Mediator Conjugates for Enhanced

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Construction of Enzyme-Cofactor/Mediator Conjugates for Enhanced in Vitro Bioelectricity Generation Haiyan Song, Chunling Ma, Wei Zhou, Chun You, Yi-Heng P. Job Zhang, and Zhiguang Zhu* Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 West Seventh Avenue, Tianjin Airport Economic Area, Tianjin 300308, China

Bioconjugate Chem. Downloaded from pubs.acs.org by TULANE UNIV on 12/03/18. For personal use only.

S Supporting Information *

ABSTRACT: Cofactor-dependent oxidoreduction and electron transfer play an important role in in vitro bioelectricity generation and many other enzyme biocatalysis reactions. To facilitate such electron generation and transfer, several approaches based on the coimmobilization of cofactors and oxidoreductases have been demonstrated. Herein, a convenient and immobilization-free approach of constructing enzyme−cofactor and enzyme−mediator conjugates was developed. The in vitro bioelectricity generation reactions via enzymatic fuel cells were evaluated. The cells equipped by the conjugates exhibited significantly improved power output and stability in contrast to those mediated by unconjugated enzymes. These results may bring a new avenue in constructing efficient in vitro electron transfer chains for various biocatalysis applications.

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quinone or nile blue-NAD+-modified electrodes,12,13 crosslinking NAD+ and glucose dehydrogenase (GDH) with chitosan and immobilizing as a whole,14 layer-by-layer adsorption of NAD+ and GDH via electrostatic interaction,15 trapping NAD+ into the micropores and GDH into the mesopores of hierarchical porous carbon electrodes,16 tethering NAD+ and two dehydrogenases with DNA scaffolds as swing arms,17 integrating enzymes and phosphorylated cofactors onto conductive polymers through ion-exchange interactions,18 and encapsulating enzymes and NAD+ in metal−organic framework nanoparticles.19 In many cases, electron mediators such as azines or quinones have been coupled with NAD+-dependent enzymes for expedited electron transfer due to their good mass transfer ability and low redox potential.20−22 Although many of these studies are enlightening, there are still concerns of reduced activity of immobilized enzymes and limited turnover of immobilized cofactors/ mediators in these systems. Besides, they require extra matrix for immobilization. PEGylation, with the remarkable therapeutic and biotechnological potential of peptides and proteins, is a process of attaching polyethylene glycol (PEG) chains to molecules of interest.23 PEGylated NAD(P) + derivatives have been developed for a long time to investigate cofactor regeneration for dehydrogenase-catalyzed reactions.24,25 For example,

INTRODUCTION In vitro bioelectricity generation can be implemented through enzymatic fuel cells (EFCs),1,2 in which the oxidoreductasecatalyzed reaction coupled with electron transfer to electrode plays a key role. With the advances in the synthesis of conductive polymers and mediators, enzyme engineering for enhanced catalytic performances, and the construction of nanomaterial-based electrodes, several EFCs relying on oxidoreductases with prosthetic groups have been reported, exhibiting high power outputs with fast direct electron transfer processes.3−5 Alternatively, oxidoreductases relying on exogenous cofactors such as nicotinamide adenine dinucleotide (NAD+) are more abundant in nature and have been intensively investigated in a plethora of EFCs.6,7 NAD+dependent enzymes, indeed, account for 80% of characterized oxidoreductases.8 Besides, some substrates such as xylose or glycerol can only be oxidized by their corresponding NAD+dependent dehydrogenases to generate electricity.9,10 Therefore, increasing the electron generation and transfer from a NAD+-mediated oxidoreductase to the electrode is of tremendous significance to the success of constructing highpower EFCs, as well as to other cofactor-dependent biocatalysis reactions.11 To facilitate cofactor-mediated electron generation and transfer, the coimmobilization of cofactors and oxidoreductases has been proposed, allowing fixed cofactors in the vicinity of enzymes without being released to the bulk and expedited cofactor regeneration by electrodes. Examples reported include affinity binding of dehydrogenases to the pyrroloquinoline © XXXX American Chemical Society

Received: October 23, 2018 Revised: November 19, 2018 Published: November 26, 2018 A

DOI: 10.1021/acs.bioconjchem.8b00766 Bioconjugate Chem. XXXX, XXX, XXX−XXX

Communication

Bioconjugate Chemistry

Scheme 1. Schematic of a Bioelectricity Generation Module Involving Unconjugated Dehydrogenases, DI, NAD+, and BV2+ or Their Conjugates

NAD+-PEG has been reported to covalently bind to GDH26 or malate dehydrogenase27 and form enzyme-PEG-NAD+ conjugates with dramatically accelerated reaction rates. Recently, Kim et al. has demonstrated a boosted biohydrogen production rate using NAD+-conjugated dehydrogenases.28 However, the preparation of enzyme-cofactor conjugates in these studies involves multisteps and are time-consuming and they have not been exploited pertaining to the bioelectricity generation. Herein, we report a convenient and immobilization-free approach to construct enzyme-cofactor/mediator conjugates, and demonstrate the benefit of the conjugation using a threeenzyme bioelectricity generation module. This module, as a key part of several enzymatic pathways, is of great importance to the complete oxidation of various sugar fuels in high-energydensity EFCs.29,30 The glucose 6-phosphate (G6P) substrate is oxidized by glucose-6-phosphate dehydrogenase (G6PDH) to generate 6-phosphogluconate (6PG) which is further oxidized by 6-phosphogluconate dehydrogenase (6PGDH) to ribulose 5-phosphate (Ru5P). Two NAD+ molecules participate in this conversion and transfer four electrons to the mediator benzyl viologen (BV2+) catalyzed by diaphorase (DI), and finally to the electrode of an EFC (Scheme 1). The redox potential of BV2+ is −359 mV (E0′ = −359 mV) which was close to that of NAD+ (E0′ = −320 mV), thus it has been demonstrated as an excellent mediator for NAD+ and DI.31 The three conjugates (G6PDH-NAD+, 6PGDH-NAD+, DI-BV2+) synthesized have been demonstrated for enhanced bioelectricity generation in EFCs in comparison with the unconjugated controls.

adopting a novel approach, in which 3-carboxyphenylboronic acid (CBA) was used as a bridge to link NAD+ and PEG molecules (Figure S2A). Previous studies have shown that CBA was an efficient linker to wire cofactors to dye-modified electrodes, and the immobilized cofactors could bind with cofactor-dependent dehydrogenases.13,33 In comparison, Ozbakir et al. explored a method of forming enzyme-PEG-NAD+ conjugates by using maleimide and carboxylic acid modified PEG to link with the amine of NAD+ and the cysteine of enzymes, in which the amide bond at the adenine moiety of NAD+ may cause significantly decreased cofactor activity;34 Kim et al. used a too complicated method to synthesize N6-(2carboxyethyl)-NAD + and then generated enzyme-NAD+ conjugates via diamino PEG.28 The measurement at UV 340 nm confirmed that the dehydrogenase-NAD+ conjugates were formed and the ratio of NAD+ to enzyme was approximately 1:1 (Figure S2B,C). In addition, the homologue structures of three enzymes were constructed in order to estimate the number of lysine with free amine groups on the surface of protein. It was found that DI may contain two surface lysines, while G6PDH and 6PGDH each had one (Figure S3), suggesting that the ratio of DI to BV2+ was 2:1 and dehydrogenase to NAD+ was 1:1. These values were close to our experimental results mentioned above. Additionally, the kinetics assay revealed that the catalytic turnover of the G6PDH or 6PGDH to NAD+-PEG was reduced to half but the km was relatively constant. This result is similar to that reported elsewhere.35 The kinetics of DI to CBV2+ did not significantly change (Table S1). Apparently, our presented method was convenient and relatively simple. Although some deactivation of the NAD+ was still observed, it was not as dramatic as the one in the approach based on the modification of the amine group in the adenine moiety of NAD+.34 To successfully construct such conjugates, it is noted that both the group chosen for conjugation and the PEG chain length can play important roles and should be carefully determined.36 Next, the synthesized conjugates were tested for bioelectricity generation in EFCs. All measurements were conducted in triplicate and the standard deviations were calculated. Within a three-electrode system (with a carbon cloth working electrode, a Ag/AgCl reference electrode and a Pt-wire counter electrode) containing 30 μM DI-BV2+ conjugates or 30 μM free DI and 90 μM free BV2+, amperometric experiments were employed to compare the conjugate and unconjugated enzymes and cofactors. After 3 mM NADH was added, the

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RESULTS AND DISCUSSION First, methods for synthesizing the conjugates were developed. 4-(Bromomethyl) phenylacetic acid was directly used to synthesize carboxyl-modified BV2+ (CBV2+), followed by an amide linkage formation between CBV2+ and DI to yield DIBV2+ (Figure S1A).32 This method was more convenient compared to the previous one by eliminating the participation of benzyl iodide,28 although the intermediate CBV2+ had two active carboxyl groups. The resulting DI-BV2+ was roughly examined by measuring the absorbance at 578 nm where the reduced BV2+ could be detected (Figure S1B). The relative molecular ratio of BV2+ moiety and DI enzyme in the conjugate was measured to be 3:1. On the other hand, dehydrogenase-PEG-NAD+ conjugates were constructed by B

DOI: 10.1021/acs.bioconjchem.8b00766 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

Figure 1. Current generation versus time in the system mediated by (A) DI and BV2+ or their conjugates; (B) G6PDH, NAD+, DI, BV2+, or their conjugates; (C) 6PGDH, NAD+, DI, BV2+, or their conjugates. (D) Power density versus current density from an EFC mediated by free G6PDH, 6PGDH, NAD+, DI, BV2+, or their conjugates. A potential of 0 V vs Ag/AgCl was employed in the amperometric experiments. A scan rate of 5 mV s−1 was used in the linear sweep voltammetric experiments. Red: conjugates, Black: their free forms.

system containing DI-BV2+ conjugates presented an increased current density of 35 ± 2 μA cm−2, while the system containing unconjugated DI and BV2+ only produced 25 ± 2 μA cm−2 (Figure 1A). A similar three-electrode system containing the same loading of DI and BV2+ as mentioned above was used to examine the effect of G6PDH-NAD+ or 6PGDH-NAD+ conjugate. The current density of the group with 10 μM conjugate can be increased to 0.04 ± 0.001 μA cm−2 or 0.18 ± 0.007 μA cm−2 after adding 3 mM G6P or 6PG (Figure 1B,C). In contrast, the control group without conjugation led to negligible current generation, possibly because the overall NAD+ concentration of 10 μM was too dilute to observe significant electricity generation. Additionally, an EFC employing this three-enzyme bioelectricity generation module on the anode and a platinum-catalyzed air-breathing cathode was assembled and characterized in terms of the power density. The linear sweep voltammetry was conducted at a scan rate of 5 mV s−1, and the corresponding power curves were drawn. The EFC mediated with 10 μM G6PDH-NAD+, 10 μM 6PGDH-NAD+, and 30 μM DI-BV2+ exhibited a power density of 0.05 ± 0.002 μW cm−2 at 3 mM G6P loading, while the one without conjugation generated almost no power if no exogenous cofactors were available (Figure 1D). This result indicates that the conjugation can promote a faster electron transfer between enzymes and cofactors or mediators, thus leading to the significantly increased current density and power density. Further, we increased the overall concentration of cofactors and substrates to examine the effect of conjugation at other conditions. Extra 2990 μM NAD+ was added into the reaction (3 mM overall NAD+), and we found that the G6PDH-NAD+ conjugate-mediated EFC yielded a current density or a power density 2- or 2.5-fold higher than that with unconjugated one

(Figure S4A,B). When the concentration of G6P was increased from 3 mM to 30 mM, the conjugate-mediated EFC still exhibited a power density 2-fold higher than that of unconjugated one (Figure S4C). Similarly, the 6PGDHNAD+ conjugate also presented the same results with even greater enhancement from the conjugated samples (Figure S4D−F). These findings suggest that enzyme−cofactor conjugates can effectively work even in the presence of a large amount of free unconjugated NAD+. To explain this, the enzyme kinetics assay of conjugated or unconjugated enzymes toward NAD+ or BV2+ was performed. It was found that the conjugates all had significantly lower km and similar kcat compared to native enzymes (Figure S5 and Table S1), indicating that at the same concentration of NAD+ or BV2+ in a certain range, the conjugation could lead to higher reaction rates. Moreover, we further calculated the relative concentration of conjugated NAD+ or BV2+ in the vicinity of enzymes by assuming the radius of the enzyme as 4.2, 3.5, and 2.4 nm for G6PDH, 6PGDH, and DI based on the estimation in Pymol, and the length of PEG linker as 5 nm (Figure S6A). Therefore, from the equation to estimate the relative volume of one enzyme-cofactor/mediator conjugate, we can obtain the relative concentration of NAD+ around G6PDH or 6PGDH conjugate as 0.50 or 0.66 mM. As a result, adding approximately 3 mM extra NAD+ can cause observable reaction rate difference between the dehydrogenase-NAD+ conjugates and unconjugated ones (Figure S5). The value for BV2+ around DI was estimated as 71 mM, representing highly concentrated BV2+ in the conjugate. In brief, we revealed the importance of the electron transfer distance and the relative concentration of the cofactor/mediator to the NAD+-dependent biocatalysis system. Notably, a recent study demonstrated the coupling of two NAD+-dependent enzymes C

DOI: 10.1021/acs.bioconjchem.8b00766 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

Figure 2. Power density versus current density from EFCs with all three conjugates (red), or with their all free forms (black). (A) The condition of 10 μM G6PDH-NAD+ or 6PGDH-NAD+, 30 μM DI-BV2+, 30 mM G6P, and 3 mM total NAD+ was used. (B) Carbon felt was used to replace carbon cloth as the anode. (C) A higher loading of 30 μM 6PGDH-NAD+, 60 μM DI-BV2+, 100 mM G6P was used. (D) The total concentration of NAD+ and BV2+ was increased to 10 mM respectively. A scan rate of 5 mV s−1 was used.

using the polymeric NAD+−polymer cofactor in metal− organic framework nanoparticles, and also exhibited that the confinement of the multienzyme cascade generated several-fold enhancements in the reaction rate compared to the diffusional system.19 In order to achieve high power densities of the EFCs, we further optimized other parameters. Under the condition of 10 μM G6PDH-NAD+ or 6PGDH-NAD+, 30 μM DI-BV2+, 30 mM G6P, and 3 mM total NAD+, the EFC equipped with conjugated enzymes produced a power density of 60 ± 4 μW cm−2 while that without conjugation generated 18 ± 0.4 μW cm−2 (Figure 2A), which was in accordance with that observed from Figure S2, a 2-fold increase in power density when conjugated. The power output of this two-dehydrogenasemediated EFC was nearly twice that of one-dehydrogenasemediated EFC, similar to a previous result claiming that the power density of EFCs was increased gradually with the increase in the number of dehydrogenases.10 To further boost the power, carbon felt anodes with a high surface area were used to replace the carbon cloth used for the above experiments. Consequently, the power densities were increased to 222 ± 13 μW cm−2 and 151 ± 8 μW cm−2 for conjugated and unconjugated enzyme-mediated EFCs (Figure 2B). The enhancement by using conjugates was reduced from 200% to ∼35%, possibly because more accessible electrode surface in the carbon felt anode had positive effects on the mass transfer of unconjugated enzymes and cofactors. With a further increased loading of 30 μM 6PGDH-NAD+, 60 μM DI-BV2+, 100 mM G6P, the power densities were increased to approximately 700 ± 31 and 400 ± 28 μW cm−2 for conjugated and unconjugated samples (Figure 2C). In

addition, when more NAD+ and BV2+ was supplemented to 10 mM, the two EFCs exhibited power densities of 822 ± 39 and 800 ± 55 μW cm−2 respectively (Figure 2D), suggesting that at high loadings of cofactors and mediators, the rate enhancement effect from the conjugation was diminished. Finally, at a higher temperature of 50 °C, with overall 10 mM NAD+ and BV2+, the power densities of these two EFCs were increased to 998 ± 62 μW cm−2 and 902 ± 51 μW cm−2, respectively (Figure S7). Our result is in accordance with the hypothesis based on the relative concentration of the cofactors and Brownian collision theory, that the more overall cofactors or mediators added, the lower the benefit from conjugation (Figure S6B). However, using a high concentration of valuable cofactors is not economically feasible for practical applications. Therefore, our conjugation strategy may allow the reduced usage of cofactors while maintaining a high reaction rate. Additionally, in comparison with other enzyme−cofactor coimmobilization approaches for bioelectricity generation, our result is comparable with that relying on NAD+, mediator, and GDH immobilized in hierarchical porous carbon electrodes;16 superior to that using cofactors and GDH covalently wired on dye-modified nanotube electrodes;13 but slightly lower than that based on layer-by-layer adsorption of NAD+, mediator, GDH, and DI onto electrodes.15 This would indicate that our scenario of forming enzyme−cofactor/mediator conjugates may be helpful to the construction of high-power EFCs. Besides the power, the durability of EFCs is also essential to the practical applications. However, cofactors including NAD+ are usually unstable, especially at evaluated temperatures. By forming enzyme−cofactor conjugates, the NAD+ moiety was D

DOI: 10.1021/acs.bioconjchem.8b00766 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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Bioconjugate Chemistry

Figure 3. (A) Current density versus time from EFCs at 50 °C without adding extra NAD+ and BV2+ (with 40 μM NAD+ and 180 μM BV2+ in total), and with 0.1 mM G6P supplemented each cycle. (B) Reusability of EFCs running under the above-mentioned condition and stored at 23 °C when not in use. Red: conjugates, Black: their free forms.

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expected to be more stable as reported previously.25,26 As we found, when no extra NAD+ and BV2+ was added (40 μM NAD+ and 180 μM BV2+ in total), at 50 °C the EFC with conjugates exhibited the repeated current peaks for each addition of 0.1 mM G6P substrate and its production continued for more than 6 cycles (Figure 3A). As a control, the one without conjugation generated much less current and could only last for less than 4 cycles. Additionally, the reusability of EFCs under the above-mentioned condition was also evaluated. EFCs with 0.1 mM G6P substrate were examined to obtain different current densities once per day and stored at 23 °C when not in use. It clearly shows that the conjugation can maintain ∼50% of the initial performance after 7 days, while the unconjugated controls lose ∼50% of the initial performance after 3 days (Figure 3B). The performance of both EFCs began to decline with time possibly caused by the decay of cofactors, since the concentration of the cofactor/ mediator added was low. These results underline the excellent operating stability of the enzyme−cofactor/mediator conjugate-mediated EFCs. In conclusion, a convenient and immobilization-free approach was developed to construct enzyme-NAD+ and enzyme-BV2+ conjugates for enhanced in vitro bioelectricity generation. The conjugates formed could lead to a multifold increase in current density and power density and much improved stability of the fuel cell under the condition of low cofactor/mediator concentration. Compared with other immobilization-based cofactor-dependent bioelectricity generation systems, our strategy not only avoids cofactor leaching or deactivating due to immobilization, but also enables designable enzyme−cofactor distance and configuration. In the future, further efforts will be focused on immobilizing these conjugates onto electrodes for better reusability and exploring more suitable biomimetic cofactors and mediators for conjugation. It is anticipated that this enzyme−cofactor/mediator conjugation strategy will impact a large number of cofactor-dependent bioelectricity generation as well as other biocatalysis systems.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: (+86)-022-2482 8797. Fax: (+86)-022-8486 1926. ORCID

Yi-Heng P. Job Zhang: 0000-0002-4010-2250 Zhiguang Zhu: 0000-0002-6625-5087 Author Contributions

H. S., Y. Z., and Z. Z. conceived the idea and designed the experiments. H. S., C. M., and W. Z. conducted the experiments. H. S., C. Y., and Z. Z. wrote the manuscript. All authors discussed the results and commented on the manuscript. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences (ZDRW-ZS2016-3S), the National Natural Science Foundation of China (21706273, 21878324), and the CAS Pioneer Hundred Talent Program (Type C, reference # 2016-081).

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.8b00766. Materials and methods and supplementary figures and tables (PDF) E

DOI: 10.1021/acs.bioconjchem.8b00766 Bioconjugate Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.bioconjchem.8b00766 Bioconjugate Chem. XXXX, XXX, XXX−XXX