An L-arabinose isomerase from Acidothermus cellulolytics ATCC 43068: cloning, expression, purification, and characterization.
The araA gene encoding an L-arabinose isomerase (L-AI) from the acido-thermophilic bacterium Acidothermus cellulolytics ATCC 43068 was cloned and overexpressed in Escherichia coli. The open reading frame of the L-AI consisted of 1,503 nucleotides encoding 501 amino acid residues. The recombinant L-AI was purified to homogeneity by heat treatment, ion-exchange chromatography, and gel filtration. The molecular mass of the enzyme was estimated to be approximately 55 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme was optimally active at 75 degrees C and pH 7.5. It required divalent metal ions, either Mn(2+) or Co(2+), for both enzymatic activity and thermostability improvement at higher temperatures. The enzyme showed relatively high activity and stability at acidic pH. It exhibited over 90% of its maximal activity at pH 6.0 and retained 80% of activity after 12 h incubation at pH 6.0. Catalytic property study showed that the enzyme had an interesting catalytic efficiency. Its apparent K (m), V (max), and catalytic efficiency (k (cat)/K (m)) for D-galactose was 28.9 mM, 4.9 U/mg, and 9.3 mM(-1) min(-1), respectively. The enzyme carried out the isomerization of D-galactose to D-tagatose with a conversion yield over 50% after 12 h under optimal conditions, suggesting its potential in D-tagatose production.
Cheng
Lifang
L
State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, Jiangsu Province, People's Republic of China.
Mu
Wanmeng
W
Zhang
Tao
T
Jiang
Bo
B
D000192:Actinomycetales / Q000201:enzymology* / Q000235:genetics; D019747:Aldose-Ketose Isomerases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D002413:Cations, Divalent / Q000494:pharmacology; D003001:Cloning, Molecular; D003067:Coenzymes; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism*; D005690:Galactose / Q000378:metabolism; D015870:Gene Expression; D006601:Hexoses / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013696:Temperature
D002413:Cations, Divalent; D003067:Coenzymes; D004271:DNA, Fungal; D005656:Fungal Proteins; D006601:Hexoses; D008670:Metals; D011994:Recombinant Proteins; D019747:Aldose-Ketose Isomerases; C019715:L-arabinose isomerase; C030192:tagatose; D005690:Galactose