L-arabinose isomerase from Acidothermus cellulolytics ATCC 43068: cloning, expression, purification, and characterization

• Published in: Applied microbiology and biotechnology 2010-04, Vol.86 (4), p.1089-1097
• Main Authors: Cheng, Lifang, Mu, Wanmeng, Zhang, Tao, Jiang, Bo
• Format: Article
• Language: English
• Subjects: Actinomycetales - enzymology; Actinomycetales - genetics; Aldose-Ketose Isomerases - chemistry; Aldose-Ketose Isomerases - genetics; Aldose-Ketose Isomerases - metabolism; Amino Acid Sequence; Amino acids; Bacteria; Berries; Biological and medical sciences; Biomedical and Life Sciences; Biotechnologically Relevant Enzymes and Proteins; Biotechnology; Cations, Divalent - pharmacology; Cloning; Cloning, Molecular; Coenzymes; DNA polymerase; DNA, Fungal - chemistry; DNA, Fungal - genetics; E coli; Electrophoresis, Polyacrylamide Gel; Enzymatic activity; Enzyme Stability; Enzymes; Escherichia coli; Escherichia coli - genetics; Food products; Food science; Fundamental and applied biological sciences. Psychology; Fungal Proteins - genetics; Fungal Proteins - metabolism; Galactose - metabolism; Gene Expression; Genetics; Genomes; Heat treatment; Hexoses - metabolism; High temperature; Hydrogen-Ion Concentration; Kinetics; Life Sciences; Metals - pharmacology; Microbial Genetics and Genomics; Microbiology; Molecular Sequence Data; Molecular Weight; Open Reading Frames; Proteins; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Sequence Analysis, DNA; Studies; Temperature
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-009-2322-z

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.