3.1.2 Influence of temperature on enzyme activity
The changes in the components of the bioactives during the extraction
process were all related to the content of indican. We hypothesized that
temperature stress during extraction induced cells to synthesize
β-glucosidase, which catalyzed the hydrolysis of indican released from
vacuoles, thereby changing the components of the bioactives.
β-Glucosidase belongs to cellulase and exists in many plants and
microorganisms. To study the influence of temperature on enzyme activity
in leaves, the leaves were treated with water of different temperature
at a solid–liquid ratio of 1:10 for 10 min. Then, the leaves were
thoroughly ground and centrifuged to obtain the crude enzyme solutions
for the determination of β-glucosidase activity. The experimental
results show that there was a great difference in the enzyme activity of
the crude enzyme solutions (Fig. 1e). The crude enzyme solution prepared
at 40 ℃ had an enzyme activity of 3348.87 ± 36.12 U/g. Compared with the
crude enzyme solutions obtained at the other temperatures, the enzyme
activity in leaves was up to 4658.69 ± 64.13 U/g at 50 ℃ and gradually
decreased with the increase in temperature. The β-glucosidase in leaves
was basically inactivated at 70 ℃, at which temperature the enzyme
activity was only 283.98 ± 9.85 U/g. A further increase in temperature
only slightly affected enzyme activity.
Taken together, it was considered that temperature-stress induced the
synthesis of intracellular β-glucosidase, which could hydrolyze indican
to indoxyl and glucose. At lower temperatures, more indoxyl condensed to
form indigo; at relatively high temperatures, indoxyl was unstable and
tended to form isatin, which eventually formed indirubin. At 100 ℃,
because of the low enzyme activity, indican cannot be hydrolyzed, so
there was only indican without indigo and indirubin.