Search

Yeast cell wall proteins can be used for surface display of various proteins of industrial interest. Generally, yeast cell wall contains proteins that are non-covalently (Scw- proteins) or covalently (Ccw-proteins) bound to β-1, 3-glucan, the latter either through GPI- anchors and β-1, 6-glucan, or by alkali labile ester linkages between γ-carboxyl groups of glutamic acid and hydroxyl groups of glucoses (Pir-proteins). Covalently bound proteins are usually used for immobilization of different recombinant proteins. Here we explore possibility of using Scw4 and Scw10 as novel immobilization platforms. Previously it was shown that Scw4 protein can be covalently and non-covalently linked to the cell wall structures. It was also shown that proteolytic processing of Scw4 by yapsins strongly affects its covalent binding. As Scw10 shares high level of similarity with Scw4, it is interesting to investigate whether Scw10 can also form both covalent and non-covalent interactions with cell wall structures. Although protein Scw10 also contains sequence charasteristic for Kex2 processing, it is not yet clearly confirmed whether Scw10 is substrate for Kex2. Furthermore, both of these proteins have potential to be used for development of new homologous and heterologous cell surface expression system for wide range of yeast species given that genomes of some other yeast species contain SCW4/SCW10 orthologs, as it is noted before. In order to develop Scw4/Scw10 based expression systems, further characterization of this two proteins is necessary. One line of the research was focused on Scw10 proteolytic processing. Results show that Scw10 undergoes processing by Kex2 and possibly by yapsins at the same site. Results also show that this processing doesn’t affect significantly Scw10 covalent binding efficiency. Other line of the research examines the level of evolutionary preservation of these proteins among different ascomycetes yeast by analysing cell wall protein profiles. Obtained results were coupled with results of in-sillico analysis of proteomes of different yeast species and it was found that Scw4 and Scw10 show high level of evolutionary preservation, given that all of analysed ascomycetes yeast, except two Schizosaccharomyces species, contained at least one protein with high similarity to them. Further research will focus on development of Scw4/Scw10 based expression system models and usage of this models in different yeast species

Xanthohumol, a prenylated chalcone from hops and beer, is a phenolic compound that has received considerable attention in recent years. This compound has a range of interesting biological properties that may have therapeutic utility: anti-inflammatory, antioxidant, antilipoperoxidative activities as well as antiangiogenic, antiproliferative and apoptotic effects, mainly assessed in vitro studies that reasonably suggest a potential chemopreventive activity. Phenomenon how xanthohumol affects brewing yeast’s metabolism, yeast viability and vitality were studied during the production of a xanthohumol enriched beer (10 mg/L xanthohumol). The results showed that yeast viability was slightly decreased by xanthohumol, but on the other hand yeast vitality in the xanthohumol enriched brewing trials was slightly better. The content of higher alcohols and esters was similar to the control in all the xanthohumol enriched brewing trials. In laboratory scale experiment about effect of xanthohumol on brewing yeast growing was done. No effect of xanthohumol on yeast growth was detected. Due to the fact that yeast viability was slightly decreased by xanthohumol, experiment with zymolyase was done. Yeast cells cultivated with xanthohumol are significantly more sensitive to zymolyase treatment.

The cell wall of yeast Saccharomyces cerevisiae contains more than 20 different manoproteins. They are considerd to play different roles in building, maintaining and modifying the wall itself when different cell cycle events and they are important for interactions of cells with their surrounding. Yeasts have evolved three different ways of attaching proteins to cell wall glucan. Some proteins are bound to β -1, 3-glucan noncovalently (Scw – soluble cell wall proteins ; extracted by hot SDS), while others are attached covalently (Ccw – covalently linked cell wall proteins ; extracted by glucanases) through GPI-anchor and β -1, 6-glucan, or directly to β -1, 3-glucan by alkali labile ester linkage between the γ -carboxyl groups of glutamic acid and hydroxyl groups of glucoses (Pir – proteins with internal repeats ; extracted by NaOH). Disruption of genes coding for the Pir-proteins was performed to investigate their potential role. After disruption of all PIR genes, 67kDa protein still remained in NaOH extract. SCW4 disruption resulted in disappearance of a 67kDa band from the extract, indicating that Scw4p could also be covalently linked to the cell wall simillar to Pir-proteins. Since it was reported previously that Scw4p was a noncovalently attached protein, this finding can be relevant for the role of Scw4p. In order to investigate the role of the Scw4p in the construction of the cell wall, yeast was transformed with a high copy number plasmid containing SCW4. In order to get further insight in the binding mechanism, a novel, simple binding assay for Pir family proteins was developed in which externally added Ccw5p/Pir4p was bound to different cell wall mutant strains. It has been shown that pir, as well as scw4 and scw10 mutants can bind externally added Ccw5p to their cell wall. Further, we investigated the conditions under which these proteins were attached to cell wall. The presence of EDTA blocked the binding of Ccw5p, indicating the cation dependence of the reaction. Both wild type and mutant cells showed enhanced binding in 0.6 M KCl. It was also shown that the native conformation of Ccw5p is required for its binding.

also as a cellular structure by which the cell corresponds with its surrounding. Budding yeast wall has a bilayered structure. The inner layer consists mainly of a network of linear ß-1, 3-glucan, and the outer layer is composed of an unusually high number of mannoproteins. Yeasts have evolved three different ways of attaching proteins to the polysaccharide network. Some proteins are bound to ß-1, 3-glucan noncovalently (Scw – soluble cell wall proteins ; extracted by hot SDS), while others are attached covalently (Ccw – covalently linked cell wall proteins ; extracted by glucanases) either through glycosylphosphatidyl inositol (GPI) anchor and ß-1, 6-glucan, or directly to ß-1, 3-glucan by an alkali labile ester linkage between the γ -carboxyl groups of glutamic acid and hydroxyl groups of glucoses (Pir – proteins with internal repeats ; extracted by mild alkalis). The exact physiological roles for most cell wall proteins are still unknown. Molecular mechanisms for incorporation of different groups of wall proteins, as well as the potential use of these mechanisms for cell surface engineering will be reviewed.

Saccharomyces cerevisiae cell wall has a bilayered structure. The inner part consists mainly of a network of linear ß-1, 3-glucan molecules, and the outer layer is composed mostly of mannoproteins. Yeasts have evolved three different ways of attaching proteins to the polysaccharide network. Some proteins are bound to ß-1, 3-glucan noncovalently (Scw – soluble cell wall proteins ; extracted by hot SDS), while others are attached covalently (Ccw – covalently linked cell wall proteins ; extracted by glucanases) through glycosylphosphatidyl inositol (GPI) anchor and ß-1, 6-glucan, or directly to ß-1, 3-glucan by an alkali labile ester linkage between the γ -carboxyl groups of glutamic acid and hydroxyl groups of glucoses (Pir – proteins with internal repeats ; extracted by NaOH). The exact physiological roles for most of the cell wall proteins are still unknown. A multiple disruption of genes coding for the Pir family proteins (ccw5 ccw6 ccw7 ccw8) was performed in order to investigate their potential role. After disruption of the genes coding for all Pir family members, 67kDa protein band still remained in the NaOH extract. Disruption of SCW4 resulted in apparent disappearance of the 67kDa band, indicating that Scw4p could also be covalently linked to the cell wall in a similar manner like Pir proteins. In order to investigate the role of the Scw4p in the construction of the cell wall, wilde type yeast was transformed with a high copy number plasmid containing SCW4 gene. Phenotypes of this strain as well as scw4, ccw5ccw6ccw7ccw8 and ccw5ccw6ccw7ccw8scw4 were examined. In order to get further insight in the binding mechanism a novel, simple binding assay for Pir family proteins, was developed. It has been shown that pir, as well as scw4 and scw10 mutant cells, can bind externally added Ccw5p to their cell wall. A study of appropriate binding conditions revealed the requirement of the native conformation of Ccw5p. The presence of EDTA blocked the binding of Ccw5p, indicating the cation dependence of the reaction. Both wild type and mutant cells showed enhanced binding in 0.6 M KCl. Further experiments should provide answers to question regarding exact conditions required, as well as reveal if the binding occurs autocatalytically or require a particular enzyme in the wall.

Yeasts have evolved three different ways regarding the glucan attached proteins of cell wall. Some proteins are bound noncovalently to the β -1, 3-glucan, while others are attached covalently to the β -1, 6-glucan through a GPI-anchor, or directly by alkali labile ester linkage between the γ -carboxyl group of glutamic acid and the hydroxyl group of glucose (named as Pir – proteins). Unusual versatility of the cell wall proteins implies different roles and reactions taking place in the wall. Different mutants lacking individual wall proteins or groups of proteins were constructed and their phenotypes were studied. After disruption of all the PIR genes (CCW5, CCW6, CCW7 and CCW8), a 67kDa protein still remained in NaOH extract. SCW4 disruption in the ccw5ccw6ccw7ccw8 mutant, however, resulted in disappearance of the 67kDa band from the extract, indicating that Scw4p could also be covalently linked to the cell wall, similarly to the Pir-proteins. Since it was reported previously that Scw4p was a noncovalently attached protein, this finding can be relevant for the role of Scw4p. A mild treatment of yeast cells with β -glucanase induced a drastic decrease in the cell surface hydrophobicity indicating that it is at least to a great extent due to mannoproteins of the outer layer of the cell wall. This phenomenon together with the obvious assumption that the outermost located mannoproteins play crucial role in the cell-cell interaction during film or floc formation motivated us to screen for the differences in cell wall protein composition between the wine yeast strains, which form a film (flor) on the wine surface and those unable to do so in order to get a better insight into the intermolecular interactions during cell attachment.

Aging of Hungarian botrytized wine Tokaji szamorodni results aromatic compounds produced as by-products of the oxidative metabolism of yeasts taht are forming a biofilm at the surface of wines. Film-forming strains show a planktonic growth during the main fermentation phase but the cells rise to the surface afetrward and form a multylayerd biofilm on it. The cells metabolize mainly ethanol and glycerol during maturation and produce caracteristic aromatic compounds like acetaldehyde. However, the mechanism of attachment and the role of cell surface moleculs in film formationare not clarified at all. Our aim was to determine the possible role of certain cell wall proteins in film formation, therefore we looked for differences in the cell wall proteins of sedimenting and film-forming Saccharomyces cerevisiae strains. We found different protein patterns among alkali extractable cell wall proteins of laboratory and film-forming Saccharomyces cerevisiae strins. In the case of film-forming strains a smaller ccw7 protein was observed and isolated. For further investigation we designed a specific primer pair for the amplification of the Ccw7p encoding gene HSP150. PCR amplification of the gene confirmed the size difference between the ccw7 proteins of the laboreatory and film-forming strains. Almost all the film-forming isolates generated a single PCR product of the same size but two of them contained additional amplicons, a shorter or a longer one. Sequence analysis of the amplified common PCR products of film-forming yeasts revealed two deletions in the internal repetitive region of the gene. In the case of TDO4 strain that has an additional longer PCR amplicon we induced meiotic segregation aiming to check whether there is any correlation between the film forming ability and the CCW7 alleles. The alleles segregated during meiosis, however, both types of the segregants maintained the film forming ability. CCW7 amplicons of different lenghts. By the use of RFLP analysis of the CCW7 amplicons we found that majority of film forming yeasts formed a separate cluster of 100% similarity, while sedimenting wine yeast strains showed an extended length polymorphism.

Yeasts have developed three different ways of attaching proteins to cell wall glucan. Some proteins are bound to betha-1, 3-glucan noncovalently, while others are attached covalently through GPI-anchor and betha-1, 6-glucan, or directly to betha-1, 3-glucan by alkali labile ester linkage between the y-carboxyl groups of glutamic acid and hydroxyl groups of glucoses (Pir-proteins). In order to get further insight into the binding mechanism, a novel, simple binding assay for Pir-family proteins was developed. It has been shown that PIR, as well as SCW4 mutants, can bind externally added Ccw5p to their cell walls. A study of appropriate binding conditions revealed the requirement of the native conformation of Ccw5p. The presence of EDTA blocked the binding of Ccw5p, indicating the cation dependence of the reaction. Both wild type and mutant cells showed enhanced binding of the Ccw5p in 0.6 M KCl. After disruption of all Pir genes (CCW5, CCW6, CCW7 and CCW8), 67 kDa protein still remained in NaOH extract. SCW4 disruption in the ccw5ccw6ccw7ccw8 mutant resulted in disappearance of the 67 kDa band from the extract, indicating that Scw4p could also be covalently linked to the cell wall by a so far unidentified alkali-labile linkage.

Saccharomyces cerevisiae cell wall has a bilayered structure. The inner part consists mainly of a network of linear ß-1, 3-glucan molecules, whereas the outer layer is composed mostly of mannoproteins. Yeasts have evolved three different ways of attaching proteins to the polysaccharide network. Some proteins are bound noncovalently while the others are covalently attached through glycosylphosphatidyl inositol (GPI) anchor and ß-1, 6-glucan, or directly to ß-1, 3-glucan (Pir – proteins with internal repeats). Pir proteins are soluble in mild alkali and do not have GPI anchor. Since these proteins are highly glycosylated their deletion changes the polysaccharide composition of the cell walls. Five PIR genes with varying number of repeating units (1-11) were identified, and the repeating unit seems to be responsible for the formation of an ester bond between the γ -carboxyl groups of glutamic acid and hydroxyl groups of glucoses. In order to get further insight in the binding mechanism, a novel, simple binding assay for Pir family proteins was developed in which externally added Ccw5p/Pir4p was bound to different cell wall mutant strains. It has been shown that pir, as well as scw4 and scw10 mutants can bind externally added Ccw5p to their cell wall. Further, we investigated the conditions under which these proteins were attached to cell wall. The presence of EDTA blocked the binding of Ccw5p, indicating the cation dependence of the reaction. Both wild type and mutant cells showed enhanced binding in 0.6 M KCl. It was also shown that the native conformation of Ccw5p is required for its binding. A multiple disruption of genes coding for the Pir family proteins (ccw5 ccw6 ccw7 ccw8) was performed in order to investigate their potential role in the cell wall biogenesis. After disruption of all Pir family genes, 67kDa protein band still remained in the NaOH extract. It was eliminated by the disruption of SCW4, indicating that Scw4p could also be covalently linked to the cell wall by an alkali sensitive linkage.

Fungal cell walls attracts attention for their use as an ideal potential target for antimycotics, but also as a cellular structure by which the cell corresponds with its surrounding. Budding yeast wall has a bilayered structure. The inner layer consists mainly of a network of linear ß-1, 3-glucan, and the outer layer is composed of an unusually high number of mannoproteins. Yeasts have evolved three different ways of attaching proteins to the polysaccharide network. Some proteins are bound to ß-1, 3-glucan noncovalently (Scw – soluble cell wall proteins ; extracted by hot SDS), while others are attached covalently (Ccw – covalently linked cell wall proteins ; extracted by glucanases) either through glycosylphosphatidyl inositol (GPI) anchor and ß-1, 6-glucan, or directly to ß-1, 3-glucan by an alkali labile ester linkage between the γ -carboxyl groups of glutamic acid and hydroxyl groups of glucoses (Pir – proteins with internal repeats ; extracted by mild alkalis). The exact physiological roles for most cell wall proteins are still unknown. Molecular mechanisms for incorporation of different groups of wall proteins, as well as the potential use of these mechanisms for cell surface engineering will be reviewed. Also, the potential of cell wall proteins for identification and characterisation of wine yeasts will be discussed.

Yeasts have evolved three different ways of attaching proteins to cell wall glucan. Some proteins are bound to betha-1, 3-glucan noncovalently, while others are attached covalently through GPI-anchor and betha-1, 6-glucan, or directly to betha-1, 3-glucan by alkali labile ester linkage between the γ -carboxyl groups of glutamic acid and hydroxyl groups of glucoses (Pir – proteins). In order to get further insight in the binding mechanism a novel, simple binding assay for Pir family proteins, was developed. It has been shown that pir, as well as scw4 and scw10 mutant cells, can bind externally added Ccw5p to their cell wall. A study of appropriate binding conditions revealed the requirement of the native conformation of Ccw5p. The presence of EDTA blocked the binding of Ccw5p, indicating that reaction is dependent on the presence of divalent cations. Both wild type and mutants cells showed higher capacity for binding Ccw5p under increased osmotic pressure indicating that reaction might be under control of the HOG pathway. On the other hand some other shock conditions do not affect capability of cells to bind Ccw5p, showing that increased binding of Pir proteins is not generally caused by a shock. After disruption of all PIR genes (CCW5, CCW6, CCW7 and CCW8), 67kDa protein still remained in NaOH extract. SCW4 disruption in the ccw5ccw6ccw7ccw8 mutant resulted in disappearance of the 67kDa band from the extract, indicating that Scw4p could also be covalently linked to the cell wall simillar to Pir-proteins. Since it was reported previously that Scw4p was a noncovalently attached protein, this finding can be relevant for the role of Scw4p. In order to investigate its role in the construction of the cell wall, yeast was transformed with a high copy number plasmid containing SCW4. Phenotypes of this strain as well as scw4, ccw5ccw6ccw7ccw8 and ccw5ccw6ccw7ccw8scw4 were examined.

A family of covalently linked cell wall proteins of Saccharomyces cerevisiae, called Pir proteins, are characterized by up to 10 conserved repeating units. Ccw5/Pir4p contains only one complete repeating sequence and its deletion caused a release of the protein into the medium. The exchange of each of three glutamines (Gln69, Gln74, Gln76) as well as one aspartic acid (Asp72) within the repeating unit leads to a loss of the protein from the cell wall. Amino acid sequencing revealed that only Gln74 is modified. Release of the protein with mild alkali, changed Gln74 to to glutamic acid, suggesting that Gln74 is involved in the linkage. Analysis by mass spectrometry showed that 5 hexoses are attached to Gln/Glu74. Sugar analysis revealed glucose as the only constituent. It is suggested that Pir proteins form novel, alkali labile ester linkages between the y-carboxyl group of glutamic acids, arising from specific glutamines, with hydroxyl groups of glucoses of betha-1, 3-glucan chains. This transglutaminase-type reaction could take place extracellularly and would energetically proceed on the account of amido group elimination.

The cell wall of the yeast S. cerevisiae has a bilayered structure. The inner part of the wall consists mainly of b-1, 3-glucan, and the outer layer is composed mostly of mannoproteins. Yeasts have evolved three different ways of attaching proteins to glucan. Some proteins are bound noncovalently, while others are attached covalently through GPI-anchor and  -1, 6-glucan, or directly to  -1, 3-glucan by a mechanism still not completely understood (Pir-proteins ; extracted from the wall by NaOH). Exact physiological role for most cell wall proteins is still unknown. Disruption of genes coding for the Pir-proteins was performed to investigate their potential role. After disruption of all PIR genes, 67kDa protein still remained in NaOH extract. SCW4 disruption resulted in disappearance of a 67kDa band from the extract, indicating that Scw4p could also be covalently linked to the cell wall simillar to Pir-proteins. Since it was reported previously that Scw4p was a noncovalently attached protein, this finding can be relevant for the role of Scw4p. In order to investigate the role of the Scw4p in the construction of the cell wall, yeast was transformed with a high copy number plasmid containing SCW4. Phenotypes of this strain as well as scw4, ccw5ccw6ccw7ccw8 and ccw5ccw6ccw7ccw8scw4 were examined.

S. cerevisiae cell wall is composed of a beta-1, 3-gucan network to which more then 20 different mannoproteins are attached (1). Besides, small amounts of chitin and beta-1, 6-glucan are also found. Some of the cell wall proteins are linked to the glucan backbone noncovalently or solely via S-S links, and can be extracted from the walls by SDS or DTT (2). Six proteins of this group have been indentified so far and it has been found that they are all either hydrolytic enzymes, or at least are homologous to known yeast glucanases (2). Covalently linked cell wall proteins can be divided according to their attachment in two groups. The first group represents proteins, which can be extracted from purified cell walls depleted of SDS-extractable proteins, using different glucanase preparations such as zymolyase, or laminarinase. Identification of nine proteins of this group has shown that they all posses a C-terminal signal for the attachment of the GPI-anchor (3, 4, 5, 6). It has, therefore, been postulated that these proteins get plasma membrane anchored through GPI, and are then translocated, by a so far unknown mechanism, to beta-1, 6-glucan, which serves as a link to the beta-1, 3-glucan network of the cell wall (7). The second group of covalently linked cell wall proteins was obtained from walls after SDS extraction with 30 mM NaOH (8, 9). Four proteins of this group were identified by N-terminal sequencing and they were designated Ccw5p to Ccw8p (Ccw for Covalently linked cell wall). It has been found that Ccw6p, Ccw7p and Ccw8p were products of PIR1, PIR2 and PIR3 genes for which it has previously been reported to contain a characteristic repetitive sequence (PIR for Proteins with internal repeats, 10). The four proteins belong to the same family sharing a high degree of identity and having several common structural features. All are proteolytically processed by the Kex2p protease so that the repetitive sequence occupies the N-terminal part of mature proteins. The number of repetitive units ranges from 2 in Ccw5p, to 9 in Ccw7p. Besides, all four proteins are extensively O-glycosylated and do not posses the signal for the addition of a GPI-anchor. The latter indicates that there must be another molecular mechanism for the incorporation of proteins into the cell wall, different from the one suggested for proteins extracted by glucanases. Therefore, the aim of this investigation was to get an insight into the way of attachment of this group of proteins to the cell wall. Since members of the Pir family are generally not found in glucanase wall extracts they may be attached to chitin in the wall, rather than to glucan. Therefore, proteins were isolated from purified cell walls by chitinase. Three distinct electrophoretic protein bands at 47, 67 and 117 kDa were obtained in the extract. The same bands were, however obteined from the walls of the mutant lacking all four proteins of the Pir family, indicating that chitinase-extractable proteins were not Pir-proteins. The same bands were also obtained from cell walls of the mutant lacking five major glucanase-extractable proteins i.e. Ccw12p, Ccw13p, Cwp1p, Icwp1p and Tip1p, suggesting that the three proteins were so far unidentified. To find out which part of Pir proteins is responsible for their attachment to the cell wall, Ccw5p was used as a model. The protein was tagged with the haemagglutinin tag at the C-trminal end, and deletions of different parts of the CCW5 gene were analysed. It was found taht the protein lacking one repetitive unit was attached to the cell wall as was the wild type. If both copies of the repetitive sequence were deleted, the protein was secreted into the medium. This strongly suggests that Pir proteins are attached to the carbohydrate network of the wall, most probably to beta-1, 3-glucan, via their repetitive sequence and that the single copy of this sequence is sufficient for the effective binding.The study of the linker which connects Pir proteins to wall carbohydrates is a subject of current investigation and the results obtained so far indicate that it most probably does not contain phosphate.

The Saccharomyces cerevisiae cell wall contains more than 20 identified mannoproteins. Some of them can be released from the wall by hot SDS/mercaptoethanol treatment and are, therefore, considered as disulphide-linked or non-covalently attached to wall structural components. A number of covalently linked cell wall proteins are released after SDS extraction. They can be divided into these extractable by glucanases and those which can be released with 30 mM NaOH. The SDS-extractable proteins either possess enzymatic activities or are homologues of enzymes, mainly glucanases. Nothing is known, however, about the function of covalently linked proteins. In order to investigate the role of NaOH-extractable cell wall proteins, genes encoding all four identified members of this family of Pir proteins, CCW5, CCW6, CCW7 and CCW8, were disrupted and the phenotype of the mutants obtained was examined. They grew somewhat more slowly, were larger and irregularly shaped, and showed pronounced susceptibility to cell wall synthesis inhibitors like Calcofluor white and Congo red. In addition, the triple and the quadruple deletants had a decreased mating ability. All these properties were more obvious the more of these genes were disrupted, indicating that probably all members of this protein family are at least functionally equivalent in the cell wall.

Surplus amounts of lignocellulosic waste materials, as corn stover, corn cobs and wheat straw have a good potential to be used as a substrate for microorganisms growth and production of biofuels and biochemicals. Pre- treatment of such lignocellulosic waste materials is necessary to obtain a substrate that is suitable for chosen microorganism. Pre- treatment was carried out in self-made high temperature, high pressure reactor with 1 % sulphuric acid as a medium for hydrolysis. Obtained liquid part was used for the growth of wild strain Saccharomyces cerevisiae. The selected wild strain yeast was isolated from Županja, Sugar factory. Obtained hydrolisate from pretreatment contains chemical compounds as furfural (among others), that has inhibitory effect on selected yeast. Set of experiments were conducted to investigate the inhibitory effect of furfural on growth and production of ethanol. Results have shown that the wild strain of selected yeast can grow in medium that has furfural concentration in amount that is present in obtained hydrolysates from pretreatment of lignocellulosic waste materials. Next step was cultivation of selected strain of yeast on liquid parts obtained from pretreatment of corn stover, corn cobs and wheat straw in high temperature, high pressure reactor. Experiments have shown that the growth and production of ethanol is possible in substrates obtained with that method of pre- treatment.