PROTEASES AND SOME OF THEIR CHARACTERISTICS IN NITROGEN FIXATION ANABAENA 7120

Subcellular fractions of the blue-green alga were prepared through the treatment with lysozyme and sonication, followed by differential centrifugation and detergent solubilization. The proteolytic activities of the subcellular fractions were determined at 280 nm based on the reaction of α-casein. The results show that there are proteolytic activities in the soluble fraction, outer membrane, intracytoplasmic membrane, and periplasmic space. The activity of the soluble protease of heterocyst was 4—5 times higher than that of the vegetative cell.The properties of the soluble protease of vegetative cell grown in combined nitrogen-free medium were also investigated. The enzyme has an optimum reactive temperature around 50—55℃, and its activity decreased rapidly at 65℃. With the presence of calcium ion, the emzyme is stable at 60℃. Preincubation of the soluble protease at 60℃ for 10 min. without calcium ion resulted in a loss of more than 80% of the original activity. The activity of the enzyme was also found to be optimal at pH 8 to 10. Moreover the levels of the proteolytic activities of the vegetative cells were markedly affected by the pH of the culture medium upon nitrogen starvation. Alkaline condition greatly enhanced the activities.Activities of proteases were found to be inhibited completely by 1 m mol/I o-phenanthrolin but slightly by 1 m mol/I EDTA. Inhibition caused by PMSF was also observed but it varied with the duration of nitrogen starvation of the cells that produced the enzyme.