3D-Printed Labware for High-Throughput Immobilization of Enzymes

Abstract

In continuous flow biocatalysis, chemical transformations can occur under milder, greener, more scalable, and safer conditions than conventional organic synthesis. However, the method typically involves extensive screening to optimize each enzyme's immobilization on its solid support material. The task of weighing solids for large numbers of experiments poses a bottleneck for screening enzyme immobilization conditions. For example, screening conditions often require multiple replicates exploring different support chemistries, buffer compositions, and temperatures. Thus, we report 3D-printed labware designed to measure and handle solids in multichannel format and expedite screening of enzyme immobilization conditions. To demonstrate the generality of these advances, alkaline phosphatase, glucose dehydrogenase, and laccase were screened for immobilization efficiency on seven resins. The results illustrate the requirements for optimization of each enzyme's loading and resin choice for optimal catalytic performance. Here, 3D-printed labware can decrease the requirements for an experimentalist's time by >95%. The approach to rapid optimization of enzyme immobilization is applicable to any enzyme and many solid support resins. Furthermore, the reported devices deliver precise and accurate aliquots of essentially any granular solid material.

Advances in 3D Gel Printing for Enzyme Immobilization

“…The scope of this review focused only on studies that directly immobilized enzymes on or in the 3D printed objects and does not include many other innovations that use 3D printing to make customizable tools and devices, such as labware [ 88 ], mass spectrometry microcolumns [ 89 ], and biomedical devices [ 90 ], where enzymes are not directly included. The review emphasized the most recent techniques and chemistry for achieving successful enzyme immobilization by 3D printing, especially by gelation mechanisms that favor high retention of enzyme activity and longevity; however, the reader is referred elsewhere for more information on the basic physics of mass transfer limitations that may occur in these systems and how they are currently being studied through modeling [ 85 ].…”

Flow Biocatalysis: A Challenging Alternative for the Synthesis of APIs and Natural Compounds

“…Membrane reactors such as the tube-in-tube device, which is used for biotransformations that involve gaseous reagents, are another type of mesoreactor [80,81]. More recently, 3D-printed continuous flow reactors have also begun to be applied to biocatalysis to create customizable bioreactors [82][83][84][85]. The characteristics of the abovementioned flow bioreactors have been extensively reviewed recently [34,36,39].…”

Biocatalysis with Laccases: An Updated Overview

“…However, the bioreactor performances were tested only in the oxidation of the artificial chemical mediator ABTS [2,20-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)] and not in other target reactions of synthetic interest. In another very recent example, laccases were included in a study of 3D-printed continuous flow bioreactors [42]. Unfortunately, when compared to other types of enzymes, e.g., alkaline phosphatase and glucose dehydrogenase, the results obtained with laccases showed very high levels of experimental error that hampered a definitive evaluation of this flow system.…”