Cloning and over-expression of inulinase gene from Aspergillus kawachii in Pichia pastoris

CHESINI, M.; ROJAS, N. L; FRATEBIANCHI, D.; CAVALITTO, S. F; GHIRINGHELLI, P. D

Mendoza

Congreso; 48º Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular (XLVIII Saib); 2012

Sociedad Argentina de Investigación Bioquímica y Biología Molecular

Inulinases comprise an important group of enzymes for fructose 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 are required for its industrial application. A. kawachii INU ORF, lacking its original signal peptide, was cloned into a Pichia pastoris expression vector under the regulation of the a-mating factor signal sequence. Since INU primary transcript presents an intron which P. pastoris is not able to remove during maturation, its in vitro deletion was developed; generating the pPICZ-a-a-INUDIDSP construction. After plasmid propagation in E. coli DH5á, this construction was used to transform P. pastoris GS115 cells. The INU gene was integrated into the yeast genomic DNA, confirmed by colony PCR. All analyzed clones induced by methanol, were able to express an inulinase after 24 hours, identified by tryptic digestion followed by MALDI TOF analysis. Nevertheless, the expressed recombinant protein was no active under standard inulinase activity assay. A new cloning strategy is under development based on bioinformatics tools, towards the analysis of intron and signal peptide sequences and/or potential post transductional modifications in order to consider a more appropriate expression system, such as S. cerevisiae.