Improved stability of urease by covalent linkage to poly (tetrafluoroethylene)

Abstract

Methods for the preparation of poly (tetrafluoroethylene) surfaces containing specific organic functionality are described. These surfaces serve as intermediates for subsequent reactions involving the enzyme urease.

     Enhanced stability of urease covalently immobilized on poly (tetrafluoroethylene) (Teflon) support 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 the solid supports with [N-[11-(trifluoroacetamido) undecanoyl] 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. For comparison, urease was also immobilized to the derivatized surface via its amino moieties using as cross-linkers cyanuric chloride as well as a new reagent, phthaloyl chloride.

     Spectrophotometric assays revealed that urease exhibited superior retention of activity after heating to temperatures up to 100^oC. 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.