2.6. Enzyme expression and purification
The daadh gene encoding the wild type D-amino acid dehydrogenase (DAADH) was synthesized by Sangon Biotech (Shanghai) Co. Ltd. DAADH originates from Ureibacillus thermosphaericus (PDB ID: 5gz6) was selected. An expression vector for DAADH_5gz6 with polypeptide linker (amino acid sequence (RTHRK)4) were constructed by amplifying the DAADH_5gz6 gene fragment infusion by PCR.
The amplified gene fragment was double-digested with NdeI and XhoI and ligated cloned into plasmid pET-28a(+), with the expression vector E.coli BL21(DE3)/pET28a. The plasmid extraction and gel recovery kit (Takara) was used. Cells were cultivated at 37 °C and 200 r/min in 10 mL of LB medium containing 50 μg/L kanamycin until the optical density (OD) at 600 nm reached 0.6-0.8. Then, gene expression was induced by adding isopropyl β-D-thiogalactopyranoside (IPTG) for an additional 12 h at 25 °C.
The harvested cells were washed twice with 20 mM Tris-HCl buffer (pH 7.0) and suspended in a buffer of 50 mM Na2HPO4 (pH 7.0). Suspended cells were disrupted by ultrasonication and centrifuged at 100,000×g for 1 h. The supernatant was purified using a Ni2+-NTA column (Seven sea biotech, Shanghai, China) to obtain samples for activity assay.