Toward Cell-Free Biofuel Production: Stable Immobilization of Oligomeric Enzymes J. Grimaldi, C. H. Collins, and G. Belfort Howard P. Isermann Dept. of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, RPI, Troy, NY 12180-3590 DOI 10.1002/btpr.1876 Published online in Wiley Online Library (wileyonlinelibrary.com)

To overcome the main challenges facing alcohol-based biofuel production, we propose an alternate simplified biofuel production scheme based on a cell-free immobilized enzyme system. In this paper, we measured the activity of two tetrameric enzymes, a control enzyme with a colorimetric assay, b-galactosidase, and an alcohol-producing enzyme, alcohol dehydrogenase, immobilized on multiple surface curvatures and chemistries. Several solid supports including silica nanoparticles (convex), mesopourous silica (concave), diatomaceous earth (concave), and methacrylate (concave) were examined. High conversion rates and low protein leaching was achieved by covalent immobilization of both enzymes on methacrylate resin. Alcohol dehydrogenase (ADH) exhibited long-term stability and over 80% conversion of aldehyde to alcohol over 16 days of batch cycles. The complete reaction scheme for the conversion of acid to aldehyde to alcohol was demonstrated in vitro by immobilizing ADH C 2014 American Institute of Chemical Engiwith keto-acid decarboxylase free in solution. V neers Biotechnol. Prog., 000:000–000, 2014 Keywords: isobutanol, immobilized enzyme, in vitro alcohol production, cell-free

Introduction With the recent discovery of abundant gas resources in the USA through “fracking” technology and recent and expected future drop in energy prices, the challenge for competitive alternate energy sources is more acute.1 Many viable fuel options are dependent on numerous external factors, including availability of a feedstock, an active industry, and competitive energy prices.2 This quantum change in the US energy mix will force funding agencies such as DOE and energy companies to reevaluate their focus. Limitations on biofuel production using cell culture (Escherichia coli,3 Clostridium,4 Saccharomyces cerevisiae,5,6 brown microalgae,7 blue-green algae,8 and others) include low product (alcohol) titers (2–5 vol%)9 due to feed-back inhibition, and instability of cells.9,10 We offer an alternate simplified biofuel production approach to cell culture with the hope of overcoming all three limitations listed above, while speeding up the process considerably and possibly reducing the cost of fuel production. Our scheme requires the following steps (Figure 1): i. E. coli cells: Recombinant production and isolation of two critical enzymes (keto-acid decarboxylase, KdcA, and alcohol dehydrogenase, ADH) using standard fermentation. ii. Starting substrate: Streptomyces cinnamonensis mutants overproduce 2-ketoisovaleric acid to titers of 2.4 g/L.11

Additional Supporting Information may be found in the online version of this article. Correspondence concerning this article should be addressed to G. Belfort at [email protected]. C 2014 American Institute of Chemical Engineers V

iii. Cell-free: Simultaneous in vitro application of the two enzymes (keto-acid decarboxylase, KdcA and alcohol dehydrogenase, ADH) attached to solid substrates to convert acid (ketoisovaleric acid) to aldehyde (isobutyraldehyde) and then to alcohol (isobutanol, the fuel). iv. Recovery: Continuous removal and recovery of isobutanol in order to drive the reactions toward isobutanol.12 This process is attractive because enzymes are produced at high titer using standard fermentation sans feed-back limitations (step (i) above), the substrate is produced in actinobacteria fermentation (step ii), protein engineering is used to stabilize the two enzymes to high product titer and high temperature, during alcohol synthesis there are no cells that undergo product feedback inhibition, enzymes are stabilized through immobilization (step (iii)), and the reaction is driven toward product through its continual removal (recovery) (step (iv)). In this manuscript, we report on optimizing the immobilization of two tetrameric enzymes: b-galactosidase (control) and ADH. Ongoing work is aimed at increasing KdcA stability and combining the two enzymes (KdcA and ADH) to generate a complete scheme with product recovery. Ethanol and butanol are leading options for biofuel production. Compared with ethanol, which has an octane value of 92 and high water affinity,13 n-butanol has a similar octane number, 96, but also a lower solubility in water.14 Isobutanol has similar properties to n-butanol with respect to water affinity; however, it has a lower octane number of 94.13 In addition, its higher energy density and the fact that it can use established fueling infrastructures,4,15 make it a promising alcohol for biofuel production. Several research groups are using metabolic engineering and systems biology (with random mutagenesis16 and gene 1

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Figure 1. Two-step enzymatic reaction to produce i-butanol from ketoisovaleric acid.

expression17) to produce alcohols and other small organic chemical intermediates.18–20 Focus has been on the valine production pathway in which a 2-ketoacid intermediate is converted into an aldehyde and then to an alcohol4,21 (Figure 1). A variety of host organisms have been examined to support this pathway, including those listed above. The major problems that arise are low volume capacity, slow reaction kinetics and eventually cell death associated with product feedback inhibition. At only 2% v/v of n-butanol, most cell hosts including those listed above drop to below 10% relative growth compared with cultures free of alcohol.9 This is a serious limitation that has forced researchers to change focus from overproduction of alcohol to adapting the cells to low to moderate levels of alcohol. The economics of such an approach with so low product concentration and the complications with cells is questionable. Immobilized enzymes have previously been used to produce energy through direct electron transfer where the enzymes are immobilized on the anodes and cathodes of fuel cells.22 In the 1970s, Katchalski et al.23 demonstrated that enzymes immobilized on surfaces and confined in cavities exhibit higher activity than in free solution. Since this discovery, many others have confirmed that tethering24,25 and adsorbing26 enzyme to suitable solid substrates not only protects27,28 but also allows enzymes to remain stable29 and highly active.30,31 Confinement of enzymes (using surface curvature32) and surface passivation with chemistry33,34 have been used to minimize losses in stability and activity of immobilized enzymes. The activity and stability of many immobilized enzymes have been measured with a variety of support materials including silica nanoparticles,35,36 mesoporous silica,37,38 self-assembled monolayers,39 acid treated single wall carbon nanotubes,24,25 agarose particles40 and diatoms.41 Immobilization of proteins on solid substrates has also been reported to cause secondary structural changes.42 The properties of the surface play a large role in proteins stability.43 More hydrophobic surfaces44 cause a decrease in the unfolding free energy barrier and an increase in the packing density.45 These changes lead to proteins losing their a-helical structure and gaining b-sheet structure.45,46 In addition, surface immobilization can affect protein leaching

Table 1. Support Material Properties Particle size Nanoparticles 38

SBA-15 Diatoms Methacrylate resin

200 nm 100 nm 1–2 mm 10–20 mm 100–300 mm

*Wide pore sized distribution.

and desorption from the surface.47,48 For covalent immobilization, the location and type of chemistry40 of the bond to the surface or tether can affect the folding stability49 and the orientation of the enzyme,50,51 which, in some nonoptimal cases, have caused enzyme to lose activity. Roughness mainly effects structure when the length scale of the rough surface is of the order of the size of the adsorbed species (monomer, dimer, oligomer, and precipitate).52 Most immobilized enzyme studies report on the behavior of monomeric enzymes. In this report, we select one oligomeric model enzyme, bgalactosidase (b-gal), and one oligomeric enzyme, alcohol dehydrogenase (ADH), which is part of the proposed twostep enzymatic production of i-butanol. The pH,53 immobilization time,54 and surface coverage53,54 can substantially impact enzymatic activity of multimeric proteins. We examined the effects of these variables on the activity of the two tetrameric enzymes when immobilized on multiple surface curvatures and chemistries, including silica nanoparticles, mesoporous silica, and methacrylate. In addition, long-term stability and conversion rates of aldehyde to alcohol were studied for immobilized ADH.

Materials and Methods Materials All materials and reagents were used as received. All buffers are at physiological pH and room temperature unless specified otherwise. Alcohol dehydrogenase from Saccharomyces cerevisiae {A3263} (ADH, 146.8 kDa, pI 5.4), bgalactosidase (b-gal, 465 kDa, pI 4.5), glutaraldehyde (GA), b-nicotinamide adenine dinucleotide (NADH), isobutyraldehyde, thiamine pyrophosphate (ThDP), ortho-nitrophenyl-bgalactoside (ONPG), 2-mercaptoethanol (BME), N-Hydroxysuccinimide (NHS), N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC), and buffer salts were purchased from Sigma-Aldrich Chemicals (Milwaukee, WI). SBA-15 was synthesized according to published protocols.38 SBA-15 is a mesoporous silica with an isoelectric point of 3 and ADH has an isoelectric point of 5.4. Diatomaceous earth (diatoms) were synthesized according to published protocols.55 Methacrylate resin (Relizyme HA403/S) was purchased from Itochu Chemicals America (White Plains, NY). Nonporous acid functionalized nanoparticles (100 and 200 nm) were purchased from Discovery Scientific (Vancouver, BC) (Table 1). Gas chromatography column: 25346 SUPELCO SPBV25 Capillary GC Column L 3 I.D. 15 m 3 0.53 mm, df 5.00 mm was purchased from SUPELCO (Milwaukee, WI). Surfaces were chosen to test two different geometries (concave vs. convex; and two different curvatures—100 vs. 200 nm particles), two different hydrophilic materials (silica vs. methacrylate), and two different immobilization methods (passive vs. covalent and direct immobilization vs. tethering). R

Surface curvature

Mean pore size

Surface area (m2/g)

Surface functionalization

Convex Convex Concave Concave Concave

N/A N/A 12 nm * 40–60 nm

15 30 370 >1300 80–90

Carboxylic acid Carboxylic acid Hydroxyl Carboxylic acid Amine

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Methods 96-Well Plate B-Gal Assay Conditions. 150 mL assay buffer (100 mM Sodium Phosphate, 10 mM KCl, 1 mM MgSO4, 50 mM BME) and 50 mL of 50 mg particles/mL assay buffer were added to each well. The assay was started by addition of 50 mL of ONPG (4 mg/mL in PBS) to each well. b-gal converts ONPG to galactose and orthonitrophenol, which absorbs light at 420 nm—yellow. Kinetic activity is tracked by observing the increase in absorbance over time. 96-Well Plate ADH Assay Conditions. 05 mL buffer A (50 mM potassium phosphate buffer, pH 6.8, 2.5 mM MgSO4, 0.1 mM ThDP), 20 mL NADH 2.5 mM in buffer A (final concentration 0.25 mM) and 25 mL 25 mg particles/ mL buffer A. The assay was started by addition of 50 mL isobutyraldehyde 120 mM in buffer A (final concentration 30 mM) to each well. ADH converts isobutyraldehyde to isobutanol using NADH as a cofactor, NADH absorbs light at 340 nm and NAD1 does not. Kinetic activity is tracked by observing the decrease in absorbance over time. Acid Functionalized Silica Nanoparticle Immobilization. Silica nanoparticles particles (50 mg) were mixed on an endover-end mixer with 1 mL (1 mg ADH/mL buffer A or 0.5, 1, or 2 mg b-gal/mL PBS) solution for 4 h. For covalent immobilization NHS/EDC succinimide chemistry was used. b-gal immobilization was studied at pH 5, 6, or 7. The particles were centrifuged and the supernatant assayed at 280 nm on a nanodrop UV–vis spectrophotometer (Thermo Fisher Scientific, Pittsburgh, PA) to determine protein concentration. Nanoparticles were washed with fresh buffer then assayed according to the 96-well plate assay conditions described above. Kinetics of immobilized enzyme was compared with those of equivalent enzyme free in solution. SBA-15 ADH Immobilization. SBA-15 particles (25 mg) were mixed on an end-over-end mixer with 1 mL (1 mg ADH/mL buffer A) solution for 4 h. SBA-15 particles were centrifuged and the supernatant assayed at 280 nm on a nanodrop UV–vis spectrophotometer (Thermo Fisher Scientific, Pittsburgh, PA) to determine protein concentration. SBA-15 particles were washed with fresh buffer A then assayed according to the 96 well plate assay conditions described above. Kinetics of immobilized enzyme was compared to those of equivalent enzyme free in solution. Diatom ADH Immobilization. Diatom particles (25 mg) were mixed on an end-over-end mixer with 1 mL (1 mg ADH/mL buffer A) solution for 4 h. The particles were centrifuged and the supernatant assayed at 280 nm in a nanodrop UV–Vis spectrophotometer (Thermo Fisher Scientific, Pittsburgh, PA) to determine protein concentration. Diatoms were washed with fresh buffer A and then assayed. The particles are opaque so the assay was performed using the same proportions as the 96 well plate assay (above) but in 1 mL cuvette format. Kinetics of immobilized enzyme was compared to those of equivalent enzyme free in solution. Methacrylate Resin Immobilization. Methacrylate resin (50 mg) was rehydrated on an end-over-end mixer with diH2O for 1 h; centrifuged and re-suspended in 1 mL (0.1% GA) and mixed similarly for 1 h. Resin was washed five times with diH2O to remove excess GA. Resin was then mixed with 1 mL (10, 5, 2.5, 1, or 0 mg ADH/mL buffer A or 4 mg b-gal/mL PBS) solution for 2 h. Resin particles were centrifuged and the supernatant assayed at 280 nm to determine protein concentration. Methacrylate resin was

3

washed with fresh buffer then assayed according to the 96well plate assay conditions described above. Kinetics of immobilized enzyme was compared to those of equivalent enzyme free in solution. Long-term ADH/Methacrylate Resin Assay. ADH immobilized on methacrylate resin (10 mg) or methacrylate resin with no ADH (10 mg) was re-suspended in 150 mL of stock reaction mixture (30 mM isobutyraldehyde, 10 mM NADH in buffer A). The mixture was put on an end-over-end mixer. Percent conversion was assessed by measuring the absorbance at 340 nm after 21 h. Fresh reaction mixture was added each day for 19 days. The contents of the reaction mixture before and after incubation with the resin were determined by gas chromatography analysis. Gas Chromatography Parameters. Gas chromatography spectra were recorded (Agilent 6890N, Agilent Technologies, Santa Clara, CA). The following parameters were used to perform the analysis: Carrier gas: Helium; Inlet temperature: 150 C, 50:1 split ratio; Constant column flow: 3.3 mL/min; Oven temperature: 35 C; Flame ionization detector temperature 275 C; Make-up gas: Helium at 45 mL/min. Each sample (1 mL) was injected into the system and the spectra recorded over 3 min.

Results and Discussion The goal of this work was to develop a viable method for immobilizing enzymes with multiple subunits. When immobilized, the enzyme must exhibit high activity or retain a large fraction of its solution activity and be able to remain immobilized and active through multiple reactions without leaching from the support. Three steps were used as part of our overall scheme: 1. A well-studied model oligomeric enzyme, b-galactosidase (b-gal), with a simple colorimetric assay was tested with various surfaces to identify the best surface and curvature (concave or convex) (Figure 2). 2. ADH was immobilized onto the optimal surface and curvature obtained from (1) and its conversion efficiency compared with that in free solution for aldehyde to alcohol conversion (Figures 3 and 4). Protein engineering has been previously used to engineer ketol-acid reductoisomerase and ADH to have a 2- to 40-fold increases in kinetic activity.56 3. Both KdcA and ADH were immobilized onto the optimal surface and conversion efficiency was compared with that in free solution for acid to aldehyde to alcohol conversion. Because KdcA is unstable when immobilized, we were able to maintain high KdcA activity in solution together with immobilized ADH in order to demonstrate the complete reaction at one time. Protein engineering to stabilize immobilized KdcA is underway in the laboratory. Below we describe the results for the steps above. b-gal Passive Immobilization (adsorption). The simplest and less expensive method of immobilizing proteins is by nonspecific attachment (using electrostatic, hydrophobic, and other interactions). b-gal and silica nanoparticles have isoelectric points of 4.5 and 3.0, respectively, so at pH 7.4, they are both negative and repulsive to each other. When bgal and 200 nm acid-functionalized silica nanoparticles are

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Figure 2. Normalized b-gal kinetic activity in solution and immobilized passively (just adsorbed) or covalently on nonporous silica, and porous methacrylate (resin) particles. (A) b-gal in solution ( ), b-gal passive immobilization on 200 nm nanoparticles ( ). (B) b-gal in solution ( ), free b-gal in a physical mixture with particles ( ), covalently immobilized b-gal ( ). (C) Reaction rate of b-gal when immobilized, pH 5 (solid), 6 (hatched), or 7 (empty) and varied surface concentration. (D) b-gal in solution ( ), b-gal covalently attached to amino functionalized methacrylate resin ( ), and amino functionalized methacrylate resin with no b-gal ( ). All absorbance readings were normalized by their initial absorbance.

allowed to mix and immobilize passively a 97% loss in activity was observed (Figure 2A) when compared with the equivalent b-gal concentration in solution. Lack of accessibility due to orientation of the enzyme’s activity site, dissociating into a dimer or monomer, and denaturing from near-neighbor interactions57 may explain this dramatic loss of activity. Covalent Attachment on Convex Surfaces. To optimize enzyme activity on a support, rather than passive immobilization, a controlled immobilization was pursued. Covalent attachment through a peptide bond would prevent leaching of the enzyme and possibly orient the protein optimally. As mentioned in the “Methods”, succinimide chemistry was used, between lysine residues and/or the N-terminus of the enzyme and the acid groups on the support, to attach the enzyme to the acid-functionalized 200 nm silica nanoparticles. After immobilization of b-gal on silica particles at pH 7, 87% of free solution activity was observed (Figure 2B). Adjusting the total amount of bound enzyme was critical for optimizing the immobilized activity. By controlling the pH at which the succinimide chemistry was performed, the peptide linkage was biased to form mainly at the N-terminus.58 Both pH and time of reaction were varied and pH 6 and 0.4 mg of b-gal/mg of particles was found to give the best activity (Figure 2C). Covalent Attachment in Concave Surfaces. Methacrylate resins provide protein structure stability when used in chromatography applications.59 Amino functionalized methacrylate resin was tested as a possible stable support system for b-gal. b-gal retained kinetic activity equivalent to enzyme free in solution (Figure 2D).

are known to be dependent on pH and NADH/substrate ratio. Experiments that show the conversion of isobutyraldehyde to isobutanol by ADH is essentially an irreversible reaction are described in the supplemental information. Covalent Attachment on Convex Surfaces. Using the techniques and results obtained as with oligomeric b-gal as a starting point, the immobilization of alcohol dehydrogenase (ADH) on silica and methacrylate particles was investigated. With the 100 and 200 nm acid-functionalized particles, the covalently attached ADH performed poorly, i.e. its activity was reduced by 92 and 84%, respectively, when compared with activity at the equivalent enzyme concentration in solution (Figure 3A). The amount of ADH covalently grafted onto the silica particles was proportionally related to the succinimide reaction time. The 100 and 200 nm particles had a total of 1.4 and 0.8 mg of ADH/m2, respectively. The difference in slope (slope is proportional to activity) for the 100 and 200 nm particles in Figure 3A suggests that the effect of near-neighbor interactions was significant.

ADH

Passive Immobilization in Concave Surfaces. Since nonporous (convex) silica nanoparticles did not provide a stable support for ADH, placing an enzyme in confinement, i.e., in a concave pore, suggests increased activity when compared with solution.38 Since ADH and SBA-15 have pI values of 5.4 and 3, respectively, both are negative at pH 7.4 and hence electrostatically repulse each other during adsorption. However, van der Waals and other attractive forces could induce binding. When ADH was allowed to adsorb nonspecifically onto SBA-15, the enzymatic activity was increased by 11% (Figure 3B). However, ADH was very loosely bound to the SBA-15 and after 3 rinse steps all the activity was lost due to ADH desorption.

Enzymatic Activity. ADH enzymatic activity is measured by following NADH consumption at 340 nm. The reactions

Covalent Attachment in Concave Surfaces. To overcome protein-leaching problems, covalent attachment was required.

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Figure 3. Normalized ADH kinetic activity in solution and immobilized passively (just adsorbed) or covalently on nonporous silica, porous SBA-15 and porous methacrylate (resin) particles. (A) ADH covalently attached on 200 ( ) and 100 ( ) nm acid functionalized silica nanoparticles, in solution ( ), and nanoparticles with no immobilized ADH ( ). (B) ADH passively immobilized on SBA-15 ( ), ADH passively immobilized on SBA-15 after multiple washes ( ), SBA-15 particles with no immobilized ADH ( ), ADH free in solution ( ). (C) ADH covalently attached to amino functionalized methacrylate resin as a function of bound ADH concentration: methacrylate resin no ADH ( ), 3 mg ADH/mg resin (X), 7 mg ADH/mg resin ( ), 15 mg ADH/mg resin ( ), 17 mg ADH/mg resin ( ), equivalent ADH free in solution ( ). All absorbance readings were normalized by their initial absorbance.

Acid-treated carbonated diatomaceous earth (diatoms) was used as a support system for ADH. Immobilized ADH was compared with equivalent enzyme free in solution, and the covalently attached enzyme lost 97% of its activity. Diatoms have acid groups on a carbon surface, which should provide good interaction with ADH. The drastic decrease in kinetic activity was probably due to the functionalization of the diatoms. Nitric acid was used to create the acid groups on the surface; if the reaction was not carried out long enough, exposed carbon would remain. Carbon is extremely hydrophobic and proteins denature rapidly at hydrophobic interfaces. Protein–surface interactions could be mitigated by introducing a spacer between the support and the enzyme and passivation of the surface with PEG6. b-gal immobilized on methacrylate resin exhibited kinetic activity equivalent to that in solution, which suggests that the immobilization conditions are benign. Amino functionalized methacrylate resin was tested as a possible stable support system for ADH because of its surface chemistry and curvature. At the maximum protein loading, ADH exhibited the best protein kinetics as well as retained kinetics equivalent to that free in solution (Figure 3C) (Table 2).

Long-term Stability. Now that a support system in which ADH retained kinetic activity was identified, long-term stability was studied. Over 20 days, new substrate was cycled into the same resin. The first 16 days showed over 80% conversion of isobutyraldehyde to isobutanol (Figure 4A). Gas chromatography was used to confirm ADH was producing the product (Figure 4B) at the reported conversions. Two-enzyme reaction scheme (ADH and KdcA: Figure 1) 18 mg enzymes/mg of resin were co-immobilized on methacrylate particles. When ketoisovaleric acid (starting substrate) was added to start the reaction, no change in absorbance was detected hence KdcA was inactive. To determine if ADH was functioning, isobutyraldehyde (intermediate product) was also added. Immobilized ADH showed enzymatic activity equivalent to that of ADH immobilized alone. To demonstrate the complete reaction scheme, the results from immobilized ADH together with KcdA free in solution are shown in Figure 5. 17 mg ADH/mg of resin was immobilized on methacrylate particles and KdcA was added to the solution. This combination of the two enzymes resulted in a larger change in absorbance

Figure 4. Long-term stability of immobilized ADH on methacrylate resin. (A) Percent conversion of isobutyraldehyde to isobutanol over 19 days. (B) Gas chromatographs of reaction mixture (top), reaction mixture after incubation with methacrylate resin without immobilized ADH (middle) and reaction mixture after incubation with ADH immobilized on methacrylate resin (bottom). N 5 NADH; N1 5 NAD1; 2 5 isobutyraldehyde; 3 5 isobutanol.

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Table 2. Enzymatic Activity of ADH at Different Protein Loadings in Resin Protein loading

Initial slope 3105 (DABS/s 3 mg)

3 mg/mg resin 7 mg/mg resin 15 mg/mg resin 17 mg/mg resin In solution

28.9 6 5 26.9 6 2 27.3 6 1.5 29.4 6 1.2 29.0 6 2.0

11

kcat 3 10

(1/s)

23.1 6 1.8 22.4 6 0.7 22.6 6 0.5 23.3 6 0.4 22.8 6 0.8 (17 mg)

than the free KdcA with no immobilized ADH present (Figure 5A). Immobilized ADH exhibited no kinetic activity in the absence of free KdcA when the initial substrate (ketoisovaleric acid) is added. Gas chromatography was used to confirm the final products of the reaction. Pure ketoisovaleric acid and pure isobutanol have the same retention time. Without the immobilized ADH on methacrylate resin, only isobutyraldehyde was produced. When immobilized ADH was added to the system, the presence of a strong isobutanol peak was observed (Figure 5B). To confirm that the peak was isobutanol, and not ketoisovaleric acid, an enlarged chromatogram depicting the loss of NADH was generated to demonstrate the cofactor was being consumed (Figure 5C).

Limitations of in situ removal from cell-based systems In situ product removal is one solution for toxicity problems associated with alcohol production. Many options for removal of n-butanol have been reported, but the number of recovery methods studied for isobutanol is much lower. Adsorption,60–62 gas stripping,63,64 and pervaporation12,65 have all been evaluated for n-butanol. The main concern that arises is not the removal of the isobutanol/media combination from the bioreactor but the methods used to purify isobutanol from the aqueous media. The method of choice is often two-phase extraction because of the properties (e.g., boiling point, and vapor pressure) of isobutanol. Recycling of media back into the fermenter system after organic extraction is often contaminated with trace amounts of the organic phase and this too may result in cellular toxicity.

Conclusions and Future Work Covalent binding has a higher cost than passive immobilization, but it leads to more stable attachment of active enzyme to the surface. The covalently attached enzyme functioned like enzymes free in solution. Changing the

Figure 5. Normalized kinetic activity of KdcA in solution and ADH immobilized on amino-functionalized methacrylate resin. (A) KdcA enzymatic activity free in solution with methacrylate resin without immobilized ADH (2), immobilized ADH enzymatic activity without free KdcA after ketoisovaleric acid is added (1), and immobilized ADH enzymatic activity with free KdcA after ketoisovaleric acid is added (3). (B) Gas chromatographs of reaction mixture without immobilized ADH (top), and reaction mixture after incubation with ADH immobilized on methacrylate resin (bottom). N 5 NADH, 1 5 ketoisovaleric acid, 2 5 isobutyraldehyde, 3 5 isobutanol. (C) Enlarged NADH peak, pure NADH (black); reaction mixture without immobilized ADH (gray), and reaction mixture after incubation with ADH immobilized on methacrylate resin (dotted).

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succinimide reaction time and pH conditions in the solution allowed us to obtain a wide range of orientations and degrees of surface coverage. Concave geometry coupled with tethering was shown to be the superior method of enzyme immobilization. KdcA demonstrated a potential to complete the conversion of ketoisovaleric acid to isobutyraldehyde; however, its ability was limited by its instability near surfaces and its propensity to self-aggregate. The development of a more robust KdcA variant that is able to withstand immobilization is attractive and a future goal for a successful in vitro system for biofuel production. The immobilization strategies we have developed have provided excellent results for stable enzymes. Kinetic activity equivalent to enzyme free in solution was observed for b-gal and ADH. Our next step is to develop a stable active mutant of KdcA. Error-prone PCR will be used to create a library of mutations in our gene for KdcA. The enzyme variants will be screened for increased activity and stability. Mutants from directed evolution will be immobilized on methacrylate resin and used with immobilized ADH to produce isobutanol from ketoisovaleric acid. Finally, the uses of cofactor NADH will need to be addressed. There are many strategies to regenerate NADH from NAD1. One example would be to use formate dehydrogenase to convert sodium formate (an inexpensive sacrificial material [$415/metric ton], Qingdao Ipolymer Chemicals Co.) to CO2. The regeneration material will result in a cost of $0.75/gal. This third enzyme could be used to make the process continuous.66 Then, the rest of the scheme described above will be pursued.

Acknowledgments We thank Dr M-O Coppens (University College London) for supplying the SBA-15 particles, Dr. Sandhage (Georgia Institute of Technology) for synthesizing the diatiom particles, and Dr. Robert DiCosimo (DuPont Corporation) for his suggestion of Relizyme resin supports. The authors want to thank the Department of Energy (Grant No. DE/ SC0006520) for funding.

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Toward cell-free biofuel production: Stable immobilization of oligomeric enzymes.

To overcome the main challenges facing alcohol-based biofuel production, we propose an alternate simplified biofuel production scheme based on a cell-...
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