Constitutive expression of AMP deaminase from Streptomyces murinus in Pichia pastoris GS115 using the GAP promoter

Abstract:

[Objective] We constructed a recombinant Pichia pastoris GS115 strain to produce AMP deaminase and optimized the fermentation conditions. [Methods] The AMPD gene was amplified from Streptomyces murinus and cloned into the expression plasmid pGAP9K, the recombinant expression plasmid was transformed into Pichia pastoris GS115 by electrotransformation. Furthermore, we determined the AMP deaminase activity of positive transformants. Finally, we optimized the fermentation conditions. [Results] We constructed a recombinant P. pastoris GS115 strain (P. pastoris GS115/pGAP9K-AMPD) that showed AMP deaminase activity. The fermentation conditions of the recombinant strain were studied. The results showed that the optimum fermentation medium of the recombinant strain contains 2% glycerin, 2% peptone, 1% yeast extract, 0.5% KH2PO4 and 0.05% MgSO4·7H2O (pH 6.0). And the recombinant enzyme activity of fermentation supernatant was 2 230±60 U/mL with 3% of inoculation amount, 24 hours of seed time, 96 hours for 200 r/min at temperature of 30 °C. [Conclusion] We constructed a recombinant P. pastoris GS115 strain that showed AMP deaminase activity about 2 230±60 U/mL under optimized fermentation conditions. This research is helpful to advance the industrial production of AMP deaminase.

Key words:AMP deaminase, Streptomyces murinus, Pichia pastoris, Recombinant expression, Fermentation optimization