冰岛硫化叶菌β-1,4-内切葡聚糖酶的同源表达、纯化与性质

Homogeneous expression,purification and characterization of endo-β-1,4-glucanase from Sulfolobus islandicus REY15A

利用PCR技术从冰岛硫化叶菌(Sulfolobus islandicus)REY15A中分别扩增得到带有和不带有信号肽编码序列的β-1,4-内切葡聚糖酶基因(S.islandicus eng),将其克隆至硫化叶菌表达载体pZC2中,构建重组表达载体pZC2-eng-YS和pZC2-eng-WS,并转化至S.islandicus E233S(△pyrEF△lacS)。重组菌株经D-阿拉伯糖诱导后,细胞破碎上清经镍离子金属鳌合亲和层析介质(Ni-NTA)柱纯化,得到重组蛋白。SDS-PAGE结果表明:不带有信号肽的葡聚糖酶(ENG-W)分子质量为41ku,带有信号肽的分子质量(ENG-SP)为43ku;酶学性质分析表明前者没有酶活性,后者酶活力为103.4U/L,最适反应温度为90℃,最适pH为4.0。耐热性试验结果表明:在90℃保温60min后酶活力稳定在最高酶活力的40%以上。金属离子试验结果表明:Mn2+对重组酶促进作用最大,使其酶活力提高了约50%,Ca2+对其抑制作用最强,使其酶活力下降了约50%。

Endo-β-1,4-glucanase gene（with or without signal peptide coding sequence） was amplified from Sulfolobus islandicus REY15A genomic DNA with PCR and inserted into the Sulfolobus expression vector pZC2,constructing the recombinant plasmid pZC2-eng-YS and pZC2-eng-WS,respectively.Then the recombinant plasmids were electro-transformed into the host strain S.islandicus E233S（△pyrEF△lacS）.After induced by D-arabinose and purified with the Ni2＋-nitrilotriacetate column,two obvious protein bands with molecular weight about 43 ku and 41 ku appeared on the SDS-PAGE.The following analysis showed the recombinant protein（ENG-W） without signal peptide does not have endo-β-1,4-glucanase activity.However,the activity of recombinant protein（ENG-SP） with signal peptide was 103.4 U/L.The optimal temperature and pH for the recombinant protein（ENG-SP） was 90 ℃ and 4.0,respectively.Further research indicated that after incubating at 90 °C for one hour,ENG-SP still had 40% of the highest activity.Mn2＋ could increase the activity of ENG-SP by 50%,while Ca2＋ could inhibit the activity of ENG-SP by 50%.