嗜热子囊菌光孢变种内切葡聚糖酶I基因在毕赤酵母中的表达及部分酶学性质  

Expression of Thermoascus aurantiacus var.levisporus Endo-β-glucanase I Gene in Pichia pastoris and Anylysis of Enzymic Properties

作　　者：张亚波[1] 刘连盟[2] 徐荣燕[1] 李多川[1] 

机构地区：[1]山东农业大学环境生物系,泰安271018 [2]中国水稻研究所,杭州310006

出　　处：《应用与环境生物学报》2009年第3期419-422,共4页Chinese Journal of Applied and Environmental Biology

基　　金：国家“863”计划项目(Nos.2006AA10Z304,2008AA05Z403)~~

摘　　要：嗜热子囊菌是一种嗜热真菌,可以产生具有很高工业价值的内切葡聚糖酶.本研究成功表达了嗜热子囊菌内切葡聚糖酶Ⅰ基因,并获得热稳定的重组内切葡聚糖酶.提取嗜热子囊菌光孢变种(Thermoascus aurantiacus var.levisporus)总RNA,通过RT-PCR方法克隆出内切-β-葡聚糖酶eg1基因的成熟肽编码序列.采用基因重组的方法构建该基因的巴斯德毕赤酵母Pichia pastoris分泌型表达载体pPIC9K-eg1,经线性化后采用电穿孔法将其导入毕赤酵母GS115中,大量筛选后获得高效表达内切葡聚糖酶Ⅰ的毕赤酵母工程菌株GpN24.该菌株采用甲醇诱导120h后,内切葡聚糖酶Ⅰ的活力可达570.7U/mL,最适温度为55℃,在90℃的条件下保温30min后仍具有60%的酶活力;最适pH为5.0,在pH3.0～5.0的条件下酶活力保持稳定.The thermophilic fungus Thermoascus aurantiacus vat. levisporus was studied and it was found able to produce thermostable endo-1,4-β-glucanase with high industrial value. The thermostable endo-1,4-β-glucanase was obtained through Pichia pastoris expression. The egl gene was isolated from T. aurantiacus var. levisporus by RTPCR. A DNA fragment coding for egl without signal sequence was cloned into P. pastoris expression vector pPIC9K for extracellular expression in P. pastoris. The resulted recombinant plasmid pPIC9K-egl was then linearized and transformed into P. pastoris GS115 by electroporation. The egl gene was integrated into the genome of P. pastoris through homologous recombination and a multi-copy expression strain, named GpN24, was obtained through high throughput screening with G418. The endo-1,4-β-glucanase secreted by GpN24 reached a final yield of 570.7 U/mL after methanol induction for 120 h. The optimum pH and temperature of the recombinant enzyme activity were 5.0 and 55 ℃, respectively. The recombinant enzyme remained 90% of its original activity after 30 min at 90 ℃.

关 键 词：纤维素酶 内切葡聚糖酶 嗜热子囊菌光孢变种 嗜热真菌 毕赤酵母 

分 类 号：Q78[生物学—分子生物学]