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The role of endocellulases and endoxylanase during liquefaction and saccharification of hydrothermally pretreated wheat straw was studied. The use of a flow-loop setup with in-line magnetic resonance imaging enabled frequent measurements of viscosity at 55°C during saccharification at 6% total solids content. Viscosity data were complemented with off-line measurements of fiber lengths and release of soluble sugars. A clear correlation between fiber attrition and a decrease in viscosity was found. Fiber lengths and viscosity dropped quickly within the first hour and then stagnated, while sugar yields increased substantially thereafter, illustrating that liquefaction and saccharification are separate mechanisms. Both endoglucanase and endoxylanase were shown to have a significant effect on viscosity during liquefaction while the addition of endoxylanase also increased sugar yield., (© 2014 American Institute of Chemical Engineers.)

Xylanase IV (XYN IV) is a new recently characterized xylanase from Trichoderma reesei. It is able to degrade several different xylans, mainly producing xylose. XYN IV has been crystallized by the hanging-drop vapour-diffusion method, using PEG 6000 as a precipitant. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 86.3, b = 137.5, c = 196.1 A, alpha = beta = gamma = 90 degrees. Assuming a molecular weight of 50.3 kDa, the V(M) values indicate there to be four XYN IV monomers in an asymmetric unit and the solvent content of the crystals to be 57%. Based on dynamic light-scattering measurements, XYN IV is a dimer in solution. A native data set to 2.8 A resolution has been collected at a home laboratory and a data set to 2.2 A resolution has been collected using synchrotron radiation.

Two minor xylanases present in Trichoderma reesei Rut C30 cultivation broth were purified as a mixture using ion-exchange, hydrophobic-interaction and gel chromatography. The purified enzyme preparation contained two active xylanases with pI values of 7.1 and 8.1. Both components had a molecular mass of 20 kDa. The purified xylanase preparation exhibited properties very similar to those of the previously isolated XYL II (pI 9.0) of T. reesei Rut C30. The activity and stability properties, apparent kinetic parameters as well as the titration curve forms were similar. The major difference in enzymic properties was the significantly lower specific activity of the pI-7.1+8.1 xylanase mixture (3350 nkat/mg) compared with the specific activity of XYL II (13500 nkat/mg). Amino acid sequences of tryptic peptides (34% of the total amino acid sequence was determined) were identical to the amino acid sequence of XYL II. Furthermore, in vitro modification of the pI-9.0 form of XYL II to pI-8.1 and pI-7.1 forms was demonstrated. Thus the purified xylanase preparation most probably contained two modified forms of XYL II. The primary amino acid sequence of XYL II contains 28 glutamine and asparagine residues and theoretically deamination of one of them lowers the pI to 8.06 and deamination of two amino acids lowers the pI to 7.02.

The crystal structure of the catalytic core domain of beta-mannanase from the fungus Trichoderma reesei has been determined at a resolution of 1.5 A. The structure was solved using the anomalous scattering from a single non-isomorphous platinum complex with two heavy-metal sites in space group P2(1). The map computed with the experimental phases was enhanced by the application of an automated model building and refinement procedure using the amplitudes and experimental phases as observations. This approach is expected to be of more general application. The structure of the native enzyme and complexes with Tris-HCl and mannobiose are also reported: the mannobiose binds in subsites +1 and +2. The structure is briefly compared with that of the homologous beta-mannanase from the bacterium Thermomonospora fusca.

Crystals of the catalytic core domain of a Trichoderma reesei beta-mannanase belonging to glycoside hydrolase family 5 have been grown by the sitting-drop method at room temperature using ammonium sulfate as precipitant. The crystals grow as thin colourless plates and belong to space group P21, with unit-cell parameters a = 50.0, b = 54.3, c = 60.2 A, beta = 111.3 degrees, and have a single monomer of mannanase in the asymmetric unit. Native data to 2.0 A resolution have been collected at room temperature using synchrotron radiation. Data for a platinum derivative have been collected to 1.65 A at 110 K in a very short time at the CCLRC Daresbury synchrotron source, using a charge-coupled device (CCD) as detector.

Production of extracellular a-galactosidases by the filamentous fungus Penicillium simplicissimum (previously P. janthinellum) VTT-D-78090 was studied on different carbon sources. Steam-exploded oat husks were chosen as the best carbon source for enzyme production. Three a-galactosidases (AGL) were purified from the culture filtrate using ion-exchange chromatography, hydrophobic interaction chromatography and gel filtration. The isoelectric points of AGLI, AGLII and AGLIII were 5.2, 4.4 and 7.0, and the molecular masses as determined by SDS/PAGE were 61, 84 and 61 kDa, respectively. All enzymes were glycosylated. The optimum pH for the activity of AGLI and AGLIII was between 3.0 and 4.5 and that of AGLII was between 4.0 and 5.0. AGLII was more stable and more resistant to product inhibition by galactose than the other two enzymes. AGLI and AGLIII were also inhibited by p-nitrophenol-a-D-galactopyranoside, the substrate used for enzyme activity assay. The gene encoding AGLI was cloned and sequenced. The gene, agl1, encodes 435 amino acids including the signal sequence. It showed similarity with the other a-galactosidases belonging to the glycosyl hydrolase family 27. The N-terminal amino acid sequence of AGLIII was also similar to the sequences of other members of family 27, whereas the N-terminus of AGLII was completely different from the sequences of other reported hydrolases.

Two cellulases of the filamentous fungus Trichoderma reesei, cellobiohydrolase II (CBHII, EC 3.2.1.91) and endoglucanase I (EGI, EC 3.2.1.4), produced in recombinant strains of the yeast Saccharomyces cerevisiae, were tested in the hydrolysis of cellulose, xylan and other polymeric substrates. Both enzymes were active against unsubstituted, insoluble cellulose. CBHII had greater activity than EGI against crystalline cellulose, whereas in the case of amorphous substrate the order was reversed. Evidence for synergism was obtained when mixtures of the two enzymes were used with a constant total protein dosage. The EGI was also active against soluble substituted cellulose derivatives, whereas the activity of CBHII against these substrates was insignificant. Both enzymes were active against barley (1-->3,1-->4)-beta-glucan, but were inactive against (1-->3,1-->6)-beta-glucan (laminarin). An apparent low mannan-degrading activity of EGI against locust-bean (Ceratonia siliqua) gum galactomannan was not confirmed when homopolymeric mannan was used as substrate in a prolonged hydrolysis test. EGI exhibited considerably greater activity against insoluble, unsubstituted hardwood xylan than against amorphous cellulose. Soluble 4-O-methyl-glucuronoxylan was also attacked by EGI, although to a somewhat lesser extent than the unsubstituted xylan. By comparison with two purified xylanases of T. reesei, EGI produced xylo-oligosaccharides with a longer mean chain length when acting on both substituted and unsubstituted xylan substrates. CBHII was inactive against xylan.