Immobilization of enzymes provides many benefits, including facile separation and recovery of enzymes from reaction mixtures, enhanced stability, and co‐localization of multiple enzymes. Calcium‐phosphate‐protein supraparticles imbued with a leucine zipper binding domain (ZR) serve as a modular immobilization platform for enzymes fused to the complementary leucine zipper domain (ZE). The zippers provide high‐affinity, specific binding, separating enzymatic activity from the binding event. Using fluorescent model proteins (mCherryZEand eGFPZE), an amine dehydrogenase (AmDHZE), and a formate dehydrogenase (FDHZE), the efficacy of supraparticles as a biocatalytic solid support was assessed. Supraparticles demonstrated several benefits as an immobilization support, including predictable loading of multiple proteins, structural integrity in a panel of solvents, and the ability to elute and reload proteins without damaging the support. The dual‐enzyme reaction successfully converted ketone to amine on supraparticles, highlighting the efficacy of this system.
In recent decades, biocatalysis has emerged as an important alternative to chemical catalysis in pharmaceutical manufacturing. Biocatalysis is attractive because enzymatic cascades can synthesize complex molecules with incredible selectivity, yield, and in an environmentally benign manner. Enzymes for pharmaceutical biocatalysis are typically used in their unpurified state, since it is time‐consuming and cost‐prohibitive to purify enzymes using conventional chromatographic processes at scale. However, impurities present in crude enzyme preparations can consume substrate, generate unwanted byproducts, as well as make the isolation of desired products more cumbersome. Hence, a facile, nonchromatographic purification method would greatly benefit pharmaceutical biocatalysis. To address this issue, here we have captured enzymes into membraneless compartments by fusing enzymes with an intrinsically disordered protein region, the RGG domain from LAF‐1. The RGG domain can undergo liquid–liquid phase separation, forming liquid condensates triggered by changes in temperature or salt concentration. By centrifuging these liquid condensates, we have successfully purified enzyme‐RGG fusions, resulting in significantly enhanced purity compared to cell lysate. Furthermore, we performed enzymatic reactions utilizing purified fusion proteins to assay enzyme activity. Results from the enzyme assays indicate that enzyme‐RGG fusions purified by the centrifugation method retain enzymatic activity, with greatly reduced background activity compared to crude enzyme preparations. Our work focused on three different enzymes—a kinase, a phosphorylase, and an ATP‐dependent ligase. The kinase and phosphorylase are components of the biocatalytic cascade for manufacturing molnupiravir, and we demonstrated facile co‐purification of these two enzymes by co‐phase separation. To conclude, enzyme capture by RGG tagging promises to overcome difficulties in bioseparations and biocatalysis for pharmaceutical synthesis.
more » « less- PAR ID:
- 10520240
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Biotechnology and Bioengineering
- ISSN:
- 0006-3592
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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