Cloning, expression and characterization of L-arabinose isomerase from Thermotoga neapolitana: Bioconversion of D-galactose to D-tagatose using the enzyme

Byoung Chan Kim, Yoon Hee Lee, Han Seung Lee, Dong Woo Lee, Eun Ah Choe, Yu Ryang Pyun

Research output: Contribution to journalArticle

114 Citations (Scopus)

Abstract

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 Co2+ and Mn2+ for its activity and thermostability. The apparent Km values of the enzyme for L-arabinose and D-galactose were 116 mM (vmax, 119 μmol min-1 mg-1) and 250 mM (vmax, 14.3 μmol min-1 mg-1), respectively, that were determined in the presence of both 1 mM Co2+ and 1 mM Mn2+. A 68% conversion of D-galactose to D-tagatose was obtained using the recombinant enzyme at the isomerization temperature of 80°C.

Original languageEnglish
Pages (from-to)121-126
Number of pages6
JournalFEMS Microbiology Letters
Volume212
Issue number1
DOIs
Publication statusPublished - 2002 Jun 18

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L-arabinose isomerase
Thermotoga neapolitana
Galactose
Organism Cloning
Arabinose
Enzymes
Thermotoga maritima
Escherichia coli
Divalent Cations
Ion Exchange Chromatography
Genes
Gel Chromatography
Amino Acid Sequence
Hot Temperature
Amino Acids
Peptides
Temperature
tagatose

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Molecular Biology
  • Genetics

Cite this

@article{34931d418e804c41a3a9d6724139e820,
title = "Cloning, expression and characterization of L-arabinose isomerase from Thermotoga neapolitana: Bioconversion of D-galactose to D-tagatose using the enzyme",
abstract = "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 Co2+ and Mn2+ for its activity and thermostability. The apparent Km values of the enzyme for L-arabinose and D-galactose were 116 mM (vmax, 119 μmol min-1 mg-1) and 250 mM (vmax, 14.3 μmol min-1 mg-1), respectively, that were determined in the presence of both 1 mM Co2+ and 1 mM Mn2+. A 68{\%} conversion of D-galactose to D-tagatose was obtained using the recombinant enzyme at the isomerization temperature of 80°C.",
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Cloning, expression and characterization of L-arabinose isomerase from Thermotoga neapolitana : Bioconversion of D-galactose to D-tagatose using the enzyme. / Kim, Byoung Chan; Lee, Yoon Hee; Lee, Han Seung; Lee, Dong Woo; Choe, Eun Ah; Pyun, Yu Ryang.

In: FEMS Microbiology Letters, Vol. 212, No. 1, 18.06.2002, p. 121-126.

Research output: Contribution to journalArticle

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T1 - Cloning, expression and characterization of L-arabinose isomerase from Thermotoga neapolitana

T2 - Bioconversion of D-galactose to D-tagatose using the enzyme

AU - Kim, Byoung Chan

AU - Lee, Yoon Hee

AU - Lee, Han Seung

AU - Lee, Dong Woo

AU - Choe, Eun Ah

AU - Pyun, Yu Ryang

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Y1 - 2002/6/18

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