Protease activity in a paddy field soil: Origin and some properties

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Abstract
Samples from the surface layer of a Gray Lowland Soil in a paddy field were treated with antibiotics and the protease activity in the soil was estimated by the analysis of reaction products from benzyloxycarbonyl-l-phenylalanyl-l-tyrosyl-l-leucine (Z-Phe-Tyr-Leu) as a substrate. Selective inhibition of bacterial and actinomycetes growth in re-moistened, oven-dried and inoculated soil indicated that Leu was the only reaction product when the soil was incubated with the substrate. Selective inhibition of fungal growth in the soil showed that Tyr-Leu and Leu were liberated from the substrate. Protease-active extracts were obtained using 0.1 mol L−1 phosphate buffer, pH 7.0, from the soil samples and treated with ammonium sulfate precipitation followed by dialysis against cold water and centrifugation. The protease activity of the extracts was lost when the preparations were kept at 80°C for 10 min and was optimum at pH 7.0 and 8.0 for Z-Phe-Tyr-Leu and benzyloxycarbonyl-l-phenylalanyl-l-leucine (Z-Phe-Leu), respectively. The preparations hydrolyzed Z-Phe-Leu to Leu and Z-Phe-Tyr-Leu to Tyr-Leu and Leu during the incubation; Tyr-Leu was the main reaction product for the latter substrate. Activity towards Z-Phe-Tyr-Leu and Z-Phe-Leu was inhibited by EDTA and p-chloromercuribenzoate but not by pepstatin and phenylmethanesulfonyl fluoride. The enzyme hydrolyzed angiotensin I (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu). Split sites in the peptide were -Tyr4-Ile5-, -Pro7-Phe8- and -Phe8-His9-. Activity towards -Tyr4-Ile5- and -Pro7-Phe8- was sensitive to EDTA but the activity towards -Phe8-His9- was almost insensitive to EDTA. These results indicate that metalloendopeptidase was the main component of the protease in the extracts from a Gray Lowland Soil in a paddy field.