Purification and characterization of a urethanase from Penicillium variabile.
Urethanase produced by Penicillium variabile was purified through ultrasonication, concentration by polyethylene glycol 20,000, and Superdex G-200 gel filtration chromatography. The molecular weight of urethanase was determined to be around 96 kDa by gel filtration. The purified enzyme showed a single band in SDS-PAGE with the molecular weight of ~13.7 kDa, which suggests that the enzyme has a multimeric structure composed of the same subunits. Peptide map fingerprinting analysis was then carried out by MALDI/TOF-TOF MS. Within the known sequences in NCBI, glucosamine-6-phosphate deaminase and 6-phosphogluconate dehydrogenase get high score as compared with urethanase. Sequence analysis informs that N-terminal sequence of urethanase is GTNTADNDAA. The Minchaelis constant (Km) and maximum reaction rate (Vm) of urethanase are 27.2 mmol/L and 156.25 μmol/L min, respectively.
Nan-di
ND
Zhou
Xiao-lei
XL
Gu
Xiao-hong
XH
Zha
Ya-ping
YP
Tian
D000581:Amidohydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D000595:Amino Acid Sequence; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010407:Penicillium / Q000201:enzymology* / Q000302:isolation & purification; D013010:Sonication; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature
D000581:Amidohydrolases; C074526:urethanase