一种新型内切-β-葡聚糖酶基因在里氏木霉中的重组与表达  

Recombination and Expression of a Novel Endo-β-glucanase Gene from Penicillium Echinulatum in Trichoderma Reesei

作　　者：夏颖[1] 赵杰[1] 夏黎明[1] 

机构地区：[1]浙江大学生物质化工教育部重点实验室,浙江大学化学工程与生物工程学院,浙江杭州310027

出　　处：《高校化学工程学报》2016年第3期626-632,共7页Journal of Chemical Engineering of Chinese Universities

基　　金：浙江省重点科技创新团队资助项目(2011R50002)

摘　　要：刺糙青霉(Penicillium echinulatum)可产生一种耐热、耐碱的新型内切-β-葡聚糖酶(EGL1),具有重要的工业应用价值。鉴于野生型刺糙青霉的产酶水平低,将该菌的内切-β-葡聚糖酶基因经过密码子优化后,在里氏木霉(Trichoderma reesei)中进行重组与表达,采用里氏木霉强启动子Pcbh1(纤维二糖水解酶Ⅰ)及其信号肽,以pCAMBIA1300为载体骨架构建重组质粒pCB-PET,并采用根瘤农杆菌介导转化技术将重组质粒pCB-PET导入里氏木霉的分生孢子中,在潮霉素抗性平板上筛选获得5个优良的重组转化子。将5个转化子重复传代培养10个批次后,分别提取染色体DNA进行PCR验证,均可检测到目的基因Egl1n(密码子优化后的Egl1)条带,说明该基因已稳定地整合到里氏木霉基因组中。采用SDS-PAGE蛋白电泳对转化子培养液进行检测,获得了与目的基因表达产物相符的蛋白条带(约41 kDa),表明Egl1n已在重组里氏木霉中成功实现了胞外表达。重组转化子在30℃,摇瓶转速200 r×min^(-1)条件下培养48 h,取发酵上清液在pH 8.0,60℃条件下,测得内切-β-葡聚糖酶活力为382.6 U×mL^(-1),是出发菌株的22.5倍。酶学性质研究结果表明:该酶耐热性能较好,在70℃以下性能稳定,其最适催化温度为60℃;该酶在pH 5.0~10.5稳定性较好、具有明显的催化活性,其最适pH为8.0,属于碱性纤维素酶。从而成功地实现了刺糙青霉内切-β-葡聚糖酶基因在里氏木霉中的重组与胞外表达,有关研究结果在里氏木霉纤维素酶的定向进化及其工业化应用中具有重要的促进作用。A novel endo-β-glucanase（EGL1） from mutant Penicillium echinulatum with high thermostability and alkali-resistance has great value in industrial applications. However, large-scale production of EGL1 is difficult due to low enzyme producing capacity of Penicillium echinulatum. In this study, a codon-optimized Egl1 gene（Egl1n） was inserted between Trichoderma reesei strong promoter Pcbh1（including secreting signal peptide sequence） and terminator, and further ligated to p CAMBIA1300 vector to construct a recombination plasmid pCB-PET with a hygromycin B resistance marker. The plasmid pCB-PET was transformed into the conidium of T. reesei ZU-02 via an optimized Agrobacterium tumefaciens mediated transformation, and 5 positive transformants were screened by hygromycin B. The chromosomal DNA from the 5 recombinant transformants was extracted separately after subculture for 10 generations. The integration of Egl1n gene in the chromosomal DNA of T. reesei was further confirmed by PCR detection. SDS-PAGE results show an obvious protein band（approximately 41 kDa） corresponding to the target gene Egl1n from the fermentation broth of the recombinant T. reesei, which proves that the Egl1n gene is extracellularly expressed. Enzyme production by the 5 recombinant transformants was performed in shaking flasks at 30℃ under agitation（200 r×min^-1） for 48 h, and the endo-β-glucanase activity in the culture broth was measured at pH8.0, 60℃. The results show that the activity can reach to 382.6 U×mL^-1, which is 22.5 times of the host strain. The study on enzymatic properties shows that the recombinant EGL1N has high thermostability. It is stable below 70℃ and the optimal temperature for reaction is 60℃. The enzyme also presents good hydrolytic activity and stability over a broad pH range（5.0-10.5）, and the optimum pH value is 8.0. These results play a significant role in the directed evolution of cellulase producing strains for industrial applications.

关 键 词：内切-β-葡聚糖酶 碱性纤维素酶 里氏木霉 基因重组 表达 

分 类 号：Q556.9[生物学—生物化学]