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Pine wood nematode cellulase gene cloning and expression, purification and characterization of the Escherichia coli

Author: RenHuiJie
Tutor: GuoDaoSen
School: Qingdao University
Course: Microbiology
Keywords: Bursaphelenchus xylophiuls cellulase gene cloning activity
CLC: S763.1
Type: Master's thesis
Year: 2011
Downloads: 37
Quote: 0
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Abstract


A gene encoding cellulase from Bursaphelenchus xylophilus was cloned by PCR, and showed 99% homology to the reported gene of cellulase BXC10. The gene was cloned into pET-15b to construct expressing vector pET-15b-BXC. Then, this recombinant plasmid was introduced into E.coli BL21(DE3) to construct engineering bacterium. Recombinant cellulase was successfully expressed in engineering bacterium induced by IPTG as analyzed by SDS-PAGE. Recombinant protein was purified by Ni2+-NTA resin and anionic (DEAE) exchange resin, its purification degree was assayed by SDS-PAGE, its cellulase activity was determined by DNS method, and its optimum temperature、the optimal pH、thermal stability and pH stability was also determined by DNS method.The results of SDS-PAGE showed that the purity of recombinant protein was approximate 50% by affinity chromatography, and there was only a uniform band of 40 kDa after purified by anionic (DEAE) exchange resin. The results determined by DNS method showed that recombinant protein expressed by engineering bacteria had good cellulose enzyme activity, the optimal temperature and pH of the recombinant cellulase were 45℃and 4.0, respectively. Experimental results proved that this protein had very good thermal stability. After treated under different temperatures for 1 h, the protein could keep more than 70 percent of the cellulase enzyme activity in a range between 45℃to 65℃, while its cellulase enzyme activity declined rapidly over 70℃. It almost lost cellulase enzyme activity completely when the temperature reached 80℃, and the rest cellulase enzyme activity of the protein was highest at 45℃, up to 98%. The pH stability of this protein was also very good. Its stability rose gradually in a range of pH3.0 to pH5.0, and the left activity was highest at pH5.0. The results can lay the foundation for further research about the role of cellulase in pine wilt disease.

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CLC: > Agricultural Sciences > Forestry > Forest Protection > Forest Pest and Disease Control > Diseases and Their Management
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