Production of tagatose by a recombinant thermostable L-arabinose isomerase from Thermus sp. IM6501.
A gene (thaI) corresponding to L-arabinose isomerase from Thermus strain IM6501 was cloned by PCR. It comprised 1488 nucleotides and encoded a polypeptide of 496 residues with a predicted molecular weight of 56019 Da. The deduced amino acid sequence had 96.8% identity with the L-arabinose isomerase of Geobacillus stearothermophilus. Recombinant ThaI with N-terminal hexa-tistidine tags was over-expressed in Escherichia coli and purified by affinity chromatography using Ni-NTA resin. The purified ThaI was thermostable with maximal activity at 60 degrees C at pH 8 for 30 min of reaction. Zn2+ and Ni2+ inactivated the catalytic activity of ThaI, 5 mM Mn2+ enhanced the bioconversion yield by 90%. The bioconversion yield of 54% from D-galactose to D-tagatose was obtained by recombinant ThaI at 60 degrees C over 3 d.
Kim
Jung-Woo
JW
National Laboratory for Functional Food Carbohydrate, and Center for Agricultural Biomaterials, Department of Food Science and Technology, Seoul National University, Suwon 441-744, Korea.
Kim
Young-Wan
YW
Roh
Hoe-Jin
HJ
Kim
Hae-Young
HY
Cha
Jae-Ho
JH
Park
Kwan-Hwa
KH
Park
Cheon-Seok
CS
D019747:Aldose-Ketose Isomerases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001406:Bacillaceae / Q000201:enzymology / Q000235:genetics; D003001:Cloning, Molecular; D004789:Enzyme Activation; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005690:Galactose / Q000737:chemistry; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006601:Hexoses / Q000138:chemical synthesis*; D006863:Hydrogen-Ion Concentration; D011786:Quality Control; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D020539:Sequence Analysis, Protein; D013045:Species Specificity; D013696:Temperature; D013824:Thermus / Q000145:classification / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism
D006601:Hexoses; D011994:Recombinant Proteins; D019747:Aldose-Ketose Isomerases; C019715:L-arabinose isomerase; C030192:tagatose; D005690:Galactose