*3.2. Adk Preparation and Concentration Determination*

The DNA fragment encoding Adk was obtained from the cDNA library by PCR from the midgut of *Bombryx mori* strain Dazao using primer sets (5 -ATGGCACCGGCCGCTGC-3 and 5 -TTACAAA GCAGACCGTGCTCTGCTG-3 ). The amplification product was gel-purified, recovered, and inserted into plasmid vectors pSKB2. The bacterial transformants containing error-free inserts were identified. Adk was expressed in *Escherichia coli* BL21(DE3) and purified by Ni-NTA affinity chromatography (GE Healthcare, Chicago, IL, USA). The fused polyhistidine tag was cleaved by Prescission protease (GE Healthcare, USA) and removed as described by Liu et al. [16]. Protein concentration was determined using the extinction coefficient of 12,950 M−1·L·cm−<sup>1</sup> at 280 nm on a NanoDrop 2000C spectrophotometer (Thermo Fisher, Waltham, MA, USA).

#### *3.3. Adk Activity Assay*

ATP and AMP were used as the substrates in the assay. The reaction mixture (1 mL) was composed of 2 mM ATP, 0.6 mM AMP, 0.1 mM glycine–NaOH (pH 9.0), 0.093 mM bromothymol blue, and 5 mM MgAC2. The initial absorbance of the freshly prepared reaction mixture at 614 nm was adjusted to 1.05 with approximately 0.5 M NaOH so that the absorbance of the mixture would not be changed after the addition of Adk. The purified Adk was exchanged into buffer A (20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 5% glycerol, and 0.1 mM dithiothreitol (DTT)) via gel filtration. The reaction was triggered by adding 15 μg Adk into the mixture. The reaction velocity was defined as the slope of the absorbance change of bromothymol blue at 614 nm in the initial 30 s, which was recorded on a DU 800 nucleic acid/protein analyzer (BeckmanCoulter, Brea, CA, USA) using a 1-cm light path plastic cuvette. For the control reaction, Adk was replaced with buffer A. All measurements were carried out at 25 ◦C. Each test was replicated at least three times.
