High-cell density cultivation of recombinant Pichia pastoris harboring fungal endoinulinase and prebiotics applications of inulo-oligosaccharides

摘要

Aim : The objective of the present study was to clone and express A. nigerendoinulinase 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.[GRAPHICS]Results : The endoinulinase gene of 1482 bp from Aspergillus nigerwas genetically engineered in the GS115 host and was secreted extracellularly using a signal sequence. As a result of fermentation with the conventional approach, recombinant endoinulinase activity was enhanced to 65.7 U ml. 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.