Facile immobilization of pyridoxal 5′-phosphate using p-diazobenzoyl-derivatized Sepharose 4B

Pyridoxal 5′-phosphate (PLP) is a ubiquitous and versatile cofactor utilized by numerous enzymes involved in amino acid biosynthetic pathways. Immobilized PLP is a valuable tool to isolate unknown PLP-dependent enzymes in nature or to perform in vitro selection or directed evolution on existing or de novo PLP-dependent enzymes. The C-6 position is preferred for covalent immobilization of PLP because it maintains all important functional groups in their native, unmodified form. Previously reported diazonium derivatization methods for C-6 immobilization utilized an azide linker compound that is hazardous and not readily available. Here we report a safer and more accessible method to synthesize p-diazobenzoyl-derivatized Sepharose 4B using the N-hydroxysuccinimide (NHS) ester chemistry. The derivative was used to immobilize PLP, and the resulting C-6 immobilized PLP had a loading of ∼2.6 µmol PLP per mL of resin, comparable to commercially available products of other immobilized cofactors.