title: Screening and identification of the levoglucosan kinase gene (lgk) from Aspergillus niger by LC-ESI-MS/MS and RT-PCR.
abstract: A protein of 75,000 Daltons with levoglucosan kinase activity was purified from Aspergillus niger. After in-gel digestion by trypsin, a 14-mer peptide was sequenced and analyzed by LC-ESI-MS/MS. Using a primer derived from the 14-mer peptide in combination with Oligo-(dT)18, a cDNA fragment was obtained by RT-PCR. A search of the GenBank database indicated that the protein had not been identified before. A similar protein named hypothetical protein FG07802.1 (EAA77996.1) was found to exist in Gibberella zeae by Blastx search. Using a primer derived from the protein, a cDNA fragment of second RT-PCR was cloned into plasmid pAJ401, which was transformed to Saccharomyces cerevisiae H158 and expressed. Two positive levoglucosan assimilating recombinants were selected. The lgk gene was screened and identified.
authors.affiliation: Department of Environmental Biotechnology, Research Center for Eco-environmental Sciences, Chinese Academy of Sciences, Beijing 100085, PR China.
authors.forename: Hui-jun
authors.initials: HJ
authors.lastname: Xie
authors.forename: Xu-liang
authors.initials: XL
authors.lastname: Zhuang
authors.forename: Hong-xun
authors.initials: HX
authors.lastname: Zhang
authors.forename: Zhi-hui
authors.initials: ZH
authors.lastname: Bai
authors.forename: Hong-yan
authors.initials: HY
authors.lastname: Qi
mesh_terms: D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis; D005947:Glucose / Q000031:analogs & derivatives* / Q000378:metabolism; D010449:Peptide Mapping / Q000295:instrumentation; D010770:Phosphotransferases / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D018076:DNA, Complementary; C014989:1,6-anhydro-beta-glucopyranose; D010770:Phosphotransferases; C506936:levoglucosan kinase, Aspergillus niger; D005947:Glucose