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Keywords:

  • porous polymer;
  • monolith;
  • immobilized enzyme bioreactor;
  • gold nanoparticles;
  • lipase;
  • biodiesel

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgments
  8. References
  9. Supporting Information

Porcine lipase has been reversibly immobilized on a monolithic polymer support containing thiol functionalities prepared within confines of a fused silica capillary and functionalized with gold nanoparticles. Use of gold nanoparticles enabled rejuvenation of the activity of the deactivated reactor simply by stripping the inactive enzyme from the nanoparticles using 2-mercaptoethanol and subsequent immobilization of fresh lipase. This flow through enzymatic reactor was then used to catalyze the hydrolysis of glyceryl tributyrate (tributyrin). The highest activity was found within a temperature range of 37–40°C. The reaction kinetics is characterized by Michaelis–Menten constant, Km = 10.9 mmol/L, and maximum reaction rate, Vmax = 5.0 mmol/L min. The maximum reaction rate for the immobilized enzyme is 1,000 times faster compared to lipase in solution. The fast reaction rate enabled to achieve 86.7% conversion of tributyrin in mere 2.5 min and an almost complete conversion in 10 min. The reactor lost only less than 10% of its activity even after continuous pumping through it a solution of substrate equaling 1,760 reactor volumes. Finally, potential application of this enzymatic reactor was demonstrated with the transesterification of triacylglycerides from kitchen oil to fatty acid methyl esters thus demonstrating the ability of the reactor to produce biodiesel. Biotechnol. Bioeng. 2014;111: 50–58. © 2013 Wiley Periodicals, Inc.


Introduction

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgments
  8. References
  9. Supporting Information

The use of enzymatic catalysis is a desirable approach to creating green and sustainable chemistry since this methodology helps to achieve high throughput under mild reaction conditions while featuring excellent chemo-, regio-, and stereo-selectivity (Koeller and Wong, 2001; Schmid et al., 2001). However, the limited operational stability of soluble enzymes due to denaturing and deactivation, as well as difficulties with their recovery and recycling, prevent their widespread use in industrial applications. This is why immobilization of soluble enzymes onto solid supports is an effective solution to those problems.

Although immobilization was first described almost 100 years ago (Nelson and Griffin, 1916), systematic investigations did not begin until the mid-1960s (Messing, 1975). Immobilization can provide for a number of benefits: (i) it often stabilizes the enzyme, (ii) it enables continuous use, (iii) the reaction is stopped after the reaction mixture leaves the reactor, and (iv) the product is not contaminated with the biocatalyst. On the other hand, immobilization may also have some disadvantages including decrease in activity due to distortion of the tertiary structure, blockage of the active site, and diffusional constrains of the mass transport (Garcia-Galan et al., 2011). The ideal immobilization technique should enable operation under mild reaction conditions. The best supports for the immobilization of free enzymes should (i) exhibit large surface areas, thus allowing immobilization of significant amounts of the enzymes, (ii) facilitate rapid mass transport of substrates and products, and (iii) provide excellent chemical and mechanical stability (Parthasarathy and Martin, 1994; Tischer and Wedekind, 1999).

A variety of techniques and solid supports have been described in the extensive literature including excellent recent reviews (Brady and Jordaan, 2009; Hernandez and Fernandez-Lafuente, 2011; Hwang and Gu, 2013; Mateo et al., 2007; Rodrigues et al., 2011). Typical immobilization techniques can be divided into two groups: physical methods such as adsorption, encapsulation, and entrapment, and chemical interactions including ion exchange and covalent attachment. In general, methods based on ion exchange are easy to implement. However, attachment of the enzymes may not be strong enough to prevent the loss of enzymes during the operation. In contrast, covalent binding is the most effective approach to averting detachment of the enzyme. However, it has been demonstrated that the chemical reaction of an enzyme with solid support leading to covalent bond may distort its three-dimensional conformation, causing partial denaturing, and a decrease in its activity (Blandino et al., 2000; Ozyilmaz and Gezer, 2010; Ozyilmaz and Yagiz, 2012). The activity of the immobilized enzymes also decreases during their operation. As a result, the solid catalyst must frequently be discarded and a new batch applied. Because of these drawbacks, new technologies are sought that will permit replacement of the enzyme in situ, that is, in the reactor, without losing the support.

This in situ replacement is currently possible by using ion exchangers and their metal complexes as a support (Ma et al., 2011; Wu et al., 2012). Most often, the bond between the support and enzyme is not sufficiently stable and use of certain substrate solutions or variations in pH lead to leak of the enzyme from the reactor. Therefore, development of novel support materials enabling strong but reversible binding of an enzyme is desirable.

Most of the supports for enzyme immobilization described in literature are porous particulates in which the mass transport of the substrates and products to and from the reaction site is achieved via diffusion through the stagnant liquid phase present in the pores. In contrast, porous polymer monoliths, which were developed in the early 1990s, are the newest contribution to the arsenal of supports (Svec and Fréchet, 1992). These monoliths are typically prepared from a mixture containing functional monomer, cross-linker, initiator, and pore forming solvents (porogens) using polymerization within the confines of a container where they are eventually used. Porous polymer monoliths contain large through-pores and exhibit high permeability to flow. These properties enable mass transport to the active site of the enzyme by fast convection rather than by slow diffusion since all the liquid phase must flow through the pores of the support. The advantages of monolithic supports have been demonstrated with several examples of immobilized enzymes used to process substrates dissolved in aqueous media (Bartolini et al., 2004; Josic and Buchacher, 2001; Krenkova et al., 2009; Logan et al., 2007; Mallik and Hage, 2006; Palms and Novotny, 2005; Pierre et al., 2006; Spross and Sinz, 2010; Sudheendran and Buchmeiser, 2010; Svec, 2006b; Svec and Fréchet, 1996; Xie et al., 1999). Recently, the application range of enzymes covalently immobilized on monolithic supports was extended to operations in both organic solvents and their biphasic mixtures with water (Kawakami et al., 2009; Urban et al., 2012). For example, Urban et al. prepared monoliths by polymerization of the reactive vinylazlactone. The azlactone functionalities readily reacted with amine groups of the porcine lipase to achieve covalent attachment of the enzyme onto the monolith. However, once the activity of the immobilized enzyme was decreased, the entire reactor had to be discarded.

Biodiesel is typically prepared by transesterification of triglycerides in fat with an alcohol in the presence of a chemical or biological catalyst (Al-Zuhair, 2007). The chemically catalyzed process can suffer from some drawbacks including slow reaction rate and necessity for high alcohol-to-oil ratios (Vasudevan and Briggs, 2008). In contrast, enzymatic transesterifications using commercially available lipases such as those from Thermomyces lanuginosus (e.g., Lipozyme TL IM), Pseudomonas antarctica (CALB, e.g., Novozym 435) or Rhizomucor miehei (e.g., RM IM) offer a number of advantages including (i) easy product separation, (ii) minimal requirements for wastewater treatment, (iii) easy glycerol recovery, and (iv) the absence of any side reactions (Jegannathan et al., 2008; Vyas et al., 2010). The high cost of lipases and contamination of the product with the enzyme are currently the major roadblocks to their wide application in large scale industrial transesterifications. Thus, immobilization of lipases on solid supports, and use of different techniques, was investigated to discover approaches that overcome these problems (Ozyilmaz and Gezer, 2010; Urban et al., 2012; Zhou et al., 2011). Garcia-Galan et al. (2011) described in a comprehensive review the potential of various enzyme immobilization strategies including covalent attachment and reversible immobilization focused on enhancement of the enzyme performance while using both porous supports and magnetic nanoparticles.

Previously, gold nanoparticles have been immobilized on monoliths and have been successfully used in the preparation of stationary phases for chromatography (Alwael et al., 2011; Cao et al., 2010; Connolly et al., 2010; Lv et al., 2012a, 2012c; Xu et al., 2010). Now, for the first time, we describe a universal approach to an easily prepared and highly efficient bioreactor using a support comprising a porous polymer monolith functionalized with gold nanoparticles. Commercially available porcine pancreatic lipase (PPL) was chosen as the model enzyme owing to its high stability and broad specificity for biotransformation of non-natural substrates (Bagi et al., 1997; Mendes et al., 2012).

Our bioreactor described in this paper consists of porous polymer monolithic supports with the pore surface coated with gold nanoparticles. The gold nanoparticles serve as an intermediate ligand on which the enzyme is immobilized (Scheme 1). In contrast to the conventional approaches, the enzyme is immobilized on gold nanoparticles via the reversible interaction of its thiol and amine functionalities with gold. The merits of this approach include: (i) high permeability and convective mass transport through the monolithic support rapidly delivering sufficient quantities of substrate to the enzyme; (ii) the increase in surface area contributed by the three dimensional gold nanoparticles attached to the otherwise “2-dimensional” pore surface enables immobilization of larger amounts of enzymes; (iii) the strong affinity of thiols and amines towards gold prevents leakage of the enzyme during operation; (iv) the interactions are achieved under very mild conditions; (v) the biocompatibility of gold nanoparticles is helpful in maintaining the native conformation and high activity of the biocatalyst; and (vi) the use of gold nanoparticles, as intermediate ligands at which the enzyme is non-covalently attached, facilitates regeneration of the activity of the bioreactor simply by removing the denatured enzyme, and subsequently recharging the bioreactor with a fresh enzyme.

image

Scheme 1. Preparation of monolith attached with gold nanoparticles, its immobilization with enzyme, and regeneration of the enzymatic bioreactor.

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Materials and Methods

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgments
  8. References
  9. Supporting Information

Complete description of chemicals, materials, and instrumentation is available in the Supporting Information. All experiments described in this report were performed at least in triplicates.

Preparation of Monolithic Capillary Columns Attached With Gold Nanoparticles

The preparation of thiol-containing monolithic capillary columns and its modification with gold nanoparticles were carried out using the procedure developed previously (Lv et al., 2012a, 2012b; Schmidt and Verger, 1998). A brief description is available in the Supporting Information.

Immobilization of Lipase

A 5 mg/mL solution of lipase in 0.25 mol/L potassium phosphate buffer pH 7.2 was pumped through the monoliths functionalized with gold nanoparticles using the high pressure 260D ISCO pump at a flow rate of 0.5 µL/min. The time used for lipase immobilization was optimized within the range of 4–30 h. The monolith was then rinsed with 0.25 mol/L potassium phosphate buffer and water. Finally, the reactor was washed with hexane and used.

Hydrolytic Reaction

The enzymatic reaction was carried out under the conditions of a continuous flow reactor. Tributyrin solution in hexane (0.85 mmol/L) with dodecane added as an internal standard was continuously pumped through the 10 cm long, 100 µm i.d. monolithic bioreactor using flow rates 0.1–0.5 µL/min. The effluent was analyzed using GC/MS and the yield of butyric acid was calculated with respect to the total tributyrin concentration in the initial solution. In kinetic experiments, the enzyme activity was calculated from the difference in tributyrin concentration in the initial solutions (0.5–7.9 mmol/L) and in the bioreactor effluent as determined using GC/MS. The lipase activity is expressed in nanomoles of converted substrate in 1 min normalized to 1 mL of bioreactor volume.

The hydrolytic reaction using free lipase in solution was performed in a magnetically stirred sealed glass vial containing a mixture of 10 µL lipase solution (5 mg/mL) in 0.25 mol/L potassium phosphate buffer pH 7.2 and 100 µL tributyrin solution in hexane at 37°C for 4 h.

Transesterification Reaction

Kitchen oil (100 µL) dissolved in a mixture containing 500 µL hexane, 500 µL tert-butanol, and 300 µL methanol was continuously pumped through the immobilized lipase bioreactor at a flow rate of 0.4 µL/min at 37°C and the effluent analyzed using GC/MS.

Regeneration of Activity

Neat 2-mercaptoethanol (400 µL) was pumped through the monolithic bioreactor using a syringe pump at a flow rate of 0.5 µL/min to remove the deactivated lipase followed by washing with water and the phosphate buffer. The activity of the monolithic bioreactor was then regenerated by immobilization of fresh lipase using procedure described above. It worth noting that 2-mercaptoethanol does not break the bonds between the gold nanoparticle and the polymer support (Xu et al., 2010).

Results and Discussion

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgments
  8. References
  9. Supporting Information

To demonstrate the advantages of the gold-coated support material for enzyme immobilization, we chose porcine lipase (triacylglycerol ester hydrolase, EC 3.1.1.3) as the model enzyme that catalyzes the hydrolytic and transesterification reactions of triacylglycerides. The lipase contains a number of functionalities available to interact with gold including a large number of amine groups and two free thiol groups with one located close to the surface of the folded protein (De Caro et al., 1981). The features and wealth of attractive applications of lipases have been summarized in an excellent review (Schmidt and Verger, 1998). We chose this enzyme since it has been immobilized covalently previously using similar monolithic support (Urban et al., 2012) and we could directly compare results achieved with conjugates prepared using different immobilization techniques.

Preparation of Monolithic Support

For the design of monolithic bioreactors, a generic porous monolithic support was first prepared in a fused silica capillary, using in situ thermally initiated polymerization of glycidyl methacrylate and ethylene dimethacrylate in the presence of cyclohexanol and 1-dodecanol as porogens. Azobisisobutyronitrile was used as the initiator (Lv et al., 2012a, 2012b). The generic monolith then reacted with 2,2′-dithiobis(ethylamine; cystamine), followed by reaction with tris(2-carboxylethyl) phosphine (TCEP), which liberated the desired thiol groups at the pore surface that were needed for the attachment of gold nanoparticles. Energy-dispersive X-ray spectroscopy (EDS) revealed 3.7 at.% sulfur in both the cystamine-modified and TCEP-cleaved monoliths. A dispersion of 15 nm gold nanoparticles was then pumped through the functionalized monoliths until a saturated surface coverage with gold was achieved. This step was visually confirmed by the pink color of the liquid leaving the capillary outlet and the deep red color of the entire monolith. EDS determined the presence of 43.2 wt% Au at the pore surface of the support. A simple calculation reveals that this amount of gold represents less than 3% of the overall volume of the support. Figure 2 shows scanning electron microscopy images of the cross-sectional segments of a generic monolith and its counterpart functionalized with gold nanoparticles. The gold nanoparticles form a dense, almost continuous layer as clearly documented with the inlay in the right panel in which the white specs are the gold nanoparticles.

image

Figure 2. Scanning electron micrographs of internal structure of the generic poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith sputtered with ca. 10 nm thick gold coating (left), and monolith functionalized with 15 nm gold nanoparticles with no sputtering applied (right).

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The flow resistance of the monoliths is related to the column permeability KF calculated using the Darcy equation inline image, where KF is the permeability, Fm is the flow rate (1.0 µL/min), η is the viscosity of used liquid (0.48 cP), ΔP is the pressure drop across the monolith, L is the monolith length (20 cm), and r is the capillary inner radius (100 µm; Guiochon, 2007; Gusev et al., 1999; Urban et al., 2008). The generic poly(glycidyl methacrylate-co-ethylene dimethacrylate) monolith had an excellent permeability characterized with a KF value of 3.23 × 10−10 cm2. After functionalization of the monolith with 15 nm gold nanoparticles, the permeability slightly decreased to 2.48 × 10−10 cm2 which can result from both the decrease in the pore size due to the partial filling of pores with nanoparticles and the change in friction within the pore necks. Immobilization of lipase for 16 h or less then did not cause any significant change in permeability.

Immobilization of Lipase

The immobilization of lipase was then achieved by pumping its solution through the gold nanoparticles containing monolithic support followed by a washing with 0.25 mol/L phosphate buffer pH 7.2. The hydrophilic polymer matrix was hydrated under these conditions and held the water that is needed for the hydrolytic reaction (Zaks and Klibanov, 1988). The bioreactor was then equilibrated with hexane and used for continuous deacylation of 1,2,3-tributyrylglycerol (tributyrin) resulting in two main products, butyric acid and dihydroxypropyl butyrate. Figure 3 shows the effect of immobilization time on the conversion of tributyrin in the bioreactor determined using gas chromatography with mass spectrometric detection (GC-MS). A maximum activity enabling a conversion of 99.0% was achieved after pumping the lipase solution through the monoliths functionalized with gold nanoparticles for 16 h at a flow rate of 0.5 µL/min. At this point, the surface of gold nanoparticles was saturated with immobilized lipase. Further extension in the reaction time decreases the size of the throughpores. This reduces permeability of the reactor and leads to an increase in back pressure.

image

Figure 3. Effect of lipase immobilization time on conversion of tributyrin hydrolysis carried out at a temperature of 37°C and with a substrate residence time of 2.5 min.

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Effect of Reaction Conditions

Reaction temperature is one of the parameters that affect activity of the enzyme in catalyzed reactions. Figure 4 shows that the immobilized lipase produced the highest tributyrin conversion and thus butyric acid yield within the temperature range 37–40°C, which is equivalent to a typical pig's body temperature of 38.8°C.

image

Figure 4. Yield of butyric acid formed from tributyrin at different temperatures. Conditions: substrate: 0.85 mmol/L tributyrin in hexane, residence time 2.0 min, lipase immobilization time 4 h.

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Literature reports indicate that the reaction rates using lipases immobilized in particles are slow, and reaction times of 4–24 h are needed to achieve high conversion (Jegannathan et al., 2008; Urban et al., 2012; Vyas et al., 2010). Also, feeding the monolith that did not contain gold nanoparticles did not lead to a reactor with significant activity. In contrast, immobilization of the lipase on porous polymer monoliths using gold nanoparticles as a spacer results in a dramatic reduction in the reaction time. The reaction time is defined as the time during which the substrate resides in the reactor and is controlled by the flow rate. We observed in our earlier experiments concerned with permanent immobilization of various enzymes on monolithic supports via a covalent bond that both the convective mass transport of the substrate and the high local concentration of the enzyme lead to a significant increase in the reaction rate (Krenkova et al., 2009; Petro et al., 1996; Svec, 2006a; Urban et al., 2012; Xie et al., 1999). Since the basic monolithic support used in this study is similar to that used previously, it is likely that these effects also enhanced the activity of lipase immobilized via the gold nanoparticles. The activation of lipase through its interaction with solid surfaces and opening the conformation has been observed recently (Schmidt and Verger, 1998; Zhou et al., 2011). In addition, some enzymes immobilized by adsorption at the surface of gold nanoparticles also exhibit enhanced activity (Deka et al., 2012). Therefore, the interaction of lipase with the gold nanoparticles is proposed as another reason for the significantly increased activity.

Figure 5 demonstrates that 87% of tributyrin was converted into products after a residence time of only 2.5 min and the maximum of conversion, 98% was achieved in 10 min. The former result is more beneficial from the point of view of the overall throughput.

image

Figure 5. Effect of residence time of tributyrin in the monolithic immobilized lipase bioreactor on conversion of the hydrolytic reaction carried out at a temperature of 37°C.

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Kinetics of Transesterification Reaction

Using the optimized reaction conditions, including a temperature of 37°C and a substrate residence time of 2.5 min, the kinetics of transesterification reaction was investigated. The activity of the immobilized enzyme was assayed by pumping 100 µL of tributyrin solutions with different concentrations ranging from 0.5 to 7.9 mmol/L through the monolithic bioreactor at a flow rate assuring the desired residence time of 2.5 min. Figure 6 shows the Lineweaver-Burk linearization that enabled calculation of the values of Michaelis–Menten constant, Km = 10.9 mmol/L, and maximum reaction rate, Vmax = 5.0 mmol/L min. While the value of Michaelis–Menten constant is very close to Km = 12.4 mmol/L, which we found previously for lipase attached to a monolithic support covalently (Urban et al., 2012), the maximum reaction rate for the present conjugate is more than 269 times faster, and 1,000 times faster compared to lipase in solution. These results clearly demonstrate the advantages derived from immobilization of the enzyme on the monolithic support using gold nanoparticles as an intermediate ligand.

image

Figure 6. Lineweaver-Burk plot for kinetics of hydrolytic reaction of tributyrin catalyzed by lipase immobilized on monoliths functionalized with gold nanoparticles. Conditions: lipase immobilization time 16 h, tributyrin solution in hexane, reaction temperature 37°C, substrate residence time 2.5 min.

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Operational Life of the Bioreactor

The operational life of the bioreactor is defined by the stability of its activity. Figure 7 demonstrates the effect of the total amount of the tributyrin substrate continuously pumped through the reactor on the yield of butyric acid as determined by GC-MS. No significant decrease in tributyrin conversion was observed until 1,760 reactor volumes were converted. After this point, the activity of immobilized lipases started to decrease more rapidly leaving tributyrin substrate either converted to butyric acid monohydroxypropyl and dihydroxypropyl esters or not converted at all. Our working hypotheses assumed that this decrease in activity could result from the leakage of the enzyme, consumption of all water present in the system, or from deactivation of the enzyme itself. Collected liquid after rinsing the reactor with different solvents including hexane, water, and mixture of hexane/methanol/tert-butanol did not exhibit any enzymatic activity and demonstrated that the first option does not apply. Washing the inactive reactor with buffer solution to rehydrate the pore surface did not regenerate the activity, thus excluding the former factor. In contrast, the activity of the reactor was rejuvenated by taking advantage of the non-covalent type of interaction of the enzyme with gold nanoparticles. The inactive lipase was stripped from the nanoparticles using 2-mercaptoethanol, followed by conditioning with water and a phosphate buffer, and subsequent immobilization of fresh lipase. This approach led to bioreactor exhibiting the original enzymatic activity and stability. It is worth noting that most of the typical immobilized enzymes must be completely discarded after the loss of activity and both new support and fresh enzymes applied to prepare another reactor. In contrast, an important advantage of our system is that the support is recycled and only the fresh enzyme must be re-applied. This regeneration was repeated several times and produced the same results each time. This finding is good evidence that the enzyme was successfully reloaded. Consequently, our bioreactor appears to be more economically viable. No decrease in the lipase activity was observed after storing the monolithic bioreactor filled with hexane in a refrigerator for 2 months.

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Figure 7. Change in activity of the immobilized lipase reactor expressed as yield of butyric acid with respect to the overall volume of substrate pumped through the reactor. The arrow shows a point at which the activity was restored after stripping the inactive enzyme and immobilizing the fresh one. Conditions: tributyrin in hexane, reaction temperature 37°C, substrate residence time 2.5 min, lipase immobilization time 16 h.

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Production of Fatty Acids Methyl Esters

Finally, our immobilized lipase bioreactor was used to produce fatty acids methyl esters, or biodiesel, from kitchen oil. Soybean oil and corn oil were dissolved in a mixture of methanol, tert-butanol, and hexane. Figure 8 confirms the successful production of biodiesel by comparing the chromatograms of the initial reaction mixtures with those obtained after passing them through the monolithic bioreactor. While no methyl esters are present in the starting oils, significant amounts of methyl esters of hexadecanoic acid and 9,12-octadecadienoic acid were detected in the effluent by GC–MS. Interestingly, no significant difference is observed between the transesterification products derived from both oils.

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Figure 8. GC–MS chromatograms of original oil (lower curves) and reaction products obtained by passing 100 µL kitchen oils dissolved in a mixture containing 500 µL hexane, 500 µL tert-butanol, and 300 µL methanol through the immobilized lipase bioreactor (upper curves). Conditions: reaction temperature 37°C, substrate residence time 2.5 min. Peaks: Hexadecanoic acid, methyl ester (1), 9,12-octadecadienoic acid, methyl ester (2), 9,12-octadecadienoic acid (3), 9,12-octadecadienoic acid, 2,3-dihydroxypropyl ester (4).

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Conclusions

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgments
  8. References
  9. Supporting Information

We have developed a novel approach for the fabrication of monolithic bioreactors. Our system is comprised of porous polymer monoliths with thiol functionalities holding gold nanoparticles on which porcine lipase is immobilized. Although the percentage of gold attached to surface of the monolithic support appears high, in reality the amount of gold related to the overall reactor volume is low. As opposed to conventional enzyme immobilization approaches, the use of gold nanoparticles as intermediate ligands or spacer arms, provides many advantages. For example, in contrast to typical covalently immobilized enzymes, with which the complete contents of the reactor including both the support and the inactive enzyme have to be discarded, our approach enables the sustainable use of the support that remains located in the reactor all the time. The regeneration of activity of our novel monolithic bioreactor can be easily achieved by removing the denatured enzyme, and immobilization of fresh one thus improving the economy of the enzymatic process. We plan to use in our future work other compounds such as 3-mercaptopropionic acid to strip the inactive enzyme from the reactor. Using lipase as a model enzyme, the bioreactor exhibited excellent catalysis efficiency and fast kinetics. This system was also successfully used for the transesterification reaction of kitchen oil for the production of biodiesel. Although the feasibility of this new concept was demonstrated with lipase, this universal immobilization technique can be easily adapted for other enzymes, and the range of applications using different immobilized enzymes extended.

Acknowledgments

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgments
  8. References
  9. Supporting Information

All experimental and characterization work was performed at the Molecular Foundry, Lawrence Berkeley National Laboratory. This work as well as Z.L. and F.S. were supported by the Office of Science, Office of Basic Energy Sciences, Scientific User Facilities Division of the U.S. Department of Energy, under Contract No. DE-AC02-05CH11231. The financial support of Y.L. by a grant from NIH (GM48364) is gratefully acknowledged. Y.L. and T.T. also gratefully acknowledge the financial support from the special assistance of 973 programs (2013CB733600, 2007CB714300, and 2007CB714302-2), the National Natural Science Foundation of China (20636010), 863 program (2006AA020102 and 2007AA10040), and Beijing Educational Committee Joint Construction.

References

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgments
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results and Discussion
  6. Conclusions
  7. Acknowledgments
  8. References
  9. Supporting Information

Additional supporting information may be found in the online version of this article at the publisher's web-site.

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