Masaaki Kuwahara

Screening of basidiomycetes for lignin peroxidase genes using a DNA probe

SummaryBasidiomycetes were screened for lignin peroxidase (LPO) genes using a DNA probe prepared from the LPO restriction fragment ofPhanerochaete chrysosporium. Southern blot analysis showed restriction fragments of chromosomal DNA ofBjerkandera adusta andCoriolus consors hybridized with the probe.Bjerkandera adusta produced LPO in a glucose-peptone medium. Ion-exchange chromatography showed that this fungus produced multiple molecular forms of LPO. One of the enzymes, LPO-2, was purified and characterized. The molecular weight of LPO-2 was 41000 with a pI of 4.2. Spectral analysis demonstrated that LPO-2 is a haem protein. The enzyme cleaved lignin model dimers mainly at the Cα-Cβ position of the side chain. The LPO-2 exhibited close similarity to LPOs ofP. chrysosporium with respect to their basic properties.

Cloning and sequencing of a ligninase gene from a lignindegrading basidiomycete, Phanerochaete chrysosporium

SummaryA ligninase gene has been cloned from a Phanerochaete chrysosporium genomic DNA library. Nucleotide sequencing of the gene has revealed that the ligninase structural gene contains 1116 bp of the protein-encoding sequence, of which 84 bp encode the signal peptide. The protein-encoding sequence is interrupted by eight introns which conform to the universal G-T/A-G splicing rule observed for the 3′ and 5′ intron boundaries. The putative eukaryotic regulatory sequences, i.e. “CAAT” and “TATA” box-like sequences, are present in the 5′ flanking region.

An extracellular NADH-oxidizing peroxidase produced by a lignin-degrading basidiomycete, Phanerochaete chrysosporium

A lignin-degrading basidiomycete, Phanerochaete chrysosporium, produces an extracellular peroxidase which in turn produces H2O2 by catalyzing the oxidation of NADH and NADPH. The high enzyme activity was observed in the culture grown under nutrient nitrogen limitation (low-N) and high oxygen tension (high-O2). The enzyme activity was absent in non-ligninolytic agitated culture and in the cultures of non-ligninolytic mutant strains of this organism. The culture method using polyurethane foam cubes as a support for the growing mycelia showed the beneficial effect of producing a large amount of the enzyme. The enzyme is capable of catalyzing the oxidation of NADH and NADPH in the absence of added H2O2, and its activity was inhibited strongly by catalase and superoxide dismutase. It is suggested that this peroxidase participates in the ligninolytic system of Phanerochaete chrysosporium as a donor of H2O2, which is required for the lignin-peroxidase reaction, by oxidizing extracellular NADH and NADPH.

Evaluation of land application using secondary effluent in a forest slope: estimation of drained water quality and discussion of the effects upon soil or plants and behavior of bacteria

Abstract The secondary effluent of domestic wastewater was watered at test facilities constructed on a slope in forest land in order to evaluate re-use of the secondary effluent and to examine changes in the chemical properties of soil or plants caused by the watering, where hydrological data in the area of the test facilities were obtained. The concentration of most cations and anions, except Ca2+, SiO2 and NO3, decreased with the accumulation of some cations in the soil layers. The substance accumulated in the soil layers was observed to transfer to plant species, where absorption of Cl− and Mn2+ by a few plant species was found. Alteration of weed species and favorable effects on tree growth under the affects of the watering were confirmed. Coliform bacteria were markedly removed during passage of the watering effluent, probably due to filtration and/or absorption effects of the soil. Estimation of the drained water quality was carried out using the relationship between the removed portion and water recovery and others, under circumstances where unfavorable effects on soil and plant growth by the watering were scarcely found. Since cost evaluation of the present procedure compared to advanced water treatment was satisfactory, the following watering mode: 30–35 kl/2 h/1100 m2/day, can be adopted as an example of re-use of secondary effluent.