Characterization and Secretive Expression in Bacillus subtilis of Endoglucanase from Bacillus safensis Isolated from Freshwater Swamp Forest

Pimpikar KANCHANADUMKERNG, Makiko SAKKA, Kazuo SAKKA, Chanpen WIWAT

Abstract


Bacillus safensis M3 was newly isolated from freshwater swamp forest soil in western Thailand. The endoglucanase gene of B. safensis M3, cel9A, had an open reading frame of 1,848 bp encoding a 616 amino acid protein. Initial expression in Escherichia coli yielded a low amount of soluble protein in the cytosolic and secreted fractions. Cel9A was successfully expressed by recombinant B. subtilis with a 4-fold greater total enzyme activity than from recombinant E. coli. By SDS-PAGE analysis, the molecular weight of Cel9A was estimated to be 70 kDa. The optimal temperature of Cel9A was 55 °C and the optimal pH was 5 - 8. Cel9A had the highest activity in the pH range from 5 - 8, and the highest stability in pH range 4 to 10, which is useful for industrial applications. Notably, Cel9A was able to hydrolyze both mixed linkage glucan (lichenan) and hemicellulose (konjac glucomannan and oat spelt xylan) better than carboxymethylcellulose. Cel9A also showed a tolerance to metal ions and surfactants. In addition, recombinant B. subtilis with endoglucanase activity has potential for biotechnological applications and benefits in the optimization of large scale enzyme production with minimal medium using agriculture wastes and other inexpensive feedstock materials.


Keywords


Bacillus safensis, cellulases, endoglucanase, recombinant expression, freshwater swamp forest

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References


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