Centre for Biotechnology,
Anna University, Chennai-600
*Corresponding Author Email :
Aim: The objective of
the present study was to clone and express A. niger endoinulinase gene
in P. pastoris for high-level expression. Further to explore high cell
density cultivation, biochemical characterization of recombinant
endoinulinase and application of inulo-oligosaccharides (IOS) as prebiotics
was also studied.
Methodology: Molecular cloning
of A. niger endoinulinase gene in P. pastoris, screening of
positive clones by genomic DNA PCR, shake flask studies, high cell density
fermentation performed with both conventional and temperature shift approach,
biochemical characterization of endoinulinase and in-vitro
fermentation of IOS was carried out to confirm prebiotic efficacy.
Results: The endoinulinase
gene of 1482 bp from Aspergillus niger was genetically engineered in
the GS115 host and was secreted extracellularly using α signal
sequence. As a result of fermentation with the conventional approach,
recombinant endoinulinase activity was enhanced to 65.7 U ml-1.
Recombinant endoinulinase showed absolute substrate specificity for inulin,
hydrolyzing inulin to IOS with the DP range 3-4.
Interpretation: Hydrolysis of
inulin by recombinant endoinulinase was characterized. In-vitro
fermentation of IOS by lactic acid and bifidogenic bacteria was studied as a
part of industrial application and functional properties of IOS was similar
to commercial prebiotics.
Key words: Aspergillus niger,
Endoinulinase, Inulin, Pichia pastoris, Prebiotics
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