Purification and characterisation of a novel enantioselective epoxide hydrolase from Aspergillus niger M200.
Purification of a novel enantioselective epoxide hydrolase from Aspergillus niger M200 has been achieved using ammonium sulphate precipitation, ionic exchange, hydrophobic interaction, and size-exclusion chromatography, in conjunction with two additional chromatographic steps employing hydroxylapatite, and Mimetic Green. The enzyme was purified 186-fold with a yield of 15%. The apparent molecular mass of the enzyme was determined to be 77 kDa under native conditions and 40 kDa under denaturing conditions, implying a dimeric structure of the native enzyme. The isoelectric point of the enzyme was estimated to be 4.0 by isoelectric focusing electrophoresis. The enzyme has a broad substrate specificity with highest specificities towards tert-butyl glycidyl ether, para-nitrostyrene oxide, benzyl glycidyl ether, and styrene oxide. Enantiomeric ratios of 30 to more than 100 were determined for the hydrolysis reactions of 4 epoxidic substrates using the purified enzyme at a reaction temperature of 10 degrees C. Product inhibition studies suggest that the enzyme is able to differentiate to a high degree between the (R)-diol and (S)-diol product of the hydrolysis reaction with tert-butyl glycidyl ether as the substrate. The highest activity of the enzyme was at 42 degrees C and a pH of 6.8. Six peptide sequences, which were obtained by cleavage of the purified enzyme with trypsin and mass spectrometry analysis of the tryptic peptides, show high similarity with corresponding sequences originated from the epoxide hydrolase from Aspergillus niger LCP 521.
Kotik
Michael
M
Laboratory of Enzyme Technology, Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídenská 1083, 142 20 Prague 4, Czech Republic. kotik@biomed.cas.cz
Kyslík
Pavel
P
D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000201:enzymology*; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D004851:Epoxide Hydrolases / Q000737:chemistry / Q000302:isolation & purification*; D004852:Epoxy Compounds / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D013237:Stereoisomerism; D013696:Temperature
D004852:Epoxy Compounds; C036354:tert-butyl glycidyl ether; D004851:Epoxide Hydrolases