Cloning and expression of a cellulase gene in the silkworm, Bombyx mori by improved Bac-to-Bac/BmNPV baculovirus expression system.
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.
Li
Xing-hua
XH
Key Laboratory of Animal Epidemic Etiology & Immunological Prevention of Ministry of Agriculture, College of Animal Sciences, Zhejiang University, 310029, Hangzhou, People's Republic of China.
Wang
Dan
D
Zhou
Fang
F
Yang
Hua-jun
HJ
Bhaskar
Roy
R
Hu
Jia-biao
JB
Sun
Chun-guang
CG
Miao
Yun-gen
YG
D000818:Animals; D015153:Blotting, Western; D012831:Bombyx / Q000235:genetics*; D002480:Cellulase / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods*; D004247:DNA / Q000378:metabolism; D007814:Larva / Q000378:metabolism; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D014242:Trichoderma / Q000201:enzymology*
D011994:Recombinant Proteins; D004247:DNA; D002480:Cellulase