Two novel GH11 endo-xylanases from Myceliophthora thermophila C1 act differently toward soluble and insoluble xylans.

Two novel GH11 endo-xylanases from Myceliophthora thermophila C1 (C1), Xyl7 and Xyl8, were purified and the influence of solubility and molecular structure of various xylans on their efficiency was investigated. Both endo-xylanases were hindered by a high degree of substitution of a xylan. The two GH11 xylanases released different products from the xylans, in which Xyl7 displayed a degradation product composition closer to GH10 xylanases. A correlation of the degradation product composition with a specific residue at position 163 in the amino acid sequence of Xyl8 is suggested: tyrosine in Xyl8; valine in Xyl7. This is confirmed with examples of various endo-xylanases reported in literature. The C1 GH11 xylanases were more efficient on self-associated xylan compared to C1 GH10 endo-xylanases and they released more small xylooligomers from these xylans. This is contrary to the general assumption that GH10 xylanases degrade xylans to a higher degree than GH11 xylanases.

Cloning, purification, and characterization of galactomannan-degrading enzymes from Myceliophthora thermophila.

Genes of ß-mannosidase 97 kDa, GH family 2 (bMann9), ß-mannanase 48 kDa, GH family 5 (bMan2), and α-galactosidase 60 kDa, GH family 27 (aGal1) encoding galactomannan-degrading glycoside hydrolases of Myceliophthora thermophila C1 were successfully cloned, and the recombinant enzymes were purified to homogeneity and characterized. bMann9 displays only exo-mannosidase activity, the K(m) and k(cat) values are 0.4 mM and 15 sec(-1) for p-nitrophenyl-ß-D-mannopyranoside, and the optimal pH and temperature are 5.3 and 40°C, respectively. bMann2 is active towards galactomannans (GM) of various structures. The K(m) and k(cat) values are 1.3 mg/ml and 67 sec(-1) for GM carob, and the optimal pH and temperature are 5.2 and 69°C, respectively. aGal1 is active towards p-nitrophenyl-α-D-galactopyranoside (PNPG) as well as GM of various structures. The K(m) and k(cat) values are 0.08 mM and 35 sec(-1) for PNPG, and the optimal pH and temperature are 5.0 and 60°C, respectively.

Two GH10 endo-xylanases from Myceliophthora thermophila C1 with and without cellulose binding module act differently towards soluble and insoluble xylans.

Xylanases are mostly classified as belonging to glycoside hydrolase (GH) family 10 and 11, which differ in catalytic properties and structures. However, within one family, differences may also be present. The influence of solubility and molecular structure of substrates towards the efficiency of two GH10 xylanases from Myceliophthora thermophila C1 was investigated. The xylanases differed in degradation of high and low substituted substrate and the substitution pattern was an important factor influencing their efficiency. Alkali-labile interactions, as well as the presence of cellulose within the complex cell wall structure hindered efficient hydrolysis for both xylanases. The presence of a carbohydrate binding module did not enhance the degradation of the substrates. The differences in degradation could be related to the protein structure of the two xylanases. The study shows that the classification of enzymes does not predict their performance towards various substrates.

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.

Structural features of ß-(1→4)-D-galactomannans of plant origin as a probe for ß-(1→4)-mannanase polymeric substrate specificity.

Statistical modeling was applied for describing structural features of ß-(1→4)-D-galactomannans. According to the model suggested theoretical ratios of limiting degrees of locust bean, tara gum and guar gum galactomannan conversions by two ß-(1→4)-mannanases of different origin (Myceliophthora thermophila and Trichoderma reesei) were calculated. Then the enzymes were tested for enzymatic hydrolysis of three considered galactomannans. Experimentally observed results were compared with theoretically calculated ones. It was shown that T. reesei ß-mannanase attacks sequences of four and more unsubstituted mannopyranosyl residues in a row, while M. thermophila ß-mannanase is a more specific enzyme and attacks sequences of five and more mannopyranosyl residues in a row. Considered statistical model and approach allows to characterize both galactomannan structures and enzyme requirements for regions of unsubstituted mannose residues for substrate hydrolysis.

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.

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).

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.

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.