[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 (SephadexG-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 ℃,pH 9.0 and polygalacturonic acid sodium as a substrate.The catalytic activity of the purified Pel was stable below 45 ℃ 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.%[目的]克隆麻类脱胶高效菌株Dickeya sp.DCE-01的果胶裂解酶基因并进行原核表达,对表达产物进行纯化和酶学性质研究.[方法]根据该菌株全基因组序列预测的果胶裂解酶基因Q59419设计引物,PCR扩增后将该基因连接到pEASY-E1和pACYCDuet-1载体上,导入E.coli BL21(DE3)进行表达.选择酶活力高的阳性克隆子进行大量诱导表达后,采用超滤和Sephadex G-100凝胶层析两步法纯化出果胶裂解酶,研究其酶学性质.[结果]克隆到果胶裂解酶基因pel (GenBank登录号:JX964997),其序列全长1 128 bp,编码375个氨基酸.pA CY CDuet-1-pel-BL表达胞外果胶裂解酶活力最高,发酵液粗酶活达298.8 IU/mL.其最适反应温度为50℃,最适pH为9.0；保温1h,酶活稳定温度≤45℃,稳定pH为9.0-10.0.酶催化作用依赖于Ca2+,其最适作用浓度为2 mmol/L; Zn2+、Ca2+和NH4+促进酶活力,Fe3+和pb2+严重抑制酶活力；聚半乳糖醛酸钠为该酶的最适底物.[结论]从麻类脱胶高效菌株中发掘到碱性果胶裂解酶基因,其表达产物在生物质加工过程中具有重要工业化应用前景.
展开▼
