Abstract:Alkaline pectate lyase (PL) from recombinant strain E.coli JM109（pHsh PL）was purified by a three-step process including (NH4)2SO4 precipitation followed by dialysis and chromatography. The purified enzyme appeared homologous on SDS-PAGE. The specific activity of the purified enzyme reached 1079 U/mg. The optimal pH and temperature were in the ranges of pH 9.0 to 10.0 and 50℃ to 66℃. The enzyme was preferable in optimal pH range in enzymatic retting of flax. Enzyme activity slightly increased in the presence of Mg2+ ion, whereas decreased in the presence of other ions, especially Fe2+. The Km of the purified enzyme for polygalacturonic acid was 20.93 mg/L, the Vmax for polygalacturonic acid hydrolysis was 105.3 μmol of unsaturated products per min and Ea was 21.74 kJ/mol. The results of the decay constant (kd ) analysis on condition of PL bonding polygalacturonic acid (kd=0.02 min-1) and PL without polygalacturonic acid ( kd=0.0342 min-1) showed the substrate was helpful to decrease thermal inactivation of PL. The products (unsaturated oligomers) from polygalacturonic acid degraded by PL were analyzed by electrospray ionization mass spectrometry(ESI-MS). The following data were obtained: ESI-MS m/z, 350.82 (unsaturated bigalacturonic acid, uG2), 527.04 (unsaturated trigalacturonic acid, uG3). However, m/z 175 (unsaturated galacturonic acid, uG1) was not found. These results indicate that the final PGA degradation products was a mixture of unsaturated oligo-galacturonides including uG3 and uG2 except for uG1. It suggests that the recombinant PL cannot degrade uG3 and uG2.
