Noncovalent Immobilization of Optimized Bacterial Cytochrome P450

Article pubs.acs.org/IECR

Noncovalent Immobilization of Optimized Bacterial Cytochrome P450 BM3 on Functionalized Magnetic Nanoparticles Atieh Bahrami,† Thierry Vincent,† Alain Garnier,† Faiçal Larachi,† John Boukouvalas,‡ and Maria C. Iliuta*,† †

Department of Chemical Engineering, Laval University, Québec, Canada, G1V 0A6 Department of Chemistry, Laval University, Québec, Canada, G1V 0A6

‡

ABSTRACT: A stable insoluble P450 BM3 system that can be easily separated from the reaction medium for recycling, while using less-expensive cofactors (NADH and BNAH) than costly NADPH, can represent a promising biocatalyst in industrial applications. In this context, the present work investigates the immobilization of double mutant cytochrome P450 BM3 (R966D/W1046S) by adsorption and cross-linking-adsorption on Ni2+-functionalized magnetic nanoparticles (MNPs). By oxidizing NADH or BNAH, the immobilized BM3 succeeded in hydroxylating the substrates (10pNCA and myristic acid) to a similar degree as the free enzyme. The adsorbed enzyme showed 88% hydroxylation residual activity after five reaction cycles (five continuous days), which was increased to 100% by cross-linking the adsorbed enzyme. In addition, the crosslinked-adsorbed enzyme kept 41% of its initial activity toward NADH after one month of storage at 4 °C, while the free enzyme showed only 31% residual activity after 1 week and was inactive afterward. The results of this work highlighted that the appropriate choice of the enzyme-support-cofactor system can result in an active, stable, and recyclable biocatalyst, which could attract growing industry interest.

1. INTRODUCTION Enzymes are becoming increasingly important in chemical industries and biotechnology. They catalyze various reactions with high selectivity under mild reaction conditions, such as physiological pH and ambient temperature.1−3 Cytochrome P450s, which are a versatile family of enzymes, present a particular ability in catalyzing the oxidation of a broad range of substrates such as xenobiotics, drugs, hydrocarbons, fatty acids, vitamins, herbicides, insecticides, and aromatic compounds. As a significant member of this family, cytochrome P450 BM3 from Bacillus megaterium is a soluble enzyme that contains both monooxygenase and reductase domains on a single polypeptide chain.4 These advantages make this protein a self-sufficient biocatalyst and an ideal model to investigate the catalytic behavior of P450s. P450 BM3 inserts a single oxygen atom from molecular oxygen into an organic substrate while reducing the other one to a water molecule.4 For instance, it ω-hydroxylates mediumto long-chain fatty acids by adding a hydroxyl group to the carbon at the ω-1 to ω-3 positions. These valuable products (ω-hydroxylated fatty acids) can be used as building blocks in the synthesis of poly ω-hydroxy fatty acids (polyethylene-like biobased plastics) or additives in the fabrication of lubricants, emulsifiers, and stabilizers.5−7 For this selective oxidation process, BM3 requires O2 and a continuous supply of electrons usually derived from cofactors such as nicotinamide adenine dinucleotide phosphate (NADPH).4,8 This particular oxidation © XXXX American Chemical Society

is not easily achievable by current organic synthesis methods, because the chemical synthesis processes (i) undergo harsh reaction conditions such as high temperature and pressure,9 (ii) require nonrenewable feedstocks and multiple steps, strong acids, toxic oxidants, and (iii) show a lack of selectivity.6,10 In the light of the growing interest of ω-hydroxylated fatty acids and chemical synthesis route’s drawbacks, the application of green and sustainable enzymatic synthesis methods could be promising alternatives. Although BM3 catalyzes many important synthesis processes, because of its low stability and continuous requirement of an expensive cofactor, NADPH, it is not extensively used in industry. To enhance the enzyme stability and reusability, enzyme immobilization on/onto/in different supports has been applied in several biotransformation processes. However, there are only a few reports on the immobilization of P450s. For example, King and co-workers11 reported successful entrapment of cytochrome P450 in calcium alginate and CNBractivated sepharose for hydroxylation of benzopyrene. In another study, encapsulation of P450 BM3 in a sol−gel matrix resulted in no loss of activity after 36 days at 4 °C.12 Molecular sieves13 have also been used to immobilize BM3; nevertheless, Received: July 12, 2017 Revised: September 3, 2017 Accepted: September 6, 2017

A

DOI: 10.1021/acs.iecr.7b02872 Ind. Eng. Chem. Res. XXXX, XXX, XXX−XXX

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Industrial & Engineering Chemistry Research

part. Moreover, myristic acid was used as a reaction model to confirm the hydroxylation at ω-(1−3) positions. The enzyme activity was reported based on cofactor (NADH or BNAH) consumption and p-nitrophenolate production. The immobilized enzyme was recovered from the reaction medium by applying a permanent magnet and reused five times (five continuous days) to evaluate its behavior in the cyclic operation, which is an essential concern in practical applications. In the same context, the storage stability of free and immobilized enzyme was examined during a month while keeping the enzyme at 4 °C. The performance of free and immobilized enzymes was evaluated under the same reaction conditions, for comparison purposes. To the best of our knowledge, a similar study is not available in the open literature. This is the first report on the adsorption/ cross-linking-adsorption of the designed R966D/W1046S BM3 on Ni2+-functionalized MNPs.

the pore diameter was found to be a limiting factor in mass transfer. To make the enzymatic process (whether with soluble or immobilized enzyme) economically efficient, different approaches can be considered. The cofactor regeneration by using dehydrogenases for NADPH or nicotinamide adenine dinucleotide (NADH)14−16 reduces the cost of process; however, as a multienzymes system, it is difficult to control. P450s have been engineered to be able to oxidize less-expensive cofactors, such as NADH8 or N-benzyl-1,4-dihydronicotinamide (BNAH).17 Direct replacement of NADPH by sodium dithionite18 or zinc dust/cobalt sepulchrate mediator system19 has also been investigated. However, they could not reach the wild-type enzyme activity in the presence of NADPH. Transferring electrons to the enzyme directly from electrodes20,21 is another alternative to replace the costly cofactor. In this case, modification of electrode surface by enzyme-friendly materials that can work as a mediator to transfer electrons from the electrode to the active site of the enzyme and prevent enzyme denaturation is required.22 Among several organic and inorganic materials that can be used as support for enzyme immobilization, magnetic nanoparticles (MNPs) have many advantages such as high specific surface area, effective enzyme loading, facile and low-cost separation from the reaction medium by applying an external magnetic field, and lower mass-transfer limitations in solution. However, bare magnetic nanoparticles do not have appropriate functional groups to adsorb, entrap, or covalently attach the enzymes. For enzyme immobilization, their surface should be modified by organic or inorganic chemicals such as polymers, surfactants, metal oxides, or silica.23,24 Modifying MNPs surface by chelated metal ions is a well-known powerful technique in enzyme purification25 that can also be applied for enzyme immobilization.26 Divalent transitional metal ions such as Ni2+, Zn2+, Co2+, and Cu2+ can interact with the thiol group of cysteine, the indole group of tryptophan, or the imidazole group of histidine.26 For example, Cu2+-chelated magnetic beads27 and cryogels25 have been successfully used to adsorb cytochrome c from its solution. Although the enzymes immobilized by metal affinity are often more active, because of the mild immobilization conditions, enzyme cross-linking is recommended to mitigate the enzyme leakage.28 A stable insoluble enzyme system that can be easily separated from the reaction medium to be reused, while using lessexpensive cofactors as source of electrons, can represent a successful biocatalyst in industry. In this context, this work investigates the immobilization of engineered cytochrome P450 BM3 (R966D/W1046S) from Bacillus megaterium by adsorption and cross-linking-adsorption on Ni2+-functionalized MNPs. The presence of Ni2+ on MNPs surface offers high affinity to adsorb the enzyme via its histidine tag, which was added to the enzyme mutant for purification and immobilization purposes. Glutaraldehyde was used to cross-link the enzyme after its adsorption on MNPs surface to improve the enzyme attachment on MNPs. The double mutant used for immobilization was designed to oxidize less-expensive cofactors (NADH and BNAH) than NADPH. In comparison with the wild-type enzyme, this mutant is not only able to consume BNAH, but shows preference to NADH rather than NADPH.17 Considering the simple method of analysis, 10-(4-nitrophenoxy) decanoic acid (10-pNCA) was used as a substrate model to evaluate the catalytic efficiency of the immobilized enzyme (adsorbed/cross-linked-adsorbed) and its free counter-

2. MATERIALS AND METHODS 2.1. Materials. The optimized cytochrome P450 BM3 from Bacillus megaterium (with R966D and W1046S mutations and the addition of a 8-his tag)29 was produced in our laboratories. The histidine tag was added to the enzyme for purification and immobilization purposes. The enzyme mutant showed the ability to consume NADH and BNAH as cofactors.17,29 Deionized water (DIW) was used for solution preparation. All reagents were used as they were received and without any further purification. Ferric chloride hexahydrate (FeCl3·6H2O, 99.4%, MP Biomedicals LLC), ferrous chloride tetrahydrate (FeCl2·4H2O, 99.0%−103.0%, Avantor Performance Materials, Inc.), ammonia (28%−30%), N-phosphonomethyl iminodiacetic acid (PMIDA) (95%), glutaraldehyde (50% solution in water), and sodium dithionite (85%) (Sigma−Aldrich), myristic acid ≥98% (Fluka), K2HPO4 99% and KH2PO4 (99%) (Caledon), Tris(hydroxylethyl) aminomethane 99.8% (Merck), methanol 99.9% (Fisher Chemicals), pure NADH disodium salt trihydrate (Amresco), and NiCl2 99% (Alfa Aesar) were used in this study. BNAH was synthesized by Oleotek, according to the procedure described by Paul et al.30 Using a route similar to that described in the literature for the synthesis of a related compound,31 the desired substrate, 10pNCA32 was prepared in three steps and with 58% overall yield from inexpensive 10-bromodecanoic acid. 2.2. Magnetic Nanoparticles Synthesis. Iron oxide nanoparticles were synthesized by coprecipitation of Fe2+ and Fe3+ in basic solution. One gram (1 g) of FeCl3·6H2O and 0.35 g of FeCl2·4H2O (Fe3+/Fe2+ ≈ 2) were stirred in 150 mL of DIW for 30 min while heating to 80 °C under nitrogen atmosphere. Then, 10 mL of aqueous ammonia were injected to the flask to increase pH to 11. The black-colored Fe3O4 started to precipitate, and stirring continued for another 30 min; particles then were separated by applying a magnet and washed with DIW until neutral pH was attained. To functionalize MNPs for enzyme adsorption, 1 g of PMIDA was added to the prepared Fe3O4 and the mixture stirred for 12 h (final pH 10.4). PMIDA-functionalized MNPs were separated by a magnet and washed with DIW several times until pH ∼7. To introduce Ni2+ to the surface of PMIDA-Fe3O4, the particles were added to NiCl2 solution (5 g/50 mL DIW) and stirred for 1 h.33 Finally, the particles were recovered, washed with DIW, filtered through a 200 nm filter paper, and dispersed in 50 mL of DIW for further use. These synthesized particles are abbreviated as Ni2+-PMIDA-MNPs. B


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Industrial & Engineering Chemistry Research 2.2.1. Ni2+-PMIDA-MNPs Characterization. Fourier transform infrared (FT-IR) spectroscopy, thermogravimetric analysis (TGA), powder X-ray diffraction (XRD), magnetization measurements, and dynamic light scattering (DLS) were used to investigate the properties of the synthesized MNPs. FT-IR spectra of the particles before and after enzyme immobilization were recorded using an FT-IR spectrometer (Nicolet Magna550, Thermo Scientific, Madison, WI) with resolution of 4 cm−1 in the range of 450−4000 cm−1 using KBr pellets. TGA measurements were performed under nitrogen flow and in the temperature range of 25−800 °C (increasing rate of 5 °C/min) using PerkinElmer Pyris Diamond TGA-DTA. The XRD patterns for bare Fe3O4 and Ni2+-PMIDA-Fe3O4 nanoparticles were obtained using a Siemens D5000 diffractometer with a Cu Kα radiation source (λ = 1.54059 Å) at room temperature at 2θ = 2°−100°. Magnetization measurement was carried out at 298 K for both bare and functionalized MNPs by MicroMag Model 2900 (Princeton Instrument Co.). The mean size/size distribution of Ni2+-PMIDA-MNPs was studied using Zetasizer Nano ZS equipment (Malvern Instruments). 2.3. P450 BM3 Immobilization. The affinity of Ni2+ for the imidazole group of histidine was used to immobilize the P450 BM3 on the synthesized particles. For that purpose, 40 mg of Ni2+-PMIDA-MNPs were added to the enzyme solution (0.2 μM) in 10 mL phosphate buffer (PBS) 0.1 M, pH 7.4. The mixture was incubated in an orbital shaker at 4 °C for 4 h. Subsequently, the immobilized enzyme (Enzyme-MNPs) was separated from the medium by applying a magnet, washed with PBS three times to remove the unbound enzyme, and then dispersed and stored in PBS at 4 °C for further use. To cross-link the adsorbed enzyme on MNPs surface, three different concentrations of glutaraldehyde (0.25%, 0.75%, and 1.25% v/v) were tested. In 10 mL of PBS solution, glutaraldehyde was added to 40 mg MNPs carrying 2 nanomoles (nmol) of enzyme. The mixture was incubated at 4 °C for 2 h. Then, particles were separated and washed thrice with PBS to remove the remaining glutaraldehyde. The crosslinked-adsorbed enzymes were dispersed in the buffer and stored at 4 °C for further use. 2.3.1. P450 BM3 Assay. The procedure of Omura and Satu34 was used to estimate the enzyme quantity (concentration). The enzyme was diluted in 0.1 M PBS pH 7.4 and saturated with carbon monoxide (CO). A few crystals of sodium dithionite were then added to the sample and the light absorbance was scanned between 400 and 500 nm at room temperature, using a Genesys 10 UV-vis spectrophotometer (Thermo Fisher). An extinction coefficient of 91 mM−1 cm−1 was applied for the CO light absorption difference in this range of absorbance. The enzyme concentration was estimated by applying the Beer− Lambert equation. Samples were taken when the enzyme was added to the particles in the buffer solution (before enzyme immobilization) and after 4 hours incubation at 4 °C (after enzyme immobilization). For immobilized enzyme samples, before and after immobilization, the particles were separated by applying a magnet and then the supernatant was analyzed to estimate initial and unbound amounts of enzyme in the solution, respectively (as described above). After each time washing the enzyme-MNPs with 10 mL of PBS, the particles were separated and the same procedure applied to estimate the washed enzyme from the MNPs. The amount of adsorbed enzyme was then calculated based on the difference between the initial amount of enzyme in the buffer and the sum of the remaining and washed enzyme in the supernatant.

2.3.2. P450 BM3 Activity Assay. To evaluate the catalytic activity of P450 BM3, myristic acid or 10-pNCA and NADH or BNAH were used as the substrate and cofactor, respectively. 2 nmol of free enzymes or 40 mg of MNPs carrying the same amount of enzyme were incubated with the substrate for a few minutes, and then the cofactor was added to the samples to start the reaction. Myristic acid, 10-pNCA, and BNAH stock solutions were prepared in methanol and were used to obtain a methanol concentration of 5% in 10 mL of final reaction volume. The light absorbance of samples at 340 or 355 nm was measured to determine NADH or BNAH consumption, respectively. The difference between the initial and remaining amounts of cofactors in the reaction medium was used to calculate the cofactor consumption. The product of 10-pNCA hydroxylation (yellow chromophore p-nitrophenolate, C6H4NO3−)35 was detected by light absorbance measurement at 435 nm. GC-MS Agilent 7890 A on a SLB-IL60 column (30 m × 0.25 mm × 0.2 μm) was employed to confirm the production of ω-hydroxylated myristic acids. The preparation of reaction samples for each substrate/cofactor system is described in more detail as follows. 2.3.2.1. Myristic Acid Hydroxylation. The tests were performed for three different concentrations of myristic acid (150, 250, and 350 μM). In experiments where NADH was used as a cofactor, the reaction medium was prepared using PBS 0.1 M, pH 7.4. To evaluate the BNAH consumption by the enzyme, 0.1 M Tris buffer, pH 8.2, was used instead, because of the instability of BNAH in PBS. NADH concentrations that were two times higher (300, 500, 700 μM) and BNAH concentrations that were three times higher (450, 750, 1050 μM) were used for myristic acid concentrations of 150, 250, and 350 μM, respectively. The reaction was left overnight at room temperature. Cofactor consumption was then measured as mentioned above and the presence of ω-hydroxylated products was determined by GC-MS analysis. Free and adsorbed enzymes activity was evaluated for these tests. These experiments were repeated four times, and the average of results is reported. 2.3.2.2. 10-pNCA Hydroxylation. Three different concentrations of 10-pNCA (150, 250, and 350 μM) were assayed for hydroxylation by free and immobilized BM3 (2 nmol or 2 nmol/40 mg MNPs) oxidizing NADH or BNAH (1 mM). The samples were prepared in 0.1 M Tris buffer, pH 8.2. The reaction samples were incubated at room temperature and left overnight. p-Nitrophenolate production and cofactor consumption were then measured as described above. Free, adsorbed and cross-linked-adsorbed enzymes were used for 10-pNCA hydroxylation. These tests were performed in triplicate, and the average of data is reported. 2.4. Immobilized BM3 Reuse. The substrate, 10-pNCA, was used to investigate reusability of the immobilized BM3. For each cycle of reaction, 10-pNCA (350 μM) was added to adsorbed/cross-linked-adsorbed enzyme samples in 10 mL of 0.1 M Tris buffer, pH 8.2. The cofactor, NADH or BNAH (1 mM), was added to the enzyme−substrate mixture to start the reaction. After overnight reaction, the immobilized enzyme was separated and washed with Tris buffer, and the supernatant was analyzed to determine the product concentration as described above. The following day, to perform another reaction cycle, the enzyme-MNPs were resuspended in the reaction medium. This process was repeated five times for five continuous days and the residual activity was determined by considering the C


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Industrial & Engineering Chemistry Research initial activity (first run) as 100% and calculating the ratio of enzyme activity in each cycle to this activity. 2.5. Storage Stability of BM3. The storage stability of free and immobilized enzyme at 4 °C was tested by determining its activity toward 10-pNCA and NADH every week for a period of one month. For each test, 10-pNCA (350 μM) and NADH (1 mM) were added to free enzyme or enzyme-MNPs stored at 4 °C (never used before) and substrate hydroxylation was carried out under the conditions described in section 2.3.2. BM3 relative activity (ratio between the enzyme weekly activity and the enzyme initial activity) was calculated for every week.

3. RESULTS AND DISCUSSION 3.1. Ni2+-PMIDA-MNPs Characterization. The presence of PMIDA and Ni2+ on the surface of iron oxide nanoparticles was confirmed by FT-IR spectra. Figure 1 shows the spectra of Figure 2. Weight loss analysis from TG curves of Ni2+-PMIDA-Fe3O4.

Figure 1. Transmission FTIR spectra of functionalized MNPs before (black) and (blue) after enzyme immobilization.

Figure 3. X-ray diffraction (XRD) patterns for bare Fe3O4 nanoparticles (red) and functionalized Fe3O4 nanoparticles (black).

Ni2+-PMIDA-Fe3O4 before and after enzyme immobilization. A strong absorption band, at ∼577 cm−1, corresponds to Fe−O vibration correlated to the magnetic core. Before enzyme immobilization, the broad band at ∼3406 cm−1 belongs to −OH groups on the surface of MNPs.33,36 The IR peaks AT ∼1385 and 1609 cm−1 are attributed to PO and CO bonds, respectively.37 The typical vibration of 1725 cm−1 assigned to −CO2H confirms the successful conjugation of Ni2+ on the surface of MNPs.33 In the IR spectra after immobilization, the broad band at 1059 cm−1 indicates the C− N stretching vibration and the peaks at 3398 and 2926 cm−1 are assigned to N−H and C−H bonds in the enzyme structure, respectively.38 Stretching of CO related to the amide bands can be recognized by its vibrational band at 1650 cm−1.39 The TGA confirmed the presence of coating on the surface of MNPs. Figure 2 shows the weight loss of Ni2+-PMIDAFe3O4 by increasing the temperature. The weight loss between 50 °C and 150 °C was assigned to the loss of physically adsorbed water on the particles. The further loss of ∼8% up to 300 °C confirmed the decomposition of the adsorbed phosphonic coupling agent.33,36 The XRD patterns for bare and functionalized MNPs show the same characteristic peaks at 2θ = 30°, 35°, 43°, 53°, and 62° (see Figure 3). It indicates that the surface modification of iron oxide nanoparticles did not lead to a phase change. Furthermore, no impurity diffraction peaks were detected, indicating a pure phase of synthesized Ni2+-PMIDA-Fe3O4. The

mean crystallite size of particles, as estimated by applying Scherrer’s equation, was 16 nm. Magnetization curves of bare and coated MNPs are plotted in Figure 4. The saturation magnetization of bare and coated Fe3O4 were found to be 72.14 and 50.84 emu/g, respectively. The reduced remanence and zero coercivity confirm that the particles are superparamagnetic. In this state, after removing the

Figure 4. Magnetization curves of (a) bare and (b) functionalized MNPs. D


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Industrial & Engineering Chemistry Research external magnetic field, the particles can be easily dispersed by gentle shaking. The size distribution graph of functionalized particles is shown in Figure 5. The average size of the particles was found to be 78 nm.

C16H 23NO5 + O2 + H+ + NADH ↓ 10 ‐ p NCA

→

C6H5NO3

+ C10H18O3 + NAD+ + H 2O

↓

4‐nitrophenole (C6H 4NO− 3 : p‐nitrophenolate)

(2)

NADH and BNAH consumption by free and immobilized enzyme hydroxylating myristic acid are shown in Figures 6 and 7, respectively. As can be seen in Figure 6, NADH consumption

Figure 5. Dynamic light scattering (DLS) result for size distribution of functionalized MNPs.

3.2. BM3 Immobilization on Ni2+-PMIDA-MNPs. The CO absorbance at 450 nm before and after enzyme immobilization confirmed that the available enzyme in the solution (2 nmol ∼238 μg) was totally adsorbed on 40 mg of Ni2+-PMIDA-Fe3O4 particles (100% efficiency of immobilization). The immobilized enzyme (5.95 mg enzyme/g support) showed a stable bond to MNPs as no enzyme leakage was observed after washing three times with PBS. Türkmen et al.27 reported 40.1 mg/g cytochrome c adsorption on magnetic beads modified by Cu2+. In the present work, a maximum enzyme capacity of 28.5 mg/g for Ni2+-PMIDA-Fe3O4 particles was obtained by increasing the enzyme initial concentration to 2200 nM (43% efficiency of immobilization). Two types of interactions possibly provided the stable enzyme bonding on the synthesized particles: (i) attractive forces between negatively charged enzyme and positively charged metal chelated on MNPs and (ii) coordination bond between histidine and Ni2+ which has two places for the imidazole group of the histidine tail of the enzyme.26,40 The effect of the concentration of glutaraldehyde used to cross-link the adsorbed enzyme on MNPs surface was studied by measuring the enzyme activity and will be discussed in section 3.3. 3.3. Free and Immobilized BM3 Catalytic Activity. To investigate the activity of free and immobilized enzyme, reaction tests were performed using two substrates: myristic acid and 10-pNCA. For both myristic acid and 10-pNCA hydroxylation processes, the cofactors (NADH and BNAH) consumption was assessed. In the case of 10-pNCA, the concentration of product (p-nitrophenolate) was measured via a photometrical method. ω-Hydroxylated myristic acids were detected by gas chromatography−mass spectrometry (GC-MS) analysis. The stoichiometric equations of myristic acid (eq 1) and 10pNCA (eq 2) hydroxylation catalyzing by BM3 are given by:

Figure 6. NADH consumption by free and immobilized enzyme versus initial myristic acid concentration (reaction performed in 0.1 M PBS, pH 7.4).

range by the immobilized enzyme was close to that of free enzyme. In all these experiments, the enzyme in free or immobilized state consumed more than 90% of available NADH in the reaction medium. Compared to NADH, the enzyme in both forms, soluble or immobilized, oxidizes BNAH in a lower range (Figure 7). However, independently of the type of cofactor, the immobilization did not affect the cofactor consumption. GC-MS analysis also revealed that both free and immobilized enzyme produced ω-1 to ω-3 hydroxylated myristic acid (as an example, Figure 8 attests the ω-1 to ω-3 products formation by immobilized BM3).

C14 H 28O2 + O2 + H+ + NADH ↓ myristic acid

→

C14 H 28O3 ↓ ω‐(1−3) hydroxylated myristic acid

Figure 7. BNAH consumption by free and immobilized enzyme versus initial myristic acid concentration (reaction performed in 0.1 M Tris buffer, pH 8.2).

+ NAD+ + H 2O (1) E


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Figure 8. Chromatographic analysis attesting to the formation of ω-1, ω-2, and ω-3 products (reaction catalyzed by immobilized P450 BM3 hydroxylating myristic acid using NADH).

Figure 9. Cofactor consumption by free and immobilized enzyme (substrate concentrations: 150, 250, and 350 μM, using 1 mM cofactor in 0.1 M Tris buffer, pH 8.2).

Cofactor (NADH and BNAH) consumption and p-nitrophenolate production by free and immobilized enzyme are illustrated in Figures 9 and 10, respectively. As can be seen, the immobilized enzyme consumes the cofactor in the range of 600−700 μM for NADH and 550−650 μM for BNAH (approximately the same range of cofactor consumption by the free BM3). Increasing the initial concentration of 10-pNCA leads to an increase of the product concentration. This rise in yield (∼10%) from 250 μM to 350 μM is lower than the increase observed from 150 μM to 250 μM (∼51% increase in product concentration). For an initial substrate concentration of 350 μM, the highest product formation (112 μM) by free enzyme oxidizing NADH was observed, which was only decreased to 92 μM by the adsorbed enzyme. By using 0.25% (v/v) glutaraldehyde to cross-link the enzyme on MNPs, the product formation yield was found to be 99 μM (see Figure

11). Applying higher concentrations of the cross-linking agent (0.75% and 1.25% (v/v)) resulted in a decrease of product concentration to 68 and 67 μM for the same initial concentration of substrate. This outcome can be due to possible agglomeration of the enzyme on MNPs or covalent bonding of glutaraldehyde to the amino acids near the enzyme active site. By replacing NADH by BNAH, 78 μM of pnitrophenolate was produced after overnight hydroxylation of 350 μM 10-pNCA by free BM3. As BNAH is not a natural cofactor for the enzyme, this decrease in hydroxylation yield was expected. For the same initial concentration of substrate, 67 μM of p-nitrophenolate was obtained with the adsorbed enzyme, compared to 70 μM for the cross-linked-adsorbed enzyme using 0.25% (v/v) glutaraldehyde. By increasing the cross-linker concentration, the product formation was decreased to 44 and 38 for 0.75% and 1.25% (v/v) of F


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Figure 10. p-nitrophenolate production by free and immobilized enzyme (substrate concentrations: 150, 250, and 350 μM, using 1 mM cofactor in 0.1 M Tris buffer, pH 8.2).

Figure 12. Enzyme residual activity versus number of cycles for (□) adsorbed enzyme consuming NADH and (◇) adsorbed enzyme oxidizing BNAH (350 μM 10-pNCA in 0.1 M Tris buffer, pH 8.2). Figure 11. Effect of glutaraldehyde concentration on enzyme activity (350 μM 10-pNCA and 1 mM cofactor in 0.1 M Tris buffer, pH 8.2).

Table 1. Free and Immobilized Enzyme Yield; Reaction Performed in 0.1 M Tris Buffer, pH 8.2, with 10-pNCA (350 μM) as the Substrate and NADH/BNAH (1 mM) as a Cofactor

glutaraldehyde, respectively. Although the addition of glutaraldehyde affected the enzyme conversion yield of 10-pNCA, it did not show a significant effect on the cofactor consumption. Remarkably, compared to available works, in this study, we found the immobilized enzyme activity very close to its free counterpart. For example, Weber et al.10 reported 75% retained activity of BM3 toward 12-para-nitrophenoxycarboxylic acid (12-pNCA) after immobilization on SBA-15 and Maurer et al.12 found the remaining activity of entrapped BM3 in sol−gel to be 52% of the free enzyme’s activity toward 10-pNCA. 3.4. Immobilized BM3 Reuse. The immobilized BM3 residual activity, with respect to the number of catalytic reaction cycles, is presented in Figure 12. After five reaction cycles, the adsorbed enzyme retained 85% and 88% of its initial activity hydroxylating 10-pNCA using NADH and BNAH, respectively. After stabilizing the immobilized enzyme by glutaraldehyde (0.25% (v/v)) on the MNPs surface, the residual activity retrieves 100% after five reuses. Table 1 shows the yield (nmol of product/nmol of enzyme) of free and immobilized enzyme. Free enzyme initial yield is higher, compared to that of the immobilized enzyme. Nevertheless, enzyme immobilization allows its reuse for at least five cycles, by which the corresponding yield increases five times, in comparison with free enzyme. Moreover, the cross-linked-adsorbed enzyme

Yield (nmol product/nmol enzyme) Adsorbed Enzyme

Cross-Linked-Adsorbed Enzyme

cofactor

free enzyme

initial run

after five reaction cycles

initial run

after five reaction cycles

NADH BNAH

564.4 367.4

466.7 339.2

2697.8 1967.1

497.2 352.9

2983.2 2117.6

presents higher yield, compared to that of the adsorbed enzyme. 3.5. Storage Stability of Free and Immobilized BM3. Figure 13 shows the immobilized BM3 relative activity toward NADH, with respect to the storage time (days). The adsorbed enzyme lost its activity toward NADH faster than its crosslinked-adsorbed counterpart. Whereas the adsorbed enzyme was

Noncovalent Immobilization of Optimized Bacterial Cytochrome P450

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