Bacteroides thetaiotaomicron is a gram-negative microbe found in the human gut system and is frequently associated with serious wound infections, peritonitis and septicaemia. Recently, a putative hydrolase with unprecedented binding properties was discovered in this microorganism. The occurrence of a binding site for flavin derivatives in addition to a mononuclear zinc and a mononuclear calcium binding site is uncommon for hydrolases. Based on structural relationships this enzyme was classified as a putative protease or molecular chaperone. A special feature of the flavin binding site is its unspecific binding of different forms of flavins (riboflavin, FMN, FAD). Not only naturally occurring, but also chemically modified flavins bind with high affinity between the two tryptophanes provided in the dimer interface. Strength and preference of cofactor binding to the enzyme was analyzed by UV/vis absorbance spectroscopy. The successful binding of flavins to the protein gave rise to a facilitated method of cofactor isolation from various biological sources, such as milk, urea and blood, as well as an improved technique for detection of flavins. Different bovine and human milk samples were used for isolation of riboflavin, FMN and FAD with apo-ppBat. All experiments showed riboflavin as the dominant isolated compound from various milk samples. Furthermore, a distinction between free and protein-bound riboflavin was established. Only a small amount of riboflavin occurred in a free state, the majority was present in a protein-bound form which was recovered by acid precipitation of milk proteins. However, the isolated amounts of riboflavin from the milk samples varied distinctly indicating a high heterogeneity of the composition of milk.