To verify whether the enzyme(s) in the earthworm extract
catalysed the aldol reaction, some control experiments were
performed (Table 1, entries 11–13). Firstly, the high temperature
(at 100uC for 24 h) pretreated earthworm extract was used to
catalyse the model reaction, which gave the product in a low yield
of 39% with 80:20 dr and 85% ee (Table 1, entry 11),
demonstrating that high temperature treatment caused a decrease
of the catalytic ability of the earthworm extract on the aldol
reaction. Secondly, the Cu2+
pretreated crude earthworm extractwas also used to catalyse the model aldol reaction, and only a low
yield of 11% was obtained with78:22 dr and 86% ee (Table 1,
entry 12). Meanwhile, to exclude the effect of Cu2+
on the
reaction, Cu2+
alone was used to catalyse the model aldol reaction,
and only a trace amount of product was observed on TLC
(Table 1, entry 13), proving that Cu2+
did not catalyse this
transformation. The above control experiments showed that high
temperature and metal ion Cu2+
, as deactivation factors of
enzyme, could greatly decrease the catalytic ability of the crude
earthworm extract on the model aldol reaction. Thus, it can be
inferred that the enzyme(s) in the crude earthworm extract
catalysed the aldol reaction.