An Earthworm Protease Hydrolyzes Triacylglycerol
2011
to those of Isozyme C, an isozyme of the earthworm-
serine proteases previously purified by the protease
purification procedures.2,5) Moreover, not only the
protease and esterase activities,4,5) but also the high
BALB- and triolein-hydrolytic activities and the weak
tristealin- and triparmitin-hydrolytic activities were
confirmed in both the purified enzyme and Isozyme C.
Thus, the enzyme showing the triacylglycerol-hydrolytic
activity in the earthworm extract was regarded to be
Isozyme C. However, the amino acid sequence of
Isozyme C showed no similarities to those of the lipase
proteins.12)
Both the purified enzyme and Isozyme C prior to
hydrolyzing the 1- or 3-ester bond of triolein to produce
1,2-diolein and oleic acid in the reaction mixture
contained only 10 mM Tris–HCl buffer (pH 8.0), and
hence, the non-regioselective hydrolysis of the substrate
was accelerated by the addition of n-octyl-ꢁ-D-gluco-
pyranoside (final 4%) to the reaction mixture to produce
1,2-diolein, 1,3-diolein, 2-monoolein, 1-monoolein, and
oleic acid (data not shown). The addition of SDS,
polyvinylalcohol 60, deoxycholate, or co-lipase (por-
cine)13) was not effective for the triolein-hydrolytic
reaction catalyzed by Isozyme C. Although Isozymes A,
B, D, E, and F did not usually represent the triacylgly-
cerol-hydrolytic activity, Isozymes A and B also showed
activity in the presence of n-octyl-ꢁ-D-glucopyranoside.
Hence, the specific activities for fibrin, a protein
substrate,2) of both the present purified enzyme and
Isozyme C were almost same, however, the activity for
triolein-hydrolysis of the purified enzyme tended to be
higher than that of Isozyme C.
The occurrences of the activities of protease, lipase,
amylase, and cellulase were demonstrated in the earth-
worm cells,2,14) however, little information has been
reported concerning the purification and characterization
of these hydrolases except for the serine proteases.2) The
cellulase activity in the earthworm cells was very weak
(our preliminary result). As described above, only the
active fraction showing the triacylglycerol-hydrolytic
activity catalyzed by Isozyme C was found, while the
other lipase proteins did not exist in the earthworm
extract. Although the fact that serine proteases represent
esterase activity is well known,2,5) the results in this
study suggest that an isozyme of the earthworm
proteases, Isozyme C, might play a role in the hydrolysis
of triacylglycerol as well as in protein decomposition.
This is an actual example that the triacylglycerol-
hydrolytic activity was derived from the catalytic
function of a serine protease, of which the amino acid
sequence resembled those of both trypsin and elastase,
but not the amino acid sequence of the lipase proteins.12)
Glass Foundation (2000–2001 and 2003–2005), Japan.
This study was also promoted as ‘‘Collaborative re-
search: type B (2003–2004)’’ in the Collaborative
Research Center of Okayama University.
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This work was supported financially by The Asahi