一种灰盖鬼伞外切β-1,3葡聚糖酶基因的克隆与表达

Cloning and Expression of an Exo-β-1,3 glucanase Gene from Coprinopsis cinerea

为了简单快速地获取大量的β-1,3葡聚糖酶以研究其在真菌形态发生过程中所发挥的作用,本研究以灰盖鬼伞的c DNA为模板克隆了一种编码外切β-1,3葡聚糖酶的基因,并将该基因插入表达质粒p ET28A(+)中,获得了重组表达质粒p ET28A(+)-exg,转化到E.coli Rosetta菌株中并进行异源重组表达.结果显示,克隆基因的c DNA全长2 415 bp,共编码786个氨基酸;SDS-PAGE电泳表明该基因在E.coli Rosetta菌株中得到了高效表达,重组表达的酶蛋白表观分子量为85 k Da;纯化后获得的表达酶经DNS法测得比活力为45 U/mg.酶学性质测定结果表明,该酶具有β-1,3葡聚糖外切酶活性,以昆布多糖为底物时,最适反应条件为p H 6.0、温度60℃,且有一定的耐热能力和较好的p H稳定性,具有较好的应用前景.

To acquire enough β- 1, 3 glucanasc for investigating its role in morphological changes of fungus, an exo- 6- 1,3 glucanase gene （exg） was cloned from eDNA of Coprinopsis cinerea okayama 7#130 and inserted into plasmid pET28A （＋）to generate the recombinant expression plasmid pET28A（ ＋ ）-exg. pET28A（ ＋ ）-exg was transformed into Escherichia coli Rosetta strain for heterologous expression. The result showed that the length of exg gene is 2 415 bp, which encodes 786 amino acids. SDS-PAGE analysis showed that the exo-β-1, 3 glucanase was effectively expressed in E. eoli Rosetta with an apparent molecular masses of 85 kDa. The specific enzyme activity was 45 U/rag after purification. Characterization of the recombinant enzyme showed that it had an exo-hydrolases activity and the optimum pH value was 6.0, and the optimum reaction temperature was at 60 ℃ when laminarin as substrate. Besides, the thermostahility and pH stability give it some prospect in the future.