title: 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 (Km), turnover number (kcat), and catalytic efficiency (kcat/Km) for substrate L-arabinose were measured to be 111.68 mM, 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.
authors: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, Jiangsu, China.
authors: Wei
authors: W
authors: Xu
authors: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, Jiangsu, China.
authors: Chen
authors: C
authors: Fan
authors: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, Jiangsu, China.|Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, 214122, China.
authors: Tao
authors: T
authors: Zhang
authors: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, Jiangsu, China.|Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, 214122, China.
authors: Bo
authors: B
authors: Jiang
authors: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, Jiangsu, China. wmmu@jiangnan.edu.cn.|Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, 214122, China. wmmu@jiangnan.edu.cn.
authors: Wanmeng
authors: W
authors: Mu
mesh_terms: D019747:Aldose-Ketose Isomerases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001089:Arabinose / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D003001:Cloning, Molecular / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics; D005690:Galactose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D062105:Molecular Docking Simulation; D031161:Pseudoalteromonas / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D011994:Recombinant Proteins; D001089:Arabinose; D019747:Aldose-Ketose Isomerases; C019715:L-arabinose isomerase; D005690:Galactose
keywords: Characterization; L-Arabinose isomerase; Molecular docking; Pseudoalteromonas haloplanktis; Substrate specificity