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.