Molecular cloning and expression of the cellulase gene from halophiles Bacillus licheniformis NTU-01
Date Issued
2011
Date
2011
Author(s)
Lin, Pei-Yin
Abstract
Endocellulase plays an important role in the degradation of fiber wastes, it can cleave the β-1,4-glycosidic bond of cellulose to release glucose, which has great potential in applications such as biomass energy. A β-1,4-endoglucanase (EC 3.2.1.4) gene belonging to the glycosyl hydrolase family 5 was cloned from the halophilic bacteria Bacillus licheniformis NTU-01, isolated from saline area in southern Taiwan. The target gene named bglC6B contains a complete ORF of 1,548 nucleotides that translates 515 amino acid residues, it showed 99% homology to Bacillus sp. NBL420 celA gene(AAK73277.1). The bglC6B gene was subcloned into pET-28b vector and heterogenously expressed by E. coli BL21 (DE3). Recombinant BglC6B, which was purified by fast protein liquid chromatography (FPLC) and based on SDS-PAGE analysis, the recombinant BglC6B showed a molecular mass of 50 kDa. The CMCase activity of purified, recombinant BglC6B is 14.97 U/ml. The enzyme showed an optimal pH and temperature of pH5 and 30℃, it was very stable at the broad temperature range from 20 to 60℃, and it retained 72% relative activity after 4 h incubation at 40℃. The effect of salinity was indicated that the enzyme was active over a range of 0-15% sodium chloride. These results indicate this recombinant BglC6B is active and stable in some extreme environment stress such as high salt concentration, which is maybe suitable for cellulose biodegration of saline agricultural wastes to cellulolytic ethanol.
Subjects
endoglucanase
bglC6B gene
halostable
heterologous expression
Type
thesis
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