Gentio-oligosaccharides (GnOS) are an emerging class of prebiotics derived from ß-1,6-glucan, a polysaccharide found within the cell walls of fungi such as Saccharomyces cerevisiae, or Brewer’s yeast (BSY). The generation of GnOS from BSY by enzymatic hydrolysis could create functional food additives, as well as minimize waste and waste-disposal costs, thereby contributing to the circular economy. In this study, a ß-1,6-glucanase from the mycoparasitic fungus Trichoderma virens was cloned and expressed in Pichia pastoris, and subsequently purified and characterized in order to assess its suitability for the production of GnOS from BSY. Tvir30 was purified by IMAC to a yield of 47.5% with a purification factor of 5.63. The pH and temperature optima of Tvir30 were determined to be 5.0 and 45oC respectively. The enzyme was found to be glycosylated, and substrate specificity analysis revealed it to be specific to ß-1,6-linkages only. Tvir30 was relatively heat sensitive, with 50% relative activity remaining after 15 min incubation at 50oC. However, at 40oC the stability of Tvir30 was improved, with activity reaching 80% at 100 h of incubation. Kinetic analysis revealed classic Michaelis-Menten kinetics, with a Vmax of 214.4 µmol mg-1 min-1, a Km of 1.873 mg mL-1, and a Kcat of 161 sec-1. Finally, analysis of hydrolysis products revealed Tvir30 to have an endolytic mode of action, capable of generating GnOS with DP>2, including from yeast ß-glucan.