Solar light-Driven photocatalyst-enzyme attached artificial photosynthetic system for regeneration and production of 1,4-NADH and L-glutamate

Cite this paper: Vietnam J. Chem., 2021, 59(2), 198-202 Article 
DOI: 10.1002/vjch.202000147 
198 Wiley Online Library © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH 
Solar light-driven photocatalyst-enzyme attached artificial 
photosynthetic system for regeneration and production of 
1,4-NADH and L-glutamate 
Chandani Singh
1
, Abhishek Kumar
2
, Rajesh K. Yadav
1*
, Vitthal L. Gole
3
, D. K. Dwivedi
4 
1
Department of Chemistry and Environmental Science, Madan Mohan Malaviya University of 
Technology, Gorakhpur-273010, U.P., India 
2
Department of Chemistry, Indian Institute of Science, BHU, Varanasi-221005, U. P., India 
3
Department of Chemical Engineering, Madan Mohan Malaviya University of Technology, Gorakhpur-
273010, U.P., India 
4
Department of Physics and Material Science, Madan Mohan Malaviya University of Technology, 
Gorakhpur-273010, U.P., India 
Submitted August 28, 2020; Accepted November 18, 2020 
Abstract 
In artificial photosynthesis process the photocatalyst-enzyme attached system is a best strategy to utilize solar 
energy for solar chemical/fuels production. Herein, we prepared a solar light active graphene-based photocatalyst 
received by the covalent attachment of 9-aminoanthracene (AA) chromophore with chemically converted graphene 
(CCG) for highly efficient 1,4-NADH regeneration (79.9 %) and conversion of α-ketoglutarate (α-KG) in to L-
glutamate (L-GM) (88.3 %) in 2 hrs. The present result is a benchmark example of highly selective solar light active 
enzyme based artificial photosynthetic system for selective formation of L-GM from α-KG. 
Keywords. CCG-AA Photocatalyst, electron microscopy, L-GM, regeneration of 1,4-NADH, photocatalysis. 
1. INTRODUCTION 
It is a well-known scientific fact that natural 
photosynthesis is an efficient process for converting 
CO2 into useful chemicals such as glucose.
[1-6] 
Therefore, many efforts have been made thus far to 
obtain artificial photosynthetic systems that can 
mimic the natural process to afford a variety of 
useful chemicals or fuels for human consumption.
[7-
9] 
Therefore an integrated platform that facilitates 
electron transfer from the covalently attached light-
harvesting parts and biocatalyst for producing 
desired products using solar light is an important 
strategy. In natural photosynthesis solar light is 
harvested by photosystem I and photosystem II 
which are composed of green pigments (scheme 
1a).
[10] 
Hence a variety of solar light harvesting 
organic and inorganic materials and metal 
complexes as photocatalysts have been used for 
multi-electron transfer in artificial photosynthetic 
system.
[11-13]
 However poor electron transfer 
efficiency from photocatalyst to biocatalyst has so 
far impeded research in this area.
[14,15]
 In this context 
graphene is an attractive option due to its well-
known high electron mobility and transfer rates. 
The covalent coupling of a light harvesting 
chromophore to graphene has been found to be a 
promising approach to obtain highly efficient 
photocatalysts for the desired photocatalyst-
biocatalyst coupled systems for solar fuel/chemical 
production. Herein, we report the successful 
development of solar light active chemically 
converted graphene (CCG) coupled to 9-
aminoanthracene (AA) photocatalyst. This visible 
light-harvesting CCG-AA photocatalyst was 
synthesized by covalent attachment of AA 
chromophore to CCG using the diazonium 
chemistry.
[16]
 In CCG-AA photocatalyst, AA acts as 
a light harvesting unit due to high molar extinction 
coefficient. Therefore the covalently attached visible 
light harvesting CCG-AA photocatalyst was 
introduced for transferring excited multi-electron to 
the reaction mediator, methyl viologen (M), that 
further leads to1,4-NADH regeneration (79.9 %). 
The 1,4-NADH regenerated was then utilized for the 
production of L-GM (88.3 %) from α-KG by using 
Vietnam Journal of Chemistry Rajesh K. Yadav et al. 
 © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 199 
glutamate dehydrogenase enzyme (GDHE). Scheme 
1b represents the photocatalyst-enzyme attached 
artificial photosynthetic system involved in the solar 
chemicals production such as L-GM under the 
irradiation of solar light. The solar light harvesting 
AA (electron donor) transfers electron to CCG 
(acceptor).
[17] 
The CCG then transfers electrons 
efficiently to reduce M. Upon reduction, M 
transfered proton and electrons for the reduction of 
NAD
+
 to 1,4-NADH cofactor. In this manner, M 
acts as a reaction mediator between CCG-AA 
photocatalyst and NAD
+
. Finally, the electrons and 
protons of 1,4-NADH are used for the exclusive 
production of L-GM with the help of GDHE. 
Scheme 1: Schematic representation of (a) natural photosynthesis which includes many electron mediator for 
the production of glucose and oxygen (b) artificial photosynthetic system using CCG-AA photocatalyst for 
carrying out L-GM production from α-KG 
2. MATERIALS AND METHODS 
2.1. Synthesis of CCG-AA photocatalyst 
The CCG-AA photocatalyst was synthesized by 
reported litrature method (see in supporting 
information figure S1).
[16] 
250 mL distilled water 
solution of CCG (1 g) was prepared in 1L round-
bottom flask (RB). The suspension was 
ultrasonicated for 1 h, 5 mL hydrazine hydrate (50 
%) was added and using ammonia solution to 
maintain pH upto 11. The resultant mixture was 
stirred at 90 
o
C for 1 h. Subsequently, AA (2.34 g) 
and isoamyl nitrite (3 mL) were added, the mixture 
was stirred vigorously overnight at 80 
o
C and then 
cooled down to room temperature. The solution was 
filtered through a membrane filter paper (0.2 mm). 
The resultant CCG-AA cake was washed with 
distilled water and dil. HCl until a clear solution was 
obtained. The resultant black solid material was 
dried under vacuum oven to receive CCG-AA in 80 
% yield. 
2.2. Photochemical 1,4-NADH regeneration 
Solar light driven artificial photosynthetic system for 
1,4-NADH regeneration was executed in a quartz 
reactor at ambient temperature by using 450W 
halogen lamp (artificial light source) equipped with 
cut-off filter (420 nm). In the above reactor 0.7 mg 
CCG-AA photocatalyst, 0.62 μmol methyl viologen 
(M), 1.24 μmol NAD+ and 1.24 mmol AsA were 
dispersed in buffer solution (2.3 ml, pH 7.0). The 
1,4-NADH regeneration was monitored at 340 nm 
by UV-Visible spectrophotometer.
[18,19]
2.3. Solar light responsive artificial 
photosynthesis of L-GM from α-KG 
The production of L-GM from α-KG also carried out 
in quartz reactor in presence of 450W halogen lamp 
(artificial light source) along with cut-off filter (420 
nm) under solar light irradiation at ambient 
temperature. For synthesis of L-GM, reaction 
solution was prepared by using 0.7 mg CCG-AA, 
0.62 μmol M, 1.24 μmol NAD+, 30 units GDH, α-10 
mmol ketoglutarate, 100 mM (NH4)2SO4 and 1.24 
mmol AsA dispersed in buffer solution (2.3 ml, pH 
7.0). The yield of L-GM was carried out by using 
Vietnam Journal of Chemistry Solar light-driven photocatalyst-enzyme... 
 © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 200 
high performance liquid chromatography (Agilent 
Technologies, USA).
[20,21]
3. RESULTS AND DISCUSSION 
3.1. Characterization 
We investigated the UV-Visible spectra of CCG and 
CCG-AA photocatalyst in DMF (figure 1a). The 
CCG-AA photocatalyst exhibited broad absorption 
band from 425 to 510 nm. It is clearly suggested 
covalent attachment of AA chromophore to CCG by 
diazonium chemistry.
The broad absorption (figure 
1a) indicated that CCG-AA photocatalyst has highly 
efficient visible light harvesting ability for the 
regeneration of 1,4-NADH and the production of 
L-GM from α-KG. 
The functionalization of CCG with AA was 
established by FTIR spectroscopy (figure 1b). In 
FTIR spectra of CCG, absorption peak at 1712 cm
-1 
was assigned to stretching (str) mode of C=O 
(carboxyl functional group), whereas the peaks at 
1622 cm
-1 
and 1422-1100 cm
-1 
were revealed to the 
C=C str and C-O str of carboxylic acid (-COOH), 
respectively.
[22,23]
 The intensities and positions of 
peaks changed significantly in the CCG-AA 
photocatalyst due to covalent attachment of AA to 
CCG. The appearance of a new peak at 1640 cm
-1 
in 
case of CCG-AA photocatalyst is due to the 
presence of -C-C- stretching of the aromatic ring 
further indicated covalent bond formation between 
CCG and AA chromophore.
[24,25] 
The functionalization of CCG with AA was also 
confirmed by Zeta-potential (ζ). As the ζ value of 
CCG-AA photocatalyst was found to be more 
negative (-43.6 mV) than CCG (-34 mV) (see in
supporting information figure S2).
[26,27]
Figure 1: (a) UV-Visible spectra of CCG (red) and 
CCG-AA photocatalyst (blue). (b) FTIR spectra of 
CCG (red) and CCG-AA photocatalyst (blue) 
The field emission scanning electron microscopy 
(FESEM) images of CCG and CCG-AA 
photocatalyst are shown in figure 2. The FESEM 
image of CCG showed wrinkled cloth like 
morphology (figure 2a),
[28-30] 
but after covalent 
attachment of CCG with AA, a clearly observable 
change in morphology was detected, as shown in 
figure 2b.
[31,32] 
Figure 2: FESEM images of (a) CCG and (b) CCG-AA photocatalyst 
3.2. Regeneration and production of 1,4-NADH 
and L-GM 
The photocatalytic activities of CCG, AA, and CCG-
AA are shown in figure 3. The 1,4-NADH 
regeneration activity was monitored by UV-Vis 
spectrophotometry.
[25]
 The 1,4-NADH regeneration 
of 79.85 %, 13.94 % and 0 %, for CCG-AA, AA and 
Vietnam Journal of Chemistry Rajesh K. Yadav et al. 
 © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 201 
CCG respectively, were obtained under visible light 
irradiation of 2 h (figure 3a). Similarly, L-GM 
production of 88.3 %, 17.5 % and 0 %, was achieved 
by CCG-AA, AA and CCG photocatalysts 
respectively, under visible light irradiation of 2 h 
(figure 3b). No other product was detected which 
indicated that the conversion proceeded in a highly 
selective manner. The higher yields of 1, 4-NADH 
and L-GM by the use of solar light harvesting CCG-
AA photocatalyst can be clearly attributed to 
improved carrier mobility.
[33] 
Figure 3: Photocatalytic activities of CCG (green), 
AA (red) and CCG-AA photocatalyst (blue) for (a) 
1,4-NADH regeneration and (b) L-GM production 
from α-KG 
4. CONCLUSION 
In summary, we have fruitfully synthesized a 
graphene based solar light active photocatalyst 
(CCG-AA) which carried out 79.9 % 1,4-NADH 
regeneration and coupling to GDHE led to 88.3 % 
L-GM production from α-KG. The photocatalyst-
enzyme coupled system is one of the most 
challenging tasks for highly selective solar 
chemical/fuels production via the route of 
mimicking natural photosynthesis process. Thus, the 
present work successfully demonstrates that a 
designed graphene based photocatalyst (CCG-AA)-
enzyme coupled system is one of the best systems to 
realize the ultimate goal of solar energy utilization 
for tailor-made synthesis of solar chemical such as 
L-GM. 
Acknowledgements. This work was supported by 
Madan Mohan Malaviya University of Technology 
(MMMUT), Gorakhpur, U.P., India. 
REFERENCES 
1. D. Gust, T. A. Moore, A. L. Moore. Solar fuels via 
artificial photosynthesis, Acc. Chem. Res., 2009, 42, 
1890-1898. 
2. A. Magnuson, M. Anderlund, O. Johansson, P. 
Lindblad, R. Lomoth, T. Polivka, S. Ott, K. Stensjo, 
S. Styring, V. Sundstrom, L. Hammarstrom. 
Biomimetic and microbial approaches to solar fuel 
generation, Acc. Chem. Res., 2009, 42, 1899-1909. 
3. A. Taglieber, F. Schulz, F. Hollmann, M. Rusek, M. 
T. Reetz. Light-driven biocatalytic oxidation and 
reduction reactions: scope and limitations, 
ChemBioChem., 2008, 9, 565-572. 
4. C. Lloyd, J. Chan. Microtubules and the shape of 
plants to come, Nat. Rev. Mol. Cell Biol., 2004, 5, 13-
22. 
5. F. B. P. Wooding, D. H. Northcote. The fine structure 
and development of the companion cell of the 
phloem of Acer pseudoplatanus, J. Cell Biol., 1965, 
24, 117-128. 
6. J. T. Greenberg.Programmed cell death: a way of life 
for plants, Proc. Natl. Acad. Sci. U. S. A, 1996, 93, 
12094-12097. 
7. C. W. Hoganson, G. T. Babcock. A metalloradical 
mechanism for the generation of oxygen from water 
in photosynthesis, Science, 1997, 277, 1953-1956. 
8. S. H. Lee, D. H. Nam, J. H. Kim, J.-O. Baeg, C. B. 
Park. Eosin Y‐sensitized artificial photosynthesis by 
highly efficient visible‐light‐driven regeneration of 
nicotinamide cofactor, ChemBioChem., 2009, 10, 
1621-1624. 
9. J. Ryu, S. H. Lee, D. H. Nam, C. B. Park. Rational 
design and engineering of quantum-dot-sensitized 
TiO₂ nanotube arrays for artificial photosynthesis, 
Adv. Mater., 2011, 23, 1883-1888. 
10. J. H. Kim, D. H. Nam, C. B. Park. Nanobiocatalytic 
assemblies for artificial photosynthesis, Curr. Opin. 
Biotechnol., 2014, 28, 1-9. 
11. D. Wang, S. H. Lee, S. Han, J. Kim, N. Trang, K. 
Kim, E-G. Choi, P. Boonmongkolras, Y. W. Lee, B. 
Shin, Y. H. Kim, C. B. Park. Lignin-fueled 
photoelectrochemical platform for light-driven redox 
biotransformation, Green Chem., 2020, 22, 5151-
5160. 
12. J. Kim, Y. W. Lee, E.-G. Choi, P. Boonmongkolras, 
S. T. Kim, S. K. Kuk, Y. H. Kim, B. Shin, C. B. Park. 
Robust FeOOH/BiVO4/Cu(In,Ga)Se2 tandem 
structure for solar-powered biocatalytic CO2 
reduction, J. Mater. Chem. A, 2020, 8, 8496-8502. 
13. E. J. Son, Y. W. Lee, J. W. Ko, C. B. Park. 
Amorphous carbon nitride as a robust photocatalyst 
Vietnam Journal of Chemistry Solar light-driven photocatalyst-enzyme... 
 © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 202 
for biocatalytic solar-to-chemical conversion, 
ACS Sustain. Chem. Eng., 2019, 7, 2545-2552. 
14. D. H. Nam, C. B. Park. Visible light-driven NADH 
regeneration sensitized by proflavine for biocatalysis, 
ChemBioChem, 2012, 13, 1278-1282. 
15. S. Kumar, R. K. Yadav, K. Ram, A. Aguiar, J. Koh, 
A. J.F.N. Sobral. Graphene oxide modified cobalt 
metallated porphyrin photocatalyst for conversion of 
formic acid from carbon dioxide, J. CO2 Util., 2018, 
27, 107-114. 
16. A. Wang, W. Yu, Z. Huang, F. Zhou, J. Song, Y. 
Song, L. Long, M. P. Cifuentes, M. G. Humphrey, L. 
Zhang, J. Shao, C. Zhang. Covalent functionalization 
of reduced graphene oxide with porphyrin by means 
of diazonium chemistry for nonlinear optical 
performance, Sci. Rep., 2016, 6, 23325. 
17. H. Wu, C. Tian, X. Song, C. Liu, D. Yang, Z. Jiang. 
Methods for the regeneration of nicotinamidecoenzymes, 
Green Chem., 2013, 15, 1773-1789. 
18. Y. Maenaka, T. Suenobu, S. Fukuzumi. Efficient 
catalytic interconversion between NADH and 
NAD
+
 accompanied by generation and consumption 
of hydrogen with a water-soluble iridium complex at 
ambient pressure and temperature, J. Am. Chem. Soc., 
2012, 134, 367-374. 
19. J. H. Kim, M. Lee, J. S. Lee, C. B. Park. 
Self‐assembled light‐harvesting peptide nanotubes 
for mimicking natural photosynthesis, Angew. Chem. 
Int. Ed., 2012, 51, 517-520. 
20. J.W. Ko, W. S. Choi, J. Kim, S. K. Kuk, S. H. Lee, 
C. B. Park. Self-assembled peptide-carbon nitride 
hydrogel as a light-responsive scaffold material, 
Biomacromolecules, 2017, 18, 3551-3556. 
21. X. F. Li, J. Zhang, L. H. Shen, Y. M. Ma, W. W. Lei, 
Q. Cui, G. T. Zou. Preparation and characterization 
of graphitic carbon nitride through pyrolysis of 
melamine, Appl. Phys. A-Mater., 2009, 94, 387-392. 
22. R. K. Yadav, J-O. Baeg, G. H. Oh, N-J. Park, K-j. 
Kong, J. Kim, D. W. Hwang, S. K. Biswas. A 
Photocatalyst-enzyme coupled artificial 
photosynthesis system for solar energy in production 
of formic acid from CO2, J. Am. Chem. Soc., 2012, 
134, 11455-11461. 
23. S. Kashyap, S. Mishra, S. K. Behera. Aqueous 
colloidal stability of graphene oxide and chemically 
converted graphene, J. Nanoparticles, 2014, Article 
ID 640281, 6 pages. 
24. V. Georgakilas, M. Otyepka, A. B. Bourlinos, V. 
Chandra, N. Kim, K. C. Kemp, P. Hobza, R. Zboril, 
K. S. Kim. Functionalization of graphene: Covalent 
and non-covalent approaches, derivatives and 
applications, Chem. Rev., 2012, 112, 6156-6214. 
25. R. K. Yadav, J. O. Baeg, A. Kumar, K. J. Kong, G. 
H. Oh, N. J. Park. Graphene-BODIPY as a 
photocatalyst in the photocatalytic-biocatalytic 
coupled system for solar fuel production from CO2, J. 
Mater. Chem. A, 2014, 2, 5068-5076. 
26. S. Gambhir, E. Murray, S. Sayyar, G. G. Wallace, D. 
L. Officer. Anhydrous organic dispersions of highly 
reduced chemically converted graphene, Carbon, 
2014, 76, 368-377. 
27. F. D. Souza, S. Gadde, M. E. Zandler, K. Arkady, M. 
E. El-Khouly, M. Fujitsuka, O. Ito. Studies on 
covalently linked porphyrin-C60 dyads: Stabilization 
of charge-separated states by axial coordination, J. 
Phys. Chem. A, 2002, 106, 12393-12404. 
28. C. Santhosh, R. Nivetha, P. Kollu, V. Srivastava, M. 
Sillanpaa, A. N. Grace, A. Bhatnagar. Removal of 
cationic and anionic heavy metals from water by 1D 
and 2D-carbon structures decorated with magnetic 
nanoparticles, Sci. Rep., 2017, 7, 14107. 
29. S. Peng, J. Liu, J. Zhang, F. Wang. An improved 
preparation of graphene supported ultrafine 
ruthenium (0) NPs: Very active and durable catalysts 
for H2 generation from methanolysis of ammonia 
borane, Int. J. Hydrog. Energy, 2015, 40, 10856-
10866. 
30. H. M. A. Hassan, V. Abdelsayed, A. E. R. S. Khder, 
K. M. AbouZeid, J. Terner, M. S. El-Shall, S. I. Al-
Resayes, A. A. El-Azhary. Microwave synthesis of 
graphene sheets supporting metal nanocrystals in 
aqueous and organic media, J. Mater. Chem., 2009, 
19, 3832-3837. 
31. J. Park, T. Jin, C. Liu, G. Li, M. Yan. Three-
dimensional graphene-TiO2 nanocomposite 
photocatalyst synthesized by covalent attachment, 
ACS Omega, 2016, 1, 351-356. 
32. M. Supur, Y. Kawashima, K. Mase, K. Ohkubo, T. 
Hasobe, S. Fukuzumi. Broadband light harvesting 
and fast charge separation in ordered self-assemblies 
of electron donor-acceptor-functionalized graphene 
oxide layers for effective solar energy conversion, J. 
Phys. Chem. C, 2015, 11924, 13488-13495. 
33. P. M. Navajas, N. G. Asenjo, R. Santamaria, R. 
Menendez, A. Corma, H. Garcia. Surface area 
measurement of graphene oxide in aqueous solutions, 
Langmuir, 2013, 29, 13443-13448. 
Corresponding author: Rajesh K. Yadav 
Department of Chemistry and Environmental Science 
Madan Mohan Malaviya University of Technology 
Gorakhpur-273010, U.P., India 
E-mail: rkyas@mmmut.ac.in.