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Abstract In order to study the enzymatic properties of purified pectate lyase, the pectate lyase gene (pel) from Dickeya sp. DCE-01, an efficient strain for bast fiber bio-extracting, will be cloned, and its prokaryotic expression system will be constructed. Primers were designed by the potential pel G403 annotated from the whole genome sequence of Dickeya sp. DCE-01. The pel was cloned, linked to pEASY-E1, and expressed in Escherichia coli BL21(DE3). The extracellular pectate lyase (Pel) was purified by the two-step process involving ultrafiltration and gel filtration (Sephadex G-100) and its enzymatic properties were studied. The results showed that a pel gene (GenBank accession No. JX964998) coding for pectate lyase (Pel) was cloned from the genome of Dickeya sp. DCE-01 and expressed successfully in Escherichia coli BL21 (DE3). The pel gene contained an ORF of 1 164 bp, encoding 387 amino acids. The pectate lyase deduced by the nucleotide sequences included a signal peptide of 35 amino acids, and a calculated molecular mass of 37.8 kD for the mature protein, and 41.4 kD for the precursor. Using polygalacturonic acid sodium as substrate, a maximum activity of 27.82 IU/mL was obtained from fermentation filtrate of E. coli harboring the pectate lyase gene. After the two-step process purification, the pectate lyase exhibited one prominent band at about 38 kD on SDS-PAGE gel. With minor impurities, the purity of pectate lyase was approximate 98.6% determined by Gel Analyzer 2010 software. The optimal reaction temperature of the pectate lyase was at 55℃ and was stable at no more than 60℃ after being incubated for 100 min. The optimal reaction pH of the pectate lyase was pH 9.5 and was stable at pH 9.0 to 10.0. Pectin from apple was the optimum substrates for this Pel comparing with pectin from citrus and polygalacturonic acid sodium. Ca2+ was indispensable for the Pel catalytic activity and the maximal activity of Pel was obtained at Ca2+ concentration of 1.5 mmol/L. However, the Pel activity was inhibited seriously by Mn2+, Pb2+ and EDTA. In summary, a pectate lyase, encoding by the pel cloned from the Dickeya sp. DCE-01, was successfully expressed in E. coli BL21(DE3) and its thermophilicity and alkalophilicity may be valuable for biomass biorefinery.
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Received: 20 November 2012
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