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Applied Microbiology and Biotechnology

, Volume 96, Issue 4, pp 951–962 | Cite as

Expression and characterization of a novel metagenome-derived cellulase Exo2b and its application to improve cellulase activity in Trichoderma reesei

  • Alei Geng
  • Gen Zou
  • Xing Yan
  • Qianfu Wang
  • Jun Zhang
  • Fanghua Liu
  • Baoli Zhu
  • Zhihua ZhouEmail author
Biotechnologically relevant enzymes and proteins

Abstract

A metagenomic fosmid library containing 1 × 105 clones was constructed from a biogas digester fed with pig ordure and rice straw. In total, 121 clones with activity of 4-methylumbelliferyl-cellobiosidase were screened from the metagenomic library. A novel GH5 cellulase gene exo2b was identified from a sequenced clone EXO02C10 and expressed in Escherichia coli BL21. The corresponding recombinant Exo2b protein showed high specific activity toward both carboxymethylcellulose (CMC; 260 U/mg protein) and β-d-glucan from barley (849 U/mg), with an optimal pH and temperature of 7.5 and 58 °C, respectively. Exo2b showed stable activity at a wide pH range from 5.5 to 9.0 and was highly thermostable at 60 °C in the presence of 60 mM cysteine. Residual activity was maintained at nearly 100% when Exo2b was incubated at 60 °C for 15 h. A thin-layer chromatography analysis of the hydrolysis products confirmed that Exo2b was an endo-β-1,4-glucanase and it could also produce oligosaccharide smaller than cellotetraose. The fragment encoding the Exo2b catalytic domain was then fused with the cbh1 gene from Trichoderma reesei, and the fused gene was successfully expressed in T. reesei Rut-C30. Compared to that of the parent strain, the filter paper activity and CMCase activity of the secreted proteins of a selected transformant A1 increased by 24% and 18%, respectively. Besides, the glucose concentration from the hydrolysis of pretreated corn stover by the A1 secreted proteins increased by 19.8%. The present study demonstrated the potential application of metagenome originated cellulase genes to modify cellulase producing fungi.

Keywords

Metagenome Cellulase Gene fusion Fungi Pretreated corn stover 

Notes

Acknowledgments

This work was financially supported by National Basic Research Program of China (973 Program) (2011CB707403) and the Knowledge Innovation Program (nos. KSCX1-YW-11B3 and KSCX2-EW-J-12) and International Joint Research Program (GJHZ1128) from the Chinese Academy of Sciences.

Supplementary material

253_2012_3873_MOESM1_ESM.doc (72 kb)
Fig. S1 Schematic representation of the fusion gene tc2bh, drawn to scale. Some expressed fusion proteins would be expected to be cleaved at the kexin site by the extracellular proteinases of T. reesei (DOC 72 kb)
253_2012_3873_MOESM2_ESM.doc (120 kb)
Fig. S2 SDS–PAGE analysis of the purified Exo2b enzyme. Lane 1 the crude Exo2b enzyme from E. coli BL 21 periplasm, lane 2 ultrafiltrates from the 250-mM imidazole washout, lane 3 ultrafiltrates from the 500-mM imidazole washout, lane 4 protein markers (DOC 119 kb)
253_2012_3873_MOESM3_ESM.doc (172 kb)
Fig. S3 The expression of the tc2bh fusion gene in Trichoderma reesei Rut-C30. Lanes 1 and 2 the total secreted proteins in the culture filtrates from the parent strain Rut-C30 and the A1 transformant, lane 3 the purified Tc2bh using a Ni-column, lane 4 verification of tc2bh expression by western blotting using an anti-His antibody, lane M protein markers (DOC 172 kb)

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Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Alei Geng
    • 1
  • Gen Zou
    • 1
  • Xing Yan
    • 1
  • Qianfu Wang
    • 1
  • Jun Zhang
    • 1
  • Fanghua Liu
    • 1
  • Baoli Zhu
    • 2
  • Zhihua Zhou
    • 1
    Email author
  1. 1.Key Laboratory of Synthetic Biology, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological SciencesChinese Academy of SciencesShanghaiChina
  2. 2.Institute of MicrobiologyChinese Academy of SciencesBeijingChina

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