Identification, characterization of levoglucosan kinase, and cloning and expression of levoglucosan kinase cDNA from Aspergillus niger CBX-209 in Escherichia coli

Abstract

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°C. This kinase is stable below 20°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 71.2 mM 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 0.20 mM. A cDNA library from A. niger was constructed in Escherichia coli DH5a. 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 DH5a.

Hybrid thermochemical processing: fermentation of pyrolysis-derived bio-oil

“…CBX 209 produces citric acid from levoglucosan (Zhuang and Zhang 2002). In both of these examples, the yield and the rate of fermentative production from levoglucosan are comparable to glucose.…”

“…The A. niger LGK was subjected to extensive biochemical characterization (Zhuang and Zhang 2002). This analysis identified the optimal temperature of 30 0 C and an optimal pH of 9.3, though the enzyme is stable at pH 6 -10.…”

“…One potential problem is the relatively high Km of LGK for levoglucosan: 71.2mM in A. niger (Zhuang and Zhang 2002) and 68 mM in L. starkeyi (Dai et al 2009). This high Km appears to result in incomplete substrate utilization (Dai et al 2009;Layton et al 2011), decreasing the overall product yield.…”

“…The fungal LGK was cloned into E. coli from an A. niger genomic library, but the resulting enzyme activity was low (Zhuang and Zhang 2002). Isolation of LGK from L. starkeyi YZ-215 and its expression in E. coli enabled utilization of levoglucosan as sole carbon source (Dai et al 2009).…”

Cloning of a novel levoglucosan kinase gene from Lipomyces starkeyi and its expression in Escherichia coli

“…In Arthrobacter bacteria, LG is transformed into glucose through at least three enzymatic reactions (Nakahara et al 1994). In yeasts and filamentous fungi, LG is directly converted to glucose-6-phosphate (one-step) by a specific levoglucosan kinase (LGK) Zhuang and Zhang 2002). As shown in Fig.…”