Covalent protein immobilization on glass surfaces: Application to alkaline phosphatase

Lyophilized alkaline phosphatase (ALPase) was immobilized on aminated glass surfaces using the in vacuo cross-linking process [Simons, B.L., King, M.C., Cyr, T., Hefford, M.A., Kaplan, H., 2002. Zero-length cross-linking of lyophilized proteins. Protein Sci. 11, 1558-1564]. In this procedure, amide bonds were formed between carboxyl groups on the protein and amino groups on the glass surface. After the non-covalently attached enzyme was removed the immobilized ALPase not only retained its activity but could also be used, washed and reused at least six times without significant loss of activity. An average of 1.4 ± 0.6 mg of reusable ALPase per gram of glass fibre was immobilized based on the activity of the soluble equivalent.