Gene cloning and catalysis features of a new mannitol-1-phosphate dehydrogenase (BbMPD) from Beauveria bassiana

A long-chain mannitol-1-phosphate dehydrogenase (MPD) was characterized for the first time from fungal entomopathogen Beauvenia bassiana by gene cloning, heterogeneous expression and activity analysis. The cloned gene BbMPD consisted of a 1334-bp open reading frame (ORF) with a 158-bp intron and the 935-bp upstream and 780-bp downstream regions. The ORF-encoded 391-aa protein (42 kDa) showed less than 75% sequence identity to 17 fungal MPD documented and shared two conserved domains with the fungal MPD family at the N- and C-terminus, respectively. The new enzyme was expressed well in the Luria-Bertani culture of engineered Escherichia coli BL21 by 16-h induction of 0.5 mM isopropyl 1-thio-beta-D-galactopyranoside at 20 degrees C after 5-h growth at 37 degrees C. The purified BbMPD exhibited a high catalytic efficiency (k(cat)/K-m) of 1.31 x 10(4) mM(-1) s(-1) in the reduction of the highly specific substrate D-fructose-6-hosphate to D-mannitol-1-phosphate. Its activity was maximal at the reaction regime of 37 degrees C and pH 7.0 and was much more sensitive to Cu2+ and Zn2+ than to Li+ and Mn2+. The results indicate a crucial role of BbMPD in the mannitol biosynthesis of B. bassiana. (C) 2009 Elsevier Ltd. All rights reserved.