Molecular Biology Reports

, Volume 37, Issue 8, pp 3721–3728 | Cite as

Cloning and expression of a cellulase gene in the silkworm, Bombyx mori by improved Bac-to-Bac/BmNPV baculovirus expression system

  • Xing-hua Li
  • Dan Wang
  • Fang Zhou
  • Hua-jun Yang
  • Roy Bhaskar
  • Jia-biao Hu
  • Chun-guang Sun
  • Yun-gen Miao
Article

Abstract

Cellulases catalyze the hydrolysis of cellulose which are mainly three types: endoglucanases, cellobiohydrolases and β-glucosidases. It can be used in converting cellulosic biomass to glucose that can be used in different applications such as production of fuel ethanol, animal feed, waste water treatment and in brewing industry. In this paper, we cloned a 1380-bp endoglucanase I (EG I) gene from mycelium of filamentous fungus Trichoderma viride strain AS 3.3711 using PCR-based exon splicing methods, and expressed the recombinant EG I mature peptide protein in both silkworm BmN cell line and silkworm larvae with a newly established Bac-to-Bac/BmNPV mutant baculovirus expression system, which lacks the virus-encoded chitinase (chiA) and cathepsin (v-cath) genes of Bombyx mori nucleopolyhedrovirus (BmNPV). An around 49-kDa protein was visualized after mBacmid/BmNPV/EG I infection, and the maximum expression in silkworm larvae was at 84 h post-infection. The ANOVA showed that the enzymes from recombinant baculoviruses infected silkworms exhibited significant maximum enzyme activity at the environmental condition of pH 7.0 and temperature 50°C. It was stable at pH range from 5.0 to 10.0 and at temperature range from 50 to 60°C, and increased 24.71 and 22.84% compared with that from wild baculoviruses infected silkworms and normal silkworms, respectively. The availability of large quantities of EG I that the silkworm provides maybe greatly facilitate the future research and the potential application in industries.

Keywords

Trichoderma viride Cellulase Bombyx mori Exon splicing Bac-to-bac/BmNPV baculovirus expression system 

Supplementary material

11033_2010_25_MOESM1_ESM.doc (252 kb)
Supplementary material 1 (DOC 252 kb)

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Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Xing-hua Li
    • 1
  • Dan Wang
    • 1
  • Fang Zhou
    • 1
  • Hua-jun Yang
    • 1
  • Roy Bhaskar
    • 1
  • Jia-biao Hu
    • 1
  • Chun-guang Sun
    • 1
  • Yun-gen Miao
    • 1
  1. 1.Key Laboratory of Animal Epidemic Etiology & Immunological Prevention of Ministry of Agriculture, College of Animal SciencesZhejiang UniversityHangzhouPeople’s Republic of China

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