Shewanella haliotis BP-1海藻酸裂解酶基因的克隆表达  

Gene Cloning and Expression of Alginate Lyase from Shewanella haliotis BP-1

作　　者：黄桂媛[1] 温顺华 李锋 卢明倩[1] 王巧贞[1] 廖威[4] 黄庶识[1] 

机构地区：[1]广西科学院生物物理实验室,广西南宁530007 [2]厦门万泰凯瑞生物技术有限公司,福建厦门361003 [3]黔南州民族师范学院化学化工学院,贵州都匀558000 [4]广西职业技术学院,广西南宁530226

出　　处：《广西科学》2016年第3期228-233,共6页Guangxi Sciences

基　　金：国家自然科学基金项目（31560017）,广西自然科学重点基金项目（2014GXNSFDA118012）和贵州省教育厅自然科学研究重点项目（黔教合KY[2014]286）资助.

摘　　要：【目的】了解海洋细菌Shewanella haliotis B P-1中海藻酸裂解酶降解海藻酸钠的生物活性。【方法】应用基因克隆和大肠杆菌异源表达技术,过量表达海藻酸裂解酶,将粗酶液通过 DEAE Sepharose FF柱分离纯化后检测其酶活性。【结果】从S.haliotis BP-1菌株的基因组DNA中克隆得到一个大小为2157 bp的海藻酸裂解酶基因Alg17S ,该基因编码的海藻酸裂解酶 Alg17S属于PL17家族的蛋白,大小为79726 Da,其中包括N端26个氨基酸的信号肽,与Saccharophagus degradans 2-40菌株产生的海藻酸裂解酶 Alg17C 具有高度同源性,相似性为52%。经纯化后获得的重组酶 Alg17S和△snAlg17S（N端不含26个氨基酸的信号肽）均具有降解海藻酸钠的活性,但△snAlg17S对海藻酸钠的催化活性比 Alg17S高,其酶比活力高达9635 U/mg。【结论】重组海藻酸裂解酶△snAlg17S兼具高表达水平及高酶活性,是进一步研究海藻酸盐糖化和生物燃料生产的潜在的优势酶。Objective]Alginate lyase in Shewanella haliotis BP-1 strains was studied illustrate its biological activity of degrading alginate.[Methods]The gene cloning technology and the Escherichia coli heterologous expression technology were applied to overexpress the alginate lyase;And the enzyme activity was analyzed after the crude enzyme was separated and purified by DEAE Sepharose FF chromatogra-phy.[Results]The alginate lyase gene Alg 1 7S , with a size of 2 1 5 7 bp,was cloned from S. haliotis BP-1 strain genomic DNA and encoded an alginate lyase Alg17S,which belonged to pol-ysaccharide lyase（PL）1 7 family and had a size of 79 726 Da protein（including an N-terminal signal peptide of 26 amino acid signal peptide）.Alg17S showed high sequence identity of 5 2% with PL-17 protein sequence Alg17C from Saccharophagus degradans 2-40.Both the purified recombi-nase Alg17S and the △snAlg17S（without the N-terminal signal peptide of 26 amino acids）can degrade alginate,but the enzymatic activity of △snAlg17S revealed a specific activity of 9 635 U/mg,which was more efficient than Alg17S.[Conclusion]The recombinant alginate lyase △s-nAlg17S that has both high-level expression and high enzymatic activity could be a potential en-zyme for further researching on the alginate saccharification and the biofuels production.

关 键 词：海藻酸裂解酶 克隆表达 酶活性测定 SHEWANELLA HALIOTIS BP-1 

分 类 号：Q74[生物学—分子生物学]