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一种果胶裂解酶基因(pel)表达体系构建及其表达产物的酶学性质
成莉凤,刘正初,段盛文,冯湘沅,郑科,郑霞,程毅
中国农业科学院麻类研究所
Construction of the Prokaryotic Expression System Carrying a Pectate Lyase Gene(pel) and Its Enzymatic Property
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摘要 本研究拟从麻类脱胶高效菌株Dickeya sp. DCE-01克隆果胶裂解酶基因(pel),并构建表达载体进行原核表达,对表达产物进行纯化和酶学性质研究。根据麻类脱胶高效菌株Dickeya sp. DCE-01全基因组序列预测的果胶裂解酶基因(pel)设计引物,PCR扩增后将该基因连接到pEASY-E1载体上,导入大肠杆菌(Escherichia coli) BL21(DE3)进行表达。采用超滤和Sephadex G-100凝胶层析两步法纯化胞外果胶裂解酶,并研究其酶学性质。结果表明:pel基因(GenBank登录号: JX 964998)序列全长1 164 bp,编码387个氨基酸,预测其N末端前35 AA为信号肽,前体蛋白分子量为41.4 kD, 成熟蛋白分子量为37.8 kD。BL21(DE3)/pEASY-E1-pel发酵液的酶活力达27.82 IU/mL。SDS-PAGE分析两步法的纯化收集液,Pel活性组分显示为38 kD的优势条带;经Gel Analyzer 2010 软件分析,显示该Pel组分纯度达98.6%。该Pel最适反应温度为55℃,在≤60℃时稳定;最适反应pH为9.5,pH 9.0~10.0时稳定。与橘子果胶和多聚半乳糖醛酸钠相比,苹果果胶为该Pel的最适底物。该果胶裂解酶的催化作用依赖于Ca2+,1.5 mmol/L的Ca2+能最大幅度提高酶活力;Mn2+、Pb2+和EDTA能严重抑制酶活力。从麻类脱胶高效菌株中克隆到果胶裂解酶基因,并在大肠杆菌成功表达;该酶的耐热耐碱性表明其在生物质加工过程中具有重要工业化应用前景。
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成莉凤
刘正初
段盛文
冯湘沅
郑科
郑霞
程毅
关键词 麻类脱胶果胶裂解酶原核表达酶学性质    
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.
Key wordsBast fiber bio-degumming    Pectate lyase    Prokaryotic expression    Enzymatic property
收稿日期: 2012-11-20     
通讯作者: 刘正初   
引用本文:   
成莉凤,刘正初,段盛文,冯湘沅,郑科,郑霞,程毅. 一种果胶裂解酶基因(pel)表达体系构建及其表达产物的酶学性质[J]. , 2013, 21(5): 546-553.
链接本文:  
http://journal05.magtech.org.cn/Jwk_ny/CN/     或     http://journal05.magtech.org.cn/Jwk_ny/CN/Y2013/V21/I5/546
 
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