Expression and characterization of the UDP-glucose 4-epimerase from Ganoderma lingzhi

Abstract:

[Background] Ganoderma lingzhi polysaccharides (GLP) are the main active component of this mushroom. UDP-glucose 4-epimerase (UGE, EC 5.1.3.2), an essential enzyme in the generation of sugar donor in synthesis of GLP, catalyzes the interconversion of UDP-glucose and UDP-galactose and thus is closely related to the content of galactose residue in GLP.[Objective] Through heterogeneous expression of the UGE gene from G. lingzhi, this study aims to further explore the enzymatic properties of important enzymes in the generation of sugar donor in GLP synthesis and gain a clearer insight into the synthesis pathway of GLP. [Methods] With the cDNA of G. lingzhi CGMCC 5.26 as template, UGE gene was cloned and then expressed in Escherichia coli BL21(DE3). The yielded recombinant enzyme was then purified and the enzymatic properties and kinetics, substrate specificity, and conversion rate of the purified enzymes were investigated. [Results] The recombinant enzyme was 45 kDa. The optimum reaction pH was 6.0, and it was stable at pH 7.0−9.0. The optimum temperature was 30℃, and it was most stable at 40℃. Fe²⁺ and Mg²⁺ could activate UGE. With UDP-glucose as substrate, the enzyme had the K_m of 0.824 mmol/L, V_max of 769.230 μmol/(L· min), k_cat of 1.333 s⁻¹, and k_cat/K_m of 1.618 L/(mmol· s). It catalyzed D-glucose, galacturonic acid, and N-acetylglucosamine. The conversion rate was increased from 16.0% to 39.4% at the optimal pH, temperature, and ratio of substrate with the addition of metal ions.[Conclusion] UGE from G. lingzhi has similar enzymatic properties to plants-derived UGE, and the catalytic efficiency was higher than most of the UGEs from bacteria. This study enriches the enzymatic properties of important enzymes involved the sugar donor synthesis in GLP production, which helps enhance the understanding of GLP synthesis pathway.

Key words:Ganoderma lingzhi;UDP-glucose 4-epimerase;enzymatic properties;substrate specificity;conversion rate