Cloning and characterization of a galactitol 2-dehydrogenase from Rhizobium legumenosarum and its application in d-tagatose production

Sujit Sadashiv Jagtap, Ranjitha Singh, Yun Chan Kang, Huimin Zhao, Jung Kul Lee

Research output: Contribution to journalArticle

10 Citations (Scopus)

Abstract

Galactitol 2-dehydrogenase (GDH) belongs to the protein subfamily of short-chain dehydrogenases/reductases and can be used to produce optically pure building blocks and for the bioconversion of bioactive compounds. An NAD+-dependent GDH from Rhizobium leguminosarum bv. viciae 3841 (RlGDH) was cloned and overexpressed in Escherichia coli. The RlGDH protein was purified as an active soluble form using His-tag affinity chromatography. The molecular mass of the purified enzyme was estimated to be 28kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 114kDa by gel filtration chromatography, suggesting that the enzyme is a homotetramer. The enzyme has an optimal pH and temperature of 9.5 and 35°C, respectively. The purified recombinant RlGDH catalyzed the oxidation of a wide range of substrates, including polyvalent aliphatic alcohols and polyols, to the corresponding ketones and ketoses. Among various polyols, galactitol was the preferred substrate of RlGDH with a Km of 8.8mM, kcat of 835min-1 and a kcat/Km of 94.9min-1mM-1. Although GDHs have been characterized from a few other sources, RlGDH is distinguished from other GDHs by its higher specific activity for galactitol and broad substrate spectrum, making RlGDH a good choice for practical applications.

Original languageEnglish
Pages (from-to)44-51
Number of pages8
JournalEnzyme and Microbial Technology
Volume58-59
DOIs
Publication statusPublished - 2014 May 10
Externally publishedYes

Fingerprint

Rhizobium
Cloning
Galactitol
Organism Cloning
Oxidoreductases
Substrates
Enzymes
Ketoses
Rhizobium leguminosarum
Affinity chromatography
Bioconversion
Molecular mass
Chromatography
Ketones
Electrophoresis
Affinity Chromatography
Sodium Dodecyl Sulfate
NAD
Escherichia coli
Gel Chromatography

Keywords

  • Galactitol 2-dehydrogenase
  • Homology modeling
  • SDR enzyme
  • Sugar alcohols

ASJC Scopus subject areas

  • Biotechnology
  • Biochemistry
  • Applied Microbiology and Biotechnology

Cite this

Cloning and characterization of a galactitol 2-dehydrogenase from Rhizobium legumenosarum and its application in d-tagatose production. / Jagtap, Sujit Sadashiv; Singh, Ranjitha; Kang, Yun Chan; Zhao, Huimin; Lee, Jung Kul.

In: Enzyme and Microbial Technology, Vol. 58-59, 10.05.2014, p. 44-51.

Research output: Contribution to journalArticle

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