Screening and identification of the levoglucosan kinase gene (lgk) from Aspergillus niger by LC-ESI-MS/MS and RT-PCR.
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.
Xie
Hui-jun
HJ
Department of Environmental Biotechnology, Research Center for Eco-environmental Sciences, Chinese Academy of Sciences, Beijing 100085, PR China.
Zhuang
Xu-liang
XL
Zhang
Hong-xun
HX
Bai
Zhi-hui
ZH
Qi
Hong-yan
HY
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
D018076:DNA, Complementary; C014989:1,6-anhydro-beta-glucopyranose; D010770:Phosphotransferases; C506936:levoglucosan kinase, Aspergillus niger; D005947:Glucose