N-Acetylneuraminate lyase produced by Escherichia coli was purified and crystallized from a genetically engineered strain (E. coli SF8/pNAL1). The enzyme showed apparent molecular masses of 105,000 Da on gel filtration and 35,000 Da on SDS/PAGE, suggesting that the enzyme is a trimer. The apparent optimum pH and temperature were found to be 6.5-7.0 and 80 degrees C respectively. The Km values for N-acetylneuraminate and N-glycollylneuraminate were 3.3 and 3.3 mM respectively. The enzyme was inhibited by reduction with NaBH4 in the presence of the substrate, indicating that the enzyme belongs to the Schiff-base-forming Class I aldolases. The enzyme was strongly inhibited by Cu2+ ions, p-chloromercuribenzoate and N-bromosuccinimide, and also inhibited competitively by the reaction product, pyruvate, and its structurally related compounds, dihydroxyacetone and DL-glyceraldehyde.

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A Trametes villosa laccase, produced as a recombinant protein in Aspergillus oryzae, was purified to an electrophoretically homogeneous state and employed in studies on the metabolism of bisphenol A. Structural analysis of the bisphenol A reaction products by the enzyme indicated that the dimer, trimer, tetramer, pentamer, and hexamer of bisphenol A with C-C and/or C-O bonds between phenol moieties, were formed as the result of successive oxidative-condensation. The reaction mixture also contained oligomers fragments, each with phenol molecules, suggesting the occurrence of cleavage of the formed oligomers to release 4-isopropenylphenol. A luciferase reporter assay using COS-7 cells revealed that both the soluble and insoluble fractions of the bisphenol A reaction products had no estrogenic activity even at rather high concentrations.

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