Rapid protein immobilization for thin film continuous flow biocatalysis

Abstract

A versatile enzyme immobilization strategy for thin film continuous flow processing is reported. Here, non-covalent and glutaraldehyde bioconjugation are used to immobilize enzymes on the surfaces of borosilicate reactors. This approach requires only ng of protein per reactor tube, with the stock protein solution readily recycled to sequentially coat >10 reactors. Confining reagents to thin films during immobilization reduced the amount of protein, piranha-cleaning solution, and other reagents by ~96%. Through this technique, there was no loss of catalytic activity over 10 h processing. The results reported here combines the benefits of thin film flow processing with the mild conditions of biocatalysis.

Hydrogen‐Borrowing Alcohol Bioamination with Coimmobilized Dehydrogenases

“…[5] Enzyme immobilization techniques include encapsulation in (in)organic microporous structures, attachment on functionalized supports, cross-linking, and coordination through ionic interactions. [5, 6]…”

“…Conversely, encapsulation (or entrapment) and noncovalent immobilization often suffer from enzyme leakage, whereas covalent enzyme immobilization can lead to random attachment between the enzyme surface and the functional groups on the support, potentially decreasing the activity. [5]…”

“…In this study, we co-immobilized an alcohol dehydrogenase from Aromatoleum aromaticum (AA-ADH)[10] and a chimeric amine dehydrogenase (Ch1-AmDH)[11] on CPG metal-ion affinity beads (EziG Fe-amber)[8c] to perform the hydrogen-borrowing amination of a panel of ( S )-configured alcohols [i.e., ( S )- 1 – 5a ] with ammonia (Scheme 1). The selected reaction proceeds with perfect inversion of configuration.…”

Ten‐Minute Protein Purification and Surface Tethering for Continuous‐Flow Biocatalysis

“…Our initial approach to immobilize proteins for continuous flow biocatalysis applied non-specific glutaraldehyde-based crosslinking to the reactor surface. [14] Although this generated a highly efficient continuous flow system for individual proteins, the method proved to be unworkable for creating the distinct enzyme zones envisioned for enzymatic assembly lines. Specifically, the relatively slow kinetics of this attachment method prevented confinement of each enzyme to a specific zone of the reactor.…”

“…Third, alkaline phosphatase and phosphodiesterase are stable to vortexing and other potentially harsh conditions. [14] Lastly, the reaction had to take place along the length of the reactor; previous experiments had shown that alkaline phosphatase and phosphodiesterase have rapid catalytic activity. [12] …”