Catalytic properties and specificity of a recombinant, overexpressed d-mannuronate lyase

Lysis of alginates and of their saturated and unsaturated fragments was monitored by 1H NMR spectroscopy. AlxMB alginate lyase performs β-elimination on the mannuronic acid (M) residues. It does not cleave the guluronic acid (G) sequences, nor the M–G or the G–M diads. In consequence, it is a true mannuronate lyase. The end product of the reaction is O-(4-deoxy-α-l-erythro-hex-4-enopyranosyl-uronic acid)-(1⃗(4)-O-(β-d-mannopyranosyluronic acid)-(1⃗4)-O-β-d-mannopyranuronic acid. Viscosity measurements made during degradation of a polymannuronate alginate showed that AlxMB behaves as an endo-enzyme. HPLC analysis of the degradation products of oligomannuronates and oligoalginates suggested that the β-elimination requires the interaction of the enzyme with at least three sequential mannuronic acid residues. The catalytic site may possess 5 sub-sites and accommodate pentamers with different M/G ratio. Kinetic measurements showed that the specificity constant Vm/Km increased with the number of mannuronic acid residues. AlxMB may be reversibly inhibited by heteropolymeric blocks in a competitive manner.