BIOTECHNOLOGICALLY RELEVANT ENZYMES AND PROTEINS A novel acid-stable, acid-active β-galactosidase potentially suited to the alleviation of lactose intolerance Shane O’Connell & Gary Walsh Received: 19 June 2009 / Revised: 18 September 2009 / Accepted: 20 September 2009 / Published online: 6 October 2009 # Springer-Verlag 2009 Abstract Extracellular β-galactosidase produced by a strain of Aspergillus niger van Tiegh was purified to homogeneity using a combination of gel filtration, ion-exchange, chroma- tofocusing, and hydrophobic interaction chromatographies. The enzyme displayed a temperature optimum of 65 °C and a low pH optimum of between 2.0 and 4.0. The monomeric glycosylated enzyme displayed a molecular mass of 129 kDa and an isoelectric point of 4.7. Protein database similarity searching using mass spectrometry-derived sequence data indicate that the enzyme shares homology with a previously sequenced A. niger β-galactosidase. Unlike currently com- mercialised products, the enzyme displayed a high level of stability when exposed to simulated gastric conditions in vitro, retaining 68±2% of original activity levels. This acid- stable, acid-active β-galactosidase was formulated, along with a neutral β-galactosidase from Kluyveromyces marx- ianus DSM5418, in a novel two-segment capsule system designed to ensure delivery of enzymes of appropriate physicochemical properties to both stomach and small intestine. When subjected to simulated full digestive tract conditions, the twin lactase-containing capsule hydrolyzed, per unit activity, some 3.5-fold more lactose than did the commercial supplemental enzyme. The acid-stable, acid- active enzyme, along with the novel two-segment delivery system, may prove beneficial in the more effective treatment of lactose intolerance. Keywords Lactase . β-galactosidase . Aspergillus niger van Tiegh . Lactose intolerance . Two-segment capsule Introduction Lactose is a disaccharide found in the milk of mammals. It is digested in vivo in the gastro-intestinal tract by lactase phlorizin hydrolase (LPH, β-galactosidase, or lactase), a membrane-bound enzyme of the small intestinal epithelial cells, yielding its constituent monosaccharides glucose and galactose (Holsinger 1988). Intestinal lactase insufficiency results in lactose maldigestion (inability to digest lactose but no symptoms experienced) and lactose intolerance (maldigestion with negative clinical symptoms). β- galactosidase deficiency is the predominantly observed situation in adult humans. It has been reported to be as high as 75% of the world’ s population with greatest prevalence amongst American and Asian populations (Shrier et al. 2008). LPH-deficient individuals have difficulty in consuming milk and other lactose-containing products as its ingestion can result in abdominal pain, diarrhoea, and flatulence (Gaska 1990; Vesa et al. 2000). Their symptoms are often ameliorated by co-consumption of exogenous β-galactosidase enzyme supplements with milk/dairy-containing meals (Medow et al. 1990; Lin et al. 1993; Kanabar et al. 2001; Gao et al. 2002). However, numerous clinical studies evaluating lactase-based digestive supplements have reported limited and variable success in this regard (Dipalma and Collins 1989; Medow et al. 1990; Sanders et al. 1992; Lin et al. 1993; Ramirez et al. 1994; Gao et al. 2002). Studies in our laboratory have indicated that these commercial products contain enzymes with physicochemical characteristics that are not ideally suited to lactose hydrolysis S. O’Connell Shannon Applied Biotechnology Centre, IT Tralee, Tralee, Co.Kerry, Ireland G. Walsh (*) Department of Chemical and Environmental Sciences, and Materials and Surface Science Institute, University of Limerick, Limerick, Ireland e-mail: Gary.Walsh@ul.ie Appl Microbiol Biotechnol (2010) 86:517–524 DOI 10.1007/s00253-009-2270-7