueous-phase system, reaction pH, accelerant (sodium taurocholate) content, reaction temperature, and membrane usage on the performance of this bioreactor was investigated
Electronic supplementary material The online version of this article (doi:10.1007/s10570-011-9593-0) contains supplementary material, which is available to authorized users. P.-C. Chen X.-J. Huang (&) F. Huang Y. Ou M.-R. Chen Z.-K. Xu MOE Key Laboratory of Macromolecular Synthesis and Functionalization, Department of Polymer Science and Engineering, Zhejiang University, 38 Zhe Da Road, Hangzhou 310027, China e-mail:
[email protected]
with the hydrolysis of olive oil. A bioreactor activity as high as 9.83 9 104 U/m2 was obtained under optimum operational conditions. Keywords Cellulose Ultrafine fiber membrane Enzyme immobilization Lipase Bioreactor
Introduction Lipases are hydrolytic enzymes capable of catalyzing a wide range of reactions, such as alcoholysis, hydrolysis, trans-esterifications, aminolysis and enantiomer resolution, therefore having been widely applied in the food, pharmaceutical, and detergent industries (Compton et al. 2006; Deng et al. 2005; Garcia-Urdiales et al. 2009; Huang et al. 2008; Ragupathy et al. 2010; Wu et al. 2010). In practical applications, like most enzymes, lipases are often attached or incorporated onto or into inert, insoluble supports, which can offer better catalytic stability, the potential for continuous operations, feasible catalyst recycling, significant reduction in operation costs, and simplified product purification process, thus overcoming the thermal stability, reusability, and recoverability limitations associated with the free enzymes (Noureddini et al. 2005; Huang et al. 2006). However, in most cases, the immobilization process for enzymes inevitably results in the loss of some activity. As the properties of supports have great impact on the performance of immobilized enzymes, thus choosing
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the suitable supporting materials for enzyme is one of the critical challenges. To maximize enzyme activity, the supports should create a specific microenvironment for the enzymes and reduce the denaturation of enzyme protein (Becher et al. 2004; Bryjak et al. 2008; Huang et al. 2009; Liu and Chang 2008; Lu and Hsieh 2010). Among different supporting materials, cellulose stands out since it is inert and biocompatible under physiological conditions, which has a dual hydrophilic/hydrophobic character that can be ‘‘tuned’’ to suit particular needs (Cunha and Gandini 2010; Kosaka et al. 2007). Additionally, cellulose is also an abundant naturally occurring polymer (Noureddini et al. 2005). All of these properties make cellulose advantageous for enzyme immobilization. Our group have been working on enzyme immobilization using cellulose as supporting material and we have comprehensively quantified the properties of immobilized lipase on oxidized cellulose electrospun fiber membrane. By applying response surface methodology to optimize the oxidation conditions of the membrane, lipase from Candida rugosa can be directly tethered on the oxidized cellulose membrane with an activity retention as high as 29.6 U/g (Huang et al. 2011). One promising characteristic of lipases is their activation in the presence of a hydrophobic interface that can induce important conformational rearrangements yielding the ‘‘open state’’ of lipases and improves their activity during immobilization (Brzozowski et al. 1991; Huang et al. 2007; Verger 1997). As a result, we deduce that appropriate modification of the supports is an efficient way to enhance the lipase activity during immobilization. By introducing a spacer to increase the distance between the support and immobilized enzyme, we can provide a moderately hydrophobic surface for lipase immobilization. Moreover, the introduction of flexible spacers is expected to enhance the activity retention of the immobilized enzymes by offering them greater freedom of movement as well as minimizing unfavorable steric hindrance posed by solid supports—in other words, turning the fixed enzyme into flexible spaced enzyme. Membrane-immobilized enzymes, that may serve as model systems for natural membrane-bound enzymes, could be practically applied to enzymeimmobilized membrane bioreactors (EMBR) as less expensive, more stable and reusable alternatives to free enzymes (Wang et al. 2008). The combination of
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enzyme-immobilization and membrane technologies can create a favorable thermodynamics and kinetics process by uniting the high selectivity of enzymatic reactions with the high efficiency of membrane separation (Lee et al. 2009; Jiao et al. 2010; Wait et al. 2010). It is well-known that the choice of various membranes is of key importance for these EMBR. Electrospun polymer non-woven fiber membranes constitute a family of ultrafine fiber membranes with promising application in EMBR. Ultrafine membranes have high specific surface area that provides high enzyme loading per unit mass, and their fine porous structure allows ready accessibility to active sites as well as low diffusion resistance necessary for high reaction rates and conversion. In addition, compared with particulates, they are easily recoverable from reaction media, showing great promise for continuous operations (Agarwal et al. 2009; Bhardwaj and Kundu 2010; Wang et al. 2006, 2009). In particular, lipaseimmobilized ultrafine fiber membranes are unique for biphasic EMBR owing to the low aqueous solubility of the substrate and the property of interfacial activation of lipases. In these EMBR, two immiscible fluids, the organic phase (for dissolving substrates) and the aqueous phase (for extracting products) are separated by a lipase-immobilized membrane that functions as a reaction interface resulting in highly efficient simultaneous biocatalysis and separation. Moreover, compared with other biphasic bioreactors such as the microfiltration membrane bioreactors (Huang et al. 2008), one particularly interesting point about ultrafine fiber membrane in biphasic EMBR is the low transmembrane pressure due to the high porosity of the electrospun mesh. Thus, these systems offer not only a high reaction rate but also low operation cost. In this paper, a high performance biphasic EMBR was assembled with the lipase-immobilized ultrafine fiber membranes. Cellulose acetate (CA) was electrospun into a non-woven ultrafine fiber membrane with wellcontrolled morphology and diameter, and then oxidized with NaIO4 to generate aldehyde groups (Liu and Hsieh 2002, 2003; Son et al. 2004a, b). Pentaethylenehexamine (PEHA) was introduced onto the oxidized cellulose (OC) ultrafine fiber membrane as a spacer to increase the distance between the support and immobilized enzyme, offering a moderately hydrophobic surface for lipase immobilization. Lipase from Candida rugosa was used as the model enzyme to covalently attach to the modified membrane using glutaraldehyde (GA) as the coupling
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agent. The effect of operating variables (namely, aqueous-phase system, pH, content of sodium taurocholate, temperature, and membrane usage) on the performance of this biphasic EMBR was investigated with the hydrolysis of olive oil as a reaction model. It is expected that this work provides a foundation for the further application of this EMBR in industry.
Experimental Materials CA (29.6%, acetyl content) was purchased from Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) and used without further purification. Lipase (from Candida rugosa) powders (1,150 units/mg solid), Bradford reagent and bovine serum albumin (BSA, molecular mass: 67,000 Da) were obtained from Sigma-Aldrich Chemical Co., (St. Louis, MO, USA) and used as received. All other chemicals were of analytical grade and used without further purification. Preparation of the PEHA-modified cellulose ultrafine fiber membrane CA was electrospun into a non-woven ultrafine fiber membrane and then reacted with NaIO4 to obtain aldehyde groups. The detailed preparation procedure and characterization of the CA and OC membrane followed the process in the Supporting Information. For the introduction of PEHA, the OC membrane was treated with PEHA solution (PEHA was dissolved in phosphate buffer solution, 0.05 M, pH 7.0) and shaken gently for 12 h at 30 °C. The resulting modified membrane was washed with phosphate buffer solution (PBS, 0.05 M, pH 7.0) until free of excess PEHA. To test the influence of PEHA contents on the immobilization effect, activity of the immobilized lipase at different PEHA contents elevated from 2.0% (v/v) to 10.0% (v/v) was measured. The relative activities of the immobilized lipases were normalized to the highest activity within this content range, respectively. Biphasic enzyme-immobilized membrane bioreactor Figure 1 shows the experimental setup of the biphasic EMBR and it consists of two cylindrical compartments
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with arrangement for holding membrane between two compartments. Cellulose ultrafine fiber membranes were subjected to oxidation and modification processes before being installed into the reactor. Lipase was immobilized by a GA activation procedure. A peristaltic pump was used between the substrate reservoir and the reactor cell for better control of feed flow rate. GA solution, washing solutions and lipase solution were successively circulated in the reactor cell by the peristaltic pump. Briefly, a volume of 200 mL GA solution was filled into the reactor to filter through the membrane at 25 °C for 2 h. Afterwards, the activated support was washed several times with PBS (0.05 M, pH 7.0) to get rid of the residual GA. Then another circulation of 200 mL lipase solution (8 mg/mL in PBS, 0.05 M, pH 7.0) was operated for 3 h at 25 °C before washing three times with 100 mL PBS (0.05 M, pH 7.0). The washings together with the reaction solution were collected for determination of protein concentration. The amount of enzyme immobilized on the membrane was measured by Bradford assay (1976) using Coomassie brilliant blue reagent. BSA was used as standard to construct the calibration curve. The amount of immobilized protein on the membranes was calculated from the protein mass balance among the initial and final lipase solutions, and the washings. The enzyme loading was defined as the amount of enzyme (mg) per gram of the membrane. Each value was the mean of three parallel experiments at least, and the standard deviation was within ca. ± 5%. The activation process was carried out in different reaction mixtures with GA content of 0.5, 1.0, 2.0, 4.0, 8.0 and 16.0% (v/v) respectively to examine the influence of GA contents on the immobilization effect. The relative activities of the immobilized lipases were normalized to the highest activity within this content range, respectively. Also, to study the effect of operating conditions, different parameters (aqueousphase system, pH, content of sodium taurocholate, temperature and membrane usage) were varied with the resulting hydrolysis conversion measured. For the continuous hydrolysis process in EMBR, a sample of 120 mL substrate emulsion was circulated using a peristaltic pump in the compartment facing the immobilized membrane side. The substrate emulsion was prepared by thoroughly mixing 130 mL olive oil with 400 mL gum arabic solution (11% (w/v) gum arabic powder and 1.25% (w/v) CaCl22H2O) for
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Fig. 1 Schematic representation for the biphasic EMBR (volume of cylindrical compartment: 120 mL each; membrane diameter: 5.0 cm; membrane shape: circular disc)
30 min with a SZ212-40A Electronic Turrax from Shanghai Zhiwei Electric Co., Ltd. and stored at 4 °C. Substrate emulsion of 120 mL was added into the organic phase compartment, and then pH value was adjusted to 8.0 with NaOH solution. In the other compartment, 120 mL aqueous solution at different pH values of 6.5, 7.0 and 7.5 was circulated simultaneously. Hydrolysis reaction occurred on the surface of lipase-immobilized membrane and the temperature of substrate reservoir or reactor cell was controlled to ±0.5 °C by circulation water through the reservoir or reactor jacket. The hydrolytic products, free fatty acid and glycerol, extracted in the aqueous phase were neutralized by continuously adding 0.05 M NaOH standard solution through an automatic titrator, holding the pH of the aqueous solution constant during the whole process. The consumed volume of NaOH standard solution was recorded periodically and used for evaluating the reaction rate. A blank run without enzymes on the membrane was tested in the same way. One lipase unit of the amount of enzymes corresponded to the release of 1 lmol fatty acid per hour under the assay conditions. The enzyme activity was the number of lipase units per gram of membrane. Specific activity was defined as the number of lipase units per milligram of protein. Activity retention value was the ratio of specific activity of immobilized lipase to that of free one. Each data point was the average of at least three parallel experiments, and the standard deviation was within ca. ±5%.
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Results and discussion Modification of membrane with PEHA According to scanning electron microscopy (SEM) images in the Supporting Information, part of the fabric shrank after deacetylation and the surface of the fibers got rough. However, the over-all structure and pore size of the membrane, and the average diameter of the fibers were maintained after hydrolysis, oxidation and modification processes (Fig. S1). From the SEM images in Fig. S1, the pore size of the cellulose membrane was less than 2 lm. ATR-FTIR of the PEHA-modified membrane (Fig. S2) confirms the removal of the aldehyde groups, in which the characteristic adsorption peak attributed to the vibration of the carbonyl group at 1,720 cm-1 (tC=O) disappeared and a new adsorption peak at *1,677 cm-1 (tC=N) in the modified membrane was observed. The oxidation degree was determined by a titration method (Liu and Hsieh 2002) and it was found that the content of aldehyde groups was 13.7%. The effect of PEHA content on the activity of the immobilized lipase was assayed over the content range of 2.0–10.0% (v/v) and the typical results are shown in Fig. 2. The activity retention of the immobilized enzyme increased with the PEHA content. To provide more enzymes with a microenvironment similar to that of the free one, a sufficient amount of modifier needs to be applied. At a PEHA content of 8.0% (v/v), the highest activity retention of the immobilized lipase
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PEHA concentration(%) Fig. 2 Effect of PEHA content on the activity of immobilized lipase (real value of lipase activity with 100% relativity activity: 52.1 U/g)
Table 1 Element concentration of PEHA-modified membrane Element (%)
N
C
H
1
4.79
43.9
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Average
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43.9
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was obtained. However, when the content went beyond 8.0% (v/v), activity retention decreased. This decrease can be attributed to the fact that when the PEHA content increased furthermore, there were more potential reaction sites for covalently coupling with lipase. As a result, one lipase molecule might be attached to the membrane by more than one activated amino groups, which hampered the free conformation of enzymes, and thus reducing their enzymatic activity (Bulmus¸ et al. 1998; Ozyilmaz 2009). The amount of PEHA that modified the fiber was studied by elemental analysis with results shown in Table 1. According to this table, the content of N element increased to 4.86% after PEHA-modification process. By calculation 16.6% aldehyde groups reacted with PEHA. This relatively low reaction efficiency was due to the steric hindrance of PEHA as well as the incompletion of macromolecular reaction. Optimization of activation
PEHA-modified membrane to study the effect of GA content on the activity of immobilized lipase. Figure 3 shows that activity increases significantly as the GA content increases and reaches the maximum activity retention at 2.0% (v/v). Above this content value, the activity retention decreased slowly. Furthermore, at [8.0% (v/v) of GA content, activity retention of the immobilized lipase tends to be constant. Investigation of the influence of GA content on enzyme loading reveals similar results. These results may be ascribed to a similar mechanism to that proposed above. There were a large number of amino groups on the membrane which provided many potential reaction sites for lipase immobilization. With the increase in GA content, more amino groups were activated and more aldehyde groups were introduced correspondingly, generating more binding sites for enzyme immobilization. Nevertheless, after over-saturated activation by excess GA, it was likely that the enzyme molecules were immobilized through multiple chemical bonds that substantially reduced their enzymatic activity. Moreover, GA may crosslink OH groups on the membrane and thus provided less binding sites for the lipase. Similar result has been reported by Bulmus¸ et al. (1998) and Ye et al. (2005). Therefore, a PEHA content of 8.0% (v/v), as well as a 2.0% (v/v) GA content is required for the modification and activation of the OC membrane. Under these optimum conditions, a residual activity of 52.1 U/g was observed with an activity retention of 54.3%, which was a 76.3% increase in residual activity compared with OC membranes without PEHA modification. 120
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To optimize the activation process of the PEHAmodified membrane, an increased GA content from 0.5% (v/v) to 16.0% (v/v) was applied to the
Fig. 3 Effect of GA content on the activity of immobilized lipase (real value of lipase activity with 100% relativity activity: 52.1 U/g)
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Effect of aqueous-phase system on performance of the biphasic EMBR The performance of biphasic EMBR is evaluated in terms of the amount of fatty acid extracted in the aqueous phase. Since the conditions of activation and immobilization were the same as described above, the amount of enzyme attached to the fiber membrane after a continuous immobilization process should also be similar. Moreover, to check for errors in the determination of reaction rate, a blank run was performed in which a membrane without enzyme was used and it was found that the pH of the aqueous phase did not change. Figure 4a shows the quantities of fatty acid with time for various aqueous-phase systems. We observed that fatty acid quantity increased with time, indicating the hydrolysis reactions were occurring during the test time. During the same time period, when pure water was used as the aqueous phase, the least amount of fatty acid was produced, with a membrane activity of
Effect of aqueous phase pH on performance of the biphasic EMBR The pH is one important parameter capable of altering enzymatic activities in aqueous solution. Fatty acid amount as a function of pH is depicted in Fig. 4b. It was observed that at pH 7.0, the hydrolysis conversion was the highest with an enzyme-immobilized membrane
(b) 1200
(a) 1200 PBS 7.0 CaCl2 solution Water
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Fatty acid(µmol)
only 8.77 9 102 U/g. Using a CaCl2 solution as the aqueous phase was better, achieving 3.82 9 103 U/g membrane activity. PBS (0.05 M, pH 7.0) was found to perform the best with a membrane activity of 9.72 9 103 U/g and may be due to the fact that the immobilized lipase needs not only some ionic strength but also a relatively stable pH to maintain its activity. Moreover, a titration of the PBS (0.05 M, pH 7.0) with standard NaOH solution showed a gradual increase in the aqueous pH without sudden change, indicating that this system was feasible for extracting and neutralizing the produced fatty acid.
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1000 ST 0.6mg/mL ST 0.4mg/mL ST 0.2mg/mL ST 0 mg/mL
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Fig. 4 Effect of operation conditions on performance of the biphasic EMBR, measured by fatty acid quantities versus time. a Aqueous solution: PBS 7.0 (filled circle), CaCl2 solution (filled square) and deionized water (filled up pointing triangle). b Aqueous phase pH value: 6.5 (filled circle), 7.0 (filled square) and 7.5 (filled up pointing triangle). c Sodium taurocholate
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solution content: 0 mg/mL (filled square), 0.2 mg/mL (filled down pointing triangle), 0.4 mg/mL (filled up pointing triangle) and 0.6 mg/mL (filled circle). d Temperature: 10 °C (filled square), 30 °C (filled circle) and 40 °C (filled up pointing triangle)
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Effect of accelerant on performance of the biphasic EMBR Sodium taurocholate is a salt mixture extracted from mammal bile that can help to promote the digestion and absorption of lipids. Figure 4c shows the fatty acid quantities versus time affected by different contents of sodium taurocholate in biphasic EMBR. It was found that the hydrolysis conversion increased with the increase in the content of accelerant. When accelerant content was 0.6 mg/mL, the activity of lipase-immobilized membrane was 60.9% higher than when no accelerant was added, significantly accelerating the hydrolysis reaction and promoting the efficiency of this bioreactor. The mechanism of bile salts resulting in accelerating lipid hydrolysis is believed that their amphiphilic nature may lead to modifications in the physical properties of the substrates, thus converting them to more appropriate forms for efficient interaction with the enzyme. It is also suggested that bile salts may change the affinity of the substrate for the enzyme via a conformational change in the enzyme to facilitate access of the substrate to the active site (Aryee et al. 2007; Gargouri et al. 1986; Wang and Hartsuck 1993; Wang and Lee 1985).
lipase-immobilized membrane increased when the temperature was increased from 10 to 40 °C. The high temperature resistance of the membrane is due to the presence of covalent bonds between the enzyme and the fibrous mesh preventing the conformational denaturation of the enzyme at higher temperatures. Effect of membrane amount on performance of the biphasic EMBR To study the effect of membrane amount on reactor performance, a membrane with the weight of 14, 20, and 33 mg respectively was installed and the amount of fatty acid produced was measured. The result was shown in Fig. 5. It is conceivable that a relatively higher conversion could be obtained by increasing the weight of the fiber membrane in the bioreactor. However, we found that the membrane weight has little impact on the hydrolysis conversion. Owing to the fact that the pores of the ultrafine fiber membrane were too small for olive oil droplets to permeate, the hydrolysis reaction only took place on the membrane surface. Moreover, to verify this speculation, we measured the droplet size of olive oil emulsion. The droplet size distribution of the prepared emulsion was determined by means of a Mastersizer 2000. Results were the average of three measurements on freshly prepared emulsions. And the average droplet size, expressed as the Sauter diameter was 15.4 lm, obviously larger than the pore size of cellulose 1400 14 mg 33 mg 20 mg
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Fatty acid (µmol)
activity of 9.72 9 103 U/g, while it was 6.33 9 103 U/g and 4.51 9 103 U/g at pH 7.5 and 6.5, respectively. The instantaneous neutralization of fatty acid by OH- at the reaction site accelerated the extraction of hydrolytic products by the aqueous phase. The increase of OH- concentration would enhance the driving force for the diffusion of OH- from bulk to reaction front, the transformation of fatty acid RCOOH to RCOO- at the reaction site and the diffusion of RCOO- from reaction front to aqueous bulk phase. It is likely that all of these factors contributed to the high catalytic efficiency at higher pH. Nevertheless, when the pH was increased, immobilized lipase on the membrane tended to be unstable and this affected the hydrolysis conversion unfavorably.
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Effect of temperature on performance of the biphasic EMBR Figure 4d shows the fatty acid quantity as a function of time and temperature. It was observed the activity of
Fig. 5 Quantities of fatty acid versus time affected by different amounts of membrane. 14 mg (filled up pointing triangle), 20 mg (filled circle) and 33 mg (filled square) (aqueous solution: PBS 7.0, aqueous phase pH value: 7.0, sodium taurocholate solution content: 0.6 mg/mL, temperature: 40 °C)
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membrane. Thus, it can be inferred that the efficiency of this biphasic EMBR can be improved by increasing the contacting area of the membrane with the substrate emulsion, resulting in increasing the amount of membrane actually in use. Therefore, aqueous phase of PBS (0.05 M, pH 7.0), an operating temperature of 40 °C together with sodium taurocholate content of 0.6 mg/mL were required for this biphasic bioreactor. Under these optimal operating conditions, an activity of 1.07 9 104 U/g of lipase-immobilized membrane, with an activity of 9.83 9 104 U/m2 was obtained. The obtained result we got is quite comparable with the related literature. First, a biphasic system was applied in our bioreactor which facilitated the interfacial activation of lipase (Huang et al. 2008). Second, cellulose ultrafine fiber membrane was used as supports with PEHA modification, which brought about a higher enzymatic activity (Huang et al. 2011). Moreover, we used ultrafine fiber membrane in the biphasic bioreactor. Compared with microfiltration membrane bioreactor which required a operating pressure of 30 kPa (Deng et al. 2005), ultrafine fiber membrane bioreactor can eliminate the influence of transmembrane pressure on bioreactor efficiency and is energy-saving. Conclusion The current study has comprehensively investigated the properties of a biphasic lipase-immobilized membrane bioreactor. PEHA was successfully introduced onto the OC ultrafine fiber membrane as a spacer for interfacial activation of lipase, resulting in a 76.26% increase in activity retention compared to membranes without PEHA modification. A biphasic EMBR was assembled with the PEHA-modified membrane for the olive oil hydrolysis, and under the optimal operating conditions a bioreactor activity of 9.83 9 104 U/m2 was obtained. Taken together these results verified the feasibility of the application of lipase-immobilized ultrafine fiber membrane bioreactor possessing high catalytic efficiency in a wide range of reactions. Acknowledgments The authors are grateful to the National Natural Science Foundation of China (Grant No. 50703034), the Opening Foundation of Zhejiang Provincial Top Key Discipline (Grant No. 20110915) and the High-Tech Research and Development Program of China (Grant No. 2007AA10Z301).
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