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.

• MeSH
• Aspergillus niger enzymologie   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• epoxid hydrolasy chemie   izolace a purifikace   MeSH
• epoxidové sloučeniny metabolismus   MeSH
• financování organizované MeSH
• gelová chromatografie MeSH
• kinetika MeSH
• koncentrace vodíkových iontů MeSH
• molekulární sekvence - údaje MeSH
• sekvence aminokyselin MeSH
• sekvenční seřazení MeSH
• stereoizomerie MeSH
• teplota MeSH

cDNA-encoding pyranose 2-oxidase (P2O) from Trametes pubescens was sequenced and cloned into Escherichia coli strain BL21/DE3 on a multicopy plasmid under the control of trc promoter. The synthesis of P2O was studied in a batch culture in M9-based mineral medium: the enzyme was synthesized constitutively at 28 degrees C in amount corresponding to 8% of the cell soluble protein (0.6 Umg(-1)). Only small portion of P2O (11%) was in the form of non-active inclusion bodies. Purified recombinant enzyme has similar physico-chemical and kinetic parameters with other P2Os. When compared to the expression of p2o of Trametes ochracea, a ratio of the mature enzyme to inclusion bodies found in the same E. coli host at 28 degrees C is as much as nine times higher. The finding makes the enzyme from T. pubescens preferable for the large-scale production by recombinant bacteria. The difference in amino acid sequences of the P2O from T. ochracea and T. pubescens may explain the favourable trait of the latter enzyme regarding protein folding.

• MeSH
• Basidiomycota enzymologie   genetika   MeSH
• buněčná inkluze enzymologie   genetika   MeSH
• druhová specificita MeSH
• Escherichia coli MeSH
• exprese genu MeSH
• financování organizované MeSH
• fungální proteiny biosyntéza   genetika   chemie   MeSH
• karbohydrátdehydrogenasy biosyntéza   genetika   chemie   MeSH
• kinetika MeSH
• sbalování proteinů MeSH

Microbial isolates from biofilters and petroleum-polluted bioremediation sites were screened for the presence of enantioselective epoxide hydrolases active towards tert-butyl glycidyl ether, benzyl glycidyl ether, and allyl glycidyl ether. Out of 270 isolated strains, which comprised bacteria, yeasts, and filamentous fungi, four were selected based on the enantioselectivities of their epoxide hydrolases determined in biotransformation reactions. The enzyme of Aspergillus niger M200 preferentially hydrolyses (S)-tert-butyl glycidyl ether to (S)-3-tert-butoxy-1,2-propanediol with a relatively high enantioselectivity (the enantiomeric ratio E is about 30 at a reaction temperature of 28 degrees C). Epoxide hydrolases of Rhodotorula mucilaginosa M002 and Rhodococcus fascians M022 hydrolyse benzyl glycidyl ether with relatively low enantioselectivities, the former reacting predominantly with the (S)-enantiomer, the latter preferring the (R)-enantiomer. Enzymatic hydrolysis of allyl glycidyl ether by Cryptococcus laurentii M001 proceeds with low enantioselectivity (E=3). (R)-tert-Butyl glycidyl ether with an enantiomeric excess (ee) of over 99%, and (S)-3-tert-butoxy-1,2-propanediol with an ee-value of 86% have been prepared on a gram-scale using whole cells of A. niger M200. An enantiomeric ratio of approximately 100 has been determined under optimised biotransformation conditions with the partially purified epoxide hydrolase from A. niger M200. The regioselectivity of this enzyme was determined to be total for both (S)-tert-butyl glycidyl ether and (R)-tert-butyl glycidyl ether.

• MeSH
• Aspergillus niger enzymologie   MeSH
• bakteriální proteiny metabolismus   MeSH
• biotransformace MeSH
• epoxid hydrolasy metabolismus   MeSH
• epoxidové sloučeniny chemie   metabolismus   MeSH
• financování organizované MeSH
• fungální proteiny metabolismus   MeSH
• grampozitivní bakterie enzymologie   izolace a purifikace   MeSH
• hydrolýza MeSH
• substrátová specifita MeSH