Yeast extract was produced from brewer's yeast of a beer factory by combined enzymatic treatments using endoprotease, exoprotease, 5'-phosphodiesterase, and adenosine monophosphate (AMP)-deaminase. Effects of enzyme combination, enzyme dosages and treatment sequence on the recovery of solid and protein, flavor and compositional characteristics were investigated. Exoprotease dosage strongly affected the recovery of protein and degree of hydrolysis (DH) and sensory characteristics. When the yeast cells were treated using optimal combination of endoprotease and exoprotease (0.6% Protamex and 0.6% Flavourzyme), high solid recovery (48.3-53.1%) and the best flavor profile were obtained. Among various treatment sequences using multiple enzymes, treatment with protease followed by nuclease resulted in the highest 5'-guanosine monophosphate (5'-GMP) content. The optimal concentrations of both 5'-phosphodiesterase and AMP-deaminase were found to be 0.03%. After treatments using optimal combination of enzyme, enzyme dosages and treatment sequence for four enzymes, a high solid yield of 55.1% and 5'-nucleotides content of 3.67% were obtained.