Satyadip
Paul
ab,
Mani
Gupta
c,
Kaushik
Dey
ab,
Ashok Kumar
Mahato
ab,
Saikat
Bag
ab,
Arun
Torris
d,
E. Bhoje
Gowd
e,
Hasnain
Sajid
f,
Matthew A.
Addicoat
f,
Supratim
Datta
*c and
Rahul
Banerjee
*ab
aDepartment of Chemical Sciences, Indian Institute of Science Education and Research, Mohanpur, Kolkata, 741246, India
bCentre for Advanced Functional Materials, Indian Institute of Science Education and Research, Mohanpur, Kolkata, 741246, India. E-mail: r.banerjee@iiserkol.ac.in
cDepartment of Biological Sciences, Center for the Climate and Environmental Sciences, Indian Institute of Science Education and Research Kolkata, Mohanpur 741246, India. E-mail: supratim@iiserkol.ac.in
dPolymer Science and Engineering Division, CSIR-National Chemical Laboratory, Dr Homi Bhabha Road, Pune 411008, India
eMaterials Science and Technology Division, CSIR-National Institute for Interdisciplinary Science and Technology, Trivandrum 695 019, Kerala, India
fSchool of Science and Technology, Nottingham Trent University, NG11 8NS Nottingham, UK
First published on 26th May 2023
Covalent organic frameworks (COFs) are ideal host matrices for biomolecule immobilization and biocatalysis due to their high porosity, various functionalities, and structural robustness. However, the porosity of COFs is limited to the micropore dimension, which restricts the immobilization of enzymes with large volumes and obstructs substrate flow during enzyme catalysis. A hierarchical 3D nanostructure possessing micro-, meso-, and macroporosity could be a beneficial host matrix for such enzyme catalysis. In this study, we employed an in situ CO2 gas effervescence technique to induce disordered macropores in the ordered 2D COF nanostructure, synthesizing hierarchical TpAzo COF-foam. The resulting TpAzo foam matrix facilitates the immobilization of multiple enzymes with higher immobilization efficiency (approximately 1.5 to 4-fold) than the COF. The immobilized cellulolytic enzymes, namely β-glucosidase (BGL), cellobiohydrolase (CBH), and endoglucanase (EG), remain active inside the TpAzo foam. The immobilized BGL exhibited activity in organic solvents and stability at room temperature (25 °C). The enzyme-immobilized TpAzo foam exhibited significant activity towards the hydrolysis of p-nitrophenyl-β-D-glucopyranoside (BGL@TpAzo-foam: Km and Vmax = 23.5 ± 3.5 mM and 497.7 ± 28.0 μM min−1) and carboxymethylcellulose (CBH@TpAzo-foam: Km and Vmax = 18.3 ± 4.0 mg mL−1 and 85.2 ± 9.6 μM min−1 and EG@TpAzo-foam: Km and Vmax = 13.2 ± 2.0 mg mL−1 and 102.2 ± 7.1 μM min−1). Subsequently, the multi-enzyme immobilized TpAzo foams were utilized to perform a one-pot tandem conversion from carboxymethylcellulose (CMC) to glucose with high recyclability (10 cycles). This work opens up the possibility of synthesizing enzymes immobilized in TpAzo foam for tandem catalysis.
The crystallinity of the TpAzo foam was confirmed through powder X-ray diffraction (PXRD), which matched well with the simulated PXRD of the pristine TpAzo COF. The peak observed at 3.3° (2θ) corresponds to a reflection from (100) planes, while the broad peak at a higher angle (2θ = 26.7°) is attributed to the reflections from (001) planes. This also signifies the π–π interaction between the COF layers in the foam matrix (Fig. 2a). To check the microporosity of TpAzo foam, we measured the N2 adsorption isotherm at 77 K, which showed a Type-I adsorption isotherm. The BET surface area and total pore volume of TpAzo foam were found to be 850 m2 g−1 and 0.594 cm3 g−1, respectively. The pore size distribution of the TpAzo foam was checked using non-local density functional theory (NLDFT), indicating an average pore size distribution centered at 2 nm (Fig. 2b and S16†). FT-IR spectra revealed intense peaks at 1619 (CO), 1567 (CC), and 1228 cm−1 (C–N), confirming the β-ketoenamine framework of the foam matrix (Fig. S17†). X-ray micro-computed tomography (micro-CT) was performed to reveal the 3D macrostructure of the TpAzo foam with interconnected pores (Fig. 2g). Within the macro-structure, we were able to isolate the three-dimensional assembly of disordered macropores (5–200 μm) and ordered lamellar COF sheets (up to 200 μm) (Fig. S19 and S20†). The void volume of the foam is approximately 75%. We could control the overall macropore size distribution (up to 400 μm) by controlling the amount of sodium bicarbonate addition. As the macropores were generated stepwise, the pore size distribution exhibited a broad range with a higher volume fraction (Fig. 2h and S21†).
We performed mass transport simulation in the 3D micro-porous structure to predict the dynamic events occurring during the entrapment of enzymes. A numerical simulator computed digital water flow experiments in all three directions (x, y, and z-axis), using an appropriate solver at 25 °C and 20 Pa pressure drop. The outcome visualized the average flow velocity profiles. The numerical simulation results predict the feasible average water flow profiles through the interconnected pore structure of the foam. The average water flow velocities in the x, y, and z directions were 6.10, 4.62, and 4.89 × 10−3 m s−1, respectively. The significant enhancement of momentum in the x-axis relative to other axes may be attributed to receptivity to flow exerted by the pore walls aligned normal to the x-axis. A higher population of flow velocity streamlines in the x-axis also supports this observation. The results also signify the interconnectivity of the pores in the foam, which should facilitate the transport of enzymes along the x-axis (Fig. S22†).
To investigate the immobilization of enzymes or proteins in TpAzo foam, we used bovine serum albumin (BSA) as a model protein. We determined the loading percentage to be 0.09 mg of BSA per 1 mg of TpAzo foam using UV-vis spectroscopy (Fig. S3†). We rinsed the solid foam 10–15 times with water to remove any BSA that was loosely bound to the foam surface, and we kept a sample for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). We then incubated the BSA@TpAzo-foam samples in water for 24 hours to check for leaching of BSA, and we collected the supernatant after centrifuging the solution. SDS-PAGE showed no band of BSA after the second round of washing of the immobilized foam. The supernatant component of the washed BSA immobilized foam showed no BSA band after being incubated in water for 24 hours (Fig. S6†). These results confirmed the absence of any BSA leaching from the TpAzo foam after a 24 hours incubation in water. We followed a similar method for immobilization of the cellulolytic enzymes β-glucosidase (BGL), cellobiohydrolase (CBH), and endoglucanase (EG). To compute the surface area and volume of the three enzymes, we placed the protein data bank (PDB) structures in a 100 Å vacuum box. We calculated the accessible surface area (ASA) and occupied volume using a 1.86 Å (N2) probe in the Zeo++ software.12 The accessible surface area of BGL, CBH, and EG are 16240, 13565, and 15489 Å2, respectively. Their accessible volumes are 97880, 74660, and 82340 Å3, respectively.
Prior to immobilization in TpAzo foam, BGL, CBH, and EG were purified (Section S-3†). We used HEPES (4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid), HEPES with 75 mM NaCl, and MES (2-(N-morpholino)ethane sulfonic acid) buffers optimized for the stability of BGL, CBH, and EG, respectively. We placed 1 mg of TpAzo foam in a reaction tube to begin the reaction.
To initiate the reaction, we added 400 μL of freshly purified 4 mg mL−1 BGL in 10 mM HEPES buffer (pH 7) to the tube. The reaction mixture was incubated for 2 hours and then centrifuged. We measured the UV-vis spectrum of the supernatant at 280 nm. The concentration of adsorbed BGL was determined using UV-vis spectroscopy to be 0.14 mg of BGL per 1 mg of foam (Fig. S4†). The BGL-immobilized TpAzo foam samples were washed several times with 10 mM HEPES buffer (pH 7) to remove any loosely bound BGL. SDS-PAGE analyses confirmed the absence of BGL from the second wash onwards. CBH and EG were similarly immobilized in TpAzo foam, and the enzyme-immobilized foams were also washed.
SDS-PAGE experiments were conducted to monitor the leaching of the enzyme from the foam (Fig. S6†). The loading percentage was calculated to be 0.22 mg CBH per 1 mg TpAzo foam and 0.33 mg EG per 1 mg TpAzo foam. SEM (Fig. 1d and S9†) and TEM (Fig. 1F and S12†) images of BGL@TpAzo-foam, CBH@TpAzo-foam, and EG@TpAzo-foam confirmed the retention of their porous morphology after enzyme immobilization. To determine the spatial distribution of the enzymes, we immobilized fluorescein isothiocyanate (FITC)-labeled enzymes in the foam. The FITC-enzyme@TpAzo-foams were excited at 552 nm, and confocal images were collected in a window range from 570 nm to 620 nm for the three enzyme-immobilized foams along the maximum intensity projection (MIP). Confocal images of FITC-EG@TpAzo-foam, FITC-BGL@TpAzo-foam, and FITC-CBH@TpAzo-foam reveal that EG, BGL, and CBH are entrapped inside the foam matrix (Fig. 1h and S14†). The PXRD patterns of the enzyme-immobilized foams are almost identical to the PXRD patterns of the TpAzo foam, indicating that the TpAzo foam retains its crystallinity in the enzymes-immobilized TpAzo foams (Fig. 2a). Micro-computed tomography of the bovine serum albumin (BSA) immobilized foam revealed a wide range of porosity distribution (up to 60 μm), with a macroporous surface area of 16110 m2 m−3. The distribution of macropore diameter of the BSA@TpAzo-foam is significantly lower (∼40 μm) compared to the pristine TpAzo foam (∼400 μm) (Fig. 2h and i), suggesting that immobilized BSA occupies the macropores preferably. The BET surface areas of the BSA@TpAzo-foam, BGL@TpAzo-foam, EG@TpAzo-foam, and CBH@TpAzo-foam dropped to 250, 280, 40, and 30 m2 g−1, respectively. The surface area of the enzyme-loaded TpAzo foam is less than that of the pristine TpAzo-foam, which is attributed to the preferential occupancy of the macropores by the enzymes, as observed through micro-computed tomography. As the macropores and micropores are interconnected, there will also be a blockage in the micropores. This blockage in the macropores led to a drop in porosity and the surface area of enzyme-loaded TpAzo foam. The extent of immobilization of BGL, CBH, and EG in the TpAzo foam is 14% (w/w), 22% (w/w), and 34% (w/w), respectively (Fig. 2b). In this context, a drop in the porosity and surface area is much higher in CBH@TpAzo-foam, and EG@TpAzo-foam followed by BGL@TpAzo-foam. Similarly, the pore volumes of BSA@TpAzo-foam, BGL@TpAzo-foam, CBH@TpAzo-foam, and EG@TpAzo-foam also decrease to 0.209, 0.205, 0.077, and 0.09 cm3 g−1 respectively. NLDFT calculations show that the enzyme-immobilized foams' average micropore size remains unchanged after enzyme immobilization (Fig. S16†). It implies that immobilized enzymes do not occupy the micropores of the foam material. FT-IR spectra of enzyme-immobilized foams exhibit a broad peak of amide-1 at 1635–1642 cm−1, which was absent in the pristine TpAzo foams (Fig. 2c–f, S17 and 18†). A series of volumes of BSA stock of concentration 4 μg μL−1 were added separately in 1 mg of TpAzo foam. The UV-vis spectra of the supernatant were collected after 2 hours of incubation. It was found that the TpAzo foam can encapsulate up to 9.0% (w/w) BSA, whereas TpAzo COF can uptake only 3.6% (w/w) BSA (Fig. 3a). It demonstrates that COF-foam has a greater uptake capability than COF, and macropores in the foam aid in BSA uptake.
Multi-enzyme tandem catalysis has emerged as an alternative to the tedious process of biocatalytic transformation.13 Cellulose, a readily available material, can be hydrolyzed into glucose by cellulases and further processed to produce biofuels.14 The major components of cellulase are endoglucanase (EG), cellobiohydrolase (CBH), and β-glucosidase (BGL). However, the multi-enzyme catalyzed hydrolysis of carboxymethylcellulose (CMC), a modified form of cellulose, to glucose with high synthetic efficiency is challenging. To address this challenge, we examined the loading of BGL, CBH, and EG with an increasing stock concentration in the foam matrix. The uptake capacity vs. stock concentration graph revealed a saturation plateau in each enzyme immobilization. The total uptake efficiency of the TpAzo foam towards different enzymes differed substantially. BGL, CBH, and EG could be immobilized in the TpAzo foam up to 14% (w/w), 22% (w/w), and 34% (w/w), respectively (Fig. 3b). However, the total uptake efficiency of BGL, CBH, and EG in the TpAzo COF was 2.8% (w/w), 7% (w/w), and 23% (w/w), respectively (Fig. S5†). The relative uptake efficiency of BGL, CBH, and EG in the TpAzo COF compared to TpAzo foam were 27% (w/w), 40% (w/w), and 73% (w/w), respectively (Fig. 3c). Weak non-covalent interactions helped to immobilize these enzymes in the foam.
We undertook molecular dynamics (MD) and hybrid QM/MM simulations to determine the preferable adsorption sites of enzymes on TpAzo foam, along with their non-covalent interaction distances and energies (Section S-15†). Each enzyme was placed directly above a 4-layer, 4 × 4 supercell of the TpAzo foam to model the enzyme-TpAzo foam wall interactions. The bottom two layers of the foam had their positions fixed. To determine the variability of the enzyme-foam binding, each enzyme was placed five times above the TpAzo foam backbone structure in randomly selected orientations. From each starting point, MD simulations were run for 50 ps at 298 K, and QM/MM enzyme-TpAzo foam binding energies were calculated on each final snapshot. The maximum binding energies were: Eint (EG, −667.58 kJ mol−1) > Eint (BGL, −554.47 kJ mol−1) > Eint (CBH, −497.27 kJ mol−1) (Fig. S28–30†). The pore environment and macroscopic pore size dictated the loading of three enzymes into the TpAzo foam. We further checked the close non-covalent interactions between the enzyme and foam by searching for all instances where atoms (including H atoms) from the enzyme and foam approached each other within 3.0 Å. EG interacted more strongly with TpAzo foam due to the significant number of observable H-bonding, followed by BGL, while CBH showed the least number of H-bonding with the TpAzo foam layers (Table S3†).
The substrate chosen for the study was p-nitrophenyl-β-D-glucopyranoside (pNPGlc), and the production of p-nitrophenol (pNP) was monitored at 405 nm using UV-vis spectroscopy to determine β-glucosidase (BGL) activity in the TpAzo foam (Fig. 3d). We added 40 μL of 100 mM pNPGlc as a substrate and 10 μL of BGL@TpAzo-foam (equivalent to 0.5 μg of free BGL) to 50 μL of 10 mM HEPES buffer (pH 7) to make the reaction volume 100 μL (Section S-6†). Hydrolysis of pNPGlc was performed at different temperatures and pH levels. The BGL@TpAzo-foam showed maximum activity at 70 °C and pH 7 (Fig. S23†). The maximum activity of BGL@TpAzo-foam under the optimized condition was set as 100%, and the activities of BGL@TpAzo-foam in other conditions were calculated relatively. The Michaelis–Menten plot of the hydrolysis of pNPGlc to pNP by BGL@TpAzo-foam at the optimized condition revealed that the Km and Vmax were 23.5 ± 3.5 mM and 497.7 ± 28.0 μM min−1, respectively (Fig. 3e), while the Km and Vmax of free BGL were 14.7 ± 0.8 mM and 1116.3 ± 18.9 μM min−1, respectively (Fig. S25†). To confirm the heterogeneity of the reaction, we performed the recyclability of BGL@TpAzo-foam. After centrifugation, we found that BGL@TpAzo-foam is recyclable for up to 10 cycles, as it retained 62% specific activity after the 10th cycle despite the loss of BGL@TpAzo-foam particles in every step during the separation (Fig. 3f). Moreover, there was no change in the morphology of the foam particles after the catalytic cycle (Fig. S10 and S13†).
We also explored the stability of free BGL and BGL@TpAzo-foam in different organic solvents. After incubation in 500 μL of various solvents for 24 hours, we checked their activity. The activity of free BGL and BGL@TpAzo-foam in the optimized buffer was set as 100%. The relative activity of BGL@TpAzo-foam in methanol, ethanol, THF, dioxane, and acetone was 2 to 8-fold higher than that of free BGL due to the confinement of the macropores in the foam matrix (Fig. 3g).15 Additionally, the interaction of BGL with the foam matrix reduces the unfolding tendency of the BGL. To compare the half-life of free BGL (73 minutes)16 with that of BGL immobilized in TpAzo foam, we measured the relative activity of the BGL@TpAzo-foam at different time intervals at 70 °C. The relative activity of the BGL@TpAzo-foam started to decrease with time and became almost half after 200 minutes, implying a higher half-life and thermotolerance of the BGL@TpAzo-foam (Fig. 3h). When free BGL and BGL@TpAzo-foam were kept at room temperature (25 °C), free BGL showed zero activity after 40 days, whereas BGL@TpAzo-foam retained 85% and 51% specific activity after 40 and 120 days, respectively (Fig. 3i). This further demonstrates the stability of BGL inside the foam matrix owing to the confinement effect of the pores.
We quantified the generated reducing sugar by UV-vis spectroscopy at 540 nm through DNS assay to determine the activity of endoglucanase (EG) and cellobiohydrolase (CBH) in the foam on carboxymethylcellulose (CMC) (Section S-6†). The maximum activity of EG@TpAzo foam was observed at 55 °C and pH 6 (Fig. S23†). We conducted the hydrolysis of CMC to reducing sugar under different temperatures and pH values. The maximum catalytic activity of EG@TpAzo-foam under the optimized condition was set as 100%, and the activities of EG@TpAzo-foam in other conditions were calculated accordingly. The steady-state kinetics revealed that the Km and Vmax of EG@TpAzo-foam were 13.2 ± 2.0 mg mL−1 and 102.2 ± 7.1 μM min−1, respectively (Fig. 4a), while the Km and Vmax of free EG were 66 mg mL−1 and 545 μM min−1 (Fig. S25†). To confirm the heterogeneity of the reaction, we tested the recyclability of EG@TpAzo-foam, and found that it can be recycled up to 10 cycles. Even though there was an unavoidable loss of EG@TpAzo-foam particles in every step during separation, the relative activity of EG@TpAzo-foam was retained at 53% after the 10th cycle (Fig. 4b). After the catalytic cycle, the morphology of the foam particles remained unchanged (Fig. S10 and S13†).
We also optimized the activity of CBH@TpAzo-foam and found that it showed the highest activity at 55 °C and pH 6 (Fig. S23†). The steady-state kinetics showed that the Km and Vmax of CBH@TpAzo-foam were 18.3 ± 4.0 mg mL−1 and 85.2 ± 9.6 μM min−1, respectively (Fig. 4c), while the Km and Vmax of free CBH were 7.8 mg mL−1 and 327 μM min−1 (Fig. S25†). Similar to EG@TpAzo-foam, CBH@TpAzo-foam was found to be recyclable up to 10 cycles with 71% activity (Fig. 4d). After the catalytic cycle, no changes were observed in the morphology of the foam particles (as shown in Fig. S10 and S13†). The catalytic activity of BGL, CBH, and EG immobilized on TpAzo foam was two to four-fold higher than their activities in TpAzo COF (as demonstrated in Fig. S24†). This lower activity in TpAzo COF can be attributed to the absence of macropores in the structure, which leads to a reduction in the flow velocity of reactants and products.
We simultaneously utilized BGL@TpAzo-foam, CBH@TpAzo-foam, and EG@TpAzo-foam in one pot to convert carboxymethylcellulose to glucose without isolating intermediates (as depicted in Fig. 5a and Section S-6†). No products were observed when the reaction was performed with only TpAzo foam. We added 10 μL each of BGL@TpAzo-foam (equivalent to 0.5 μg of free BGL), CBH@TpAzo-foam (equivalent to 0.5 μg of free CBH), and EG@TpAzo-foam (equivalent to 0.5 μg of free EG), and the conversion of CMC to glucose was monitored by GOD-POD (glucose oxidase-peroxidase) assay. The maximum conversion rate under these conditions was set as 100% to compare the conversion rates in other conditions (as shown in Fig. 5b).
We conducted a control experiment to determine the contribution of each enzyme@TpAzo-foam to glucose conversion. In an equimolar mixture of BGL@TpAzo-foam and EG@TpAzo-foam (equivalent to 0.75 μg of free BGL and 0.75 μg of free EG), the relative conversion dropped to 80%. Similarly, in an equimolar mixture of BGL@TpAzo-foam and CBH@TpAzo-foam (equivalent to 0.75 μg of free BGL and 0.75 μg of free CBH), the relative conversion was 26%. The relative conversion was 10% for an equimolar combination of EG@TpAzo-foam and CBH@TpAzo-foam (equivalent to 0.75 μg of free EG and 0.75 μg of free CBH). In the presence of EG@TpAzo-foam (equivalent to 1.5 μg of free EG), the relative conversion was only 22% (Fig. 5b). The enzymes@TpAzo-foam demonstrated high recyclability (10 cycles) with excellent relative conversions (∼66%) (Fig. 5c). When we immobilized BGL, CBH, and EG simultaneously in the foam (BGL + CBH + EG@TpAzo-foam) in different combinations for CMC conversion, we found that the trend of the relative activity of CMC hydrolysis was similar to that of a mixture of BGL@TpAzo-foam, CBH@TpAzo-foam, and EG@TpAzo-foam (Fig. S26†). However, the relative activity dropped to 62% in the presence of BGL + CBH + EG@TpAzo-foam (Fig. 5d). The different ratios of immobilized enzymes in the same foam (BGL + CBH + EG@TpAzo-foam) compared to the individual mixtures (BGL@TpAzo-foam + CBH@TpAzo-foam + EG@TpAzo-foam) likely caused a drop in relative activity.
EG@TpAzo-foam degrades CMC from the chain end by binding to the substrate until a minimum chain length is achieved. After that, CBH@TpAzo-foam starts to hydrolyze one or a few accessible bonds in the chain from the surface. Finally, BGL@TpAzo-foam hydrolyzes cellobiose and small soluble oligomers to glucose.17 We analyzed the conversion of CMC to glucose by EG@TpAzo-foam + CBH@TpAzo-foam + BGL@TpAzo-foam mixture and EG + CBH + BGL@TpAzo-foam using HRMS. The HRMS value confirmed (observed value M + Na: 203.0553, theoretical value: 203.06) the presence of glucose. Thus, the synergistic action of these three enzymes embedded in foam enables the one-pot production of glucose.
Footnote |
† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d3sc01367g |
This journal is © The Royal Society of Chemistry 2023 |