Open AccessCloning, expression and characterization of l ‐arabinose isomerase from Thermotoga neapolitana : bioconversion of d ‐galactose to d ‐tagatose using the enzyme
Author(s) -
Kim ByoungChan,
Lee YoonHee,
Lee HanSeung,
Lee DongWoo,
Choe EunAh,
Pyun YuRyang
Publication year - 2002
Publication title -
fems microbiology letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.899
H-Index - 151
eISSN - 1574-6968
pISSN - 0378-1097
DOI - 10.1111/j.1574-6968.2002.tb11254.x
Subject(s) - thermotoga maritima , thermostability , isomerase , biochemistry , arabinose , chemistry , enzyme , galactose , escherichia coli , gene , fermentation , xylose
Gene araA encoding an l ‐arabinose isomerase (AraA) from the hyperthermophile, Thermotoga neapolitana 5068 was cloned, sequenced, and expressed in Escherichia coli . The gene encoded a polypeptide of 496 residues with a calculated molecular mass of 56 677 Da. The deduced amino acid sequence has 94.8% identical amino acids compared with the residues in a putative l ‐arabinose isomerase of Thermotoga maritima . The recombinant enzyme expressed in E. coli was purified to homogeneity by heat treatment, ion exchange chromatography and gel filtration. The thermophilic enzyme had a maximum activity of l ‐arabinose isomerization and d ‐galactose isomerization at 85°C, and required divalent cations such as Co 2+ and Mn 2+ for its activity and thermostability. The apparent K m values of the enzyme for l ‐arabinose and d ‐galactose were 116 mM ( v max , 119 μmol min −1 mg −1 ) and 250 mM ( v max , 14.3 μmol min −1 mg −1 ), respectively, that were determined in the presence of both 1 mM Co 2+ and 1 mM Mn 2+ . A 68% conversion of d ‐galactose to d ‐tagatose was obtained using the recombinant enzyme at the isomerization temperature of 80°C.