Biodegradation of chlorobenzoic acids by ligninolytic fungi.

We investigated the abilities of several perspective ligninolytic fungal strains to degrade 12 mono-, di- and trichloro representatives of chlorobenzoic acids (CBAs) under model liquid conditions and in contaminated soil. Attention was also paid to toxicity changes during the degradation, estimated using two luminescent assay variations with Vibrio fischeri. The results show that almost all the fungi were able to efficiently degrade CBAs in liquid media, where Irpex lacteus, Pycnoporus cinnabarinus and Dichomitus squalens appeared to be the most effective in the main factors: degradation and toxicity removal. Analysis of the degradation products revealed that methoxy and hydroxy derivatives were produced together with reduced forms of the original acids. The findings suggest that probably more than one mechanism is involved in the process. Generally, the tested fungal strains were able to degrade CBAs in soil in the 85-99% range within 60 days. Analysis of ergosterol showed that active colonization is an important factor for degradation of CBAs by fungi. The most efficient strains in terms of degradation were I. lacteus, Pleurotus ostreatus, Bjerkandera adusta in soil, which were also able to actively colonize the soil. However, in contrast to P. ostreatus and I. lacteus, B. adusta was not able to significantly reduce the measured toxicity.

Phosphorylation of Type IA restriction-modification complex enzyme EcoKI on the HsdR subunit.

Phosphorylation of Type I restriction-modification (R-M) enzymes EcoKI, EcoAI, and EcoR124I - representatives of IA, IB, and IC families, respectively - was analysed in vivo by immunoblotting of endogenous phosphoproteins isolated from Escherichia coli strains harbouring the corresponding hsd genes, and in vitro by a phosphorylation assay using protein kinase present in subcellular fractions of E. coli. From all three R-M enzymes, the HsdR subunit of EcoKI system was the only subunit that was phosphorylated. Further, evidence is presented that HsdR is phosphorylated in vivo only when coproduced with HsdM and HsdS subunits - as part of assembled EcoKI restriction endonuclease, while the individually produced HsdR subunit is not phosphorylated. In vitro phosphorylation of the HsdR subunit of purified EcoKI endonuclease occurs on Thr, and is strictly dependent on the addition of a catalytic amount of cytoplasmic fraction isolated from E. coli. So far this is the first case of phosphorylation of a Type I R-M enzyme reported.

Purification of a new manganese peroxidase of the white-rot fungus Irpex lacteus, and degradation of polycyclic aromatic hydrocarbons by the enzyme.

The white-rot fungus Irpex lacteus has been reported to be an efficient degrader of polycyclic aromatic hydrocarbons, polychlorinated biphenyls and pentachlorophenol. The fungus produces ligninolytic enzymes laccase, lignin peroxidase and manganese peroxidase (MnP), the latter being the major one produced. MnP was purified using anion exchange and size exclusion chromatography. SDS-PAGE showed the purified MnP to be a monomeric protein of 37 kDa (37.5 kDa using MALDI-TOF) with an isoelectric point at 3.55. The pH optimum was relatively broad, from 4.0 to 7.0 with a peak at pH 5.5. Kinetic constants K(m) were 8 microM for H(2)O(2) and 12 or 31 microM for Mn(2+) depending on the substrate. The enzyme did not perform oxidation in the absence of H(2)O(2) or Mn(2+). MnP was active at 5-70 degrees C with an optimum between 50-60 degrees C. At temperatures above 65 degrees C the enzyme rapidly lost activity. Degradation of four representatives of PAHs (phenanthrene, anthracene, fluoranthene, and pyrene) was tested and the enzyme showed the ability to degrade them in vitro. Major degradation products of anthracene were identified. The results confirm the role of MnP in PAH degradation by I. lacteus, including cleavage of the aromatic ring.