Expression and characterization of an endo-1,4-β-galactanase from Emericella nidulans in Pichia pastoris for enzymatic design of potentially prebiotic oligosaccharides from potato galactans.
Potato pulp is a high-volume side-stream from industrial potato starch manufacturing. Enzymatically solubilized β-1,4-galactan-rich potato pulp polysaccharides of molecular weights >100 kDa (SPPP) are highly bifidogenic in human fecal sample fermentations in vitro. The objective of the present study was to use potato β-1,4-galactan and the SPPP as substrates for enzymatic production of potentially prebiotic compounds of lower and narrower molecular weight. A novel endo-1,4-β-galactanase from Emericella nidulans (anamorph Aspergillus nidulans), GH family 53, was produced in a recombinant Pichia pastoris strain. The enzyme was purified by Cu(2+) affinity chromatography and its optimal reaction conditions were determined to pH 5 and 49°C via a statistical experimental design. The specific activity of the E. nidulans enzyme expressed in P. pastoris was similar to that of an endo-1,4-β-galactanase from Aspergillus niger used as benchmark. The E. nidulans enzyme expressed in P. pastoris generated a spectrum poly- and oligo-saccharides which were fractionated by membrane filtration. The potential growth promoting properties of each fraction were evaluated by growth of beneficial gut microbes and pathogenic bacteria. All the galactan- and SPPP-derived products promoted the growth of probiotic strains of Bifidobacterium longum and Lactobacillus acidophilus and generally did not support the propagation of Clostridium perfringens in single culture fermentations. Notably the growth of B. longum was significantly higher (p<0.05) or at least as good on galactan- and SPPP-derived products as fructooligosaccharides (FOS). Except in one case these products did not support the growth of the pathogen Cl. perfringens to any significant extent.
Center for BioProcess Engineering, Department of Chemical and Biochemical Engineering, Building 229, Technical University of Denmark, 2800 Kgs. Lyngby, Denmark.
Malwina
M
Michalak
Lise V
LV
Thomassen
Henna
H
Roytio
Arthur C
AC
Ouwehand
Anne S
AS
Meyer
Jørn D
JD
Mikkelsen
D001419:Bacteria / Q000145:classification / Q000254:growth & development / Q000378:metabolism; D001709:Biotechnology / Q000379:methods*; D003470:Culture Media; D032842:Emericella / Q000201:enzymology* / Q000235:genetics; D005685:Galactans / Q000737:chemistry / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007422:Intestines / Q000382:microbiology; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism*; D010843:Pichia / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D010936:Plant Extracts / Q000737:chemistry / Q000378:metabolism; D056692:Prebiotics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D011198:Solanum tuberosum / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
D003470:Culture Media; D005685:Galactans; D009844:Oligosaccharides; D010936:Plant Extracts; D056692:Prebiotics; D011994:Recombinant Proteins; D006026:Glycoside Hydrolases; C094192:arabinogalactan endo-1,4-beta-galactosidase