TY - JOUR
T1 - Cloning and characterization of a galactitol 2-dehydrogenase from Rhizobium legumenosarum and its application in d-tagatose production
AU - Jagtap, Sujit Sadashiv
AU - Singh, Ranjitha
AU - Kang, Yun Chan
AU - Zhao, Huimin
AU - Lee, Jung Kul
N1 - Funding Information:
This research was supported by the Converging Research Center Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2011-50210), and a grant from the Intelligent Synthetic Biology Center of Global Frontier Project (2011-0031955) funded by the Ministry of Science, ICT and Future Planing, Republic of Korea. This work was also supported by WTU Joint Research Grants of Konkuk University.
PY - 2014/5/10
Y1 - 2014/5/10
N2 - 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.
AB - 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.
KW - Galactitol 2-dehydrogenase
KW - Homology modeling
KW - SDR enzyme
KW - Sugar alcohols
UR - http://www.scopus.com/inward/record.url?scp=84897903207&partnerID=8YFLogxK
U2 - 10.1016/j.enzmictec.2014.02.012
DO - 10.1016/j.enzmictec.2014.02.012
M3 - Article
C2 - 24731824
AN - SCOPUS:84897903207
SN - 0141-0229
VL - 58-59
SP - 44
EP - 51
JO - Enzyme and Microbial Technology
JF - Enzyme and Microbial Technology
ER -