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Cloning of the Cellulase Gene and Characterization of the Enzyme from a Plant Growth Promoting Rhizobacterium, Bacillus licheniformis K11  

Woo, Sang-Min (Department of Applied Microbiology, Yeungnam University)
Kim, Sang-Dal (Department of Applied Microbiology, Yeungnam University)
Publication Information
Applied Biological Chemistry / v.50, no.2, 2007 , pp. 95-100 More about this Journal
Abstract
The cellulase gene of Bacillus licheniformis K11 which has plant growth-promoting activity by auxin and antagonistic ability by siderophore was cloned in pUC18 using PCR employing heterologous primers. The 1.6kb PCR fragment contained the full sequence of the cellulase gene, denoted celW which has been reported to encode a 499 amino acid protein. Similarity search in protein data base revealed that the cellulase from B. licheniformis K11 was more than 97% identical in amino acid sequence to those of various Bacillus spp. The cellulase protein from B. licheniformis K11, overproduced in E. coli DH5${\alpha}$ by the lac promoter on the vector, had apparent molecular weight of 55 kDa upon CMC-SDS-PAGE analysis. The protein not only had enzymatic activity toward carboxymethyl-cellulose (CMC), but also was able to degrade insoluble cellulose, such as Avicel and filter paper (Whatman$^{\circledR}$ No. 1). In addition, the cellulase could degrade a fungal cell wall of Phytophthora capsici. Consequently B. licheniformis K11 was able to suppress the peperblight causing P. capsici by its cellulase. Biochemical analysis showed that the enzyme had a maximum activity at 60$^{\circ}C$ and pH 6.0. Also, the enzyme activity was activated by Co$^{2+}$ of Mn$^{2+}$ but inhibited by Fe$^{3+}$ or Hg$^{2+}$. Moreover, enzyme activity was not inhibited by SDS or sodium azide.
Keywords
PGPR; Cloning; Cellulase; Bacillus licheniformis K11; PCR(polymerase chain reaction);
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Times Cited By KSCI : 7  (Citation Analysis)
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