This article will describe the procedure for synthesizing a hydrophobically modified Nafion enzyme immobilization membrane and how to immobilize proteins and/or enzymes within the membrane and test their specific activity.
Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel cells.1-3 In most bioelectrochemical applications, enzymes or organelles are immobilized onto an electrode surface with the use of some type of polymer matrix. This polymer scaffold should keep the enzymes stable and allow for the facile diffusion of molecules and ions in and out of the matrix. Most polymers used for this type of immobilization are based on polyamines or polyalcohols – polymers that mimic the natural environment of the enzymes that they encapsulate and stabilize the enzyme through hydrogen or ionic bonding. Another method for stabilizing enzymes involves the use of micelles, which contain hydrophobic regions that can encapsulate and stabilize enzymes.4,5 In particular, the Minteer group has developed a micellar polymer based on commercially available Nafion.6,7 Nafion itself is a micellar polymer that allows for the channel-assisted diffusion of protons and other small cations, but the micelles and channels are extremely small and the polymer is very acidic due to sulfonic acid side chains, which is unfavorable for enzyme immobilization. However, when Nafion is mixed with an excess of hydrophobic alkyl ammonium salts such as tetrabutylammonium bromide (TBAB), the quaternary ammonium cations replace the protons and become the counter ions to the sulfonate groups on the polymer side chains (Figure 1). This results in larger micelles and channels within the polymer that allow for the diffusion of large substrates and ions that are necessary for enzymatic function such as nicotinamide adenine dinucleotide (NAD). This modified Nafion polymer has been used to immobilize many different types of enzymes as well as mitochondria for use in biosensors and biofuel cells.8-12 This paper describes a novel procedure for making this micellar polymer enzyme immobilization membrane that can stabilize enzymes. The synthesis of the micellar enzyme immobilization membrane, the procedure for immobilizing enzymes within the membrane, and the assays for studying enzymatic specific activity of the immobilized enzyme are detailed below.
1. Modification of Nafion with Quaternary Ammonium Salts
2. Immobilization of Enzymes into TBAB-Modified Nafion for Activity Assays
3. Assay of Immobilized NAD-Dependent Dehydrogenase Enzyme
4. Assay of Immobilized PQQ-Dependent Dehydrogenases
5. Assay of Immobilized Glucose Oxidase
6. Representative Results
The micellar structure of the modified Nafion polymer can be disrupted by drying the original salt/polymer co-casted film too fast. Figure 2 shows a salt/polymer mixture that has been dried correctly resulting in a transparent, light brown film. A film that dries too fast can result in opaque, white flakes of polymer due to the fact that the drying process can destroy the micellar structure.
Once the modified Nafion polymer and enzyme have been mixed and co-cast onto the bottom of a cuvette, enzymatic activity assays can be used to assess the stability of the enzyme within the polymer film. Tables 2-4 show assay results of two dehydrogenase enzymes and glucose oxidase immobilized into various modified Nafion films, respectively. Note the higher activity of the enzymes that are immobilized vs. the enzymes in buffer solution, showing that modified Nafion polymers can actually enhance the activity of certain enzymes (called superactivity). Other enzymes have transport limitations that decrease their specific activity when immobilize them in the polymer (i.e. cellulases and amylases, whose substrates are quite large macromolecules).
Quaternary ammonium salt used | 3 fold excess |
T3A (tetrapropylammonium bromide) | 32.37 mg/ml |
TBAB (tetrabutylammonium bromide) | 39.19 mg/ml |
TPAB (tetrapentylammonium bromide) | 46.01 mg/ml |
TEHA (triethylhexylammonium bromide) | 32.37 mg/ml |
TMHA (trimethylhexylammonium bromide) | 27.25 mg/ml |
TMOA (trimethyloctylammonium bromide) | 30.66 mg/ml |
TMDA (trimethyldecylammonium bromide) | 34.07 mg/ml |
TMDDA (trimethyldodecylammonium bromide) | 37.48 mg/ml |
TMTDA (trimethyltetradecylammonium bromide) | 40.89 mg/ml |
TMHDA (trimethylhexadecylammonium bromide) | 44.31 mg/ml |
TMODA (trimethyloctadecylammonium bromide) | 47.71 mg/ml |
Table 1. Amounts of tetra-alkyl ammonium salts to use for Nafion polymer modification.
Type of Nafion | Enzyme activity (U/g) |
Buffer (no polymer) | 16.63 ± 8.11 |
Nafion (un-mod.) | 9.25 ± 2.21 |
TMTDA | 3.23 ± 2.92 |
TBAB | 3.93 ± 3.33 |
TMDDA | 4.19 ± 1.04 |
TMOA | 3.51 ± 1.11 |
TMDA | 8.00 ± 4.53 |
TMHA | 1.68 ± 1.39 |
TMHDA | 4.83 ± 0.99 |
TMODA | 10.45 ± 3.20 |
Table 2. NAD-dependent glucose dehydrogenase activity immobilized in selected modified Nafion polymers (note: immobilized activity is a function of initial specific activity of the enzyme).
Type of Nafion | Enzyme activity (mU/g) |
Buffer (no polymer) | 7.18 ± 0.51 |
Nafion (un-mod.) | 70.1± 0.5 |
TMTDA | 133 ± 6 |
TBAB | 244 ± 4 |
TMDDA | 221 ± 6 |
TMOA | 1.78 ± 0.63 |
TMDA | 206 ± 5 |
TEHA | 40.1 ± 50.6 |
TMHDA | 0 |
TMODA | 1.45 ± 0.06 |
Table 3. PQQ-dependent glucose dehydrogenase activity immobilized in selected modified Nafion polymers (note: immobilized activity is a function of initial specific activity of the enzyme).
Type of Nafion | Enzyme activity (U/g) |
Buffer (no polymer) | 103.61 ± 3.15 |
Nafion (un-mod.) | 19.93 ± 10.10 |
TMTDA | 247.25 ± 12.49 |
TBAB | 152.27 ± 5.29 |
TMDDA | 262.05 ± 6.26 |
TMOA | 129.18 ± 2.31 |
TMDA | 141.23 ± 1.97 |
TMHA | 131.75 ± 2.89 |
TMHDA | 132.50 ± 1.18 |
TMODA | 136.50 ± 0.96 |
Table 4. Representative glucose oxidase specific activity immobilized in selected modified Nafion polymers (note: immobilized activity is a function of initial specific activity of the enzyme).
Figure 1. Schematic of TBAB incorporation into Nafion polymer and subsequent use in enzyme immobilization.
Figure 2. Optical photograph of initial co-cast films of Nafion and TBAB. Slow drying yields a transparent, light brown film covering the bottom of the weighing tray.
In the described procedure, tetra-alkyl ammonium salts are used to modify commercial Nafion to create micellar polymers that can be used to immobilize and stabilize enzymes. The assays described in the procedure show that the polymer can be used to immobilize a wide variety of enzymes with a high retention of activity. If the enzyme of interest has very low activity or is impure, a higher concentration may be required and should not affect the immobilization process, unless immobilizing enzymes in concentrations greater than 10 mg/ml. The simplicity of the procedure separates it from other enzyme immobilization techniques in that no synthetic steps (such as polymer synthesis or cross-linking) are required. Also, these synthetic steps frequently denature proteins or dramatically decrease their activity. The protein concentration of 1mg/mL is merely a suggested concentration for high enzyme loading. Lower enzyme concentrations can always be employed, but will result in less volumetric catalytic activity. In theory, higher enzyme concentrations can be used as long as the enzyme dissolves.
Because the polymers are soluble in lower aliphatic alcohols such as methanol, ethanol, and propanol, a high percentage of alcohol must be present when mixing the polymer suspension with an enzyme. In many cases, this is not a problem as long as the ethanol is evaporated in a timely manner once the enzyme-encapsulated film is cast. However, one limitation of this immobilization can occur if an enzyme is not at all alcohol-tolerant and denatures or precipitates upon the addition of the modified Nafion suspension. In rare cases, enzymes will precipitate or denature when mixed with the modified Nafion suspension, usually indicating that the enzyme has become denatured and will not function. It is possible to decrease the alcohol content in the suspension by resuspending the polymers in alcohol/water mixtures, but lower aliphatic alcohols are required in significant concentrations (>25%) in the resuspension solution, so this immobilization technique does not work for enzymes/enzyme solutions that cannot tolerate these concentrations of alcohol.
The enzymatic assays for each of the polymers with each of the three enzymes show that the trends in relative specific activity compared to enzyme in solution is a function of the enzyme system. This is expected, since each of the enzymes is a different size, different pI, different optimal pH, as well as the fact that PQQ-dependent glucose dehydrogenase is a membrane associated protein and therefore needs a very different chemical microenvironment that cytosolic proteins. Therefore, the hydrophobically modified micellar Nafion gives a more membrane-like environmental for stabilizing the active PQQ-dependent glucose dehydrogenase than buffer and shows superactivity, which is rare in immobilized enzymes. Another issue to consider is that polymer membranes decrease transport of large molecules and although glucose (the substrate for all three tests shown here) is small, the coenzyme NAD needs to diffuse in and out of the membrane for NAD-dependent dehydrogenases and this decreases the observed enzymatic activity. Overall, it is important to note that the exact polymer needed for each enzyme has to be optimized, because of the differences in size, charge, optimal pH, and transport of substrate/cofactors for each of the enzyme systems.
Other than immobilized enzyme assays, the primary application that has been explored with this enzyme immobilization method is the fabrication of enzymatic biosensors and biofuel cells. When modified Nafion polymers containing encapsulated redox enzymes are cast onto electrode surfaces, bioelectrocatalytic processes can occur in the presence of appropriate substrates and cofactors, producing an electric current response. Bioanodes fabricated with modified Nafion have been used in biofuel cells that utilize ethanol, methanol, pyruvate, and glycerol, as described in the introduction.
The authors have nothing to disclose.
The authors acknowledge the Office of Naval Research, United Soybean Board, and National Science Foundation for funding.
Name of the reagent | Company | Catalog number |
Nafion | Sigma-Aldrich | 70160 |
Tetra alkylammonium bromide salts | Sigma-Aldrich | n/a |
Alcohol dehydrogenase | Sigma-Aldrich | A3263 |
Nicotinamide adenine dinucleotide (NAD) | Simga-Aldrich | N7004 |
Sodium pyrophosphate | Sigma-Aldrich | P8010 |
Phenazine methosulfate (PMS) | Sigma-Aldrich | P9625 |
2,6-Dichloroindophenol (DCIP) | Sigma-Aldrich | D1878 |
Glucose oxidase | Sigma-Aldrich | G7141 |
4-Hydroxybenzoic acid | Sigma-Aldrich | 240141 |
Sodium azide | Sigma-Aldrich | S8032 |
Peroxidase | Sigma-Aldrich | P8375 |
4-aminoantipyrine | Sigma-Aldrich | 06800 |
UV/Vis Spectrophotometer | Thermo | Evolution 260 Bio or Spectronic Genesys 20 |
Vortex Genie | ||
Analytical balance |