Formula omitted]: Synthesis and application in affinity chromatography of exo-type cellulases

Abstract

p- Aminophenyl 1-thio-β- d-cellobioside (APTC) is shown to be a functional affinity ligand for the separation of exo-(cellobiohydrolases) and endo-(endoglucanases) acting cellulases. APTC is prepared by direct attachment of p-aminobenzenethiol to 2,3,6- tri-O- acetyl-4-O-(2,3,4,6- tetra-O- acetyl-β- d-glucopyranosyl)-(β- d-glucopyranosyl bromide, and subsequent deacetylation. APTC has been coupled to N-hydroxysuccinimide- activated agarose for affinity chromatography. Trichoderma reesei cellulases were used as representative enzymes. The behavior of these enzymes on APTC-affinity columns was essentially equivalent to that reported for the same enzymes on p- aminobenzyl 1-thio-β- d-cellobioside (ABTC)-columns; ABTC being the traditional ligand for affinity chromatography of exocellulase. The major cellobiohydrolases are retained on these columns, whereas the major endoglucanases are not. The cellobiohydrolases may be eluted from the columns by the addition of cellobiose to the mobile phase. The primary advantage of the APTC-ligand over other affinity ligands is its ease of preparation; the preparation of APTC requires approximately one-half the number of synthetic steps as required for the preparation of ABTC.