Expression and characterization of fumarase (FUMR) from Rhizopus oryzae

Abstract

Fumarase catalyzes the reversible hydration of fumarate to l-malate in Rhizopus oryzae. A recombinant pET22b-fumR harboring a fumarase gene (fumR) from R. oryzae was constructed for high level expression in E. coli BL21 (DE3). The FUMR activity was optimal at 30°C and pH 7.2. The enzyme was stable below 45°C and at pH 3.0-9.0. No effects of Zn(2+), Fe(2+), or EDTA were observed on enzyme activity. A slight inhibition of FUMR activity was seen with Mg(2+), while Ca(2+) had a small stimulatory effect. The K(m) for l-malic acid and fumaric acid were 0.46 mM and 3.07 mM, respectively. The activity of FUMR catalyzing hydration of fumarate to l-malate was completely inhibited by 2mM fumaric acid. The unique enzymatic properties suggested that overexpression of FUMR could enhance fumaric acid accumulation in R. oryzae.