Abstract:The alkaline pectate lyase gene pel from Paenibacillus campinasensis BL-11 was synthesized by total gene synthesis, cloned on a pHKA carrier, and actively expressed in Pichia pastoris GS115. The signal peptide and promoter were optimized, so that the pectate lyase activity reached 432.36 U/mL after induction was carried out by 168-h shake flask fermentation. The basic enzymatic properties of the recombinant pectate lyase were preliminarily measured, and the optimal pH and temperature were 10.0 and 60 ℃, respectively. The residual enzyme activity was still higher than 90% when it was incubated at 50 ℃ for 300 min, while the residual enzyme activity was only about 50% after the treatment was carried out at 60 ℃ for 210 min. The enzyme activity remained more than 90% after the recombinant pectate lyase was treated with different buffer solutions ranging from pH 9~12 for 16 h. This enzyme had strong alkali resistance, but the enzyme activity was slightly decreased after being treated in slightly acidic buffers. Meanwhile, the effects of divalent metal ions on pel expression were also determined. The results showed that Ca2+ was essential for the trans-elimination reaction, and Fe2+, Mg2+ and Ni2+ had activating effects on pel activity, while the enzyme was completely inactivated with metal ion-ethylenediaminetetraacetic acid complexes. In summary, pectate lyase exhibits potential industrial application owing to its good stability under alkali conditions.
