Culture filtrates of different fungi of the genus Rhizopus forming tempe (i.e. traditional Indonesian food) were grown on a soybean protein-raffinose-phytate medium and investigated for protease activity using soyprotein as substrate. All isolates belonging to the species R. oryzae, R. stolonifer, R. oligosporus, and R. microsporus var. chinensis, formed the wellknown Rhizopus-pepsin (aspartic proteinase, 35 kD, isoelectric points: 5.9, 5.0, <4) and an additional protease mainly active under alkaline conditions. The new protease (33 kD, isoelectric points: variable and isolate specific) was purified approximately 300-fold and shown to be a serine protease (inhibitor studies). During fungal culture (12 -135 h) the aspartic proteinase is expressed first followed by the serine protease. Both proteases are insensitive to the soybean Kunitz and Bowman-Birk inhibitors. The best rate of soyprotein degradation is achieved by the coordinate action of both proteases at pH 6.5. The examined Rhizopus isolates differ in the time course and intensity of the expression of the alkaline protease

Expression of proteolytic activities by nine Rhizopus strains during soybean fermentation to produce Tempeh has been determined. All strains showed activity at acidic, neutral, and alkaline pH values. In protein solubilizates prepared from Tempeh pronounced differences in the time course and intensity of maximum protease activities were observed between the isolates when measured at pH 7, the approximate pH of Tempeh during ripening. Proteases expressed by fungi during growth on Tempeh were solubilized and the enzymes were enriched by acetone precipitation and chromatographies with Phenylsuperose, Mono S, and Mono P, respectively. They were identified as one aspartic‐ (∼35 kD) and one serine (∼33 kD) protease, each existing in different isoforms. Proteases isolated from Tempeh were identical to those expressed during fungal growth in a soyprotein‐raffinosephytate liquid medium. The temperature stability of the Tempeh‐ and the cell wall‐isolated proteases is very low in contrast to the proteases secreted into the medium. Proteases isolated from Tempeh, cell walls and liquid media exhibit differences in the number of alkaline isoforms.