This work represents the first report on the ability of autochthonous fungi from Tunisia to produce ligninolytic enzymes. Three hundred and fifteen fungal strains were isolated from different Tunisian biotopes. These fungal strains were firstly screened on solid media containing Poly R-478 or ABTS as indicator compounds that enabled the detection of lignin-modifying enzymes as specific color reactions. Of the 315 tested strains, 49 exhibited significant ABTS-oxidation activity expressed within the first week of incubation and only 18 strains decolorized the Poly R-478. Liquid cultivations and laccase, manganese peroxidase and lignin peroxidase activity assays of positive strains confirmed that eight efficient enzyme producers were found in the screening. These strains were attributed to the most closely related species using PCR amplification and sequencing of the internal transcribed spacer 'ITS' regions of the ribosomal DNA. The identification results showed fungal genera such as Oxyporus, Stereum and Trichoderma which have been only rarely reported as ligninolytic enzyme producers in the literature. Culture conditions and medium composition were optimized for the laccase producer Trametes trogii CTM 10156. This optimization resulted in high laccase production, 367 times more than in non-optimized conditions and which reached 110 U ml -1 within 15 days of incubation.
The phenol-degrading strain Pseudomonas pseudoalcaligenes MH1, identified by the rRNA approach, was isolated from wastewater enrichment culture. It utilized phenol up to 1.5 g/L as the sole source of carbon and energy. In addition, cresols (o-, m-, p-), 4-hydroxybenzoic acid, syringic acid, and vanillic acid were metabolized as sole substrates by phenol-grown cells of strain MH1. Using primers, designed on the basis of the sequence of the dmp operon of P. putida strain CF600, a gene encoding phenol hydroxylase, which catalyzes the hydroxylation of phenol to catechol, was detected in strain MH1. The whole phenol hydroxylase operon of strain MH1 was amplified in a polymerase chain reaction fragment of 5.207 kb that was cloned and sequenced. The total sequence revealed a cluster of six ATG starting open reading frames (ORFs). Analysis of the regulatory signals showed a putative promoter region, 40 bp upstream from the transcriptional start of ORF1, which have a strong homology to a set of positively controlled promoters. Comparison of the MH1 phenol hydroxylase gene sequence with those of other Pseudomonas strains revealed higher homology except in the 5' region. Thus, the deduced amino acid sequence of the first subunit of phenol hydroxylase of P. pseudoalcaligenes strain MH1 exhibited a difference at the N-terminal region (the first 10 amino acids) compared with that of known phenol hydroxylase of Pseudomonas strains.
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