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Literature summary for 2.7.1.17 extracted from

  • Akinterinwa, O.; Cirino, P.C.
    Heterologous expression of d-xylulokinase from Pichia stipitis enables high levels of xylitol production by engineered Escherichia coli growing on xylose (2009), Metab. Eng., 11, 45-55.
    View publication on PubMed
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Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli strain PC07, xyl3 gene from Pichia stipitis Escherichia coli

Protein Variants

Protein Variants Comment Organism
additional information Pichia stipitis xylulokinase gene (XYL3) is PCR-amplified from Pichia stipitis (strain UC7) genomic DNA, genes of interest are cloned downstream of a tac promoter and upstream of a transcription termination sequence into the medium copy vector pLOI3809, transforming plasmid into a DELTAxylB mutant Escherichia coli strain (PC07), determine whether an alternative xylulokinase (one not expected to act on xylitol) could functionally replace XylB in Escherichia coli, Xyl3 shares only 23% amino acid sequence identity with Escherichia coli XylB Escherichia coli

Organism

Organism UniProt Comment Textmining
Escherichia coli
-
 DELTAxylB mutant strain (PC07) with introduced xyl3 from Pichia stipitis, xylB encodes XK in Escherichia coli
-

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
additional information
-
 3.1 U/mg protein (native substrate 5 mM xylulose), with 0.14 U/mg protein activity when using 75 mM xylitol as substrate (activity in range of background level), activities measured from cell lysates by monitoring NADH depletion, using an assay in which pentulokinase activity is coupled to pyruvate kinase and lactate dehydrogenase activities, reaction mixture (final concentrations indicated): 71 mM Tris–HCl pH 7.5, 7.1 mM MgCl2, 1 mM EDTA, 50 mM KCl, 7.1 mM KF, 5 mM KCN, 1.4 mM ATP, 1 mM phosphoenolpyruvic acid, 0.3 mM NADH, 0.7 U/ml pyruvate kinase, 1 U/ml lactate dehydrogenase, and 5 mM D-xylulose or 75 mM xylitol Escherichia coli

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
-
 Escherichia coli

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
-
 Escherichia coli

Cofactor

Cofactor Comment Organism Structure
ATP
-
 Escherichia coli