Molecular and biochemical characterization of the thermoactive family 1 pectate lyase from the hyperthermophilic bacterium Thermotoga maritima

摘要

pThe ability of the hyperthermophilic bacterium iThermotoga maritima/i to grow on pectin as a sole carbon source coincides with the secretion of a pectate lyase A (PelA) in the extracellular medium. The ipel/iA gene of iT. maritima/i was functionally expressed in iEscherichia coli/i as the first heterologously produced thermophilic pectinase, and purified to homogeneity. Gel filtration indicated that the native form of PelA is tetrameric. Highest activity (422units/mg, with a iK/isubm/sub of 0.06mM) was demonstrated on polygalacturonic acid (PGA), whereas pectins with an increasing degree of methylation were degraded at a decreasing rate. In the tradition of pectate lyases, PelA demonstrated full dependency on Casup2+/sup for stability and activity. The enzyme is highly thermoactive and thermostable, operating optimally at 90°C and pH9.0, with a half-life for thermal inactivation of almost 2h at 95°C, and an apparent melting temperature of 102.5°C. Detailed characterization of the product formation with PGA indicated that PelA has a unique eliminative exo-cleavage pattern liberating unsaturated trigalacturonate as the major product, in contrast with unsaturated digalacturonate for other exopectate lyases known. The unique exo-acting mode of action was supported by progression profiles of PelA on oligogalacturonides (degree of polymerization, 3—8) and the examination of the bond cleavage frequencies./p