Isolation of Alkaline and Neutral Proteases from Aspergillus flavus var. columnaris , a Soy Sauce Koji Mold

Abstract

Two different extracellular proteases, protease I (P-I), an alkaline protease, and protease II (P-II) a neutral protease, from Aspergillus flavus var. columnaris were partially purified by using (NH4)2SO4 precipitation, diethylaminoethyl-Sephadex A-50 chromatography, carboxymethylcellulose CM-52 chromatography, and Sephadex G-100 gel filtration. The degree of purity was followed using polyacrylamide gel electrophoresis. The activity of P-I was completely inhibited by 0.1 mM phenylmethylsulfonyl fluoride, and that of P-II was completely inhibited by 1 mM ethylenediaminetetraacetate. By using these inhibitors with extracts of wheat bran koji, the proportions of total activity that could be assigned to P-I and P-II were 80 and 20%, respectively. This compared favorably with activities estimated by using polyacrylamide gel electrophoresis slices (82 and 18%, respectively). Extracts from factory-run soybean koji gave comparable results. Both enzymes demonstrated maximum activity at 50 to 55°C and only small changes in activity between pH 6 and 11. For P-I, activity was somewhat higher from pH 8.0 to 11.0, whereas for P-II it was somewhat higher from pH 6 to 9. In the presence of 18% NaCl, the activities of both P-I and P-II dropped by approximately 90 and 85%, respectively. P-I was inferred to possess aminopeptidase activity since it could hydrolyze L-leucyl-p-nitroanilide hydrochloride. P-II was devoid of such activity. The ramifications of the results for factory-produced soy sauce koji are discussed.

Development of a process for producing ethyl caproate- and ethyl lactate-rich riceshochu

“…The flourishing enzyme activities of koji contribute to the rich flavours of rice shochu and they support yeast fermentation . The type of koji is a significant factor in the shochu production process because different koji produce different metabolites and enzymes . The effects of white and yellow koji on shochu production were investigated with the addition of both the CAPB consortium and LAB on the first day of the second‐stage fermentation.…”

Purification and properties of an alkaline proteinase of Fusarium culmorum

“…The enzyme thus functions at pH 6, the physiological pH of grain, but not at its maximal rate. The broad pH optimum of this proteinase resembles those of the␣alkaline proteinases from some Aspergillus species [15–18], but those of Fusarium sp. ‘S‐19‐5’[19] and F. graminearum [20] have somewhat sharper pH optima, at approximately pH 10.…”

Comparative biochemistry of the proteinases of eucaryotic microorganisms.

“…A study has recently been initiated on the proteinases of A. flavus var. columnaris used for the manufacture of soy sauce in Thailand (125).…”