Properties of protease extracted from tea-field soil

Crude enzyme extract was obtained from a low-pH soil from a tea field by sh aking with 0.1 M PO43- buffer (pH 7.0). Hydrolytic activity toward benzylox y-carbonyl-L-Phe-L-Leu (Z-L-Phe-L-Leu) and Z-L-PheL-Tyr-L-Leu showed two pH optima, at about pH 5 and 9, suggesting that the soil contained at least t wo protease components. The acid-type protease in the extract was assumed t o be Ser-carboxypeptidase because phenylmethanesulphonyl fluoride and diiso propylphosphoro fluoridate inhibited its activity. Peptide bonds in the C-t erminal residues of Leu-enkephalin and angiotensin I were split more by pro tease than those in the N-terminal residue. The apparent molecular weight o f the acid-type protease was estimated to be 75 kDa by Sephadex G-100 gel f iltration and the isoelectric point 4.4 by isoelectric focusing. A neutral- type protease in the extract was assumed to be a metallocarboxypeptidase be cause only o-phenanthrorine inhibited its activity. Peptide bonds in the C- terminal residues of Leu-enkephalin and angiotensin I were hydrolyzed to a greater extent than those in the N-terminal residues. The apparent molecula r weight of the neutral-type protease was estimated to be 37 kDa and the is oelectric point 5.8, 8.0 and 9.4. The isoelectric point 9.4 fraction showed the highest relative activity.