Purification and Some Properties of Carboxyl Proteinase in Extract from Lentinus edodes Fruit-bodies

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To clarify the function of carboxyl proteinase inhibitor (S-PI, Pepstatin Ac) in the fruit-body formation of Basidiomycetes, we purified the carboxyl proteinase in the extract from Lentinus edodes fruit-bodies. About 70mg of purified enzyme was obtained from 6kg of wet fruit-bodies, with 20% recovery. The enzyme showed a single protein band on polyacrylamide gel electrophoresis. The molecular weight and isoelectric point were 42, 000 and pH 4.5, respectively. The enzyme contained no arginine, and the contents of lysine and histidine residues were higher than those of other carboxyl proteinases in vegetative mycelium or culture filtrates. The enzyme was most active between pH 2.5-2.8, and stable over a range of pH 3.1-5.7 when incubated at 37°C, for 3 hr. The enzyme was inhibited by S-PI, DAN, and EPNP, which are specific inhibitors for carboxyl proteinases. The rate of inhibition by S-PI was very different from that in other carboxyl proteinases described above. The enzyme preferentially split the Leu(15)-Tyr(16) bond of oxidized insulin B-chain at the rate of 70% for total hydrolysis. These characteristics are compared with those of other carboxyl proteinases.
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