Recombination and expression of a novel endo-β-glucanase gene from penicillium echinulatum in trichoderma reesei

被引：0
|
作者：
Xia Y. [1 ]
Zhao J. [1 ]
Xia L.-M. [1 ]
机构：
[1] Key Laboratory of Biomass Chemical Engineering of Ministry of Education, College of Chemical and Biological Engineering, Zhejiang University, Hangzhou
来源：
Gao Xiao Hua Xue Gong Cheng Xue Bao/Journal of Chemical Engineering of Chinese Universities | 2016年 / 30卷 / 03期
关键词：
Alkali cellulase; Endo-β-glucanase; Expression; Gene recombination; Trichoderma reesei;
D O I：
10.3969/j.issn.1003-9015.2016.00.010
中图分类号：
学科分类号：
摘要：
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 pCAMBIA1300 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. © 2016, Editorial Board of “Journal of Chemical Engineering of Chinese Universities”. All right reserved.
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页码：626 / 632
页数：6
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