Cloning, Expression, and Characterization of a Novel L-Arabinose Isomerase from the Psychrotolerant Bacterium Pseudoalteromonas haloplanktis.

Abstract

L-Arabinose isomerase (L-AI, EC 5.3.1.4) catalyzes the isomerization between L-arabinose and L-ribulose, and most of the reported ones can also catalyze D-galactose to D-tagatose, except Bacillus subtilis L-AI. In this article, the L-AI from the psychrotolerant bacterium Pseudoalteromonas haloplanktis ATCC 14393 was characterized. The enzyme showed no substrate specificity toward D-galactose, which was similar to B. subtilis L-AI but distinguished from other reported L-AIs. The araA gene encoding the P. haloplanktis L-AI was cloned and overexpressed in E. coli BL21 (DE3). The recombinant enzyme was purified by one-step nickel affinity chromatography . The enzyme displayed the maximal activity at 40°C and pH 8.0, and showed more than 75% of maximal activity from pH 7.5-9.0. Metal ion Mn2+ was required as optimum metal cofactor for activity simulation, but it did not play a significant role in thermostability improvement as reported previously. The Michaelis-Menten constant (K m), turnover number (k cat), and catalytic efficiency (k cat/K m) for substrate L-arabinose were measured to be 111.68mM, 773.30/min, and 6.92/mM/min, respectively. The molecular docking results showed that the active site residues of P. haloplanktis L-AI could only immobilize L-arabinose and recognized it as substrate for isomerization.