Abstract:

Objective: To construct a recombinant strain to express adenosine deaminase (AMPD) efficiently. Methods: The
AMPD gene from Streptomyces murinus was expressed in E. coli DE3/pET28α-AMPD. Specific primers were designed
to amplify the AMPD gene. The PCR product was cloned into the plasmid pHY-WZX, and the recombinant plasmid was
transformed into Bacillus subtilis WB600. Results: The recombinant Bacillus subtilis WB600/pHY-AMPD was successfully
constructed to express AMPD. Furthermore, the fermentation medium was optimized to contain 10 g/L glucose, 30 g/L yeast
extract, 0.5 g/L CaCl2, 1 g/L sodium citrate, 10 g/L NaH2PO4, 10 g/L K2HPO4, and 0.5 g/L (NH4)2SO4. The enzyme activity
reached (2 230 ± 50) U/mL after shake flask cultivation at 37 ℃ for 60 h with a shaking speed of 200 r/min. Conclusion: The
AMPD gene from Streptomyces murinus has been successfully expressed in Bacillus subtilis.

Key words: adenosine deaminase, Streptomyces murinus, Bacillus subtilis, recombinant expression