Dpni enzyme

E. coli JM109 served as the host strain for site-specific mutagenesis. E. coli Rosetta gami-B (DE3) (Weidi Biotech, Shanghai, China) was used to express various mutant enzymes. The gene for BbPETase^CD was synthesized by Genewiz (Suzhou, China) and using pET-22b(+) plasmid as gene vector. DNA sequencing was performed by Genewiz (Suzhou, China). 5 mL His Trap^TM FF affinity chromatography column was purchased from GE Healthcare (9 × 15.7 mm, 90 μm, Marlborough, MA, USA). Fast pfu enzyme and DpnI enzyme were purchased from TransGen Biotech (Beijing, China). A plasmid extraction kit was purchased from Beyotime (Shanghai, China). A BCA kit was purchased from Dingguo Biotech (Tianjin, China). A sulfhydryl assay kit was purchased from Bestbio (Shanghai, China). The crystallinity of PET films was measured using DSC by Standard (Qingdao, China). Other reagents were purchased from Sangon Biotech (Shanghai, China), Heowns (Tianjin, China), or Meryer (Shanghai, China).

pET28(a+)-GAD1407 was constructed in our laboratory [8 (link)]. Pfu DNA polymerase and Dpn I enzyme was purchased from TransGen Biotech (Beijing, China). The pET-28a (+)/Escherichia coli BL21 (DE3) system was used for the expression of GAD and subsequent mutants.