We studied the effect of manganese and various organic chelators on the distribution, depolymerization, and mineralization of synthetic '4C-labeled lignins (DHP) in cultures of Phanerochaete chrysosporium. In the presence of high levels of manganese [Mn(II) or Mn(III)], along with a suitable chelator, lignin peroxidase (LiP) production was repressed and manganese peroxidase (MnP) production was stimulated. Even though partial lignin depolymerization was observed under these conditions, further depolymerization of the polymer to smaller compounds was more efficient when low levels of manganese were present. LiPs were prevalent under these latter conditions, but MnPs were also present. Mineralization was more efficient with low manganese. These studies indicate that MnP performs the initial steps of DHP depolymerization but that LiP is necessary for further degradation of the polymer to lower-molecular-weight products and mineralization. We also conclude that a soluble Mn(II)-Mn(III) organic acid complex is necessary to repress LiP.
Nitrogen, carbon, and manganese are potent regulators of lignin degradation, but although nitrogen and carbon elicit a generalizated response when cells are starved, manganese is a relatively specific regulator of lignin and manganese peroxidase (LiP and MnP, respectively). At high manganese levels, MnP is induced, and LiP is repressed. At low Mn levels, MnP is repressed, and LiP is induced. Organic acid chelators are very important in attaining LiP repression with high Mn. Both mineralization and lignin depolymerization are regulated by manganese in the presence of organic acid chelators. As long as the chelators keep Mn(II) and Mn(III) in solution, repression is observed, but eventually, dismutation reactions cause the formation and precipitation of Mn (IV) as MnO2. Repression is immediately relieved, and depolymerization and mineralization proceed at a high rate.
Recently, Mn(II) has been shown to induce manganese peroxidases (MnPs) and repress lignin peroxidases (LiPs) in defined liquid cultures of several white rot organisms. The present work shows that laccase is also regulated by Mn(II). We therefore used Mn(II) to regulate production of LiP, MnP, and laccase activities while determining the effects of Mn(II) on mineralization of ring-labeled synthetic lignin. At a low Mn(II) level, Phanerochaete chrysosporium and Phlebia brevispora produced relatively high titers of LiPs but only low titers of MnPs. At a high Mn(II) level, MnP titers increased 12to 20-fold, but LiPs were not detected in crude broths. P. brevispora formed much less LiP than P. chrysosporium, but it also produced laccase activity that increased more than sevenfold at the high Mn(II) level. The rates of synthetic lignin mineralization by these organisms were similar and were almost seven times higher at low than at high Mn(II). Increased synthetic lignin mineralization therefore correlated with increased LiP, not with increased MnP or laccase activities.
n‐Propanol was the most effective solvent for extracting antibacterial substances from olive oil mill waste water (‘alpechines’). Several phenolics were detected in propanol extracts that had bactericidal effects on Bacillus megaterium ATCC 33085, inhibiting sporulation and germination at 5.6 mmol/l total phenolics (expressed as syringic acid). The biological effect was increased in the presence of high glucose and NaCl concentrations and after β‐glucosidase hydrolysis.
Apurinic/apyrimidinic (AP) sites in DNA are considered to be highly mutagenic and must be corrected to preserve genetic integrity. We have isolated cDNAs from the Trypanosomatidae Leishmania major and Trypanosoma cruzi capable of complementing the deficiency of exonuclease III and dUTPase in the Escherichia coli mutant BW286. This double mutant is non-viable at 37 degreesC due to an accumulation of non-repaired sites following excision of uracil from DNA. The genes were expressed as beta-galactosidase-AP endonuclease fusion proteins and as such are active in repair of AP sites in E. coli. The Trypanosoma and Leishmania sequences have unique N-termini containing sequences that correspond to probable nuclear transport signals, while the C-terminal domains exhibit pronounced similarity to exonuclease III. The L.major gene was overexpressed as a histidine-tagged protein and recombinant enzyme exhibited endonuclease activity on AP DNA in vitro. Furthermore, expression of the enzymes in AP endonuclease-deficient E.coli mutants conferred significant resistance to killing by methylmethane sulphonate and peroxides. This study constitutes one of the first descriptions of DNA repair enzymes in these pathogenic organisms where oxidative stress is an important mechanism of both drug-mediated and intracellular killing.
In addition to excreting lignin-degrading peroxidases, the white rot fungus Phanerochaete flavido-alba also excretes a laccase. This protein was purified to homogeneity and found to have a molecular weight of 94,000 and an isoelectric point lower than 3.55. Its UV-visible spectrum is typical of copper-containing proteins.
The antibacterial effects of olive oil wastes from traditional installations (TM), wastes from modern installations (CM), and wastes from an evaporation pool (EP) were assayed with a soil bacterium (Bacillus megaterium ATCC 33085). Irrespective of the origin, fresh wastes showed higher antimicrobial activity than those from evaporation pools. Among several extractants, ethyl acetate and n‐propanol were the most effective organic solvents in the recovery of the antimicrobial activity from wastes. No antimicrobial activity was recovered by hexane extraction. Extracts from evaporation pool wastes showed no antibacterial activity. A good correlation between phenolic content and antimicrobial activity was found.
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