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KMID : 0903520070500020095
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Journal of the Korean Society of Agricultural Chemistry and Biotechnology
2007 Volume.50 No. 2 p.95 ~ p.100
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Cloning of the Cellulase Gene and Characterization of the Enzyme from a Plant Growth Promoting Rhizobacterium, Bacillus licheniformis K11
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Woo Sang-Min
Kim Sang-Dal
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Abstract
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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 1.6 kb 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 DH5α 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® 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 60oC and pH 6.0. Also, the enzyme activity was activated by Co2+ or Mn2+ but inhibited by Fe3+ or Hg2+. Moreover, enzyme activity was not inhibited by SDS or sodium azide.
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KEYWORD
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PGPR, Cloning, Cellulase, Bacillus licheniformis K11, PCR (polymerase chain reaction)
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