Enhanced expression of endoinulinase from Aspergillus niger by codon optimization in Pichia pastoris and its application in inulooligosaccharide production

Abstract

In the present study, the endoinulinase gene (EnInu) from Aspergillus niger CICIM F0620 was optimized according to the codon usage of Pichia pastoris and both the native and the optimized gene were expressed in P. pastoris. Use of the optimized gene resulted in the secretion of recombinant endoinulinase activity that reached 1,349 U ml(-1), 4.18 times that observed using the native gene. This is the highest endoinulinase activity reported to date. The recombinant enzyme was optimally active at pH 6.0 and 60 °C. Moreover, inulooligosaccharides production from inulin was studied using the recombinant enzyme produced from the optimized gene. After 8 h under optimal conditions, which included 400 g l(-1) inulin, an enzyme concentration of 40 U g(-1) substrate, 50 °C and pH 6.0, the inulooligosaccharide yield was 91 %. The high substrate concentration and short reaction time described here should reduce production costs distinctly, compared with the conditions used in previous studies. Thus, this study may provide the basis for the industrial use of this recombinant endoinulinase for the production of inulooligosaccharides.

Genetic Modification of the Marine-Isolated Yeast Aureobasidium melanogenum P16 for Efficient Pullulan Production from Inulin

“…So far, inulinase genes from different sources have been overexpressed in the Saccharomyces cerevisiae mutant (Δgal80), Saccharomyces sp. W0, Pichia pastoris, and Yarrowia lipolytica ACA-DC 50109 Liu et al 2011;Wang et al 2011;Zhang et al 2010;Liu et al 2012;He et al 2014). Therefore, this is the first time to show that the inulinase gene from K. maximum can be actively expressed in A. melanogenum.…”

Cloning, Expression, Characterization, and Mutagenesis of a Thermostable Exoinulinase From Kluyveromyces cicerisporus

“…Inulin is a kind of natural storage carbohydrate that exists in the roots and tubers of many plants, such as Jerusalem artichoke, chicory, and dahlia [1,2]. Inulin contains glucosyl and fructosyl moieties linked by a β-2,1-glycosidic bond and terminates with a glucose residue at the reducing end [3].…”

Expression of recombinant myostatin propeptide pPIC9K-Msp plasmid in Pichia pastoris

“…; 3) protein expression is flexible, can be either intracellular or extracellular, with high density growth; 4) the exogenous gene can be integrated into the yeast chromosome by homologous recombination, resulting in genetic stability; 5) P. pastoris has low levels of protein secretion, thus allowing the exogenous protein to become the dominant protein in the growth media; 6) Pichia pastoris has low levels of glycosylation compared with Saccharomyces cerevisiae. In Pichia pastoris, the sugar chain contains 8-14 mannose residues in each side chain, which is much shorter than the 50-150 mannose residues on each side chain in Saccharomyces cerevisiae (Han 2006;Liu et al, 2013;He et al, 2014).…”