摘要
目的原核表达枯草芽孢杆菌Expansin蛋白,并分析其促进多糖糖化的活性。方法以过夜培养的Bacillus subtilis subsp. Subtilis subtilis str. 168基因组为模板合成expansin基因;在引物的N-端添加6-his tag,PCR合成融合基因6his-expansin,构建重组质粒6his-expansin-6his-p ET28a(+)[heh-pET28a(+)],转化大肠埃希菌BL21(DE3),IPTG诱导表达融合蛋白6His-Expansin-6His(HEH);并对表达条件进行优化。产物经Ni-NTA纯化,检测其与多糖降解酶(polysaccharide degrading enzyme,PDE)(纤维素酶、木聚糖酶、果胶酶)及多糖复合酶(polysaccharide complex enzyme,PCE)制剂的协同活性(synergistic activity,SA)。结果经双酶切鉴定,重组质粒heh-p ET28a(+)构建正确;融合蛋白HEH在0. 05 mmol/L IPTG 30℃诱导30 h的最适表达条件下,表达量约为1. 2 mg/m L,其相对分子质量为26 400,目的蛋白纯度达95%;该蛋白与纤维素酶、木聚糖酶、果胶酶的SA分别为171. 53%、61. 95%、230. 00%,与PCE降解滤纸(filter paper)、秸秆(rice straw)的SA分别为312. 83%、356. 59%。结论重组的融合蛋白HEH与PDE均具有较高的协同作用,为Expansin在生物质木质纤维素糖化领域的应用奠定了基础。
Objective To express the Expansin of Bacillus subtilis in prokaryotic cells and analyze its activity in promoting the saccharification of polysaccharide.Methods Using Bacillus subtilis subsp.subtilis str.168 genome as template,expansin gene was synthesized.The 6-his tag was added to the N-terminus of expected protein by primer design,based on which a fusion gene 6-his-expansin was synthesized by PCR,based on which recombinant plasmid 6 his-expansin-6 his-pET28 a(+)[heh-p ET28 a(+)]was constructed,transformed to E.coli BL21(DE3)and induced with IPTG for expression of fusion protein 6 His-Expansin-6 His(HEH).The final IPTG concentration,temperature and time for induction were optimized.The expressed product was purified by Ni-NTA chromatography and determined for synergistic activities with various polysaccharide degrading enzyme(PDE)preparations(cellulose,xylanase and pectinase)and poly-saccharide complex enzyme(PCE).Results Restriction analysis proved that recombinant plasmid heh-pET28 a(+)was con-structed correctly.After induction with 0.05 mmol/L IPTG at 30℃for 30 h,the expression level of HEH reached about 1.2 mg/m L,while the relative molecular mass was 26 400,and the purity reached 95%after purification.The synergistic activities of the expressed protein with cellulose,xylanase and pectinase were 171.53%,61.95%and 230.00%,while those with PCE in degrading filter paper and rice straw were 312.83%and 356.59%,respectively.Conclusion Recombinant fusion protein HEH showed high synergistic activities with various PDEs,which laid a foundation of application of Expansin in the saccharification of biomass lignocellulose.
作者
叶凯霞
朱华琛
赵庆新
陈洪生
YE Kai-xia;ZHU Hua-chen;ZHAO Qing-xin;CHEN Hong-sheng(College of Biotechnology and Pharmaceutical Engineering,Nanjing Tech University,Nanjing 211800,Jiangsu Province,China)
出处
《中国生物制品学杂志》
CAS
CSCD
2019年第9期977-982,共6页
Chinese Journal of Biologicals
基金
国家自然科学基金(41773103)
江苏省盐土生物资源研究重点实验室开放基金(JKLBS2017012)
关键词
枯草芽孢杆菌
EXPANSIN
原核细胞
基因表达
多糖糖化
协同活性
Bacillus subtilis
Expansin
Prokaryotic cells
Gene expression
Saccharification of polysaccharide
Syner gistic activity
