US 2025/0198980 A1 · COMBINATORIAL HIGH-THROUGHPUT SCREENING OF COMPLEX POLYMERIC ENZYME IMMOBILIZATION SUPPORTS

Abstract

A novel combinatorial and high-throughput platform that enables rapid screening of complex and heterogeneous copolymer brushes as enzyme immobilization supports named Combinatorial High-throughput Enzyme Support Screening (CHESS). Using a 384 well-plate format, we synthesized arrays of three-component polymer brushes in the microwells using photo-activated surface-initiated polymerization, and immobilized enzymes in situ. The utility of CHESS to identify optimal immobilization supports under thermally and chemically denaturing conditions was demonstrated using Bacillus subtilis Lipase A (LipA). The identification of supports with optimal compositions was validated by immobilizing LipA on polymer-brush modified biocatalyst particles. We further demonstrated that CHESS could be used to predict the optimal composition of polymer brushes a priori for the previously unexplored enzyme, alkaline phosphatase (AlkP). Our findings demonstrate that CHESS represents a predictable and reliable platform for dramatically accelerating the search of chemical compositions for immobilization supports and further facilitate the discovery of biocompatible and stabilizing materials.

Claims25

Dependent claims

A method of screening copolymer compositions as supports for enzymes comprising the steps of:

providing a bis[bromo]benzophenone-functionalized (2BrBP-functionalized) multi-well plate wherein a plurality of the wells of the multi-well plate have a defined and unique copolymer brush affixed to the bis[bromo]benzophenone (2BrBP) in a well and wherein the copolymer brush is created by systematically varying the relative concentration of the constituent monomers or other constituents used to create the copolymer brush, whereby the performance of the protein of interest can then be assayed in the unique polymer brush environment of the well;
adding an enzyme to the copolymer brushes of the wells;
adding an enzyme substrate to the wells;
exposing the plate to a defined set of conditions or chemicals;
and
measuring the conversion of the enzyme substrate in each of the plurality of wells responsive to the exposure to the defined set of conditions or chemicals.

3 more elements

A method of preparing a polymer-functionalized welled plate for enzyme immobilization comprising the steps of:

depositing a solution of bis[bromo]benzophenone (2BrBP) in toluene into the wells of the plate, whereby the toluene causes the well material to swell which allows the 2BrBP to intercalate into the walls of the plate;
irradiating the wells of the plate with UV-light to crosslink the 2BrBP;
depositing a mixture of monomers in the wells of the plate, wherein the mixture of monomers in a plurality of the wells systematically varies in relative proportion across a series of the wells;
and
irradiating the wells of the plate with green light to initiate the polymerization of the copolymer brushes.

2 more elements

Dependencies are inferred from the claim text.

Combinatorial high-throughput screening of complex polymeric enzyme immobilization supports

Life of this patent

Combinatorial high-throughput screening of complex polymeric enzyme immobilization supports

Life of this patent

Abstract

Claims25