Thermostable L-arabinose isomerase from Bacillus stearothermophilus IAM 11001 for D-tagatose production: gene cloning, purification and characterisation.
BACKGROUND
D-Tagatose, as one of the rare sugars, has been found to be a natural and safe low-calorie sweetener in food products and is classified as a GRAS substance. L-Arabinose isomerase (L-AI, EC 5.3.1.4), catalysing the isomerisations of L-arabinose and D-galactose to L-ribulose and D-tagatose respectively, is considered to be the most promising enzyme for the production of D-tagatose.


RESULTS
The araA gene encoding an L-AI from Bacillus stearothermophilus IAM 11001 was cloned, sequenced and overexpressed in Escherichia coli. The gene is composed of 1491 bp nucleotides and codes for a protein of 496 amino acid residues. The recombinant L-AI was purified to electrophoretical homogeneity by affinity chromatography. The purified enzyme was optimally active at 65 degrees C and pH 7.5 and had an absolute requirement for the divalent metal ion Mn(2+) for both catalytic activity and thermostability. The enzyme was relatively active and stable at acidic pH of 6. The bioconversion yield of D-galactose to D-tagatose by the purified L-AI after 12 h at 65 degrees C reached 36%.


CONCLUSIONS
The purified L-AI from B. stearothermophilus IAM 11001 was characterised and shown to be a good candidate for potential application in D-tagatose production.
Cheng
Lifang
L
State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu, China.
Mu
Wanmeng
W
Jiang
Bo
B
D019747:Aldose-Ketose Isomerases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005690:Galactose / Q000378:metabolism; D015870:Gene Expression; D005798:Genes, Bacterial; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D006601:Hexoses / Q000096:biosynthesis*; D006863:Hydrogen-Ion Concentration; D008345:Manganese
D006601:Hexoses; D008345:Manganese; D019747:Aldose-Ketose Isomerases; C019715:L-arabinose isomerase; C030192:tagatose; D005690:Galactose