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

• Published in: Journal of the American Chemical Society 2024-01, Vol.146 (1), p.858-867
• Main Authors: Paul, Satyadip, Gupta, Mani, Kumar Mahato, Ashok, Karak, Shayan, Basak, Ananda, Datta, Supratim, Banerjee, Rahul
• Format: Article
• Language: English
• ISSN: 0002-7863; 1520-5126; 1520-5126
• DOI: 10.1021/jacs.3c11169

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 β-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-β-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.