A Fasciclin Protein Is Essential for Laccase-Mediated Selective Phenol Coupling in Sporandol Biosynthesis.

The biaryl scaffold, often showing axial chirality, is a common feature of various fungal natural products. Their biosynthesis requires an oxidative phenol-coupling reaction usually catalyzed by laccases, cytochrome P450 enzymes, or peroxidases. The combination of a laccase and a fasciclin domain-containing (fas) protein is encoded in many biosynthetic gene clusters of biaryls from ascomycetes. However, such phenol-coupling systems including their regio- and stereoselectivity have not been characterized so far. Elucidating the biosynthesis of the antiparasitic binaphthalene sporandol from Chrysosporium merdarium, we demonstrate the combination of a laccase and a fas protein to be crucial for the dimerization reaction. Only the heterologous coproduction of the laccase and the fas protein led to a functional phenol-coupling system, whereas the laccase alone showed no coupling activity. Thus, the laccase/fas protein combination forms an independent group of phenol-coupling enzymes that determines the coupling activity and selectivity of the reaction concurrently and applies to the biosynthesis of many fungal natural products with a biaryl scaffold.

Comparative characterization of extracellular enzymes secreted by Phanerochaete chrysosporium during solid-state and submerged fermentation.

Influence of water content on the expression of lignocellulolytic enzymes by Phanerochaete chrysosporium remains unclear. This work compares the enzyme production profiles of P. chrysosporium during solid-state and submerged fermentation. There were 110 and 64 extracellular carbohydrate-active enzymes identified in solid-state and submerged fermentation respectively, among which 57 enzymes were common to both of the secretomes. P. chrysosporium secreted more cellulases (especially lytic polysaccharide monooxygenase) and hemicellulases during solid-state fermentation while the proportion of enzyme containing carbohydrate-binding module was higher for submerged fermentation. Although its activities were weaker, the enzyme cocktail from submerged fermentation was surprisingly more effective in hydrolysis at low substrate loading. This advantage of enzymes from submerged fermentation was mainly attributed to carbohydrate-binding module because more xylanases bound with substrate at the beginning of hydrolysis. These results reveal the influence of fermentation conditions on enzyme produced by P. chrysosporium for the first time and show the importance of carbohydrate-binding module in the hydrolysis process of lignocellulose.

Characterization of a GH family 3 ß-glycoside hydrolase from Chrysosporium lucknowense and its application to the hydrolysis of ß-glucan and xylan.

The Bxl5-gene encoding a GH3 glycoside hydrolase of Chrysosporium lucknowense C1 was successfully cloned, the homologous recombinant product was secreted, purified and characterized. Bxl5 (120 ± 5 kDa) was able to hydrolyze low molecular weight substrates and polysaccharides containing ß-glucosidic as well as ß-xylosidic residues. The K(m) and V(max)/E values were found to be 0.3mM and 88 s(-1) on p-nitrophenyl-ß-d-glucopyranoside (PNPG), and 13.5mM and 1.8s(-1) on p-nitrophenyl-ß-d-xylopyranoside (PNPX). Optimal pH and temperature for Bxl5 were 4.6 and 75°C for the PNPG hydrolysis, and 5.0-5.5 and 70°C for PNPX hydrolysis. The enzyme was quite stable when incubated at elevated temperatures up to 65°C. Bxl5 hydrolyzes polymeric ß-glucans by the exo-mechanism allowing their complete conversion to d-glucose and is effective for xylan hydrolysis in combination with endo-acting xylan-degrading enzymes. The enzyme seems to be a very promising for bioconversion purposes.

The ferulic acid esterases of Chrysosporium lucknowense C1: purification, characterization and their potential application in biorefinery.

Three ferulic acid esterases from the filamentous fungus Chrysosporium lucknowense C1 were purified and characterized. The enzymes were most active at neutral pH and temperatures up to 45 °C. All enzymes released ferulic acid and p-coumaric acid from a soluble corn fibre fraction. Ferulic acid esterases FaeA1 and FaeA2 could also release complex dehydrodiferulic acids and dehydrotriferulic acids from corn fibre oligomers, but released only 20% of all ferulic acid present in sugar beet pectin oligomers. Ferulic acid esterase FaeB2 released almost no complex ferulic acid oligomers from corn fibre oligomers, but 60% of all ferulic acid from sugar beet pectin oligomers. The ferulic acid esterases were classified based on both, sequence similarity and their activities toward synthetic substrates. The type A ferulic acid esterases FaeA1 and FaeA2 are the first members of the phylogenetic subfamily 5 to be biochemically characterized. Type B ferulic acid esterase FaeB2 is a member of subfamily 6.

A comparative characterization of indigenous keratinase enzymes from district Khairpur, Sindh, Pakistan.

To isolate and characterize keratinolytic fungi and bacteria from indigenous soils, a total of 80 samples were collected from Ghari Mori District. Khairpur, and these organisms were isolated using standard microbiological technique. The isolated keratinolytic microorganisms comprised: Absidia sp., Chrysosporium asperatum, Chrysosporium keratinophilum, Entomophthora coronata, Bacillus subtilis and Staphylococcus aureus and their keratinolytic properties were distinguished from the production of keratinase by measurement of zone of hydrolysis on skimmed milk agar (p<0.05). C.keratinophylum and B. subtilis produced largest zone among all the isolated species. The crude keratinase revealed that the optimum time for production of the enzyme was seven days, optimum temperature 30°C and optimum pH 9 for C.keratinophylum but for B. subtilis, the optimum time was three days, optimum temperature 37°C and optimum pH 7. The enzyme activity of C. keratinophylum and B. subtilis were determined to be 220 U/ml and 260 U/ml respectively (P<0.05).

Combined HILIC-ELSD/ESI-MS(n) enables the separation, identification and quantification of sugar beet pectin derived oligomers.

The combined action of endo-polygalacturonase (endo-PGII), pectin lyase (PL), pectin methyl esterase (fungal PME) and RG-I degrading enzymes enabled the extended degradation of methylesterified and acetylated sugar beet pectins (SBPs). The released oligomers were separated, identified and quantified using hydrophilic interaction liquid chromatography (HILIC) with online electrospray ionization ion trap mass spectrometry (ESI-IT-MS(n)) and evaporative light scattering detection (ELSD). By MS(n), the structures of galacturonic acid (GalA) oligomers having an acetyl group in the O-2 and/or O-3 positions eluting from the HILIC column were elucidated. The presence of methylesterified and/or acetylated galacturonic acid units within an oligomer reduced the interaction with the HILIC column significantly compared to the unsubstituted GalA oligomers. The HILIC column enables a good separation of most oligomers present in the digest. The use of ELSD to quantify oligogalacturonides was validated using pure GalA standards and the signal was found to be independent of the chemical structure of the oligomer being detected. The combination of chromatographic and enzymatic strategies enables to distinguish SBPs having different methylesters and acetyl group distribution.

Characterization and mode of action of two acetyl xylan esterases from Chrysosporium lucknowense C1 active towards acetylated xylans.

Two novel acetyl xylan esterases, Axe2 and Axe3, from Chrysosporium lucknowense (C1), belonging to the carbohydrate esterase families 5 and 1, respectively, were purified and biochemically characterized. Axe2 and Axe3 are able to hydrolyze acetyl groups both from simple acetylated xylo-oligosaccharides and complex non-soluble acetylglucuronoxylan. Both enzymes performed optimally at pH 7.0 and 40 °C. Axe2 has a clear preference for acetylated xylo-oligosaccharides (AcXOS) with a high degree of substitution and Axe3 does not show such preference. Axe3 has a preference for large AcXOS (DP 9-12) when compared to smaller AcXOS (especially DP 4-7) while for Axe2 the size of the oligomer is irrelevant. Even though there is difference in substrate affinity towards acetylated xylooligosaccharides from Eucalyptus wood, the final hydrolysis products are the same for Axe2 and Axe3: xylo-oligosaccharides containing one acetyl group located at the non-reducing xylose residue remain as examined using MALDI-TOF MS, CE-LIF and the application of an endo-xylanase (GH 10).

Chrysosporium lucknowense C1 arabinofuranosidases are selective in releasing arabinose from either single or double substituted xylose residues in arabinoxylans.

Two novel arabinofuranosidases, Abn7 and Abf3 from Chrysosporium lucknowense (C1), belonging to the glycoside hydrolase family 43 and 51 were purified and characterized. Abn7 is exclusively able to hydrolyze arabinofuranosyl residues at position O-3 of double substituted xylosyl residues in arabinoxylan-derived oligosaccharides, an activity rarely found thus far. Abf3 is able to release arabinose from position O-2 or O-3 of single substituted xyloses. Both enzymes performed optimal at pH 5.0 and 40°C. Combining Abn7 and Abf3 resulted in a synergistic increase in arabinose release from arabinoxylans. This synergistic effect is due to the action of Abf3 on the remaining arabinose residues at position O-2 on single substituted xylosyl residues resulting from the action of Abn7 on double substituted xylosyl residues. Arabinose release was further increased when an endo-1,4-ß-xylanase was present during digestion. The efficiency of these arabinohydrolases from C1 on insoluble arabinoxylan substrates is discussed.

Isolation, optimization, and partial purification of amylase from Chrysosporium asperatum by submerged fermentation.

A potent fungus for amylase production, Chrysosporium asperatum, was isolated from among 30 different cultures obtained from wood samples collected in the Junagadh forest, India. All of the isolated cultures were screened for their ability to produce amylase by submerged fermentation. Among the selected cultures, C. asperatum (Class Euascomycetes; Onygenales; Onygenaceae) gave maximum amylase production. In all of the different media tested, potato starch was found to be a good substrate for production of amylase enzyme at 30 degrees C and pH 5.0. Production of enzyme reached the maximum when a combination of starch and 2% xylose, and organic nitrogen (1% yeast extract) and ammonium sulfate were used as carbon and nitrogen sources, respectively. There was no significant effect of metal ions on enzyme activity. The enzyme was relatively stable at 50 degrees C for 20 min, and no inhibitory effect of Ca+2 ions on amylase production was observed.

Mode of action of Chrysosporium lucknowense C1 α-l-arabinohydrolases.

The mode of action of four Chrysosporium lucknowense C1 α-L-arabinohydrolases was determined to enable controlled and effective degradation of arabinan. The active site of endoarabinanase Abn1 has at least six subsites, of which the subsites -1 to +2 have to be occupied for hydrolysis. Abn1 was able to hydrolyze a branched arabinohexaose with a double substituted arabinose at subsite -2. The exo acting enzymes Abn2, Abn4 and Abf3 release arabinobiose (Abn2) and arabinose (Abn4 and Abf3) from the non-reducing end of reduced arabinose oligomers. Abn2 binds the two arabinose units only at the subsites -1 and -2. Abf3 prefers small oligomers over large oligomers. It is able to hydrolyze all linkages present in beet arabinan, including the linkages of double substituted residues. Abn4 is more active towards polymeric substrate and releases arabinose monomers from single substituted arabinose residues. Depending on the combination of the enzymes, the C1 arabinohydrolases can be used to effectively release branched arabinose oligomers and/or arabinose monomers.

PRP8 intein in Ajellomycetaceae family pathogens: sequence analysis, splicing evaluation and homing endonuclease activity.

Inteins are intervening sequences that are transcribed and translated with flanking host protein sequences and then self-excised by protein splicing. Bi-functional inteins also contain a homing endonuclease responsible for their genetic mobility. The PRP8 intein, the most widespread among fungi, occurs in important pathogens such as Histoplasma capsulatum and Paracoccidioides brasiliensis, from the Ajellomycetaceae family. Herein, we describe the bi-functional PRP8 intein in two other Ajellomycetacean pathogens, Blastomyces dermatitidis and Emmonsia parva. Sequence analysis and experimental evidence suggest that the homing endonuclease from PbrPRP8 is inactive. The splicing activity of the PRP8 intein from the B. dermatitidis, E. parva and P. brasiliensis species complex was demonstrated in a non-native protein context in Escherichia coli. Since the PRP8 intein is located in a functionally essential nuclear protein, it can be considered a promising therapeutic target for anti-fungal drugs, because inhibition of intein splicing should inhibit proliferation of intein-containing pathogens.

Chrysosporium lucknowense arabinohydrolases effectively degrade sugar beet arabinan.

The filamentous fungus Chrysosporium lucknowense (C1) is a rich source of cell wall degrading enzymes. In the present paper four arabinose releasing enzymes from C1 were characterized, among them one endoarabinanase, two arabinofuranosidases and one exoarabinanase. Combinations of these enzymes released up to 80% of the arabinose present in sugar beet arabinan to fermentable monosugars. Besides the main product arabinobiose, unknown arabinose oligomers are produced from highly branched arabinan when endoarabinanase was combined with exoarabinanase and/or arabinofuranosidase. All described arabinose releasing enzymes are temperature stable up to 50 degrees C and have a broad pH stability. This makes C1 arabinohydrolases suitable for many biotechnical applications, like co-fermentation bioethanol production.

Branched arabino-oligosaccharides isolated from sugar beet arabinan.

Sugar beet arabinan consists of an alpha-(1,5)-linked backbone of L-arabinosyl residues, which can be either single or double substituted with alpha-(1,2)- and/or alpha-(1,3)-linked L-arabinosyl residues. Neutral branched arabino-oligosaccharides were isolated from sugar beet arabinan by enzymatic degradation with mixtures of pure and well-defined arabinohydrolases from Chrysosporium lucknowense followed by fractionation based on size and analysis by MALDI-TOF MS and HPAEC. Using NMR analysis, two main series of branched arabino-oligosaccharides have been identified, both having an alpha-(1,5)-linked backbone of L-arabinosyl residues. One series carries single substituted alpha-(1,3)-linked L-arabinosyl residues at the backbone, whereas the other series consists of a double substituted alpha-(1,2,3,5)-linked arabinan structure within the molecule. The structures of eight such branched arabino-oligosaccharides were established.

Degradation of the insecticidal toxin produced by Bacillus thuringiensis var. kurstaki by extracellular proteases produced by Chrysosporium sp.

AIMS: Some Cry proteins produced by the soil bacterium Bacillus thuringiensis (Bt) or by transgenic Bt plants persist in agricultural soils for an extended period of time, which may pose a hazard for nontarget soil organisms. The aims of our study were to screen for soil fungi capable of degrading the Cry1Ac toxin and to identify the mechanisms that lead to the inactivation of this protein. METHODS AND RESULTS: Of the eight fungal strains screened, only one, Chrysosporium sp., was found to produce extracellular proteases capable of degrading the 66-kDa Cry1Ac at the N-terminal end of amino acid 125 (alanine). The proteolytic products of the Cry1Ac toxin did not exhibit any insecticidal activity against Helicoverpa armigera, in contrast to its high toxicity exhibited in the native form. CONCLUSIONS: Proteases elaborated by the Chrysosporium sp. degrade the Cry1Ac toxin in a way that it looses its insecticidal activity against H. armigera. SIGNIFICANCE AND IMPACT OF THE STUDY: Chrysosporium sp., a specific soil micro-organism capable of producing proteases that degrade the Cry1Ac toxin into inactive products under controlled conditions is being reported for the first time. Application of this observation needs to be further tested in field conditions.

N-Glycosylation in Chrysosporium lucknowense enzymes.

Twenty-eight enzymes, encoded by different genes and secreted by different mutant strains of Chrysosporium lucknowense, were subjected to MALDI-TOF MS peptide fingerprinting followed by analysis of the MS data using the GlycoMod tool from the ExPASy proteomic site. Various N-linked glycan structures were discriminated in the C. lucknowense proteins as a result of the analysis. N-Glycosylated peptides with modifications matching the oligosaccharide compositions contained in the GlycoSuiteDB were found in 12 proteins. The most frequently encountered N-linked glycan, found in 9 peptides from 7 proteins, was (Man)(3)(GlcNAc)(2), that is, the core pentasaccharide structure forming mammalian-type high-mannose and hybrid/complex glycans in glycoproteins from different organisms. Nine out of 12 enzymes represented variably N-glycosylated proteins carrying common (Hex)(0-4)(HexNAc)(0-6)+(Man)(3)(GlcNAc)(2) structures, most of them being hybrid/complex glycans. Various glycan structures were likely formed as a result of the enzymatic trimming of a 'parent' oligosaccharide with different glycosidases. The N-glycosylation patterns found in C. lucknowense proteins differ from those reported for the extensively studied enzymes from Aspergilli and Trichoderma species, where high-mannose glycans of variable structure have been detected.

Design of highly efficient cellulase mixtures for enzymatic hydrolysis of cellulose.

An extremely highly active cellobiohydrolase (CBH IIb or Cel6B) was isolated from Chrysosporium lucknowense UV18-25 culture filtrate. The CBH IIb demonstrated the highest ability for a deep degradation of crystalline cellulose amongst a few cellobiohydrolases tested, including C. lucknowense CBH Ia, Ib, IIa, and Trichoderma reesei CBH I and II. Using purified C. lucknowense enzymes (CBH Ia, Ib, and IIb; endoglucanases II and V; beta-glucosidase, xylanase II), artificial multienzyme mixtures were reconstituted, displaying an extremely high performance in a conversion of different cellulosic substrates (Avicel, cotton, pretreated Douglas fir wood) to glucose. These mixtures were much or notably more effective in hydrolysis of the cellulosic substrates than the crude multienzyme C. lucknowense preparation and other crude cellulase samples produced by T. reesei and Penicillium verruculosum. Highly active cellulases are a key factor in bioconversion of plant lignocellulosic biomass to ethanol as an alternative to fossil fuels.

Specific xyloglucanases as a new class of polysaccharide-degrading enzymes.

Three specific xyloglucanases (XGs) were isolated from Aspergillus japonicus (32 kDa, pI 2.8), Chrysosporium lucknowense (78 kDa, pI 3.8) and Trichoderma reesei (75-105 kDa, pI 4.1-4.3). The characteristic feature of these enzymes was their high specific activity toward tamarind xyloglucan, whereas the activity against carboxymethylcellulose (CMC) and barley beta-glucan was absent or very low. Peptide mass fingerprinting using MALDI-TOF mass spectrometry showed that the T. reesei XG represents Cel74A, whose gene has been discovered recently (GenBank accession no. AY281371 ), but the enzyme has not been characterized and described elsewhere. Tryptic peptides from A. japonicus and C. lucknowense xyloglucanases did not show any identity to those from known glycoside hydrolases. All enzymes produced XXXG, XXLG/XLXG and XLLG oligosaccharides as the end products of xyloglucan hydrolysis. A. japonicus XG displayed an endo-type of attack on the polymeric substrate, while the mode of action of two other xyloglucanases was similar to the exo-type, when oligosaccharides containing four glucose residues in the main chain were split off the ends of xyloglucan molecules. These results together with growing literature data allow concluding that specific xyloglucanases may represent a new class of glycoside hydrolases, which are different from regular endo-1,4-beta-glucanases.

Cellulase complex of the fungus Chrysosporium lucknowense: isolation and characterization of endoglucanases and cellobiohydrolases.

Using different chromatographic techniques, eight cellulolytic enzymes were isolated from the culture broth of a mutant strain of Chrysosporium lucknowense: six endoglucanases (EG: 25 kD, pI 4.0; 28 kD, pI 5.7; 44 kD, pI 6.0; 47 kD, pI 5.7; 51 kD, pI 4.8; 60 kD, pI 3.7) and two cellobiohydrolases (CBH I, 65 kD, pI 4.5; CBH II, 42 kD, pI 4.2). Some of the isolated cellulases were classified into known families of glycoside hydrolases: Cel6A (CBH II), Cel7A (CBH I), Cel12A (EG28), Cel45A (EG25). It was shown that EG44 and EG51 are two different forms of one enzyme. EG44 seems to be a catalytic module of an intact EG51 without a cellulose-binding module. All the enzymes had pH optimum of activity in the acidic range (at pH 4.5-6.0), whereas EG25 and EG47 retained 55-60% of the maximum activity at pH 8.5. Substrate specificity of the purified cellulases against carboxymethylcellulose (CMC), beta-glucan, Avicel, xylan, xyloglucan, laminarin, and p-nitrophenyl-beta-D-cellobioside was studied. EG44 and EG51 were characterized by the highest CMCase activity (59 and 52 U/mg protein). EG28 had the lowest CMCase activity (11 U/mg) amongst the endoglucanases; however, this enzyme displayed the highest activity against beta-glucan (125 U/mg). Only EG51 and CBH I were characterized by high adsorption ability on Avicel cellulose (98-99%). Kinetics of Avicel hydrolysis by the isolated cellulases in the presence of purified beta-glucosidase from Aspergillus japonicus was studied. The hydrolytic efficiency of cellulases (estimated as glucose yield after a 7-day reaction) decreased in the following order: CBH I, EG60, CBH II, EG51, EG47, EG25, EG28, EG44.

Molecular taxonomy of dermatophytes and related fungi by chitin synthase 1 (CHS1) gene sequences.

In the present study, the nucleotide sequences of the CHS1 gene from dermatophytes and related fungi in the genera Chrysosporium, Epidermophyton, Microsporum and Trichophyton were investigated using molecular methods. About 440-bp genomic DNA fragments of the CHS1 gene from 21 species were amplified by polymerase chain reaction (PCR) and sequenced. The CHS1 nucleotide sequences of these fungi showed more than 83% similarity. The molecular taxonomy of the CHS1 gene sequences revealed that Microsporum was genetically distinct from Chrysosporium and Trichophyton, as classified by morphological characteristics.