Cloning and characterization of a novel l-arabinose isomerase from Bacillus licheniformis

Ponnandy Prabhu, Manish Kumar Tiwari, Marimuthu Jeya, Paramasamy Gunasekaran, In-Won Kim, Jung-Kul Lee

Research output: Contribution to journalJournal articleResearchpeer-review

Abstract

Based on analysis of the genome sequence of Bacillus licheniformis ATCC 14580, an isomerase-encoding gene (araA) was proposed as an l-arabinose isomerase (L-AI). The identified araA gene was cloned from B. licheniformis and overexpressed in Escherichia coli. DNA sequence analysis revealed an open reading frame of 1,422 bp, capable of encoding a polypeptide of 474 amino acid residues with a calculated isoelectric point of pH 4.8 and a molecular mass of 53,500 Da. The gene was overexpressed in E. coli, and the protein was purified as an active soluble form using Ni-NTA chromatography. The molecular mass of the purified enzyme was estimated to be similar to 53 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 113 kDa by gel filtration chromatography, suggesting that the enzyme is a homodimer. The enzyme required a divalent metal ion, either Mn2+or Co2+, for enzymatic activity. The enzyme had an optimal pH and temperature of 7.5 and 50C, respectively, with a k (cat) of 12,455 min-1 and a k (cat)/K (m) of 34 min-1 mM-1s for l-arabinose, respectively. Although L-AIs have been characterized from several other sources, B. licheniformis L-AI is distinguished from other L-AIs by its wide pH range, high substrate specificity, and catalytic efficiency for l-arabinose, making B. licheniformis L-AI the ideal choice for industrial applications, including enzymatic synthesis of l-ribulose. This work describes one of the most catalytically efficient L-AIs characterized thus far.
Original languageEnglish
JournalApplied Microbiology and Biotechnology
Volume81
Issue number2
Pages (from-to)283-290
Number of pages8
ISSN0175-7598
DOIs
Publication statusPublished - 2008
Externally publishedYes

Keywords

  • Aldose-Ketose Isomerases
  • Arabinose
  • Bacillus
  • Chromatography, Gel
  • Cloning, Molecular
  • Cobalt
  • Coenzymes
  • DNA, Bacterial
  • Dimerization
  • Electrophoresis, Polyacrylamide Gel
  • Enzyme Stability
  • Escherichia coli
  • Gene Expression
  • Hydrogen-Ion Concentration
  • Isoelectric Point
  • Kinetics
  • Manganese
  • Molecular Weight
  • Open Reading Frames
  • Recombinant Proteins
  • Sequence Analysis, DNA
  • Temperature
  • 3G0H8C9362 Cobalt
  • 42Z2K6ZL8P Manganese
  • B40ROO395Z Arabinose
  • EC 5.3.1.- Aldose-Ketose Isomerases
  • EC 5.3.1.4 L-arabinose isomerase
  • Amines
  • Amino acids
  • Bacteriology
  • Chromatographic analysis
  • Chromatography
  • Cloning
  • Colloids
  • DNA sequences
  • Electrophoresis
  • Encoding (symbols)
  • Enzymes
  • Gel permeation chromatography
  • Gelation
  • Gels
  • Genetic engineering
  • Industrial applications
  • Manganese alloys
  • Manganese compounds
  • Metal ions
  • Metal refining
  • Molecular mass
  • Nucleic acids
  • Organic acids
  • Polymers
  • Porous materials
  • Purification
  • Sodium sulfate
  • Sugar (sucrose)
  • Sugars
  • Water pollution
  • Gene encoding
  • cobalt
  • isomerase
  • levo arabinose isomerase
  • manganese
  • unclassified drug
  • bacterium
  • catalysis
  • clone
  • electrokinesis
  • enzyme activity
  • gene expression
  • genetic analysis
  • substrate
  • article
  • Bacillus licheniformis
  • bacterial gene
  • controlled study
  • enzyme analysis
  • enzyme kinetics
  • enzyme purification
  • enzyme specificity
  • enzyme stability
  • enzyme structure
  • gene identification
  • gene overexpression
  • genetic code
  • isoelectric point
  • molecular cloning
  • molecular weight
  • nonhuman
  • open reading frame
  • pH
  • protein quaternary structure
  • sequence analysis
  • thermostability
  • Bacillus licheniformis ATCC 14580
  • Amino acid residues
  • Catalytic efficiencies
  • Divalent metal ions
  • DNA sequence analyses
  • E. coli
  • Encoding genes
  • Enzymatic activities
  • Enzymatic syntheses
  • Gel filtrations
  • Genome sequences
  • Homodimer
  • Isoelectric points
  • Isomerase
  • L-Arabinose isomerase
  • Open reading frames
  • Polyacrylamide gel electrophoreses
  • Purified enzymes
  • Rare sugar
  • Ribulose
  • Sodium dodecyl sulfates
  • Substrate specificities
  • Substrate specificity
  • Wide ph ranges
  • Characterization
  • l-Arabinose isomerase
  • BIOTECHNOLOGY
  • D-TAGATOSE
  • ESCHERICHIA-COLI
  • D-GALACTOSE
  • GEOBACILLUS-STEAROTHERMOPHILUS
  • PROTEIN STABILITY
  • GENOME SEQUENCE
  • PH OPTIMUM
  • ACIDIC PH
  • PURIFICATION
  • STRAIN
  • Eubacteria Bacteria Microorganisms (Bacteria, Eubacteria, Microorganisms) - Endospore-forming Gram-Positives [07810] Bacillus licheniformis species strain-ATCC 14580
  • Facultatively Anaerobic Gram-Negative Rods Eubacteria Bacteria Microorganisms (Bacteria, Eubacteria, Microorganisms) - Enterobacteriaceae [06702] Escherichia coli species
  • Bacillus licheniformis araA gene [Endospore-forming Gram-Positives]
  • L-arabinose isomerase 9023-80-7 EC 5.3.1.4
  • 03502, Genetics - General
  • 10062, Biochemistry studies - Nucleic acids, purines and pyrimidines
  • 10802, Enzymes - General and comparative studies: coenzymes
  • 31000, Physiology and biochemistry of bacteria
  • 31500, Genetics of bacteria and viruses
  • Biochemistry and Molecular Biophysics
  • DNA sequence analysis laboratory techniques, genetic techniques
  • genome scan laboratory techniques, genetic techniques
  • nickel-nitrilo triacetic acid chromatography laboratory techniques, chromatographic techniques
  • Enzymology
  • Molecular Genetics
  • ARABINOSE ISOMERASES
  • BACILLUS
  • BACILLUS LICHENIFORMIS
  • ESCHERICHIA
  • ESCHERICHIA COLI
  • GENE CLONING
  • GENE EXPRESSION
  • ISOMERASES
  • Biotechnology
  • Enzyme systems

Fingerprint Dive into the research topics of 'Cloning and characterization of a novel l-arabinose isomerase from Bacillus licheniformis'. Together they form a unique fingerprint.

Cite this