Covalent Organic Frameworks for the Purification of Recombinant Enzymes and Heterogeneous Biocatalysis.

Recombinant enzymes have gained prominence due to their diverse functionalities and specificity and are often a greener alternative in biocatalysis. This context makes purifying recombinant enzymes from host cells and other impurities crucial. The primary goal is to isolate the pure enzyme of interest and ensure its stability under ambient conditions. Covalent organic frameworks (COFs), renowned for their well-ordered structure and permeability, offer a promising approach for purifying histidine-tagged (His-tagged) enzymes. Furthermore, immobilizing enzymes within COFs represents a growing field in heterogeneous biocatalysis. In this study, we have developed a flow-based technology utilizing a nickel-infused covalent organic framework (Ni-TpBpy COF) to combine two distinct processes: the purification of His-tagged enzymes and the immobilization of enzymes simultaneously. Our work primarily focuses on the purification of three His-tagged enzymes ß-glucosidase, cellobiohydrolase, and endoglucanase as well as two proteins with varying molecular weights, namely, green fluorescent protein (27 kDa) and BG Rho (88 kDa). We employed Ni-TpBpy as a column matrix to showcase the versatility of our system. Additionally, we successfully obtained a Ni-TpBpy COF immobilized with enzymes, which can serve as a heterogeneous catalyst for the hydrolysis of p-nitrophenyl-ß-d-glucopyranoside and carboxymethylcellulose. These immobilized enzymes demonstrated catalytic activity comparable to that of their free counterparts, with the added advantages of recyclability and enhanced stability under ambient conditions for an extended period, ranging from 60 to 90 days. This contrasts with the free enzymes, which do not maintain their activity as effectively over time.

Covalent Organic Framework Cladding on Peptide-Amphiphile-Based Biomimetic Catalysts.

Peptide-based biomimetic catalysts are promising materials for efficient catalytic activity in various biochemical transformations. However, their lack of operational stability and fragile nature in non-aqueous media limit their practical applications. In this study, we have developed a cladding technique to stabilize biomimetic catalysts within porous covalent organic framework (COF) scaffolds. This methodology allows for the homogeneous distribution of peptide nanotubes inside the COF (TpAzo and TpDPP) backbone, creating strong noncovalent interactions that prevent leaching. We synthesized two different peptide-amphiphiles, C10FFVK and C10FFVR, with lysine (K) and arginine (R) at the C-termini, respectively, which formed nanotubular morphologies. The C10FFVK peptide-amphiphile nanotubes exhibit enzyme-like behavior and efficiently catalyze C-C bond cleavage in a buffer medium (pH 7.5). We produced nanotubular structures of TpAzo-C10FFVK and TpDPP-C10FFVK through COF cladding by using interfacial crystallization (IC). The peptide nanotubes encased in the COF catalyze C-C bond cleavage in a buffer medium as well as in different organic solvents (such as acetonitrile, acetone, and dichloromethane). The TpAzo-C10FFVK catalyst, being heterogeneous, is easily recoverable, enabling the reaction to be performed for multiple cycles. Additionally, the synthesis of TpAzo-C10FFVK thin films facilitates catalysis in flow. As control, we synthesized another peptide-amphiphile, C10FFVR, which also forms tubular assemblies. By depositing TpAzo COF crystallites on C10FFVR nanotubes through IC, we produced TpAzo-C10FFVR nanotubular structures that expectedly did not show catalysis, suggesting the critical role of the lysines in the TpAzo-C10FFVK.

Landscaping Covalent Organic Framework Nanomorphologies.

The practical utilization of covalent organic frameworks (COFs) with manipulation at the atomic and molecular scale often demands their assembly on the nano-, meso-, and macroscale with precise control. Consequently, synthetic approaches that establish the ability to control the nucleation and growth of COF crystallites and their self-assembly to desired COF nanomorphologies have drawn substantial attention from researchers. On the basis of the dimensionality of the COF morphologies, we can categorize them into zero- (0-D), one- (1-D), two- (2-D), and three-dimensional (3-D) nanomorphologies. In this perspective, we summarize the reported synthetic strategies that enable precise control of the COF nanomorphologies' size, shape, and dimensionality and reveal the impact of the dimensionalities in their physicochemical properties and applications. The aim is to establish a synergistic optimization of the morphological dimensionality while keeping the micro- or mesoporosity, crystallinity, and chemical functionalities of the COFs in perspective. A detailed knowledge along the way should help us to enrich the performance of COFs in a variety of applications like catalysis, separation, sensing, drug delivery, energy storage, etc. We have discussed the interlinking between the COF nanomorphologies via the transmutation of the dimensionalities. Such dimensionality transmutation could lead to variation in their properties during the transition. Finally, the concept of constructing COF superstructures through the combination of two or more COF nanomorphologies has been explored, and it could bring up opportunities for developing next-generation innovative materials for multidisciplinary applications.

Crystallizing Sub 10 nm Covalent Organic Framework Thin Films via Interfacial-Residual Concomitance.

Synthesis of covalent organic framework (COF) thin films on different supports with high crystallinity and porosity is crucial for their potential applications. We have designed a new synchronized methodology, residual crystallization (RC), to synthesize sub 10 nm COF thin films. These residual crystallized COF thin films showcase high surface area, crystallinity, and conductivity at room temperature. We have used interfacial crystallization (IC) as a rate-controlling tool for simultaneous residual crystallization. We have also diversified the methodology of residual crystallization by utilizing two different crystallization pathways: fiber-to-film (F-F) and sphere-to-film (S-F). In both cases, we could obtain continuous COF thin films with high crystallinity and porosity grown on various substrates (the highest surface area of a TpAzo COF thin film being 2093 m2 g-1). Precise control over the crystallization allows the synthesis of macroscopic defect-free sub 10 nm COF thin films with a minimum thickness of ∼1.8 nm. We have synthesized two COF thin films (TpAzo and TpDPP) using F-F and S-F pathways on different supports such as borosilicate glass, FTO, silicon, Cu, metal, and ITO. Also, we have investigated the mechanism of the growth of these thin films on various substrates with different wettability. Further, a hydrophilic support (glass) was used to grow the thin films in situ for four-probe system device fabrication. All residual crystallized COF thin films exhibit outstanding conductivity values. We could obtain a conductivity of 3.7 × 10-2 mS cm-1 for the TpAzo film synthesized by S-F residual crystallization.