Properties of protease extracted from tea-field soil

 Crude enzyme extract was obtained from a low-pH soil from a tea field by shaking with 0.1 M PO₄ ^3– buffer (pH 7.0). Hydrolytic activity toward benzyloxycarbonyl-L-Phe-L-Leu (Z-L-Phe-L-Leu) and Z-L-Phe-L-Tyr-L-Leu showed two pH optima, at about pH 5 and 9, suggesting that the soil contained at least two protease components. The acid-type protease in the extract was assumed to be Ser-carboxypeptidase because phenylmethanesulphonyl fluoride and diisopropylphosphoro fluoridate inhibited its activity. Peptide bonds in the C-terminal residues of Leu-enkephalin and angiotensin I were split more by protease than those in the N-terminal residue. The apparent molecular weight of the acid-type protease was estimated to be 75 kDa by Sephadex G-100 gel filtration and the isoelectric point 4.4 by isoelectric focusing. A neutral-type protease in the extract was assumed to be a metallocarboxypeptidase because 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 molecular weight of the neutral-type protease was estimated to be 37 kDa and the isoelectric point 5.8, 8.0 and 9.4. The isoelectric point 9.4 fraction showed the highest relative activity. Received: 12 January 1999 / Accepted: 22 June 1999