麻类脱胶高效菌株果胶裂解酶基因克隆与表达

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

摘要:

【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。pACYCDuet-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 °C, 最适pH为9.0; 保温1 h, 酶活稳定温度≤45 °C, 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。

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.