title: Cloning, expression and characterization of xylose isomerase from the marine bacterium Fulvimarina pelagi in Escherichia coli.
abstract: Production of a xylose isomerase (XI) with high tolerance to the inhibitors xylitol and calcium, and high activity at the low pH and temperature conditions characteristic of yeast fermentations, is desirable for a simultaneous isomerization/fermentation process for cellulosic ethanol production. A putative XI gene (xylA) from the marine bacterium Fulvimarina pelagi was identified by sequence analysis of the F. pelagi genome, and was PCR amplified, cloned, and expressed in Escherichia coli. The rXI was produced in shake flask and fed-batch fermentations using glucose as the growth substrate. The optimum pH for rXI was approximately 7, although activity was evident at pH as low as 5.5. The purified rXI had a molecular weight in 160 kDA, a Vmaxof 0.142 U/mg purified rXI, and a KMfor xylose in the range of 1.75-4.17 mM/L at pH 6.5 and a temperature of 35°C. The estimated calcium and xylitol KIvalues for rXI in cell-free extracts were 2,500 mg/L and >50 mM, respectively. The low KMof the F. pelagi xylose isomerase is consistent with the low nutrient conditions of the pelagic environment. These results indicate that Ca2+and xylitol are not likely to be inhibitory in applications employing the rXI from F. pelagi to convert xylose to xylulose in fermentations of complex biomass hydrolysates. A higher Vmaxat low pH (<6) and temperature (30°C) would be preferable for use in biofuels production. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1230-1237, 2016.
authors.affiliation: School of Chemical, Biological, and Environmental Engineering, Oregon State University, 101 Covell Hall, Corvallis, OR, 97331-2701.
authors.forename: Curtis A
authors.initials: CA
authors.lastname: Lajoie
authors.affiliation: Trillium FiberFuels, Inc., 720 NE Granger Ave., Corvallis, OR, 97330-9660.
authors.forename: Joshua B
authors.initials: JB
authors.lastname: Kitner
authors.affiliation: Trillium FiberFuels, Inc., 720 NE Granger Ave., Corvallis, OR, 97330-9660.
authors.forename: Stephen J
authors.initials: SJ
authors.lastname: Potochnik
authors.affiliation: School of Chemical, Biological, and Environmental Engineering, Oregon State University, 101 Covell Hall, Corvallis, OR, 97331-2701.
authors.forename: Jakob M
authors.initials: JM
authors.lastname: Townsend
authors.affiliation: Trillium FiberFuels, Inc., 720 NE Granger Ave., Corvallis, OR, 97330-9660.
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Beatty
authors.affiliation: School of Chemical, Biological, and Environmental Engineering, Oregon State University, 101 Covell Hall, Corvallis, OR, 97331-2701.
authors.forename: Christine J
authors.initials: CJ
authors.lastname: Kelly
mesh_terms: D019747:Aldose-Ketose Isomerases / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D012230:Rhizobiaceae / Q000201:enzymology*
chemical_list: D004269:DNA, Bacterial; D019747:Aldose-Ketose Isomerases; C023298:xylose isomerase
keywords: ethanol; marine; xylose isomerase; xylulose