To study MK-2461 stability of immobilized enzyme, immobilized 7α- or 7β-HSDH and immobilized preparations were incubated in different pH values using the following buffers: 50 mM Tris–HCl (pH 7.0–9.0) or 50 mM Gly–NaOH (pH 9.0–10.0) at 25 °C for 5 h, respectively. After incubation, substrates were added and the residual activity of the enzyme was determined as described in Section 2.5 and the initial activity without incubation defined as 100%.
2.7. The effects of pH and temperature on the enzymatic reaction
The TCDCA conversion rate and the TUDCA yield rate were assayed through the generation and consumption of NADPH, respectively. Change of absorbance at 340 nm was recorded to measure enzyme activity. To determine the TCDCA conversion rate, the immobilized 7α-HSDH (3 g) assay mixture in a follicles (ovary) total volume of 6 mL contained 2 mM TCDCA in 50 mM Gly–NaOH buffer and 2 mM NADP+. The TUDCA yield rate was determined based on the catalysis of immobilized 7β-HSDH (3 g) in a 6 mL total volume of assay mixture containing 2 mM T-7-KLCA (synthesized according to Supplementary material 3) in 50 mM Gly–NaOH buffer and 2 mM NADPH. All the reactions were performed in triplicate, and both mean values and standard deviations were reported.