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Enzyme assay, cloning and sequencing of novel β-glucosidase gene from Aspergillus niger f321 (unidentified Nigerian strain)

R Auta, A Campbell, I Radecka, P Hooley

Abstract


β-glucosidase is a cellulase enzyme under intense investigation for its potential role in cellulose degradation for the generation of fermentable sugars used in biofuels production. Ten catalytic sites have been identified that are conserved in β- glucosidases from a range of prokaryotes and eukaryotes. NCBI Primer BLAST was used in this study to design primers that successfully clone a partial β-glucosidase gene from an uncharacterised Nigerian strain of the filamentous fungi Aspergillus niger F321 (A. niger F321). Two β-glucosidase genes from A. niger F321 denoted as ANRA12.6 and ANRA12.9 were amplified from genomic DNA using PCR techniques and the amplicons gave estimated PCR products of 1,190 bp and 1,950 bp respectively. Subsequent cloning into E. coli produced positive results for blue/white screening of transformed colonies while the colony PCR of their pDNA gave estimated sizes of 860 bp and 1,600 bp respectively. DNA sequencing confirmed that the chosen A. niger F321 partial β-glucosidase sequences had been successfully cloned. Bioinformatics studies also suggested that the cloned β-glucosidases share some characteristics with their bacterial counterparts. The findings in this study highlight the increasing need for more information on β-glucosidase structure and function.

Keywords: Aspergillus niger, β-glucosidase, cellulase, PCR, sequencing, Bioinformatics




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