title: Heterologous expression of a bioactive β-hexosyltransferase, an enzyme producer of prebiotics, from Sporobolomyces singularis.
abstract: Galacto-oligosaccharides (GOS) are indigestible dietary fibers that are able to reach the lower gastrointestinal tract to be selectively fermented by health-promoting bacteria. In this report, we describe the heterologous expression of an optimized synthetically produced version of the β-hexosyltransferase gene (Bht) from Sporobolomyces singularis. The Bht gene encodes a glycosyl hydrolase (EC 3.2.1.21) that acts as galactosyltransferase, able to catalyze a one-step conversion of lactose to GOS. Expression of the enzyme in Escherichia coli yielded an inactive insoluble protein, while the methylotrophic yeast Pichia pastoris GS115 produced a bioactive β-hexosyltransferase (rBHT). The enzyme exhibited faster kinetics at pHs between 3.5 and 6 and at temperatures between 40 and 50°C. Enzyme stability improved at temperatures lower than 40°C, and glucose was found to be a competitive inhibitor of enzymatic activity. P. pastoris secreted a fraction of the bioactive rBHT into the fermentation broth, while the majority of the enzyme remained associated with the outer membrane. Both the secreted and the membrane-associated forms were able to efficiently convert lactose to GOS. Additionally, resting cells with membrane-bound enzyme converted 90% of the initial lactose into GOS at 68% yield (g/g) (the maximum theoretical is 75%) with no secondary residual (glucose or galactose) products. This is the first report of a bioactive BHT from S. singularis that has been heterologously expressed.
authors.affiliation: Department of Microbiology, North Carolina State University, Raleigh, North Carolina, USA.
authors.forename: Suzanne F
authors.initials: SF
authors.lastname: Dagher
authors.forename: M Andrea
authors.initials: MA
authors.lastname: Azcarate-Peril
authors.forename: José M
authors.initials: JM
authors.lastname: Bruno-Bárcena
mesh_terms: D001487:Basidiomycota / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005700:Galactosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D005947:Glucose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007785:Lactose / Q000378:metabolism; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D056692:Prebiotics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013696:Temperature
chemical_list: D004791:Enzyme Inhibitors; D056692:Prebiotics; D011994:Recombinant Proteins; D005700:Galactosyltransferases; D005947:Glucose; D007785:Lactose