title: Cloning and characterization of a galactitol 2-dehydrogenase from Rhizobium legumenosarum and its application in D-tagatose production.
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(-1)mM(-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.
authors.affiliation: Department of Chemical Engineering, Konkuk University, Seoul 143-701, Republic of Korea.
authors.forename: Sujit Sadashiv
authors.initials: SS
authors.lastname: Jagtap
authors.affiliation: Department of Chemical Engineering, Konkuk University, Seoul 143-701, Republic of Korea.
authors.forename: Ranjitha
authors.initials: R
authors.lastname: Singh
authors.affiliation: Department of Chemical Engineering, Konkuk University, Seoul 143-701, Republic of Korea.
authors.forename: Yun Chan
authors.initials: YC
authors.lastname: Kang
authors.affiliation: Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA; Department of Biochemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA; Department of Bioengineering, University of Illinois at Urbana-Champaign, 1206 West Gregory Drive, Urbana, IL 61801, USA; Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA; Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 1206 West Gregory Drive, Urbana, IL 61801, USA. Electronic address: zhao5@illinois.edu.
authors.forename: Huimin
authors.initials: H
authors.lastname: Zhao
authors.affiliation: Department of Chemical Engineering, Konkuk University, Seoul 143-701, Republic of Korea. Electronic address: jkrhee@konkuk.ac.kr.
authors.forename: Jung-Kul
authors.initials: JK
authors.lastname: Lee
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D002384:Catalysis; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D004376:Galactitol / Q000378:metabolism; D005798:Genes, Bacterial; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D016961:Rhizobium leguminosarum / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013401:Sugar Alcohol Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D004376:Galactitol; D013401:Sugar Alcohol Dehydrogenases; C095946:galactitol 2-dehydrogenase
keywords: Galactitol 2-dehydrogenase; Homology modeling; SDR enzyme; Sugar alcohols