鲍鱼褐藻胶裂解酶基因的原核表达及酶学性质

Characterization of an Alginate Lyase，from Abalone and Its Expression inEscherichia coli

DOI：10.3969/j.issn.1673-1689.2018.06.010

中文关键词: 鲍鱼 褐藻胶裂解酶 基因克隆表达 酶学性质

英文关键词: abalone,alginate lyase,gene cloning and expression,enzyme properties

基金项目:

摘要点击次数: 82

全文下载次数: 199

中文摘要:

褐藻胶寡糖因其内在的生物功能具有良好的应用前景，褐藻胶裂解酶可将褐藻胶降解为褐藻胶寡糖，基因工程技术为高效生产褐藻胶裂解酶提供了技术方法。从鲍鱼肝胰腺中提取RNA，通过RT-PCR得到cDNA，以cDNA为模板进行PCR扩增得到褐藻胶裂解酶基因algl，将其与pET-28a（+）载体连接，并在大肠杆菌BL21菌株中进行高效诱导表达。将工程菌体超声波破碎后进行十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS- PAGE）检测，利用Ni-NTA琼脂糖凝胶柱纯化重组蛋白Algl，继而对其进行酶学特性研究。细胞破碎物酶活检测及SDS-PAGE分析表明，重组蛋白为包涵体，相对分子量约为32 000。包涵体纯化、复性后的酶活为15.6 U/mL，比酶活为644.6 U/mg。纯化后的工程酶酶学性质研究表明：该酶最适温度35 ℃；最适pH值为8.0；只能降解多聚甘露糖醛酸（polyM）而不能降解多聚古洛糖醛酸（polyG）；催化褐藻胶的Km值为1.71 mg/mL，Vmax为19.08 U/mL。重组酶Algl对多聚甘露糖醛酸的底物特异性和适冷性具有潜在的生物技术和工业应用。

英文摘要:

Alginate oligosaccharides have good prospects because of their useful and biologically important functions. Alginate lyase can degrade alginate to alginate oligosaccharides，genetic engineering technology provides technology for efficient production of alginate lyase. Extraction of RNA from the hepatopancreas of abalone cDNA obtained by RT-PCR，cDNA as template for PCR amplification to obtain alginate lyase gene algl，connection with pET-28a（+） vector. The recombinant plasmid was transformed into E.coli BL21，induced and expressed. The new protein bands about 32 000 showed on polyacrylamide gel electrophoresis（SDS-PAGE） and the protein form of inclusion bodies. Inclusion body purification，refolding and determination of its activity 15.6 U/mL，specific activity 644.6 U/mL. The optimum temperature and pH were 35 ℃ and 8，respectively. Alyl only degradation poly mannuronate（polyM） and not degradable poly guluronic acid（polyG）. The Km and Vmax values for alginate were 1.71 mg/mL and 19.08 U/mL，respectively. Recombinase Algl poly mannuronic acid substrate specificity and cold adaptation has the potential for biotechnology and industral applications.

查看全文 查看/发表评论 下载PDF阅读器