Cloning and expression of a pectate lyase gene from an efficient strain for bast fiber bio-extracting

Abstract:

[Objective] We cloned and expressed the pectate lyase gene (pel) from Dickeya sp. DCE-01, a strain for bast fiber bio-extracting, in E. coli BL21(DE3). Then we characterized the purified pectate lyase (Pel). [Methods] Primers were designed by the potential pel Q59419 annotated from the whole genome sequence of Dickeya sp. DCE-01. The pel was cloned, linked to pEASY-E1 and pACYCDuet-1, and expressed in E. coli BL21(DE3). After comparing the activities of extracellular Pel from the engineering strains, the Pel with highest activity was purified by the two-step process involving ultrafiltration and gel filtration (Sephadex G-100) and then characterized. [Results] The pel (GenBank: JX964997) was 1 128 bp and encoded 375 amino acids. The highest activity of the extracellular Pel from pACYCDuet-1-pel-BL strain was 298.8 IU/mL. The optimal condition for the purified enzyme was at 50 °C, pH 9.0 and polygalacturonic acid sodium as a substrate. The catalytic activity of the purified Pel was stable below 45 °C for 1 h at pH 9.0?10.0. It was dependent on Ca2+, while it was promoted by Zn2+ and NH4+ and inhibited seriously by Fe3+ and Pb2+. The maximal activity of the purified enzyme was obtained at Ca2+ concentration of 2 mmol/L. [Conclusion] An efficient alkaline pectate lyase gene has been excavated from strain DCE-01, and its expression product may be available for biorefinery.