High level production of fungal recombinant inulinase in high-cell-density cultures of Pichia pastoris

CHESINI, M.; VITA, C.E.; WAGNER, E.; ROJAS, N.L.; HOURS, R.A.; GHIRINGHELLI, P.D.

Mar del Plata

Congreso; LI Reunión de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB); 2015

Sociedad Argentina de Investigación Bioquímica y Biología Molecular (SAIB)

Inulinases comprise an important group of enzymes for fructose syrups and fructooligosaccharides production, extensively used as sweeteners and functional food additives. Aspergillus kawachii produces inulinases (INU), but due to its low expression levels, its cloning and over expression were required for its industrial application. A. kawachii INU ORF, was cloned into Pichia pastoris expression vector pPICZαA and expressed in X-33 P. pastoris cells. After the selection of a high expressing clone, it was possible to design an suitable fermentation process in order to obtain high levels of inulinase activity. This process was carried out in fed batch system, limited by methanol concentration, regulated by oxygen consumption. The yield of recombinant inulinase in a 5-L bioreactor reached 681 U/mL after 27,8 h of induction, which means 7000 times higher enzyme production than that obtained from wild type A. kawachii cultures. After production process, the enzyme was purified to homogeneity using chromatographic techniques, and the biochemical characterization was performed. By the strategy proposed, it was possible to obtain a stable and robust biocatalyst in appropriate concentrations levels to be applied in industrial processes for the production of high fructose syrup.