2.8 Preparation of recombinant protein
The methods of protein expression, purification and Western blot followed by the previously reported; more specific parameters were showed in supplement figure (Supplementary Table 2).
2.9 Enzyme activity assays
The α-amylase activity was tested using an amylase activity assay kit (Sigma-Aldrich, MO, USA) according to the manufacturer’s protocol. Briefly, 20 uL of purified α-amylase expressed in the Escherichia. coli expression system and 30 µL of Amylase assay buffer was added to each well of the microplate. The reaction was initiated by adding 100 µL of the Master reaction mix and mixed using a horizontal shaker. After 3 min, an initial optical density was read at 405 nm. The plate was incubated at 25 °C and measured the absorbance (405 nm) every 5 minutes. One unit is the amount of amylase that cleaves ethylidene-pNP-G7 to generate 1.0 µmole of p-nitrophenol (p-NP) per minute at 25 °C.
The CYP6AE76 activity was assessed according to the method reported by Qian et al. (Qian et al., 2008) and Shabbir et al. (Shabbir et al., 2021) with slight modification. A 125 µL of 2 mM p-nitroanisole (p-NA) solution and 50 µL cytochrome P450 monooxygenase expressed in E. coli were added to each well of a microplate and mixed. This mixture was incubated at 27 °C for 2 min, and the reaction was initiated by the addition of 25 uL of 9.6 mM NADPH. The optical density at 405 nm was recorded using a microplate reader (FlexStation 3, Molecular Devices, CA, USA).