Publications

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

  Zhuang X., Zhang H.

  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 spectrome ... >> More

  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. << Less

  Protein Expr Purif 26:71-81(2002) [PubMed] [EuropePMC]

• Microbial conversion of pyrolytic products to biofuels: a novel and sustainable approach toward second-generation biofuels.

  Islam Z.U., Zhisheng Y., Hassan e.l. B., Dongdong C., Hongxun Z.

  This review highlights the potential of the pyrolysis-based biofuels production, bio-ethanol in particular, and lipid in general as an alternative and sustainable solution for the rising environmental concerns and rapidly depleting natural fuel resources. Levoglucosan (1,6-anhydrous-β-D-glucopyran ... >> More

  This review highlights the potential of the pyrolysis-based biofuels production, bio-ethanol in particular, and lipid in general as an alternative and sustainable solution for the rising environmental concerns and rapidly depleting natural fuel resources. Levoglucosan (1,6-anhydrous-β-D-glucopyranose) is the major anhydrosugar compound resulting from the degradation of cellulose during the fast pyrolysis process of biomass and thus the most attractive fermentation substrate in the bio-oil. The challenges for pyrolysis-based biorefineries are the inefficient detoxification strategies, and the lack of naturally available efficient and suitable fermentation organisms that could ferment the levoglucosan directly into bio-ethanol. In case of indirect fermentation, acid hydrolysis is used to convert levoglucosan into glucose and subsequently to ethanol and lipids via fermentation biocatalysts, however the presence of fermentation inhibitors poses a big hurdle to successful fermentation relative to pure glucose. Among the detoxification strategies studied so far, over-liming, extraction with solvents like (n-butanol, ethyl acetate), and activated carbon seem very promising, but still further research is required for the optimization of existing detoxification strategies as well as developing new ones. In order to make the pyrolysis-based biofuel production a more efficient as well as cost-effective process, direct fermentation of pyrolysis oil-associated fermentable sugars, especially levoglucosan is highlly desirable. This can be achieved either by expanding the search to identify naturally available direct levoglusoan utilizers or modify the existing fermentation biocatalysts (yeasts and bacteria) with direct levoglucosan pathway coupled with tolerance engineering could significantly improve the overall performance of these microorganisms. << Less

  J Ind Microbiol Biotechnol 42:1557-1579(2015) [PubMed] [EuropePMC]

• Engineering ethanologenic Escherichia coli for levoglucosan utilization.

  Layton D.S., Ajjarapu A., Choi D.W., Jarboe L.R.

  Levoglucosan is a major product of biomass pyrolysis. While this pyrolyzed biomass, also known as bio-oil, contains sugars that are an attractive fermentation substrate, commonly-used biocatalysts, such as Escherichia coli, lack the ability to metabolize this anhydrosugar. It has previously been s ... >> More

  Levoglucosan is a major product of biomass pyrolysis. While this pyrolyzed biomass, also known as bio-oil, contains sugars that are an attractive fermentation substrate, commonly-used biocatalysts, such as Escherichia coli, lack the ability to metabolize this anhydrosugar. It has previously been shown that recombinant expression of the levoglucosan kinase enzyme enables use of levoglucosan as carbon and energy source. Here, ethanologenic E. coli KO11 was engineered for levoglucosan utilization by recombinant expression of levoglucosan kinase from Lipomyces starkeyi. Our engineering strategy uses a codon-optimized gene that has been chromosomally integrated within the pyruvate to ethanol (PET) operon and does not require additional antibiotics or inducers. Not only does this engineered strain use levoglucosan as sole carbon source, but it also ferments levoglucosan to ethanol. This work demonstrates that existing biocatalysts can be easily modified for levoglucosan utilization. << Less

  Bioresour. Technol. 102:8318-8322(2011) [PubMed] [EuropePMC]

• Producing glucose 6-phosphate from cellulosic biomass: structural insights into levoglucosan bioconversion.

  Bacik J.P., Klesmith J.R., Whitehead T.A., Jarboe L.R., Unkefer C.J., Mark B.L., Michalczyk R.

  The most abundant carbohydrate product of cellulosic biomass pyrolysis is the anhydrosugar levoglucosan (1,6-anhydro-β-d-glucopyranose), which can be converted to glucose 6-phosphate by levoglucosan kinase (LGK). In addition to the canonical kinase phosphotransfer reaction, the conversion requires ... >> More

  The most abundant carbohydrate product of cellulosic biomass pyrolysis is the anhydrosugar levoglucosan (1,6-anhydro-β-d-glucopyranose), which can be converted to glucose 6-phosphate by levoglucosan kinase (LGK). In addition to the canonical kinase phosphotransfer reaction, the conversion requires cleavage of the 1,6-anhydro ring to allow ATP-dependent phosphorylation of the sugar O6 atom. Using x-ray crystallography, we show that LGK binds two magnesium ions in the active site that are additionally coordinated with the nucleotide and water molecules to result in ideal octahedral coordination. To further verify the metal binding sites, we co-crystallized LGK in the presence of manganese instead of magnesium and solved the structure de novo using the anomalous signal from four manganese atoms in the dimeric structure. The first metal is required for catalysis, whereas our work suggests that the second is either required or significantly promotes the catalytic rate. Although the enzyme binds its sugar substrate in a similar orientation to the structurally related 1,6-anhydro-N-acetylmuramic acid kinase (AnmK), it forms markedly fewer bonding interactions with the substrate. In this orientation, the sugar is in an optimal position to couple phosphorylation with ring cleavage. We also observed a second alternate binding orientation for levoglucosan, and in these structures, ADP was found to bind with lower affinity. These combined observations provide an explanation for the high Km of LGK for levoglucosan. Greater knowledge of the factors that contribute to the catalytic efficiency of LGK can be used to improve applications of this enzyme for levoglucosan-derived biofuel production. << Less

  J. Biol. Chem. 290:26638-26648(2015) [PubMed] [EuropePMC]