CATALYTIC PROPERTIES AND SPECIFICITY OF A RECOMBINANT, OVEREXPRESSED D-MANNURONATE LYASE

Lysis of alginates and of their saturated and unsaturated fragments wa s monitored by H-1 NMR spectroscopy. AlxM(B) alginate lyase performs b eta-elimination on the mannuronic acid (M) residues. It does not cleav e the guluronic acid (G) sequences, nor the M-G or the G-M diads. In c onsequence, it is a true mannuronate lyase. The end product of the rea ction is (4-deoxy-alpha-L-erythro-hex-4-enopyranosyl-uronic acid)-(1-- >(4)-O-(beta-D-mannopyranosyluronic acid)-(1-->4)-O-beta-D-mannopyranu ronic acid. Viscosity measurements made during degradation of a polyma nnuronate alginate showed that AlxM(B) behaves as an endo-enzyme. HPLC analysis of the degradation products of oligomannuronates and oligoal ginates suggested that the beta-elimination requires the interaction o f the enzyme with at least three sequential mannuronic acid residues. The catalytic site may possess 5 sub-sites and accommodate pentamers w ith different M/G ratio. Kinetic measurements showed that the specific ity constant V-m/K-m increased with the number of mannuronic acid resi dues. AlxM(B) may be reversibly inhibited by heteropolymeric blocks in a competitive manner. (C) 1998 Elsevier Science Ltd. All rights reser ved.