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

作　　者：成莉凤[1] 刘正初[1] 段盛文[1] 冯湘沅[1] 郑科[1] 郑霞[1] 程毅[1] 

机构地区：[1]中国农业科学院麻类研究所,湖南长沙410205

出　　处：《微生物学通报》2013年第8期1403-1413,共11页Microbiology China

基　　金：国家863计划项目(No.2006AA02Z249);国家麻类产业技术体系建设专项项目(No.CARS-19-E24)

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。[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 pACY- CDuet-l-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 Ca＆2＋, while it was promoted by Zn^2＋ and NH^4＋ and inhibited seriously by Fe^3＋ and Pb^2＋. The maximal activ- ity of the purified enzyme was obtained at Ca＆2＋ concentration of 2 mmol/L. ]Conclusion] An efficient alkaline pectate lyase gene has been excavated from strain DCE-01, and its expres- sion product may be available for biorefinery.

关 键 词：麻类脱胶 高效菌株 果胶裂解酶 基因克隆 酶学性质 

分 类 号：S182[农业科学—农业基础科学] Q93[生物学—微生物学]