Heterologous expression of a deacetylase and its application in L-glufosinate preparation

Abstract

With potent herbicidal activity, biocatalysis synthesis of L-glufosinate has drawn attention. In present research, NAP-Das2.3, a deacetylase capable of stereoselectively resolving N-acetyl-L-glufosinate to L-glufosinate mined from Arenimonas malthae, was heterologously expressed and characterized. In Escherichia coli, NAP-Das2.3 activity only reached 0.25 U/L due to the formation of inclusive bodies. Efficient soluble expression of NAP-Das2.3 was achieved in Pichia pastoris. In shake flask and 5 L bioreactor fermentation, NAP-Das2.3 activity by recombinant P. pastoris reached 107.39 U/L and 1287.52 U/L, respectively. The optimum temperature and pH for N-acetyl-glufosinate hydrolysis by NAP-Das2.3 were 45 °C and pH 8.0, respectively. The K_m and V_max of NAP-Das2.3 towards N-acetyl-glufosinate were 25.32 mM and 19.23 μmol mg^-1 min^-1, respectively. Within 90 min, 92.71% of L-enantiomer in 100 mM racemic N-acetyl-glufosinate was converted by NAP-Das2.3. L-glufosinate with high optical purity (e.e._P above 99.9%) was obtained. Therefore, the recombinant NAP-Das2.3 might be an alternative for L-glufosinate biosynthesis.

Keywords: Deacetylase; Enantioselectively hydrolysis; L-glufosinate; Pichia pastoris; Soluble expression.