Pakistan Journal of Biological Sciences1028-88801812-5735Asian Network for Scientific Information10.3923/pjbs.2008.2001.2006Pseudomonas fluorescens DSM 50106 in Different Strains of E. coli]]>HaghighatianM. MofidM.R. NekoueiM.K. YaghmaeiP. TafreshiA.H. 1220081116A NADH-dependent mannitol dehydrogenase gene (mtlD)
was cloned from Pseudomonas fluorescens, subcloned into an expression
vector (pDEST110) and entered into different strains of E. coli to
compare their protein expression and the enzyme specific activity. Purifications
were accomplished by Ni2+-NTA affinity chromatography. Using
this approach, the efficiency of purification process significantly increased
(up to 90%) so that the purified enzyme gave a sharp single band (55 kDa)
in SDS-PAGE. The results showed that among the strains, BL21 (DE3) plysS
exhibited the maximum expression level of MDH(mannitol dehydrogenase)
(11 mg L-1). Results from activity assay with fructose as substrate
also showed that in this strain the specific activity of 63 U mg-1
protein monitored for the enzyme, the record not reported before. Resazurin
staining also indicated that the enzyme reduced fructose, whereas oxidized
other substrates including mannitol, sorbitol and arabitol under optimal
assay condition. From HPLC analysis it was showed for the first time that
the enzyme could convert substrate isomaltulose to the specific products,
GPM and GPS. Interestingly, because of the high specificity of the enzyme
for substrate, the method can be used as an alternative approach to substitute
non-specific conventional method of isomalt production.]]>Brünker, P., J. Altenbuchner, K.D. Kulbe and R. Mattes,1997udomonas fluorescens 50106 in Escherichia coli.]]>1351157167Conde, C., P. Silva, A. Agasse, R. Lemoine, S. Delrot, R.M. Tavares and H. Gerós,2007Olea europaea and implications for salt stress tolerance. ]]>484253Horer, S., J. Stoop, H. Mooibroek, U. Baumann and J. Sassoon,2001Agaricus bisporus.]]>2762755527561Jennings, D.B., M. Ehrenshaft, D.M. Pharr and J.D. Williamson,1998951512915133Kavanagh, K.L., M. Klimacek, B. Nidetzky and D.K. Wilson,2002Pseudomonas fluorescens mannitol 2-dehydrogenase binary and ternary complexes. Specificity and catalytic mechanism.]]>2774343343442Kavanagh, K.L., M. Klimacek, B. Nidetzky and D.K. Wilson,2003Pseudomonas fluorescens mannitol 2-dehydrogenase: Evidence for a very divergent long-chain dehydrogenase family.]]>143-144551558Lee, J.K., B.S. Koo, S.Y. Kim and H.H. Hyun,2003Candida magnoliae.]]>6944384447Ndindayino, F., C. Vervaet, G. Van Den Mooter and J.P. Remon,2002235159168Nidetzky, B., D. Shmidt, A. Weber and K.D. Kulbe,1996144765Otte, S. and J.W. Lengeler,2001Klebsiella pneumoniae Kay2026.]]>194221227Perrot, S., H. Dutertre-Catella, C. Martin, P. Rat and J.M. Warnet,200372122139Ronda, F., M. Gomez, C.A. Blanco and P.A. Caballero,200590549555Sambrook and Russell,20013rd Edn.,pp: 4-55pp: 4-55Soetaert, W., P.T. Vanhooren and E.J. Vandamme,199910261275Stoop, J.M.H. and D.M. Pharr, 1994106503511Voegele, R.T., M. Hahn, G. Lohaus, T. Link, I. Heiser and K. Mendgen,2005Uromyces fabae.]]>137190198Whitford, D.,20071st Edn.,pp: 326-333pp: 326-333