Behavior of Endopeptidases of Detergent in Natural Waters

Keiko MIYAMOTO, Shin-ichiro HOSHI and Kensei KOBAYASHI*

Department of Chemistry and Biotechnology, Faculty of Engineering, Yokohama National University; 79-5 Tokiwadai, Hodogaya-ku, Yokohama-shi 240-8501 Japan

Many enzymes are reported to be dissolved in natural waters, and most of them are believed to be released by organisms living in situ. In these days, enzymes are used in industrial products such as detergents, but the behavior of these enzymes is unknown. We studied behavior of endopeptidase activity possibly dissolved in natural waters, since endopeptidases are now artificially added to detergents. For assay of endopeptidase activity, succinyl-L-Ala-Ala-Pro-Phe-p-nitroanilide was used as a substrate, and reaction product, p-nitroaniline, was monitored by spectrophotometrically at 380 nm at 30 °C. The detection limit of the present method was 100 mU dm−3(1 U=1 μmol min−1). Endopeptidase activity of commercial detergents tested is 6.4--11 U g−1. Their activity was completely inhibited by addition of phenylmethanesulfonyl fluoride (known as an inhibitor of serine proteases), and partly inhibited by addition of ethylenediaminetetraacetic acid (known as an inhibitor of metal proteases). Activity is preserved when the detergents were dissolved in river water than in pure water. It was suggested that enzymes added to detergents could be kept active in natural waters for days particularly in winter time. The activity level in river waters is lower than the detection limit by the present methods. Thus more sensitive methods must be developed to determine endopeptidase activity dissolved in river waters.

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