A new subfamily of fungal subtilases: structural and functional analysis of a Pleurotus ostreatus member

Pleurotus ostreatus produces several extracellular proteases which are believed to be involved in the regulation of the ligninolytic activities of this fungus. Recently, purification and characterization of the most abundant P. ostreatus extracellular protease (PoSl) have been reported. The sequence of the posl gene and of the corresponding cDNA has been determined, allowing the identification of its pre- and pro-sequences. A mature protein sequence has been verified by mass spectrometry mapping, the N-glycosylation sites have been identified and the glycosidic moieties characterized. Mature PoSl shows a cleaved peptide bond in the C-terminal region, which remains associated with the catalytic domain in a non-covalent complex. Reported results indicate that this enzyme is involved in the activation of other P. ostreatus secreted proteases, thus suggesting its leading role in cascade activation mechanisms. Analyses of the PoSl sequence by homology search resulted in the identification of a DNA sequence encoding a new protease, homologous to PoSl, in the Phanerochaete chrysosporium genome. A new subgroup of subtilisin-like proteases, belonging to the pyrolysin family, has been defined, which includes proteases from ascomycete and basidiomycete fungi

quorum sensing involvement in response surface methodology for optimisation of sclerotiorin production by penicillium sclerotiorum in shaken flasks and bioreactors

Sclerotiorin, an azaphilone produced by some filamentous fungi including Penicillium sclerotiorum, is a pigment with variety of biological activities including lipoxygenase inhibition, reduction of cholesterol levels, and anti-cancer properties. Sclerotiorin has potential use in pharmaceutical as well as food industries. In this context, the purpose of this study was to provide a simple and robust procedure for optimised production of sclerotiorin by P. sclerotiorum using a central composite design developed through response surface methodology (RSM) and to identify the molecule(s) involved in the signalling mechanism in P. sclerotiorum. The optimisation of sclerotiorin production was carried out using RSM in shaken flasks and the obtained results were then replicated using a 2-L stirred tank bioreactor. Penicillium sclerotiorum ethyl acetate culture extract was analysed using thin layer chromatography (TLC) and potential signalling molecules were identified using Gas chromatography-mass spectrometry (GC-MS). The experimental studies suggested an increase in the sclerotiorin production by 2.1-fold and 2.2-fold in shaken flasks and stirred tank bioreactors respectively. Further analysis of P. sclerotiorum ethyl acetate culture extract reported the presence of ricinoleic acid, an oxylipin, belonging to a family of signalling molecules tentatively involved in the enhancement of sclerotiorin production. This paper has highlighted the positive effect of the optimal supplementation of P. sclerotiorum culture extracts for enhanced production of sclerotiorin. It has also examined potential molecules involved in the signalling mechanism in P. sclerotiorum culture extract for the overproduction of sclerotiorin

Repurposing designed mutants: a valuable strategy for computer-aided laccase engineering – the case of POXA1b

The broad specificity of laccases, a direct consequence of their shallow binding site, makes this class of enzymes a suitable template to build specificity toward putative substrates. In this work, a computational methodology that accumulates beneficial interactions between the enzyme and the substrate in productive conformations is applied to oxidize 2,4-diamino-benzenesulfonic acid with POXA1b laccase. Although the experimental validation of two designed variants yielded negative results, most likely due to the hard oxidizability of the target substrate, molecular simulations suggest that a novel polar binding scaffold was designed to anchor negatively charged groups. Consequently, the oxidation of three such molecules, selected as representative of different classes of substances with different industrial applications, significantly improved. According to molecular simulations, the reason behind such an improvement lies in the more productive enzyme–substrate binding achieved thanks to the designed polar scaffold. In the future, mutant repurposing toward other substrates could be first carried out computationally, as done here, testing molecules that share some similarity with the initial target. In this way, repurposing would not be a mere safety net (as it is in the laboratory and as it was here) but rather a powerful approach to transform laccases into more efficient multitasking enzymes.This work was funded by INDOX (KBBE-2013-7-613549) European project and CTQ2013-48287-R Spanish National Project. V. G. and E. M. acknowledge Università degli Studi di Napoli and Generalitat de Catalunya for their respective predoctoral fellowships.Peer ReviewedPostprint (author's final draft

Recombinant protein expression system in cold loving microorganisms

Soluble and functional proteins are of high demand in modern biotechnology. Although many recombinant proteins have been successfully obtained from common prokaryotic and eukaryotic hosts, these systems result to be often unproductive due to the peculiar properties of the protein to be produced. Incorrect folding of the nascent polypeptide chains is one of the main problems occurring during heterologous protein production in bacteria. Since formation of inclusion bodies often impairs the recombinant production of valuable proteins, many experimental approaches have been explored to minimize this undesirable effect [1, 2]. Expression of "difficult" proteins has also been carried out by lowering the temperature at the physiological limit allowed for the growth of mesophilic host organisms (between 15 and 18°C for Escherichia coli). Lowering the temperature, in fact, has a pleiotropic effect on the folding process, destabilising the hydrophobic interactions needed for intermediates aggregation [3]. On the basis of the above considerations, a rational alternative to mesophilic organisms is the use of naturally cold-adapted bacteria as hosts for protein production at low temperature (even at around 0°C)

A Novel White Laccase from Pleurotus ostreatus

Abstract Two laccase isoenzymes (POXA1 and POXA2) produced by Pleurotus ostreatus were purified and fully characterized. POXA1 and POXA2 are monomeric glycoproteins with 3 and 9% carbohydrate content, molecular masses of about 61 and 67 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, of about 54 and 59 kDa by gel filtration in native conditions, and of 61 kDa by matrix-assisted laser desorption ionization mass spectrometry (only for POXA1) and pI values of 6.7 and 4.0, respectively. The N terminus and three tryptic peptides of POXA1 have been sequenced, revealing clear homology with laccases from other microorganisms, whereas POXA2 showed a blocked N terminus. The stability of POXA2 as a function of temperature was particularly low, whereas POXA1 showed remarkable high stability with respect to both pH and temperature. Both enzymes oxidize syringaldazine and ABTS (2, 2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) together with a variety of different substituted phenols and aromatic amines with the concomitant reduction of oxygen, but POXA1 is unable to oxidize guaiacol. Both enzymes were strongly inhibited by sodium azide and thioglycolic acid but not by EDTA. UV/visible absorption spectra, atomic adsorption, and polarographic data indicated the presence of 4 copper atoms/mol of POXA2 but only one copper, two zinc, and one iron atoms were found/mol of POXA1. The neutral pI and the anomalous metal content of POXA1 laccase render this enzyme unique in its structural characteristics. The lack of typical absorbance at 600 nm allows its classification as a "white" laccase

Induction and Transcriptional Regulation of Laccases in Fungi

Fungal laccases are phenol oxidases widely studied for their use in several industrial applications, including pulp bleaching in paper industry, dye decolourisation, detoxification of environmental pollutants and revalorization of wastes and wastewaters. The main difficulty in using these enzymes at industrial scale ensues from their production costs. Elucidation of the components and the mechanisms involved in regulation of laccase gene expression is crucial for increasing the productivity of native laccases in fungi. Laccase gene transcription is regulated by metal ions, various aromatic compounds related to lignin or lignin derivatives, nitrogen and carbon sources. In this manuscript, most of the published results on fungal laccase induction, as well as analyses of both the sequences and putative functions of laccase gene promoters are reviewed. Analyses of promoter sequences allow defining a correlation between the observed regulatory effects on laccase gene transcription and the presence of specific responsive elements, and postulating, in some cases, a mechanism for their functioning. Only few reports have investigated the molecular mechanisms underlying laccase regulation by different stimuli. The reported analyses suggest the existence of a complex picture of laccase regulation phenomena acting through a variety of cis acting elements. However, the general mechanisms for laccase transcriptional regulation are far from being unravelled yet