The boosting effect of recombinant hemicellulases on the enzymatic hydrolysis of steam-treated sugarcane bagasse.

To increase the efficiency of enzyme cocktails in deconstructing cellulose and hemicelluloses present in the plant cell wall, a combination of enzymes with complementary activities is required. Xylan is the main hemicellulose component of energy crops and for its complete hydrolysis a system consisting of several enzymes acting cooperatively, including endoxylanases (XYN), ß-xylosidases (XYL) and α-l-arabinofuranosidases (ABF) is necessary. The current work aimed at evaluating the effect of recombinant hemicellulolytic enzymes on the enzymatic hydrolysis of steam-exploded sugarcane bagasse (SEB). One recombinant endoxylanase (HXYN2) and one recombinant ß-xylosidase (HXYLA) from Humicola grisea var thermoidea, together with an α-l-arabinofuranosidase (AFB3) from Penicillium pupurogenum, all produced in Pichia pastoris, were used to formulate an efficient enzyme mixture for SEB hydrolysis using a 23 Central Composite Rotatable Design (CCRD). The most potent enzyme for SEB hydrolysis was ABF3. Subsequently, the optimal enzyme mixture was used in combination with commercial cellulases (Accellerase 1500), either simultaneously or in sequential experiments. The supplementation of Accellerase 1500 with hemicellulases enhanced the glucose yield from SEB hydrolysis by 14.6%, but this effect could be raised to 50% when hemicellulases were added prior to hydrolysis with commercial cellulases. These results were supported by scanning electron microscopy, which revealed the effect of enzymatic hydrolysis on SEB fibers. Our results show the potential of complementary enzyme activities to improve enzymatic hydrolysis of SEB, thus improving the efficiency of the hydrolytic process.

Penicillium purpurogenum produces a novel, acidic, GH3 beta-xylosidase: Heterologous expression and characterization of the enzyme.

Xylan, a component of plant cell walls, is composed of a backbone of ß-1,4-linked xylopyranosyl units with a number of substituents. The complete degradation of xylan requires the action of several enzymes, among them ß-xylosidase. The fungus Penicillium purpurogenum secretes a number of enzymes participating in the degradation of xylan. In this study, a ß-xylosidase from this fungus was expressed in Pichia pastoris, and characterized. This enzyme (Xyl2) is a member of glycoside hydrolase family 3; it consists of a sequence of 792 residues including a signal peptide of 20 residues, with a theoretical molecular mass for the mature protein of 84.2 KDa and an isoelectric point of 5.07. The highest identity with a characterized fungal enzyme, is with a ß-xylosidase from Aspergillus oryzae (70%). The optimal activity of Xyl2 is found at pH 2.0 and 28 °C. The enzyme is most stable at pH 2.0 and conserves 40% of activity at 42 °C (after 1h incubation). The kinetic parameters for p-nitrophenyl-ß-d-xylopyranoside are: KM 0.53 mM, kcat 1*107 s-1 and kcat/KM 1.9*1010 M-1 s-1. The enzyme is about 10% active on p-nitrophenyl-α-l-arabinofuranoside. Xyl2 exhibits a high hydrolytic activity on xylooligosaccharides; it liberates xylose from beechwood and birchwood glucuronoxylan and it acts synergistically with endoxylanases in the degradation of xylan. Its low pH optimum make this enzyme particularly useful in potential applications requiring a low pH such as increasing the flavor of wine.

Corncob and sugar beet pulp induce specific sets of lignocellulolytic enzymes in Penicillium purpurogenum.

Penicillium purpurogenum is a filamentous fungus, which grows on a variety of natural carbon sources and secretes a large number of enzymes involved in cellulose, hemicelluloses and pectin biodegradation. The purpose of this work has been to identify potential lignocellulolytic enzymes and to compare the secreted enzymes produced when the fungus is grown on sugar beet pulp (rich in cellulose and pectin) and corn cob (rich in cellulose and xylan). Culture supernatants were subjected to two-dimensional nano-liquid chromatography/tandem mass spectrometry. Using MASCOT and a genome-derived protein database, the proteins present in the supernatant were identified. The putative function in the degradation of the polysaccharides was determined using dbCAN software. The results show that there is a good correlation between the polysaccharide composition of the carbon sources and the function of the secreted enzymes: both cultures are rich in cellulases, while sugar beet pulp induces pectinases and corncob, xylanases. The eventual biochemical characterisation of these enzymes will be of value for a better understanding of the biodegradation process performed by the fungus and increase the availability of enzymes for biotechnological methods associated with this process.

Penicillium purpurogenum Produces a Set of Endoxylanases: Identification, Heterologous Expression, and Characterization of a Fourth Xylanase, XynD, a Novel Enzyme Belonging to Glycoside Hydrolase Family 10.

The fungus Penicillium purpurogenum grows on a variety of natural carbon sources and secretes a large number of enzymes which degrade the polysaccharides present in lignocellulose. In this work, the gene coding for a novel endoxylanase has been identified in the genome of the fungus. This gene (xynd) possesses four introns. The cDNA has been expressed in Pichia pastoris and characterized. The enzyme, XynD, belongs to family 10 of the glycoside hydrolases. Mature XynD has a calculated molecular weight of 40,997. It consists of 387 amino acid residues with an N-terminal catalytic module, a linker rich in ser and thr residues, and a C-terminal family 1 carbohydrate-binding module. XynD shows the highest identity (97%) to a putative endoxylanase from Penicillium subrubescens but its highest identity to a biochemically characterized xylanase (XYND from Penicillium funiculosum) is only 68%. The enzyme has a temperature optimum of 60 °C, and it is highly stable in its pH optimum range of 6.5-8.5. XynD is the fourth biochemically characterized endoxylanase from P. purpurogenum, confirming the rich potential of this fungus for lignocellulose biodegradation. XynD, due to its wide pH optimum and stability, may be a useful enzyme in biotechnological procedures related to this biodegradation process.

The genome sequence of the soft-rot fungus Penicillium purpurogenum reveals a high gene dosage for lignocellulolytic enzymes.

The high lignocellulolytic activity displayed by the soft-rot fungus Penicillium purpurogenum has made it a target for the study of novel lignocellulolytic enzymes. We have obtained a reference genome of 36.2 Mb of non-redundant sequence (11,057 protein-coding genes). The 49 largest scaffolds cover 90% of the assembly, and Core Eukaryotic Genes Mapping Approach (CEGMA) analysis reveals that our assembly captures almost all protein-coding genes. RNA-seq was performed and 93.1% of the reads aligned to the assembled genome. These data, plus the independent sequencing of a set of genes of lignocellulose-degrading enzymes, validate the quality of the genome sequence. P. purpurogenum shows a higher number of proteins with CAZy motifs, transcription factors and transporters as compared to other sequenced Penicillia. These results demonstrate the great potential for lignocellulolytic activity of this fungus and the possible use of its enzymes in related industrial applications.

Identification, heterologous expression and characterization of a novel glycoside hydrolase family 30 xylanase from the fungus Penicillium purpurogenum.

Penicillium purpurogenum grows on a variety of natural carbon sources and secretes to the medium a large number of enzymes that degrade the polysaccharides present in lignocellulose. In this work, the gene coding for a novel xylanase (XynC) belonging to family 30 of the glycoside hydrolases (GH), has been identified in the genome of the fungus. The enzyme has been expressed in Pichia pastoris and characterized. The mature XynC has 454 amino acid residues and a calculated molecular weight of 49 240. The purified protein shows a molecular weight of 67 000, and it is partially deglycosylated using EndoH. Its pH optimum is in the range of 3-5, and the optimal temperature is 45 °C. It is active on both arabinoxylan and glucuronoxylan, similarly to other fungal GH 30 xylanases. It liberates a set of oligosaccharides, which have been detected by thin-layer chromatography, thus indicating that it is an endo-acting xylanase. It hydrolyzes xylooligosaccharides, releasing mainly xylobiose, in contrast to other fungal GH family 30 enzymes which generate chiefly xylose. Highest sequence identity to a characterized family 30 xylanase is found with the enzyme from the fungus Bispora sp (53%). This is the first GH 30 xylanase described from a Penicillium.

Penicillium purpurogenum produces a novel endo-1,5-arabinanase, active on debranched arabinan, short arabinooligosaccharides and on the artificial substrate p-nitrophenyl arabinofuranoside.

Penicillium purpurogenum secretes numerous lignocellulose-degrading enzymes, including four arabinofuranosidases and an exo-arabinanase. In this work, the biochemical properties of an endo-arabinanase (ABN1) are presented. A gene, coding for a potential ABN was mined from the genome. It includes three introns. The cDNA is 975 bp long and codes for a mature protein of 324 residues. The cDNA was expressed in Pichia pastoris. The enzyme is active on debranched arabinan and arabinooligosaccharides. In contrast to other characterized ABNs, inactive on p-nitrophenyl-α-L-arabinofuranoside (pNPAra), ABN1 is active on this substrate. The enzyme has an optimal pH of 4.5 and an optimal temperature of 30-35 °C. Calcium does not activate ABN1. ABN1 belongs to GH family 43 sub-family 6, and a Clustal alignment with sequences of characterized fungal ABNs shows highest identity (54.6%) with an ABN from Aspergillus aculeatus. A three-dimensional model of ABN1 was constructed and the docking with pNPAra was compared with similar models of an enzyme very active on this substrate and another lacking activity, both from GH family 43. Differences in the number of hydrogen bonds between enzyme and substrate, and distance between the substrate and the catalytic residues may explain the differences in activity shown by these enzymes.

Biochemical and structural characterization of Penicillium purpurogenum α-D galactosidase: Binding of galactose to an alternative pocket may explain enzyme inhibition.

The fungus Penicillium purpurogenum degrades plant cell walls by the action of cellulolytic, xylanolytic and pectinolytic enzymes. The α-D-galactosidase is one of the enzymes which may act on pectin degradation. This enzyme has several biotechnological and medical applications. The aim of this work was to better understand the molecular mechanism of α-D-galactosidase from P. purpurogenum (GALP1). For this purpose, a gene coding for the enzyme was identified from the fungal genome and heterologously expressed in Pichia pastoris. The enzyme belongs to glycosyl hydrolase family 27. The protein of 435 amino acids has an optimum pH and temperature for activity of 5.0 and 50 °C, respectively. The KM for p-nitrophenyl-α-D-galactopyranoside (GalαpNP) is 0.138 mM. The enzyme is inhibited by GalαpNP at concentrations higher than 1 mM, and by the product galactose. A kinetic analysis of product inhibition shows that it is of mixed type, suggesting the presence of an additional binding site in the enzyme. To confirm this hypothesis, a structural model for GALP1 was built by comparative modelling methodology, which was validated and refined by molecular dynamics simulation. The data suggest that galactose may bind to an enzyme alternative pocket promoting structural changes of the active site, thus explaining its inhibitory effect. In silico site-directed mutagenesis experiments highlighted key residues involved in the maintenance of the alternative binding site, and their mutations for Ala predict the formation of proteins which should not be inhibited by galactose. The availability of an α-galactosidase with different kinetic properties to the existent proteins may be of interest for biotechnological applications.

Heterologous expression, purification and characterization of three novel esterases secreted by the lignocellulolytic fungus Penicillium purpurogenum when grown on sugar beet pulp.

The lignocellulolytic fungus, Penicillium purpurogenum, grows on a variety of natural carbon sources, among them sugar beet pulp. Culture supernatants of P. purpurogenum grown on sugar beet pulp were partially purified and the fractions obtained analyzed for esterase activity by zymograms. The bands with activity on methyl umbelliferyl acetate were subjected to mass spectrometry to identify peptides. The peptides obtained were probed against the proteins deduced from the genome sequence of P. purpurogenum. Eight putative esterases thus identified were chosen for future work. Their cDNAs were expressed in Pichia pastoris. The supernatants of the recombinant clones were assayed for esterase activity, and five of the proteins were active against one or more substrates: methyl umbelliferyl acetate, indoxyl acetate, methyl esterified pectin and fluorescein diacetate. Three of those enzymes were purified, further characterized and subjected to a BLAST search. Based on their amino acid sequence and properties, they were identified as follows: RAE1, pectin acetyl esterase (CAZy family CE 12); FAEA, feruloyl esterase (could not be assigned to a CAZy family) and EAN, acetyl esterase (former CAZy family CE 10).

Penicillium purpurogenum produces a highly stable endo-ß-(1,4)-galactanase.

The polysaccharides of galactose present in the pectin of the plant cell wall are degraded by endo-ß-1,4-galactanases. The filamentous fungus Penicillium purpurogenum, which grows on a number of natural carbon sources, among them sugar beet pulp which contains pectin, has a gene (ppgal1) coding an endo-ß-1,4-galactanase (PpGAL1). This enzyme was expressed heterologously in Pichia pastoris. It has a molecular mass of 38 kDa, a pH optimum of 4-4.5, and an optimal temperature of 60 °C. It is 100 % stable for up to 24 h at pH 4-4.5 and 40 °C. These stability properties, which exceed those from other endo-ß-1,4-galactanases reported to date, make it particularly suitable for industrial processes requiring acidic conditions and temperatures up to 40 °C. PpGAL1 is, therefore, a potentially effective tool in the food industry and in other biotechnological applications.

Aspergillus fumigatus Produces Two Arabinofuranosidases From Glycosyl Hydrolase Family 62: Comparative Properties of the Recombinant Enzymes.

The genes of two α-L-arabinofuranosidases (AbfI and II) from family GH 62 have been identified in the genome of Aspergillus fumigatus wmo. Both genes have been expressed in Pichia pastoris and the enzymes have been purified and characterized. AbfI is composed of 999 bp, does not contain introns and codes for a protein (ABFI) of 332 amino acid residues. abfII has 1246 bp, including an intron of 51 bp; the protein ABFII has 396 amino acid residues; it includes a family 1 carbohydrate-binding module (CBM) in the N-terminal region, followed by a catalytic module. The sequence of ABFI and the catalytic module of ABFII show a 79 % identity. Both enzymes are active on p-nitrophenyl α-L-arabinofuranoside (pNPAra) with KM of 94.2 and 3.9 mM for ABFI and II, respectively. Optimal temperature for ABFI is 37 °C and for ABFII 42 °C, while the pH optimum is about 4.5 to 5 for both enzymes. ABFII shows a higher thermostability. When assayed using natural substrates, both show higher activity over rye arabinoxylan as compared to wheat arabinoxylan. ABFII only is active on sugar beet pulp arabinan and both are inactive towards debranched arabinan. The higher thermostability, higher affinity for pNPAra and wider activity over natural substrates shown by ABFII may be related to the presence of a CBM. The availability of the recombinant enzymes may be useful in biotechnological applications for the production of arabinose.

Properties of Two Novel Esterases Identified from Culture Supernatant of Penicillium purpurogenum Grown on Sugar Beet Pulp.

BACKGROUND: The filamentous fungus Penicillium purpurogenum grows on a variety of natural carbon sources, such as sugar beet pulp, and secretes to the medium a large number of enzymes that degrade the carbohydrate components of lignocellulose. Sugar beet pulp is rich in pectin, and the purpose of this work is to identify novel esterases produced by the fungus, which may participate in pectin degradation. METHODS AND FINDINGS: Partially purified culture supernatants of the fungus grown on sugar beet pulp were subjected to mass spectrometry analysis. Peptides thus identified, which may be part of potential esterases were probed against the proteins deduced from the fungal genome sequence. The cDNAs of two putative esterases identified were expressed in Pichia pastoris and their properties studied. One of these enzymes, named FAET, is a feruloyl esterase, while the other, PE, is classified as a pectin methyl esterase. CONCLUSIONS: These findings add to our knowledge of the enzymology of pectin degradation by Penicillium purpurogenum, and define properties of two novel esterases acting on de-esterification of pectin. Their availability may be useful as tools for the study of pectin structure and degradation.

Heterologous expression of a Penicillium purpurogenum exo-arabinanase in Pichia pastoris and its biochemical characterization.

Arabinan is a component of pectin, which is one of the polysaccharides present in lignocelluose. The enzymes degrading the main chain of arabinan are the endo- (EC 3.2.1.99) and exo-arabinanases (3.2.1.-). Only three exo-arabinanases have been biochemically characterized; they belong to glycosyl hydrolase family 93. In this work, the cDNA of an exo-arabinanase (Arap2) from Penicillium purpurogenum has been heterologously expressed in Pichia pastoris. The gene is 1310 bp long, has three introns and codes for a protein of 380 amino acid residues; the mature protein has a calculated molecular mass of 39 823 Da. The heterologously expressed Arap2 has a molecular mass in the range of 60-80 kDa due to heterogeneous glycosylation. The enzyme is active on debranched arabinan with optimum pH of 4-5.5 and optimal temperature of 40 °C, and has an exo-type action mode, releasing arabinobiose from its substrates. The expression profile of arap2 in corncob and sugar beet pulp follows a different pattern and is not related to the presence of arabinan. This is the first exo-arabinanase studied from P. purpurogenum and the first expressed in yeast. The availability of heterologous Arap2 may be useful for biotechnological applications requiring acidic conditions.

Heterologous expression and characterization of α-L-arabinofuranosidase 4 from Penicillium purpurogenum and comparison with the other isoenzymes produced by the fungus.

Penicillium purpurogenum secretes at least four arabinofuranosidases. In this work, the gene of α-L-arabinofuranosidase 4 (ABF4) has been sequenced and expressed in Pichia pastoris. The gene is 1521 pb long, has no introns and codes for a protein of 506 amino acid residues including a signal peptide of 26 residues. Mature protein has a calculated molecular mass of 55.4 kDa, shows 77% identity with α-L-arabinofuranosidase 1 from P. purpurogenum and belongs to family 54 of the glycosyl hydrolases. Purified enzyme has a molecular mass near 68 kDa, is active on p-nitrophenyl α-L-arabinofuranoside and p-nitrophenyl-ß-D-galactofuranoside, and follows Michaelis-Menten kinetics with KM of 1.58 ± 0.13 mM and 5.3 ± 1.18 mM, respectively. The pH optimum is 4.6 and optimal temperature is 50 °C. The enzyme is active on sugar beet arabinan and wheat flour arabinoxylan but does not act on short arabinooligosaccharides or debranched arabinan. It shows synergistic effect on arabinose liberation from wheat arabinoxylan when combined with endoxylanase from P. purpurogenum. The properties of ABF4 have been compared with those of the other arabinofuranosidases produced by the fungus. P. purpurogenum is the first fungus possessing four biochemically characterized arabinofuranosidases. The availability of four different ABFs may be valuable for biotechnological applications.

Heterologous expression of a Penicillium purpurogenum pectin lyase in Pichia pastoris and its characterization.

Lignocellulose is the major component of plant cell walls and it represents a great source of renewable organic matter. One of lignocellulose constituents is pectin. Pectin is composed of two basic structures: a 'smooth' region and a 'hairy' region. The 'smooth' region (homogalacturonan) is a linear polymer of galacturonic acid residues with α-(1→4) linkages, substituted by methyl and acetyl residues. The 'hairy' region is more complex, containing xylogalacturonan and rhamnogalacturonans I and II. Among the enzymes which degrade pectin (pectinases) is pectin lyase (E.C. 4.2.2.10). This enzyme acts on highly esterified homogalacturonan, catalysing the cleavage of α-(1→4) glycosidic bonds between methoxylated residues of galacturonic acid by means of ß-elimination, with the formation of 4,5-unsaturated products. In this work, the gene and cDNA of a pectin lyase from Penicillium purpurogenum have been sequenced, and the cDNA has been expressed in Pichia pastoris. The gene is 1334 pb long, has three introns and codes for a protein of 376 amino acid residues. The recombinant enzyme was purified to homogeneity and characterized. Pectin lyase has a molecular mass of 45 kDa as determined by SDS-PAGE. It is active on highly esterified pectin, and decreases 40% the viscosity of pectin with a degree of esterification ≥85%. The enzyme showed no activity on polygalacturonic acid and pectin from citrus fruit 8% esterified. The optimum pH and temperature for the recombinant enzyme are 6.0 and 50 °C, respectively, and it is stable up to 50 °C when exposed for 3 h. A purified pectin lyase may be useful in biotechnological applications such as the food industry where the liberation of toxic methanol in pectin degradation should be avoided.

Cold-active xylanase produced by fungi associated with Antarctic marine sponges.

Despite their potential biotechnological applications, cold-active xylanolytic enzymes have been poorly studied. In this work, 38 fungi isolated from marine sponges collected in King George Island, Antarctica, were screened as new sources of cold-active xylanases. All of them showed xylanase activity at 15 and 23 °C in semiquantitative plate assays. One of these isolates, Cladosporium sp., showed the highest activity and was characterized in detail. Cladosporium sp. showed higher xylanolytic activity when grown on beechwood or birchwood xylan and wheat bran, but wheat straw and oat bran were not so good inducers of this activity. The optimal pH for xylanase activity was 6.0, although pH stability was slightly wider (pH 5-7). On the other hand, Cladosporium sp. showed high xylanase activity at low temperatures and very low thermal stability. Interestingly, thermal stability was even lower after culture media were removed and replaced by buffer, suggesting that low molecular component(s) of the culture media could be important in the stabilization of cold-active xylanase activity. To the best of our knowledge, this study is the first report on extracellular xylanase production by fungi associated with Antarctic marine sponges.

Penicillium purpurogenum produces two GH family 43 enzymes with ß-xylosidase activity, one monofunctional and the other bifunctional: Biochemical and structural analyses explain the difference.

ß-Xylosidases participate in xylan biodegradation, liberating xylose from the non-reducing end of xylooligosaccharides. The fungus Penicillium purpurogenum secretes two enzymes with ß-D-xylosidase activity belonging to family 43 of the glycosyl hydrolases. One of these enzymes, arabinofuranosidase 3 (ABF3), is a bifunctional α-L-arabinofuranosidase/xylobiohydrolase active on p-nitrophenyl-α-L-arabinofuranoside (pNPAra) and p-nitrophenyl-ß-D-xylopyranoside (pNPXyl) with a KM of 0.65 and 12 mM, respectively. The other, ß-D-xylosidase 1 (XYL1), is only active on pNPXyl with a KM of 0.55 mM. The xyl1 gene was expressed in Pichia pastoris, purified and characterized. The properties of both enzymes were compared in order to explain their difference in substrate specificity. Structural models for each protein were built using homology modeling tools. Molecular docking simulations were used to analyze the interactions defining the affinity of the proteins to both ligands. The structural analysis shows that active complexes (ABF3-pNPXyl, ABF3-pNPAra and XYL1-pNPXyl) possess specific interactions between substrates and catalytic residues, which are absent in the inactive complex (XYL1-pNPAra), while other interactions with non-catalytic residues are found in all complexes. pNPAra is a competitive inhibitor for XYL1 (Ki = 2.5 mM), confirming that pNPAra does bind to the active site but not to the catalytic residues.

Characterization of a recombinant α-glucuronidase from Aspergillus fumigatus.

The degradation of xylan requires the action of glycanases and esterases which hydrolyse, in a synergistic fashion, the main chain and the different substituents which decorate its structure. Among the xylanolytic enzymes acting on side-chains are the α-glucuronidases (AguA) (E.C. 3.2.1.139) which release methyl glucuronic acid residues. These are the least studies among the xylanolytic enzymes. In this work, the gene and cDNA of an α-glucuronidase from a newly isolated strain of Aspergillus fumigatus have been sequenced, and the gene has been expressed in Pichia pastoris. The gene is 2523 bp long, has no introns and codes for a protein of 840 amino acid residues including a putative signal peptide of 19 residues. The mature protein has a calculated molecular weight of 91,725 and shows 99 % identity with a putative α-glucuronidase from A. fumigatus A1163. The recombinant enzyme was expressed with a histidine tag and was purified to near homogeneity with a nickel nitriloacetic acid (Ni-NTA) column. The purified enzyme has a molecular weight near 100,000. It is inactive using birchwood glucuronoxylan as substrate. Activity is observed in the presence of xylooligosaccharides generated from this substrate by a family 10 endoxylanase and when a mixture of aldouronic acids are used as substrates. If, instead, family 11 endoxylanase is used to generate oligosaccharides, no activity is detected, indicating a different specificity in the cleavage of xylan by family 10 and 11 endoxylanases. Enzyme activity is optimal at 37 °C and pH 4.5-5. The enzyme binds cellulose, thus it likely possesses a carbohydrate binding module. Based on its properties and sequence similarities the catalytic module of the newly described α-glucuronidase can be classified in family 67 of the glycosyl hydrolases. The recombinant enzyme may be useful for biotechnological applications of α-glucuronidases.

Α-L-arabinofuranosidase 3 from Penicillium purpurogenum (ABF3): potential application in the enhancement of wine flavour and heterologous expression of the enzyme.

An α-l-arabinofuranosidase (ABF3) from Penicillium purpurogenum was purified and its possible biotechnological application in the enhancement of wine flavour combined with P. purpurogenum ß-glucosidase was studied. A must from Muscat of Alexandria was used to isolate the glycosides. The total monosaccharide (glucose, arabinose and xylose) levels in the glycosides were determined after acid hydrolysis, and were compared with the result of enzymatic hydrolysis. These results were analogous to those obtained in similar experiments using a commercial preparation, thus suggesting that the enzyme from P. purpurogenum may prove useful in this particular application. This prompted us to express the enzyme heterologously. The abf3 gene was thus expressed in Pichia pastoris. The recombinant enzyme was purified and it shows the same properties of the native ABF3 (substrate specificity, kinetic constants, pH and temperature optima and antibody cross-reactivity).

The effect of acetylated xylan and sugar beet pulp on the expression and secretion of enzymes by Penicillium purpurogenum.

Sugar beet pulp is a natural carbon source composed mainly of pectin and cellulose, which is utilized and degraded by the ascomycete Penicillium purpurogenum. The fungus also grows on and degrades acetylated xylan which lacks cellulose and pectin. Both carbon sources have been used in our laboratory to grow the fungus and to purify different enzymes secreted to the medium. The enzymes involved in the complex process of degradation of these carbon sources by the fungus have been explored previously under non-denaturing conditions; multienzyme complexes were separated and some subunits identified by Western blots and mass spectrometry. In this work, proteomic profiles show that the secretome is composed of numerous proteins varying in pI and molecular weight. Some enzymes are common to both growth conditions, while others are specific for each carbon source. The results show that the carbon sources utilized exert strong regulatory control over the proteins secreted. This is the first secretome study from a lignocellulolytic Penicillium.