An L-arabinose isomerase from Acidothermus cellulolytics ATCC 43068: cloning, expression, purification, and characterization |
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Authors: | Lifang Cheng Wanmeng Mu Tao Zhang Bo Jiang |
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Institution: | (1) State Key Laboratory of Food Science and Technology, Jiangnan University, 1800 Lihu Avenue, Wuxi, 214122, Jiangsu Province, People’s Republic of China;(2) School of Food Science & Technology, Jiangnan University, Wuxi, 214122, People’s Republic of China; |
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Abstract: | 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°C and pH 7.5. It required divalent metal ions, either Mn2+ or Co2+, 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. |
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Keywords: | |
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