Secretory Expression and Characterization of Prolyl Aminopeptidase in Bacillus subtilis

Abstract:

Construct recombinant Bacillus subtilis to over-expression Aspergillus oryzae prolyl minopeptidase，then purified and characterized its basic enzyme properties. The pap gene was cloned from pMD19-pap vector and ligated into pMA5 to generate recombinant pMA5-pap expression vector. Then the vector was transformed into Bacillus subtilis WB600 competent cells and secretory expressed. By adding 5% D-sorbitol and 2 mmol/L CaCl2 in TB medium，the enzyme activity in fermented supernatant increased from 7.5 to 36.0 U/mL. The recombinant prolyl aminopeptidase was purified by ammonium sulfate，Hitrap Q anion exchange chromatography and SuperdexTM 75 gel chromatography，the purification factor was 8.8 and the specific activity of the purified enzyme was 247.3 U/mg. The purified enzyme has the highest activity at pH 7.5 and 50 ℃，the K m and V max was estimated to be 0.171 mmol/L and 55.99 μmol/（L·min） respectively.