Exceptionally high stability of urease covalently immobilized on silica, tungsten, and poly-(tetrafluoroethylene) (Teflon) supports has been observed by using a novel immobilization protocol which links the enzyme via its surface-exposed carboxylic groups (rather than conventionally used amino groups) to phospholipid-coated surfaces. Silanization of each of the three solid supports with [N-[11-(trifluoroacetamido)unde-canoyl]amino]propyltriethoxysilane, followed by removal of the trifluoroacetyl protective group, furnished the required amino-functionalized surfaces. The supports were linked to amino-functionalized phospholipids, which, in turn, were coupled to the carboxylic moieties of urease through diimide activation of the latter. For comparison, aminated supports, without the phospholipid coatings, were linked directly to carboxylic groups of urease. Also for comparison, urease was immobilized to the derivatized surfaces via its amino moieties using as cross-linkers, cyanuric chloride, as well as a new reagent, phthaloyl chloride. All of the surfaces were characterized by FTIR and X-ray photoelectron spectroscopy as well as by solid-state NMR in the case of the silica surfaces. Spectrophotometric assays revealed that the urease carboxyl-based protocols gave higher total and active immobilized enzyme yields. Among the non-lipid surfaces, urease was the least stable when immobilized on tungsten. On the lipid-coated surfaces, urease exhibited superior retention of activity after heating to temperatures up to 100-degrees-C. This stability was largely independent of the nature of the support material. It was found that urease bound to lipid-coated silica or Teflon could be boiled in aqueous solution for 1 h with minimal loss of activity.
