Recombinant production, functional characterization and biotechnological application of fungal oxidoreductases
Author(s)
Lezzi, Chiara
Date Issued
March 21, 2011
Type
Doctoral Thesis
Abstract
The oxido-reductases are enzymes able to catalyze the electron removal from oxidizer to
reducer. Laccase and tyrosinase, multicopper enzymes oxidizing phenolic compounds and
aromatic diamines by using molecular oxygen as the electron acceptor, belong to the
oxido-reductases group. They are enzymes commonly found in plants, fungi, bacteria and
insects. Laccase substrates have large versatility and this makes these enzymes highly
interesting for various biotechnological and industrial applications.
Though laccases and tyrosinases can be directly purified from natural producers, protein
recombinant expression in a suitable host organism is the most profitable procedure to
obtain, industrially, these enzymes in high quantities and with high purity levels.
The presence of oxidative enzyme activities has been also associated to microbial
detoxification of olive mill wastewaters (OMWs) after their treatment with selected
basidio- and asco-mycetes.
The objectives of this study were to characterize genes coding for oxidoreductases in
Basidiomycetes, in particular the ERY3 and ERY4 laccase genes of Pleurotus eryingii and
the PPO2 gene coding for the Agaricus bisporus tyrosinase, and to select non-conventional
yeast able to produce new oxidoreductase enzymes.
The ERY3 gene, coding for a P. eryngii laccase, was isolated, cloned and transformed in
Saccharomyces cerevisiae cells. The production of recombinant laccase was assayed with a
colorimetric test in presence of ABTS, on SDS-PAGE and Western blot analysis.
The recombinant yeasts cells were immobilized in calcium alginate beads and the
immobilization parameters (inoculum, sodium alginate and CaCl2 concentration) were
optimized.
The ERY4 laccase gene of P. eryngii was expressed in S. cerevisiae and the recombinant
laccase resulted to be not biologically active. This gene was thus modified in order to study
the role of its N- and C-terminal regions in determining enzyme catalytic properties. ERY4
gene was subjected to a mutational analysis following these approaches: i) C-terminal
progressive deletion to study the role of specific amino acid residues at the C-terminus of
Ery4 protein, ii) site-directed mutagenesis of the C-terminal region, iii) chimerical laccases
derived from the substitution of both its terminal regions with the corresponding regions of
ERY3 gene. The fourteen recombinant genes were separately expressed in S. cerevisiae.
2
Almost all mutant recombinant isoforms showed that they possess enzymatic activity and
affinities for different substrates.
Genes encoding the laccase isoforms which showed the best enzymatic performances on
different substrates, were expressed in S. cerevisiae by displaying the recombinant enzyme
on yeast cell surface. The biochemical characterization, immunodetection and Western blot
analysis of displayed proteins were performed. To our knowledge, this is the first example
of a functional laccase immobilized on yeast surface.
The PPO2 tyrosinase gene of Agaricus bisporus was expressed in S. cerevisiae cells and
the produced protein was purified by using affinity chromatography and biochemically
characterized. To our knowledge, this is the first time that an A. bisporus tyrosinase was
expressed in heterologous host S. cerevisiae and in biologically active form.
In order to select, from biotechnologically relevant habitat, microorganisms producing
oxidoreductases, the effluents from 5 different local olive mills were collected. Three
hundred isolates were identified according to their rDNA sequence. Twelve on the 300
identified isolates, belonging to Candida membranifaciens, C. tropicalis, Geotricum
candidum, Pichia fermentans, P. holstii and S. cerevisiae species, demonstrated that they
were able to use OMW as unique nutrient source for their growth. Phenolic compound
concentration and antimicrobial activity was rapidly and significantly decreased by the G.
candidum strains. The physiological properties of the described G. candidum isolates
confirmed the potential of these yeasts to produce a wide range of extracellular enzymes.
One of the selected G. candidum strains was chosen as a model to set up an immobilized
system of viable cells for mill waste bioremediation. Concentration of G. candidum as free
cells in the medium, the COD values, the concentration of phenolic compounds, the
medium decolourization and the antimicrobial activity were measured at estabished
interval times for both immobilized and free G. candidum biomasses. Indeed, the COD
removal and phenolics degradation by calcium alginate entrapped cells were higher than
those of free cells, because the immobilization dramatically reduces the extracellular
protease(s) activity, thus increasing the stability of microbial secreted oxidases.
Additional information
Dottorato di ricerca in Biotecnologie vegetali
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