Identification, characterization of levoglucosan kinase, and cloning and expression of levoglucosan kinase cDNA from Aspergillus niger CBX-209 in Escherichia coli.
The first enzyme responsible for assimilating levoglucosan in Aspergillus niger CBX-209 was corroborated to be levoglucosan kinase that catalyzes the transfer of a phosphate group from ATP to levoglucosan to yield a glucose 6-phosphate in the presence of magnesium ion and ATP by FAB-mass spectrometric method combined with previous observations from HPLC and enzymological experiments. Levoglucosan kinase was purified to apparent homogeneity by using a combination of seven purification steps. SDS-PAGE revealed a single protein band of 56 KDa. It is a monomeric enzyme and maximal enzyme activity was measured at pH 9.3 and 30 degrees C. This kinase is stable below 20 degrees C at a quite broad pHs ranging from 6 to 10 and levoglucosan could protect the enzyme from thermal inactivation. Exclusive substrate specificity for levoglucosan suggested that not only the structure of the intramolecular glucosidic linkage but also the configuration of the pyranose frame would be specific for recognition by levoglucosan kinase. The K(m) values of this enzyme were 71.2mM for levoglucosan and 0.25 mM for ATP, determined by double reciprocal plottings and ADP inhibited on the enzyme activity competitively with a Ki value of 0.20mM. A cDNA library from A. niger was constructed in Escherichia coli DH5alpha. The library was screened for levoglucosan kinase gene on NCE selective medium and three positive recombinants were selected after a five day culture. Detection of activities of levoglucosan kinase in the cell extracts indicated that levoglucosan kinase gene (lgk) was expressed by the recombinant strain of E. coli DH5alpha.
D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D005947:Glucose / Q000031:analogs & derivatives* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D010770:Phosphotransferases / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity; D013696:Temperature
D018076:DNA, Complementary; C014989:1,6-anhydro-beta-glucopyranose; D010770:Phosphotransferases; D005947:Glucose