SE537440C2 - Process for enzymatic hydrolysis of glucuronoxylane - Google Patents

Abstract

..c1flß20] Û/ UÛl]72 4 ' f? ABSTRACT The present invention relates to an isolated polypeptide having a-glucuronidaseactivity and that can degrade glucuronoxylan molecules by hydrolysis of aglycosidic Iinkage between a MeGlcA residue (glucuronic side chain) and a non-terminal xylopyranosyl residue. The isolated polypeptide is capable of cleavingglycosidic linkages within glucuronoxylans from plant biomass, thereby removinginternal glucuronic side chains. The invention also relates to amino acidsequences of the isolated polypeptide, and homologous thereto, as well asisolated' polynucleotides having nucleic acid sequences encoding thepolypeptides. The invention further relates to methods of isolating thepolypeptides from microbial cultures, such as that of Pichia stipitis, usingchromatographic techniques, and to methods of producing substantially enriched preparations of the polypeptides.

Description

FIELD OF THE INVENTION This invention relates generally to the enzymatic degradation of plant biomass. More specifically, the invention relates to an isolated polypeptide having α-glucuronidase activity, a method for isolating the polypeptide and a substantially enriched formulation thereof.

Background of the Invention The pulp and paper industry processes large quantities of plant biomass or tra. This biomass includes complex carbohydrates, such as cellulose, hemicellulose and lignin, which must be partially degraded and treated to give a paper product. Tra contains about 20% hemicelluloses, of which xylan forms an essential part of bAcle lovtra and conifers. The main group of hemicelluloses present in the heart are glucuronone xylans. These include a 13-, 1.4-15-membered D-xylopyranose backbone with 4-O-methyl-D-glucuronic acid substituents linked α-1,2. In addition, the 2,3-positions p5 xylose backbone may be partially acetylated. The glucuronoxylan content in lovtra is typically between 15 and 30% by weight based on ptraet.

During pulp production, hemicelluloses undergo various changes. Hemicelluloses such as xylan do not form densely packed crystalline structures as do cellulose, due to the presence of side chains in the xylan structure. This characteristic of hemicelluloses means that these polysaccharides are broken down more easily than cellulose. While some hemicelluloses and degraded hemicellulose products are dissolved in boiling liquids, such as slightly alkali-soluble hemicelluloses, others are broken down into lower molecular weight products that can be retained in an insoluble form within the fiber matrix or released in the boiling liquids. The fraction of hemicellulose and degraded hemicellulose products that are dissolved in the boiling liquids are then lost to the process, resulting in loss of exchange of the wood. Hemicelluloses are believed to contribute to the swelling of the pulp and thus the formability of the straight fibers during sheet formation as a result of their non-crystalline hydrophilic nature. During the degradation of xylan, it is therefore desirable to minimize the loss of hemicellulose and degraded hemicellulose products in the boiling liquids, and to maximize the preservation of the hemicellulose in the fiber matrix. 1 537 4 In order to produce unwanted degraded hemicellulose products, selective degradation of hemicellulose is performed using enzymes. Enzymatic degradation of the hemicellulosaxylan is a complex process that requires the action of various enzymes that generally fall into tv5 categories: i) enzymes that degrade the polysaccharide backbone, such as endo-13-1,4-xylanase (EC 3.2.1.8) and 13-xylosidase (EC 3.2.1.37); and ii) enzymes such as free & side chains, the main chain substituents, so-called accessory xylanolytic enzymes, which include α-glucuronidase (EC 3.2.1.139), αL-arabinofuranosidase (EC 3.2.1.55), acetylxyl anesterase (EC 3.1.1.72) and feruloyl esterase (3.1.1.73). While the endoxylanase attacks the main chain of xylans and 13-xylosidase, xylooligosaccharides hydrolyze to xylose, the α-arabionfuranosidase and the α-glucuronidase degrade the arabinoset and the 4-0-methyl-glucuronic acid substituents, respectively, of Iran. The esterase hydrolyzes ester linkages between xylose units p5 xylanet and attic acid (acetylxylanesterase) or between arabinose side chain residues and phenolic acids, such as ferulic acid (ferulic acid esterase) and β-coumaric acid (β-coumaric acid esterase).

The removal of MeGIcA or GIcA side chains from xylan is proposed to increase the retention of the xylan in the fiber matrix. At present, only one GH family, GH67, contains exclusively α-glucuronidases. However, the activity of these enzymes is limited because they release MeGIcA or GIcA only from those fragments of glucuronxylan (alduronic acids) in which the uronic acid is linked to non-reducing terminal xylopyranosyl residues. These α-glucuronidases do not cleave glycoside linkages within polymeric substrates, as within glucuronoxylanes. The only α-glucuronidase described so far to have the ability to release MeGIcAsid chains Iran glucuronxylan from the praised Jr enzyme present in the cellulolytic system of the root rot fungus Schizophyllum commune. Formulas 1 to 4 show the glycoside linkers in fragments of glucuronxylan that are attacked () or not attacked (x) by GH67-α-glucuronidases.

Xylft1w4Xylp1.4Xylp1-4, Xy1-Xylfi1-4Xylp1.4Xyl * Xylp1-4Xy101-4Xy1p14xy. 22222Aryl kix al0.1alalalal MeGIcAMeWcAMeGicAGicAGicAriteGcA Forme! 1Forme! 2Forme! 3Forme! These enzymes also do not hydrolyze aryl-α-glucuronides, which act as substrates for the non-hemicellulolytic family of 4-α-glucuronidases. Although an α-glucuronidase that hydrolyzes aryl-α-D-glucuronosides Jr edge in GH4, this enzyme does not recognize glucuronone xylan or its fragments as a substrate. The types of glycoside linkages that are cleaved by α-glucuronidases to date are therefore limited.

There is a need for an improved process for enzymatic hydrolysis of glucuron xylan to increase the retention of glucuron xylan in the fiber matrix during degradation of plant biomass, and for enzymes for use therein.

It is an object of the present invention to provide an alternative process for the enzymatic hydrolysis of glucuronxylan which, at least to some extent, can alleviate the problems described above.

Summary of the Invention In accordance with the invention, there is provided an isolated polypeptide which has α-glucuronidase activity and which can degrade a glucuronoxylan molecule by hydrolysis of a glycosidic link between a MeGIcA residue and a non-terminal xylopyranosyl residue.

The invention also provides an isolated polypeptide having an amino acid sequence selected from the following group: i. The amino acid sequence of SEQ ID NO: 1; II. an amino acid sequence that is at least 95% homologous to SEQ. ID NO: 1 or part clarav; an amino acid sequence as Jr at least 85% homologous to SEQ. ID NO: 1 or part clarav; an amino acid sequence as Jr 5 at least 75% homologous to SEQ. ID NO: 1 or part clarav; An amino acid sequence as Jr 5 at least 65% homologous to SEQ ID NO: 1 or a portion thereof; an amino acid sequence as Jr 5 at least 50% homologous to SEQ ID NO: 1 or a portion thereof; vii. a functional variant of n5gon of the amino acids listed in i-vi.

Additional features of the invention mediate that the polypeptide has a molecular weight of about 120 kDa and that the polypeptide is a biologically active fragment of the polypeptide.

The invention extends to an isolated polynucleotide encoding a polypeptide of the invention, the polynucleotide having a nucleotide sequence selected from the following group: the nucleotide sequence of SEQ ID NO: 2; a nucleotide sequence as Jr 5 at least 95% homologous to SEQ ID NO: 2 or a portion of clarav; iii. a nucleotide sequence as Jr 5 at least 85% homologous to SEQ ID NO: 2 or a portion thereof; and iv. a nucleotide sequence as Jr 5 at least 75% homologous to SEQ ID NO: 2 or a portion thereof.

The invention also provides a method of isolating a polypeptide of the invention, the method comprising the steps of: culturing a microbe capable of expressing the polypeptide in induction medium and creating microbial biomass; separating the microbial biomass from the induction medium; iii. fractionating the induction medium by anion exchange chromatography to give a first eluent; fractionating the first eluent by hydrophobic interaction chromatography to obtain a second eluent; fractionating a second eluent by anion exchange chromatography to obtain a third eluent; and 4,537 4 vi. fractionating the third eluent by anion exchange chromatography to obtain a fraction containing the isolated polypeptide.

Further features of the invention provide the microbe to be selected from the group including Pichia stipitis, Schizophyllum commune, Aspergillus clavatus, Neosartorya fischeri, Aspergillus fumigatus, Aspergillus terreus, Aspergillus oryzae, Sclerotinelia sclerotiropia, Sclerotinelia sclerotiropia, Coprinopsis cinerea okayama, Magnaporthe grisea, Fusarium sporotrichioides, Cryptococcus neoformans var. neoformans, Cellvibriojaponicus, Saccharophagus degradans, Opitutus terrae, Phaeosphaeria nodorum, Bacteroides ovatus and Streptomyces pristinaespiralis; and for the microbe to be preferably Pichia stipitis CBS 6054.

Other jars of the present invention show a method of isolating the polypeptide to further include the step of concentrating one or more of the induction medium; the first eluent, the second eluent, the third eluent and the fraction comprising the isolated peptide.

The invention further provides a substantially enriched formulation of a polypeptide of the invention.

Additional features of the invention allow purification of the polypeptide from a culture of a microbe selected from the group including Schizophyllum commune, Aspergillus clavatus, Neosartorya fischeri, Aspergillus fumigatus, Aspergillus terreus, Aspergilfus oryeaisia tria, Sclerophylaia, Sclerophysia. , Gibberella zeae, Podospora hellerina, Coprinopsis cinerea okayama, Magnaporthe grisea, Fusarium sporotrichioides, Cryptococcus neoformans var. neoformans, Cellvibriojaponicus, Saccharophagus degradans, Opitutus terrae, Phaeosphaeria nodorum, Bacteroides ovatus and Streptomyces pristinaespiralis; and that the microbe is preferably Pichia stipitis CBS 6054. Other features of the invention allow the induction medium to be glucose YNB medium supplemented with xylooligosaccharides and methyl-β-xylopyranoside; for xylooligosaccharides to be in a concentration of 0.5 mg / ml; for the methyl β-xylopyranoside to be in a concentration of 0.33 mg / ml; for the fractionation of the induction medium by anion exchange chromatography to obtain a first eluent is performed using a HiTrap-DEAE-FF column; for the first eluent to be obtained by supplying a first elution buffer to the HiTrap-DEAE-FF column, the first elution buffer comprising a NaCl gradient of 0 to 1.0 M in about 50 mM sodium phosphate buffer at about pH 7.0; for fractionating the first eluent by hydrophobic interaction chromatography to obtain a second eluent is performed using a butyl FF column; for the second eluent to be obtained by adding a second elution buffer to the butyl FF column, the second elution buffer comprising reducing the (NH 4) 2 SO 4 gradient of 1.1M to 0.61M in about 50 mM acetate buffer at an approximate pH of 4.0; for the fractionation of the second eluent by anion exchange chromatography to obtain a third eluent to be performed using a "Tricorn MonoQ5 / 50GL" column; for the third eluent to be obtained by adding a third elution buffer to the Tricorn MonoQ 5 / 50GL column, the third elution buffer comprising an increasing NaCl gradient from 0 to 1.0 M in about 50 mM sodium acetate buffer at about pH 4.0; fractionating the third eluent by anion exchange chromatography to obtain a substantially pure enzyme fraction to be performed using a Tricron MonoQ 5 / 50GL column; for the substantially pure enzyme fraction to be obtained by supplying a fourth elution buffer to the Tricron MonoQ 5 / 50GL column, the fourth buffer comprising an unknown NaCl gradient Iran 0 to 1.0 M in about 50 mM sodium phosphate buffer at about pH 7.0.

Additional features of the invention make it possible to obtain the polypeptide from a culture of a microbial group selected from the group including Schizophyllum commune, Aspergillus clavatus, Neosartorya fischeri, Aspergillus fumigatus, Aspergillus terreus, Aspergillus oryzae, Sclerotinelia sclerotinia sclerotinia sclerotinia sclerotinia sclerotinia crassa, Gibberella zeae, Podospora hellerina, Coprinopsis cinerea okayama, 6 537 4 Magnaporthe grisea, Fusarium sporotrichioides, Cryptococcus neoformans var. neoformans, Cellvibriojaponicus, Saccharophagus degradans, Opitutus terrae, Phaeosphaeria nodorum, Bacteroides ovatus and Streptomyces pristinaespirali; and for the microbe to be preferably Pichia stipitis CBS 6054.

Brief Description of the Drawings Additional features of the invention will now become apparent from the following description, taken by way of example only, with reference to the accompanying drawings and SEQ. IDNO: s.

In the drawings: Figure 1 is a summary of α-glucosinidase purification from the induction medium of P. stipitis CBS 6054; Figure 2 shows an SDS-PAGE gel of purified P. stipitis α-glucosinidase, row 1 - protein markers (Fermantase #SM 0431), row 2 - α-glucuronidase, 10 lig protein, row 3 - α-glucuronidase, 20 lig; row 4— protein markers (SERVA # 39216); Figure 3 is a chromatogram of the TLC analysis of products formed from aldopentauric acid (Xyl-Xyl (MeGIcA) -Xyl-Xyl) (A) and glucuronxylan (B) "under the influence" of purified P. stipitis α-glucuronidase, A : pray: row 1 and row 8 - xylose and xylooligosaccharide standards, 2 - aldopentauronic acid (Xyl-Xyl- (MeGIcA) -Xyl-Xyl), row 3 - enzyme blank, row 4-7 - products of enzyme reaction after 1, 10, 60 min and 18 h, B: pray: row 1 and row 5 - xylose and xylooligosaccharide standards, row 2 —glucuronxylan control, row 3 and row 4 — products of enzyme reaction after 4 h and 18 h, respectively.

Figure 4 is a summary of a BLAST search challenge using a sequence of P. stipitis. The sequence of microorganisms used for the alignment and construction of phylogenetic tra is shown in bold; Figure 5 is a schematic representation of the homologous alignment of the amino acid sequence of P. stipitis CBS 6054 α-glucuronidase with the nine protein sequences Iran Aspergillus fumigatus A1293, Pyrenophora tritici-repentis Pt-1C-BFP, Neurospora gassa 0 -1, Cellvibrio japonicus Ueda107, Coprinopsis cinerea okayama 7 # 130, Aspergillus oryzae RI B40, Bacteroides ovatus ATCC 8483, Streptomyces Pistinaespiralis ATCC 25486 which showed more than 50% identity; Figure 6 shows a summary of xylan substrates used to evaluate enzymatic substrate specificity and degree of deposition of xylan side chains; Figure 7 shows the experimental set-up of Box-Behnken for the removal of 4-O-methyl-D-glucuronic acid -Iran birch xylan by α-glu; Figure 8 shows a central composite design for effect on concentration of xylan from oat cleft and enzyme doses on arabinose removal; Figure 9 shows a bar graph of the content (% OD biomass) of the extracts and ash of bagasse, pine (Pinus patula) and bamboo (Bambusidae balcooa); Figure 10 shows a bar graph of Clason lignin (% OD biomass) of bagasse, pine (Pinus patula) and bamboo (Bambusidae balcooa); Figure 11 shows a bar graph of the content (% OD biomass) of cellulose and the pentosa of bagasse, pine (Pinus patula) and bamboo (Bambusidae balcooa); Figure 12 shows a bar graph of xylan yield (% pentosan) extracted using ultra purification and ethanol precipitation protocol Iran bagasse, pine (Pinus patula) and bamboo (Bambusidae balcooa); Figure 13 shows a 1-3 C-CPMAS NMR solid phase spectrum showing the effect of mild alkali xylan extraction on the integrity of cellulose fibers in (A) Pinus patula, (B) bagasse, (C) Eucalyptus grandis and (D) jatte bamboo. Spectrum 1, 2 and 3 indicate: r5material, free extract material and material after xylane extraction and ** denote peaks for carbon resonances in glucose units of lower ordered cellulose; Figure 14 shows a comparison of neutral sugar composition of lignocellulosic material before (EF) and after xylane extraction (Pxyl) for (A) Pinus patula (pine), (B) bagasse (bag), (C) Eucalyptus grandis (EU) and (D) bamboo (BM).

Figure 15 shows a summary of the profile for neutral sugars and uronic acid of pre-extracted xylan.

Figure 16 shows elution profiles for xylan p5 HPAEC-PAD (Dionex) CarboPac P10 column from (A) monomeric sugars, (B) xylitol, (C) birch xylan (Roth) and (D) xylan from oat cleft; Figure 17 shows the elution profiles of xylan p5 HPAEC-PAD (Dionex) CArboPac column P from (A) mildly alkali-extracted H 2 O 2 bleached bagasse (Bag B), (B) mildly alkali-extracted ultra-purified bagasse (Bag H) and (C) mildly alkali metal extra bagasse (Bag L); Figure 18 shows the elution profiles for xylan on HPAEC-PAD (Dionex) CarboPac column P from (A) Eucalyptus grandis H [EU H] and (B) Eucalyptus grandis L [EU L] (C) bamboo and (D) Pin us patula ; Figure 19 shows a bar graph of the insoluble fraction obtained after 72% acid hydrolysis of mildly alkali-extracted xylan H 2 O 2 bleached bagasse (Bag B), ultra-purified bagasse (Bag Fib ethanol precipitated bagasse (Bag L), bamboo, ultra-purified E.grandis (EU H), ethanol precipitated E.grandis (EU L) and P. patula (Pine) with reference to birch xylan (Roth); Figure 20 shows the characterization of xylan by (A) 1 H-NMR and (B) 13 C-NMR analysis of birch xylan, (C ) 1 H-NMR and (D) 1 H-NMR analysis of H 2 O 2 -bleached bagasse (Bag B) and (E) 1 H-NMR and (F) 13 H-NMR analysis of xylan from oat column, Me represents methyl group from glucuronic acid and Ac = Acetyl group, Ar = arabinose group; Figure 21 shows the characterization of xylan by (A) 11-1 NMR and (B) 13 C-NMR analysis of bagasse, (C) 1-H-NMR and ( D) 13 C-NMR analysis of E.grandis xylan: In spectra (1) = Lopez-extracted xylan (Bag L or EU L), and (2) High ultra-purified xylan (Bag H or Eu H); Figure 22 shows the characterization of xylan by (A) 11-1 NMR and (B) 13 C-NMR analyzes of bam bu, (C) 1 H-NMR and (D) 13 C-NMR analyzes of P. patula-xylan; Figure 23 shows the FTIR spectrum of xylan extracted from different types of lignocellulosic materials Iran batten (iv) birch **, (F) ethanol precipitated bagasse [Bag L] (2), (E) ultra-purified bagasse [Bag H] (1), ( D) xylan frail oat split *, (C) bamboo, (B) ethanol precipitated E.grandis [EU L] (2), (B) ultrapureed E. grandis [EU H] (1) and (A) P.patula; Figure 24 shows the deposition of 4-0-MeGIcA by AbfB and a-glu from oat cleft / birch, mild alkali pre-extracted bagasse Hoije (BH), H202-bleached bagasse (BB), bamboo (BM) and Pinus patula (PP) xylan and by a-glu Iran mildly alkaline-for-extracted Eucalyptus grandi (EH), Eucalyptus grandis-gel (ES) extracted from pulp; Figure 25 shows response area diagrams for deposition of glucuronic acid as a function of (A) time (h) and temperature (° C) at 16500 nKat g-1 substrate, (B) temperature (° C) and enzyme dose (nKat g-1 substrate) at 9 h and (C) time (h) and enzyme dose (nKat g-1 substrate) at 33.5 ° C; Figure 26 shows interaction effects between time, temperature and enzyme dose on glucuronic acid deposition. The first columns above show the interaction between temperature (Temp) and time, enzyme dose (ABIB / a-glu) and time, and enzyme dose (a-glu) and temperature. In the right part of the second column, the cell shows the size and significance of the treatment and interaction effects measured by the size of the bar graph. The t (l44) values are displayed at the end of each bar chart in each Pareto chart. The vertical dotted line in the Pareto diagram is a measure of statistical significance at p = 0.05; Figure 27 shows a summary of regression coefficients for glucuronic acid release as a function of the hydrolysis parameters (coded variable); 537 4 SEQ. ID NO 1 Jr the deduced amino acid sequence of the α-glucuronidase gene of P. stipitis, available in Genbank as accession number XP 001385893; and SEQ. ID NO 2 The DNA sequence of the α-glucuronidase gene of P. stipitis, such as the Jr available in Genbank as accession number XM 001385893 and published P. stipitis genome sequence (Vrargka et al 2007).

Detailed Description Referring to the Drawings The invention relates to an isolated polypeptide which has α-glucuronidase activity and is designed to degrade glucuronyl xylan molecules found in plant biomass. Unsurprisingly, this polypeptide exhibits benefits for enzymatic activity in the degradation of glucuronxylan molecules that cannot be seen in other enzymes previously reported to exhibit α-glucuronidase activity. Scanning other α-glucuronidases Jr limited in their hydrolysis of glycoside linkers of glucuronyl xylamolecules because they have only the ability to hydrolyze a glycoside linker between a MeGIcA residue and a terminal xylopyranosyl residue. In contrast, the isolated polypeptide of the invention has been found to be unexpectedly capable of hydrolyzing a glycoside linkage between a MeGIcA residue and a non-terminal xylopyranosyl residue. In addition, the aglucuronidase activity of the isolated polypeptide of the invention is widely applicable to a number of glucuronon xylan molecules obtained from various plant biomass sources.

The present invention provides an isolated polypeptide having an amino acid sequence which is the amino acid sequence of SEQ. ID NO 1; or one that is substantially similar to that of a sequence that is at least 95% homologous to SEQ. ID NO 1 or part thereof, 5 at least 85% homologous to SEQ. ID NO 1 or part thereof; At least 75% homologous to SEQ. ID NO 1 or part thereof; At least 65% homologous to SEQ ID NO 1 or part thereof; At least 50% homologous to SEQ. ID NO 1 or part clarav; a functional variant of the flake of these amino acid sequences. The identity of the full-length isolated polypeptide can be confirmed with reference to its molecular weight of about 120 kDa, using techniques such as SDS-PAGE or α-glucuronidase activity assay for the identification of biologically active fragments of the polypeptide. The polypeptide Jr is typically isolated from the induction medium of a culture of Pichia stipitis CBS 6054, although it will be appreciated that other microbes expressing substantially similar polypeptides may also be used. S5dana microbes include ra r Schizophyllum commune, Aspergillus davatus, Neosartorya fischeri, Aspergillus fumigatus, Aspergillus terreus, Aspergillus oryzae, Sderotinia sderotiorum, Botryotinia fuckeliana, Pyrenophora crippina, Gypsies, Fusarium sporotrichioides, Cryptococcus neoformans var. neoformans, Cellvibriojaponicus, Saccharophagus degradans, Opitutus terrae, Phaeosphaeria nodorum, Bacteroides ovatus and Streptomyces pristinaespiralis.

Since the polypeptide is secreted into the induction medium of the microbial culture, various chromatographic techniques can be used to isolate the polypeptide from the induction medium such as anion exchange chromatography, hydrophobic chromatography and anion exchange chromatography. The polypeptide is typically isolated from the induction medium by first separating microbial biomass from the induction medium, fractionating the induction medium by anion exchange chromatography to obtain a first eluent; fractionating the first eluent by hydrophobic interaction chromatography to obtain a second eluent; fractionating the second eluent by anion exchange chromatography to obtain a third eluent; and fractionating the third eluent by anion exchange chromatography to obtain a fraction comprising the isolated polypeptide. Additional steps for concentrating one or more of the induction medium; the first eluent; the second eluent; the third eluent; and the fraction comprising the isolated polypeptide is also excreted.

The isolated polypeptide of the invention may be provided in the form of a substantially enriched formulation of the polypeptide. A substantially enriched formulation prepared according to the invention generally means that the predominant protein species or components of the formulation are the polypeptide of SEQ. IDNO 1, or a substantially similar one. However, it will be appreciated that more purified forms of the substantially enriched formulation are included within the scope of the invention, such as formulations comprising at least 75% of the polypeptide; preparations comprising at least 80% of the polypeptide; preparations comprising at least 90% of the polypeptide. A substantially enriched formulation prepared according to the invention also generally refers to the essential presence of biologically active enzymes capable of hydrolyzing the backbone of a glucuronoxylan molecule.

Example 1 Materials and Methods P. Stipitis Strains and Their Cultivation P. Stipitis CBS 6054 were grown in vials in medium containing YNB (Difco, 6.7 g / l), L-asparagine (2 g / l), KH 2 PO 4 (5 g / l) and carbonaceous (glucuronoxylan from glucose or birch bark, 10 g / l) at a temperature of 30 ° C and a stirring of 180 rpm. Exponentially grown cells were harvested at a cell density of 0.15-0.20 mg / ml (dry weight). α-glucuronidase substrate and products Deacetylated glucuronxylan was extracted from sawdust [Ebringerova et al., 1967, Holzforschung 21: 74-77], aldotetrauronic acid Xyl (MeGIcA) -Xyl (MeGIcA) Xyl-Xyl, the shortest product of glucuronoxyl anhydrolysis by family 11 endoxylanases, was prepared as previously described [Biely et al., 1997, J. Biotechno. 57: 151-166]. 4-O-methyl-F-glucuronic acid (MeGIcA) was prepared by transesterification of its methyl ester by S.commune glucuronoyl esterases [panikova and Biely, 2006, FEBS Lett. 580: 4597-4601].

P. Stipitis α-glucuronidase preparation in fronyaron ay xylan The enzyme, which became a formula for purification, was prepared in induction experiments which are challenged as follows: cells from exponential phase grown in a YNB medium with 1% glucose were collected by centrifugation, washed with basalt YNB medium (without carbon cold) and suspended in the same medium supplemented with 0.5 mg / ml xylooligosaccharide mixture (XYLO-OLIGO 70, Suntory Limited, Japan) and 0.33 mg / ml of methyl-13-xylo-13 537 4 pyranoside. The cell concentration was 0.6-0.8 mg / ml dry weight (105 ° C). After 24 hours of incubation on a shaker (180 rpm) at 30 ° C for 24 hours, the mixture was centrifuged and the clear supernatant used to purify extracellular α-glucuronidase which was co-induced with endo-13-1.4-xylanase.

Purification of P. Stipitis-α-glucuronidase The clear induction medium (600 ml) was concentrated 300-fold p5 Amicon 10 kDa spruce membrane. The secreted proteins were first fractionated by anion exchange chromatography on a HiTrap DEAE-FF column (GE Healthcare, Sweden) using elution with a NaCl gradient (0-1.0 M) in 50 mM sodium phosphate buffer (pH 7.0). Fractions containing α-glucuronidase, eluting as a peak between 0.2-0.26 M NaCl, were combined, concentrated and desalted, then equilibrated in 50 mM acetate buffer (pH 4.0) containing 2M (NH 4) 2 SO 4. The resulting eluent was redissolved by hydrophobic interaction chromatography on a butyl FF column (5 ml) (GE Healthcare) eluting with a decreasing gradient of (NH 4) 2 SO 4 in the same buffer. α-glucuronidase was eluted at a concentration of between 1.1 and 0.61 M (NH 4) 2 SO 4. The fractions having α-glucuronidase activity were combined, desalted, concentrated and redissolved by two additional ion exchange chromatography steps using a Tricorn MonoQ 5 / 50GL column (Amersham, UK) (polystyrene / divinylbenzene). In the first step, the column was equilibrated with 50 mM sodium acetate buffer (pH 4.0) and eluted with an increasing gradient of NaCl (0-1.0 M). In the second elution step, the acetate buffer was replaced with 50 mM sodium phosphate buffer (pH 7.0). Fractions having α-glucuronidase activity were desalted and concentrated by membrane filtration p5 Microcon (10 kDa grans, Millipore Co., USA).

Sequence analysis of purified protein Purified α-glucuronidase was redissolved by SDS PAGE on 5% acrylamide gels and electroblotted onto a polyvinylidene difluoride membrane (Millipore Corp., USA). The sequence of the 15 N-terminal amino acids was determined using an HP G105A protein sequencer (Hewlett Packard, Palo Alto, Ca, USA). 14 537 4 α-glucuronidase assay Purification fractions having the α-glucuronidase activity were qualitatively identified by TLC analysis using a silica gel (Merck silica gel 60 p5 aluminum plates) in ethyl acetate acetic acid: 1-propanol: formic acid: water (1: 15, 5: 5, based p5 volume). The narvaron of free MeGIcA derived from alduronic acid (10 mg / ml) or glucuronxylan (2%) dissolved 10.05 M sodium acetate buffer (pH 4.4) indicated α-glucuronidase activity. The enzyme is usually used at a concentration of 50 μg protein / ml.

A quantitative assay based on the determination of free MeGIcA released from aldopentauronic acid (Xyl-Xyl (MeGIcA) -Xyl-Xyl, 10 mg / ml) or glucuronxylan from birchwood (2%) according to the procedure described by Milner and Avigad [Milner and Avigad , 1967, Carbohyd. Res. 4: 359-361] was used to detect substrates and products with α-glucuronidase activity. The substrate and products were detected using N- (naphthylethylenediamine) dihydrochloride reagent [Buonias, 1980, Anal. Biochem. 106: 291-295]. The reagent is brown to the color in the narvaron of MeGIcA and purple to the color in the narvaron of xylose-containing compounds. Protein samples (1-10 μg, depending on purity) were incubated for 10-60 minutes in 0.1 ml of reaction mixture containing the substrate in 50 mM acetate buffer (pH 4.4). The reaction was stopped by adding 0.3 ml of copper reagent and boiling for 10 minutes at 100 ° C, followed by the addition of 0.2 ml of Nelson reagent and 0.4 ml of water. The absorbance was matted at 600 nm using GIcA calibration. One unit of α-glucuronidase was defined as the amount of enzyme producing 1-phenol uronic acid p5 1 min from aldopentauronic acid (Xyl-Xyl (MeGIcA) -Xyl-Xyl.

Protein determination Protein concentration was determined according to the method of Bradford [Bradford, 1976, Anal.

Biochem. 72, 248-254] using bovine serum albumin as standard. Protein molecular weight was estimated using SDS-PAGE (Laemmli, 1970, Nature 227: 680685] and compared with similar redissolved unflaked protein molecular weight markers (FERMENTAS, Canada) and colored protein markers (SERVA, GmnH). IEF challenged p5 Multiphor-II system ( GE Healthcare, Sweden) using SERVALYT PRECOTES 3-6 precast gel and IEF markers 3-10 (SERVA, GmbH) 537 4 Results Isolation of α-glucuronidase A new α-glucuronidase was observed during the growth of several P. stipitis strains p5 different types of xylan The highest growth of the yeast was observed p5 glucuronxylan Although glucuronxylan was used as the main carbon source to a limited degree IA it was compared with the use of D-xylose, D-glucose or 6-1,4-xylooligose , there was no accumulation of acid oligosaccharides in the medium. All fragments released from glucuronxylan were fully utilized, indicating that the yeast secreted an α-glucuronidase into the medium. one of the yeast after growth of p5 glucuronxylan revealed strong α-glucuronidase activity.

The partially purified enzyme was found to be capable of removing branches of glucuronxylan and released MeGIcA Iran alduronic acid in which MeGIcA was linked to internal xylopyranosyl residues. The enzyme could not be purified from a partially used glucuronene xylan medium because the presence of xylan residues increased the viscosity of the concentrated medium and increased the enzyme purification.

However, the extracellular α-glucuronidase was successfully purified from the medium when it was washed, glucose-grown cells Iran exponential phase were incubated in a synthetic medium provided with a mixture of xylobiosis, xylotriosis and methyl 1-6-D-xylopyranoside, which .stipitis. The level of extracellular α-glucuronidase induced under these conditions was 0.015 U / ml, which represented approximately 20% of the activity observed during incubation of cells with 1% glucuronxylan. This level of α-glucuronidase was sufficient for its purification from the induction medium containing only dialyzable nutrient components. The enzyme was successfully purified from a concentrated induction medium using a combination of ion exchange and hydrophobic interaction chromatography (Figure 1). The enzyme was probed into a single band by analysis by SDS PAGE, corresponding to a protein of approximately 120 kDa (Figure 2) and of sufficient purity to be used for N-terminal amino acid sequence analysis.

Catalytic Properties of P. Stipitis α-Glucuronidase The purified α-glucuronidase was found to be substantially free of endoxylanes or 13-xylosidase activity. The only reaction shown by the enzyme with aldopenturonic acid having the structure Xyl-Xyl (MeGIcA) -Xyl-Xyl * or with birch-glucuronxylan was the release of MeGIcA residues (Figure 3). The enzyme also released MeGIcA from aldotetrauronic acid Xyl (MeGIcA) -Xyl-Xyl * (not shown) which similarly acts as a substrate for family 67 of α-glucuronidases (Biely, 2003, Xylanolytic enzymes, In: Handbook of Food Enzymology, p. 879-916, Marcel Dekker, Inc. NY). The initially clear glucuronoxyl solution showed increased opalescence and increased viscosity due to sufficient branch removal. The amount of uronic acid released from birchwood-glucuronxylan by the enzyme after a long-term treatment was 0.35 kmol per 1 mg of glucuronxylan, representing 75% of the total MeGIcA inneal in the polysaccharide.

Optimal conditions for enzyme activity The optimal pH value for enzyme activity was found to be 4.4. At pH 4.0 and pH 5.5, the activity represented 26.5% and 51.6%, respectively, of the activity at the optimum pH value. The optimum temperature for the enzyme activity was 60 ° C (18.43 U / mg with polymeric glucuronxylan), but the protein was found to be unstable at this temperature, with a 50% loss of activity for 30 minutes. The protein was found to be stable for at least 3 hours at 40 ° C, with specific activity on glucuronxylan 3.01 U / mg. The pI value of α-glucuronidase calculated from sequence (ExPASy online ProtParam tool) was 4.64, but isoelectric focusing data indicated that the pI value of the protein was closer to 4.0.

N-terminal amino acid sequence and homology analysis Edman analysis of purified α-glucuronidase revealed an amino acid sequence including the N-terminal LGGLQNIVFKNSKDD sequence which exactly corresponded to the P. stipitis gene XP 0013855930 which has been deduced from the available amino acid sequence, number 19, encoding a protein with unknown function but which has a similar molecular mass as the isolated α-glucuronidase (SEQ. ID NO 1). The portion of the gene encoding the first 19 amino acids apparently corresponds to the secreted signal sequence of the enzyme, which is not protected in the fully developed extracellular protein comprising 957 amino acids.

A BLAST search was performed using the sequence of P. stipitis α-glucuronidase which revealed similar genes in the genome of many other microorganisms, mostly fungi. The list of microorganisms with orthologs of the P. stipitis α-glucuronidase gene having an identity higher than 34% and similarity higher than 51% is shown in Figure 4. All orthologs correspond to proteins with about 1000 amino acids, which corresponds to a molecular mass of about 120 kDa, in all respects it is proteins with unknown function. The highest identity (54%) and the highest similarity (69%) were shown by the gene sequence for Aspergillus davatus.

The alignment of eight selected homologous sequences (shown in bold in Figure 4) with the β-stipitis aglucuronidase sequence is shown in Figure 5. The alignment shows 6 conserved glutamic acid residues and 12 conserved aspartic acid residues, of which tv5 may be the amino acid involved in catalysis of the reaction. In addition, 3 tyrosines and 6 tryptophans are preserved. If the aromatic amino acid is surface exposed, they may play an important role in the recognition of the xylan backbone as one of the conditions for the enzyme to act on the polymer substrate.

The P. stipitis enzyme releases MeGIcA residues that are linked to terminal or internal xylopyranosyl residues of glucuronxylan and aldouronic acids generated from the polysaccharide under the action of endoxylanases. The sequence for P. stipitis CBS 6054 is phylogenetically derived from both GH families 67 and 4.

The xylosfermenting yeast Pichia stipitis is unique in that it also has a limited form of utilizing xylan as a carbon cold. The yeast was found to preferentially hydrolyze glucuronxylan from lovtra, possibly due to the fact that the uptake in its xylanolytic enzyme is limited to the production of only three enzymes, endo13-1.4-xylanase, α-glucuronidase and 13-xylosidase. This property has the xylanolytic system for yeast corresponding to its natural environment, i.e. the one for the digestive tract has Passalide beetles which feed on biomass from lovtrad rich in acetylglucuronexylan. During growth of this deacetylated glucuronxylan, the first enzymes were secreted by the yeast into the growth medium and no accumulation of acidic oligosaccharides (alduronic acids) could occur during growth of this calcium source. The level of secreted endoxylanase by this yeast has previously been found to be such that it did not encourage further studies of the xylanolytic system of this yeast species. It is therefore surprising that this depleted xylanase secretes a new and useful α-glucuronidase.

The newly described type of α-glucuronidase has the ability to release MeGIcA residues from Iran polymer substrates. Its action on aldotetrauronic acid Xyl (MeGIcA) Xyl-Xyl, the shortest acid product Iran glucuronoxylanhydrolysis by family endoxylanases confirms that the new α-glucuronidase family exhibits catalytic activity for G H 67 enzyme. The N-terminal amino acid sequence of α-glucuronidase by S.commune did not match any sequence for the enzymes grouped in the new family.

The ability of this new α-glucuronidase enzyme to branch off glucuronxylan is believed to affect the physicochemical properties of the polysaccharide. Deacetylation of acetylglucuronexylan or the removal of side chains of α-L-arabinofuranosyl in arabinoxylan is suggested to reduce the looseness of the polysaccharides and possibly result in the precipitation of branched polymers. Treatment of plant biomass using the new α-glucuronidase enzyme described has been proposed to reduce the loss of glucuronxylan in the boiling of liquids created during papermaking and maximize the preservation of glucuronxylan in the fiber matrix.

The removal of methyl glucuronic acid and glucuronic acid from glucuronxylan is expected to create compositions that have useful applications in the paper and pulp industry and the pharmaceutical field. Increased amounts of glucuronxylan in papermaking not only contribute to the conservation of plant biomass in papermaking processes, but can also provide unique properties in paper strength, paper coating and ink retention. The re-precipitation of glucuroxylan can be used in the pharmaceutical field for coating drugs to increase their storage capacity and facilitate the slow release of medical compounds. The hydrolysis of glucuronxylan by synergistic activities of α-glucuronidase together with β-xylanases and 13-xylosidases can release fermentable sugars Iran glucuronxylan for conversion to commercial products, such as ethanol, lactate and other fine chemicals. 19 537 4 EXAMPLE 2 Materials and methods Statistical analysis Unless otherwise stated, tests are challenged three times. Analysis of variance (ANOVA) including test means and standard deviations is challenged using Microsoft Excel and Statistica 2007.

Optimization of xylane extraction for analysis of α-glucuronidase activity The selectivity and efficiency of the mild alkalixylane extraction methods borrowed from Hoije et al. [2005, Carbohydr. Polym. 61: 266-275] and De Lopez et al. [1996, Biomass and Bioenergy, 10: (4): 201-211] were evaluated for potential use in the integrated production of substantially pure xylan biopolymers and pulp production from the raw material commonly found in South Africa. The evaluation was based on four factors as follows: (1) xylane extraction efficiency, (2) degree of polymerization and substitution, (3) chemical composition of the extracted xylan, (4) purity of the extracted xylan, and (5) the structural integrity and chemical composition of r5material after xylane extraction. A summary of the xylan substrates used to evaluate enzymatic substrate specificity and degree of removal of xylan side chains is shown in Figure 6.

Raw material designation The raw material used includes Eucalyptus (Eucalyptus grandis), pine (Pinus patula), jatte bamboo (Bambusa balcooa) and sugar cane (Saccharum officinarum L) bagassee. Wood chips from E.grandis were supplied by "The Transvaal Wattle Cooperatives" from Piet Retif, Mpumalanga Province, while the P.patu / a trade was harvested from Stellenbosch University's forest plantations in the Western Cape province of South Africa. Jatte bamboo stems (plants that were 1.5 5r) were supplied from mature plantations located in Paarl in the Western Cape province of South Africa. Bagasse was a by-product of the sugar manufacturing industry donated by TBS Company located in the Nkomazi region of the Cyclostra Lewveld in the province of Mpumalanga in South Africa. Xylan from oat clef (Sigma), birch xylan (Roth) and mild alkali-extracted H 2 O 2 bleached 537 4 bagassexylan (donated by Prof. A.M.F. Milagres, University of Sao Paulo, Brazil) were used as reference xylans.

Materials from the raw material were prepared for analysis according to the TAPPI test methods (TAPP !, T264 cm-97 (2002-2003)) and NREL Laboratory Analytical Procedures (NREL LAP) [Hammes et al., 2005, Laboratory Analytical Procedure (LAP), version 2005 , NREL Biomass Program, National Bioenergy Center]. Chips derived from the various raw materials were dried to a moisture content (mc) of z: 10% and then conditioned to a relative humidity of 55% at 20 ° C for at least 24 hours before further size reduction. The chips were successively reduced in size by Condux hammer mill, a Retch and a Wiley laboratory mill and fractionated by sieving using stackable sieves (ASTM) of 850 μm / 20 mesh size, 425 pm / 40 mesh size and 250 pm / 60 mesh size with lid and v5gsk51. The particles that passed through 425 μm / 40 mesh size but were retained p5 a 250 μm / 60 mesh size were collected for chemical composition analysis and those retained p5 425 μm / 40 mesh size were used for xylane extraction. The moisture content of the raw material was determined using the National Renewable Energy Laboratory Analytical Procedure (NREL LAP) to determine the total amount of solid mass in the biomass [Hammes et al., 2005, Laboratory Analytical Procedure (LAP), NREL Biomass Program, National Bioenergy Center]. The percentage moisture content was calculated as a percentage of kiln dry (o.d.) weight for biomass.

The extracts were determined in two sequential steps, starting with cyclohexane / ethanol (2: 1) followed by hot water extraction, using a soxhlet apparatus. Both extractions were performed according to TAPPI test method T264 om-88 and NREL LAP methods [Suiter et al., 2005, Analytical Procedure (LAP), version 2006, National Biomass Program, National Bioenergy Center]. The extracts were quantified on a moisture-free base.

The content of Clason lignin (acid-insoluble) in the raw material was determined following a NREL LAP method for the determination of structural carbohydrates and lignin in biomass [Suiter et al., 2005, Analytical Procedure (LAP), version 2006, National Biomass Program, National Bioenergy Center] and TAPP! test procedures (1249 cm-85). The clasonic lignin was calculated on an oven-dry mass. 21 537 4 The inneal of Seifertcellulosa was stocked according to the analytical methods summarized by Browning [1967, Methods of wood chemistry, Vol. II Interscience publishers] and Fengel and Wegner [1989, Wood Chemistry, Ultrastructure, Reactions, Walter de Gruyter, Berlin, Germany]. Materials free from the extracts weighing 1.1 g were oven dried and treated with a mixture of acetylacetone (6 ml), dioxin (2 ml) and 32% HCl (2 ml) in a round bottom flask followed by incubation in a boiling water bath for 30 minutes. . Treated samples were quantitatively transferred to pretreated sintered glass crucibles for vacuum filtration and washing. The residue was washed successively with 100 ml each of methanol cyclodioxane, hot water (80 ° C), methanol and diethyl ether and then dried at 105 ° C for 2 hours. The content of Seifertcellulose was defined as the dry weight of the dried Aterstoden presented as a percentage of material free from the extracts.

Monomer sugar composition of the acid hydrolyzate was analyzed after storage at -20 ° C for at least 24 hours. The analysis is challenged in high performance anion exchange chromatography coupled to pulsed amperometric detection (HPAEC-PAD, Dionex) equipped with a GP50 gradient pump, a Carbopac PA 10 column mm x 250 mm) and electrochemical detector (ED40). Data follow-up and analysis are challenged with the use of PEAKNET software packages. The eluents were 250 mM NaOH and Milli-Q water in a ratio of 1.5: 98.5 at a flow rate of 1 ml of sodium acetate (1M NaOAc) eluent using acidic sugars (glucuronide / methylglucuronic acid) was analyzed. The samples were filtered p5 filter with 0.22 h pore size before analysis p5 HPAEC-PAD. The quantity of sugars was determined from the standard curve of the respective analytical grade of sugar (arabinose, rhaminos, galactose, glucose, mannose, xylose and glucuronic acid). The amount of sugar was presented as a percentage relative to the oven dry (o.d.) weight of the substrate. The pentosanine content of the raw material was determined according to TAPP! standard methods for feeding the pentosan in tra and pulp (T223 cm-84). The xylan content was calculated from a standard curve of kiln dry biomass [Xyl = xyl x cf, where: Xyl = xylan content (mg), xyl = xylose content (mg), cf = correction factor (0.88)]. The ash content was determined by a thermogravimetric method. Lignocellulose sample (0.5 g) is incinerated to ash in a muffle furnace at 575 ± 25 ° C for 4 hours or until a constant weight is obtained. Ash content was calculated as a percentage of initial kiln-dry biomass.

Xylane extraction and characterization Extraction of xylan from the raw material is challenged using two mild alkali extraction methods described in section 2.9 above. The Hoije method involved xylane extraction after ultra-purification using membrane dialysis (MWCO 12-14 kDa) while the Lopez method involved fractionation of the hydrolysates by ethanol precipitation. In both methods, xylane extraction was performed without prior removal of solvents and hot water extracts. Extracts were concentrated before ultrafiltration or fractionation to one third of the initial volume using a rotary evaporator (Rotavapor Buchi R-124, Switzerland) under vacuum at 40 ° C. The extraction efficiency was defined as the yield of xylan per theoretical content of pentosanes in the material. The Lopez method was restricted to extraction of xylan only from E.grandis and bagasse.

The structure and chemical composition of the raw material before xylane extraction were analyzed before and after xylane extraction by solid state 13C nuclear magnetic resonance cross-polarization / MaGIcAl angular rotation (13C-NMR CP / MAS) p5 a Varian VNMRS 500 at diameter fixed frequency NMR frequency 125 MHz for 1-3C, using a 6 mm T3 probe temperature of 25 ° C. Dry pray was loaded into 6 mm zirconia rotors for analysis. Spectrum was collected using TV polarization and magic angle spinning (CP / MAS). The rotation speed was .5 kHz, the proton 90 ° pulse was 5 [ice, the contact pulse 1500 ice and the delay between repetitions S. Chemical shifts were determined relative to TMS by setting the downstream peak for an external diamond reference to 38.3 ppm. The carbon resonances in the solid phase NMR spectrum were assigned according to Larsson et al. [1999, Solid State Nuclear Magnetic Resonance 15: 31-40], Renard and Jarvis [1999, Plant Physiology 119: 1315-1322], Lahaye et al. [2003, Carbohydrate Research 338: 1559-1569]; Atalla and Isogai [2005, Recent developments in spectroscopic and chemical characterization of cellulose, In Dumitriu, S (ed) Marcel Dekker, New York, p. 123-157], Virkki et al [2005, Carbohydrate Polymers 59: 357-366], Geng et al. [2006, 23,537 International Journal of Polymer Characterization 11: 209-226] and Oliviera et al. [2008, Chemical composition and lignin structural features of banana plant leaf sheath and rachis, In Hu, T.Q.Q. (ed) chapter 10: 171-188].

The extracted xylan samples were analyzed by solid phase 1-3 C nuclear magnetic resonance transpolarization / MaGIcAl angular rotation (13 C-NMR CP / MAS) and liquid 13 C and 1 H-NMR and Fourier Transform Infrared (FTIR) spectroscopy. The xylan samples were subjected to a 13 C and an 11 INMR grain on either a Variable 400 or 600 NMR spectrometer. The 13 C-NMR spectrum was collected using a 1.3s tracking time and a 1s pulse displacement at ° C. 13 C spectra were collected overnight (minimum 19000 scans). 1-1-1 NMR spectra were collected after filtering the sample with a 4.8 s tracking time at 50 ° C. I-1 spectrum was collected with 64 scans and pre-fed HDO peaks. The 13 C and 1 H NMR spectra were interpreted according to the orientation of characteristic signals for related raw material presented by Ebringerova et al. [1988, Carbohydrate Polymers 37: 231-230], Vignon and Gey [1998, Carbohydrate Research 307: 107-111], Renard and Jarvis [1999, Plant Physiology 119: 1315-1322], Teleman et al. [2002, Carbohydrate Research 337: 373-377], Grondahl et al. [2003, Carbohydr. Polym. 53: 359-366], Lahaye et al. [2003, Carbohydrate Research 338: 1559-1569], Sun et al. [2004, Carbohydrate Research 339: 291-300; Polym. Degrad. and Stability 84: 331-337; Carbohydrate Polymers 56: 195-204], Sims and Newman [2006, Carbohydrate Polymers 63: 379-384]; Habibi and Vignon [2005, Carbohydrate Research 340: 1431-1436], Pinto et al. [2005, Carbohydrate Polymers 60: 489-497], Geng et al. [2006, International Journal of Polymer Characterization 11: 209-226], Maunu [2008. 13C CPMAS NMR Studies of wood, cellulose fibers, and derivatives. In Hu, T.O. (ed)], Shao et al. [2008, Wood Science Technology 42: 439-451].

FTIR spectroscopy, dry solid samples of the xylan were recorded on a Nexus 670 spectrometer from Thermo Nicolet with Smart Golden Gate ATR food accessories installed. This single reflective food accessory features a diamond ATR crystal bonded to a tungsten carbide support equipped with ZnSe focusing lenses. The spectra were collected over the spectral range 4000 to 650 cm 1 using 16 scans at 6 cm 1 resolution and calibrated against a previously recorded background. Thermo Nicolets OMNIC® software was used to collect and process the infrared spectrum. Spectrum signals for FTIR were interpreted according to pitcher bands presented in Fengel and Wegener (1989); Sun et al (2004); Xu et al (2000), Sims and Newman (2006).

The degree of polymerization of the extracted xylan fractions was evaluated on HPAEC (Dionex) using a CarbopacTM PA100 column (4 x 25 mm) and a guard column, and an electrochemical detector (ED40) for pulsed amphoteric detection (PAD). The PA 100 column separates monomers and oligomers up to a degree of polymerization (DP) which is usually eluted within a retention time of 25 minutes. HPAEC PA100 columns base their separation on the degree of substitution, i.e. the longer the retention time, the higher the DP or degree of substitution (Combined CarboPac manual, pages 52-56). Samples (10 μl) were injected into the column and eluted with helium degassed 0.25M NaOH, Milli-O. H 2 O and 1M NaOAc at a flow rate of 1 mlmin-1. Elution profiles for the samples referred to elution profiles for monomeric sugars (arabinose, raminose, galactose, glucose, xylose and mannose) and polymeric xylan (xylan from birch and oat column) and H2 O2-bleached bagasse. Samples with lower peak intensity <20 nC or no peaks eluted within the retention time of 25 minutes were considered polymeric with DP> 10 sugar units.

The composition of neutral sugars in extracted xylan samples was determined on HPAEC-PAD (Dionex) p5 Carbopac PA 10 column after mild acid hydrolysis described by Yang et al. [2005, LWT 38: 677-682]. Samples (0.1 g) were placed in Schott flasks (50 ml) to which 1 ml of 72% H 2 O 2 was added. The mixture was incubated at 30 ° C in a water bath for 1 hour. Deionized water (30 ml) was added after autoclaving at 121 ° C for 1 hour. The samples were cooled to room temperature before filtration. The filter cake was dried at 105 ° C to determine the residual Clasonic lignin. The liquid fraction was filtered through a 0.22 μm pore filter before being subjected to the HPAEC-PAD (Dionex) p5 Carbopac PA 10 column. Monomeric sugars were quantified from standard graphs of analytical grade (arabinose, raminose, galactose, glucose, xylose and mannose). The total content of neutral sugars in the samples was presented in comparison with the initial oven-sized xylan mass. 537 4 Determination of uronic acid composition The content of uronic acid composition in the xylan samples and the raw material was quantified using chromatographic and colorimetric methods. The chromatographic method used a two-step acid hydrolysis method from Prof. A.M.F. Milagres at the University of So Paulo in Brazil. Xylan sample (150 mg oven dry mass) was hydrolyzed in 0.75 ml of 72% (w / w) H 2 SO 4 in McCartney flasks. The mixture was incubated at 45 ° C for 7 minutes in a water bath after which 22.5 ml of distilled water was added. The bottles were unloaded and autoclaved at 121 ° C for 30 minutes. After cooling to room temperature, the liquid fraction was separated by vacuum filtration through a glass microfiber filter (GF / A-Whatman).

The liquid fraction was further filtered through a 0.22 μm filter and Wills frozen overnight at -20 ° C before analysis for glucuronic acid content 511 using HPAEC-PAD (Dionex) p5 Carbopac PA 10 column. Quantification of uronic acid was based on standard graphs for glucuronic acid (Sigma). Uric acid losses during autoclaving were taken into account by autoclaved glucuronic acid at 121 ° C for 1 hour in 4% H2504.1. The calorimetric method used carbazole-sulfuric acid analysis from Li et al. [2007, Carbohydr. Res. 342 (11): 1442-1449]. Total uric acid concentration was determined from standard curve graphs for D-galacturonic acid (Merck) and in the following methods uronic acid content was presented as a percentage of the initial xylan amount.

Deposition of xylan side chains The degree of selective deposition of side groups p5 4-0-methylglucuronic acid (4-0-MeGIcA) by αD-glucuronidase in Schizophyllum commune (a-glu) was determined using xylan harletted from Eucalyptus balis, Pinucalyptus grandis, and bagassee from South Africa. a-glu from S.Commune was evaluated for selective removal of 4-0-MeGIcA side chains from xylan harlett from calves for lovtra, conifer and grass (including grain) with the aim of developing a controlled enzymatic technology for diversification of functional xylan properties. The effects on hydrolysis time, temperature and specific dosage of enzyme xylan on the removal of 4-O-MedIcA side chains, and subsequent modification of viscosity, solubility, precipitation and aggregation of xylan were therefore underestimated. Xylan samples replaced with side chains for arabinose and / or 4-O-methylglucuronic acid (4-O-MeGIcA) (Figure 7), xylan from oat cleft (Sigma) and birch xylan (Roth) were used as model xylan. Xylan solution (1% w / v) for each material was prepared in deionized water (dH 2 O). The xylanate which showed limited solubility in water was first prepared by dissolving in ethanol and subsequent heating according to de Wet et al. [2008, Appl. Microbiol. Biotechnol. 77: 975-983]. Xylan solutions were made in bulk and stored in ampoules at -20 ° C. Xylan from oat column (Sigma) with a sugar composition of 10:15:75 (arabinose: glucose: xylose) and birch xylan (Roth) with sugar composition of 8.3: 1.4: 89.3 (4-0-MeGIcA: glucose and xylose) [Kormelik and Voragen, 1993, Carbohydr. Res. 249: 345-353] made p5 similarly used as model xylan. The enzyme used was α-D-glucuronidase (α-glu) with specific activity = 300 nKat with purified wild-type Schizophyllum commune (VTT-D-88361-ATCC 38548 (donated by Prof. Matti Siika-aho at VTT Biotechnology Institute in Finland) ) for selective removal of 4-O-methyl-D-glucuronic acid / D-glucuronic acid side groups. The enzyme aliquots were stored at 4 ° C.

A xylan solution (1% w / v) prepared from 4-O-MeGIcA-substituted substrates was treated with α-glu (9000 nKat g-1) in 5 ml reaction volumes consisting of 2.5 ml of the substrate and boiled up to 5 ml with 0.05M acetate buffer, pH 4.8. The reactions proceeded for 16 hours at 2C.

Sugar release from 4-0 MeGIcA side chains was analyzed using (HPAECPAD) on Carbopac PA 10 column eluted with helium degassed Mill-Q. H 2 O, 250 mM NaOH and 1M NaOAc (acidic sugars only). G-glucuronic acid was used as the standard sugar. Insolubility, precipitation and aggregation of xylan hydrogels were confirmed by visual inspection (photographs taken) and quantified by feeding the viscosity using a Rheometer (MCR501). The degree of xylan precipitation was quantified by determining residual xylose in solution using phenol-sulfur analysis for total sugar [Dubois et al., 1956, Annul. Chem. 28 (3): 350-356].

A Box-Behnken statistically designed three-factor experiment with 3 central points totaling 15 grains was run twice: Statistica 7.0 software program (StatSoft, Inc., 27 537 4 1984-2005) was used for design and analysis using response surface method, RSM) as shown in Figure 8. Regression and ANOVA assays are challenged to determine the magnitude and significance of individual and interacting effects of the hydrolysis parameters on viscosity. Optimal conditions were determined using the adversity function. Response area diagrams were fitted with a second degree polynomial as follows: 2 = 6o ÷ 61X1-I- 622X22 622X22 63X3 633X32 £ whereby: Z = viscosity (mPa. $), [30+ 131 13n = lines regression coefficient,, ". •• ••• [jinn = quadratic regression coefficient, E = error, and X1, X2, x3 = hydrolysis time, temperature and enzyme xylan-specific dose.

Optimal side chain deposition conditions The optimal combination levels for hydrolysis parameters were determined: time, temperature and dosage of α-D-glucuronidase to remove side chain from 4-0 methylglucuronic acid.

The effect of xylan charge, enzyme charge, hydrolysis time and temperature, and their interaction on aglu-removal of side chains from 4-0-MeGIcA from oat clef and birch xylan, were determined using the response surface method (RSM). Time, temperature and enzyme xylan specific dosage constituted the independent variable while the series of side chain deposition and viscosity change formed the dependent variables. The software program Statistica 7.0 (StatSoft, Inc., 1984-2005) was used for design and analysis of the experiments. Statistical analysis included regression and ANOVA analysis. Pareto diagram curves were used to show size and significance effects while the unobtrusiveness and profiling functions were used to determine the optimal setpoint for the hydrolysis parameters. Xylan from oat column (1% w / v) was prepared according to de Wet et al. [2008, App !. Microbiol. Biotechnol. 77: 975-983]. The solution was made in bulk and stored in ampoules at -2C. αD-glucuronidase (α-glu) was purified from wild-type Schizophyllum commune (VTT-D-88362- ATCC 38548) with specific activity of 300 nKat mg-1 (donated by Prof. Matti Siika-aho p5 VTT Biotechnology Institute in Finland), was used for the selective removal of 4-O-methyl-D-glucuronic acid / side chain groups of D-glucuronic acid. The enzyme aliquots were stored at 42 ° C. D-glucuronic acid (Sigma) was used as the standard sugar.

Optimization of hydrolysis parameters Optimal setpoints for time, temperature and enzyme dosing of α-glu deposition from 4-0-MegIcA from xylan from birch were determined in a Box-Behnken statistical three-factor design with 3 central points totaling 15 grains in duplicate. Hydrolysis parameters were each tested at two levels and the midpoints with the highest, middle and lowest levels were noted as 1, 0 and -1, respectively. The temperature was tested at 2C and 2C, time at 1 hour and 16 hours, enzyme dosage of α-glu was 2000 nKat g 'and 18000 nKat g-1. The central points for temperature and time were 2C and 8.5 h, while the specific dosage of α-glucylane was 360000 nKatrespective 11000 nKatVariables were coded according to the equation: - Xdar: [x, = coded value for variable , X, = natural value, AX, = scaling factor (half the range of the independent variables that constituted time, temperature and specific dosage of enzyme xylan)]. 4-0 MegIcA side chains were analyzed using (HPAEC-PAD) p5 Carbopac PA column eluted with helium degassed Mill-Q H 2 O, 250 mM NaOH and 1M NaOAc (for acidic sugars only). D-glucuronic acid was used as a standard sugar.

The response area diagram was fitted with a second degree polynomial that included both linear and quadratic interactions as follows: ZA is 4.): 3 4- Afirr-z iftz43-v..1 fl..44 + 2X1-X, 1 4 'A: 11 -1 'xl 4- A .3: -. Tot: 4- APcp-t4-xi AuxIxi'fcKvic + 44 Anytii +6 dar: Z = response (degree of side chain removal), Bo + 131 13n = Linear regression coefficient, 1311 13nn = quadratic regression coefficient, Xi, X2, x3 = hydrolysis time, temperature and enzyme xylan-specific dose or xylan and enzyme load, E = error. 29 537 4 RESULTS Extraction and characterization of xylan from lignocellulosic material The chemical composition of bagasse, pine (Pinus patula) and bamboo (Bambusidae balcooa) is shown in Figures 9-11. Bagasse had the highest content of ash (8.6%) and the extracts in solvents (6.2%) (Figure 9), lignin (30.0%) (Figure 10), cellulose (53.80%) and pentosanes ( 22.00%) (Figure 11). Both E.grandis and P.patula contained ash and the extracts were less than 3% (Figure 9). However, P. patula showed the lowest pentosan level (8.49%). The cellulose level in E.grandis and bamboo was in the range of 40.43% (Figure 11) while the lignin content was about 23% (Figure 10). Extraction of xylan from P.patula, bagasse, E.grandis and bamboo by the Hoije method gay extraction efficiency values of 71.20, 65.50, 35.20 and 20.20% respectively (Figure 12) while extraction of xylan from Bagasse and E .grandis using the Lopez method gay extraction efficiency values of 28.00 and 12.00% respectively (Figure 12). 13C-CP / MAS NMR solid phase spectrum for crude, extract-free and xylan-extracted residues of P. patula, bagasse, E.grandis and bamboo material showed characteristic signals derived from the six carbon resonances of anhydrous glucose ring in cellulose which were 2005, Recent developments in spectroscopic and chemical characterization of cellulose. In Dumitriu, S. (ed.) Marcel Dekker, New York, p. 123-157] (Figures 13A-D, spectrum 1). Starting at the upper part of the spectrum C6 for the primary alcohol group at chemical shift (5) 60-70 ppm and resonances for a cluster of C2, C3 and C5 from the ring carbon, other than those anchoring the glycoside link, were shown at 5- 70-81 ppm , The C4 resonance at 5,8193 ppm and Cl at 5,102-108 ppm. In addition, typical duplicates in C4 and C6 resonances (over the range) representing cellulose in less orderly form (amorphous) and cellulose in orderly form (crystalline) (lower range) (Atalla and lsogai, 2005) were shown in the spectrum over all lignocellulosic raw material (Figures 13A-D). However, duplicates of C6 in the spectrum of P. patula (Figure 13A, spectrum 1) were more resolved than in bagasse (Figure 13B), E.grandis (Figure 13C) and bamboo (Figure 13D). Characteristic signals for acetyl groups at 20-22 ppm, aliphatic groups at 30-40 ppm, methyl (CH3) arising from lignin residues at 50-60 ppm, Cl from arabinose residues at 5-110-120 ppm, aromatic compounds from lignin residues at 5 160 ppm 537 4 and C6 from uronic acid residues or carbonyl groups at 170-190 ppm were also in accordance with Liitia et al. [2001, Wood Research SS: 503-510]; Maunu [2002, Progress in Nuclear Magnetic Response Spectroscopy 40: 151-174]; La haye etal. [2003, Carbohydrate Research 338: 1559-1569]; Oliveira et al. [2008, Chemical composition and lignin structural features of banana plant leaf sheath and rachis. In Hu, T.Q. (ed). Chapter 10: 171-188] identified in the spectrum of unprocessed raw material (Figures 13A-D, spectrum 1). 13 C-CP / MAS NMR spectra of the material in which the extracts were deposited showed changes in line and cleaved samples for signals in the upper region of C4 and C6 and the resonances between 81-93; 60-70 and 20-22 ppm, respectively (Figures 13A-D, spectrum 2). While the 13 C-CP / MAS NMR spectrum of the product from which xylan has been precipitated showed disappearance or reduction in the intensity of signals involving Iran acetyl groups, aliphatic groups, methyl groups, aromatic groups, C6 Iran uron / carbonyl groups at , 50-60, 140-160 and 170-190 ppm, respectively (Figures 13A-D, spectrum 3). 1-3C-CP / MAS NMR spectra for bagasse from which xylan has been deposited showed sharper signals especially in resonance between 5 and 40 ppm, which originated from aliphatic groups (Oliveira, 2008) and complete disappearance of resonances arising from methyl groups at 5 40-50 ppm (Figure 13B, spectrum 3). While in the 1-3C-CP / MAS NMR spectrum of P.patula, E.grandis and bamboo, a reduced intensity of the signal from methyl groups was noted (Figure 13A, C, D, spectrum 3).

The initial glucose levels in extract-free bagasse, bamboo, P.patula and E.grandis were 68.0, 66.0, 61.0 and 59.0, respectively. During xylan extraction using the Hoije method, the glucose content of the raw materials increased to 75.0, 76.0, 65.0 and 79.0%, respectively, while the xylose concentration decreased from 14.0 to 10.0%, 27.0 to 25%. to 19%, 30.0 to 22.0% in P.patula, bagasse, E.grandis and bamboo, respectively (Figure 14A-D). In addition, xylan extraction corresponds to a decrease in arabinose and galactose content in all raw materials (Figure 14A-d).

The concentration of mannose (16.0%) detectable only in the P.patu / a r5 product increased to 18% in xylan-extracted residue (Figure 14A). The presence of uronic acids was detectable in all four raw materials in the lignocellulose (Figure 15). The highest and lowest uronic acid content were found in E.grandis and bagasse products, respectively (Figure 15). 31 537 4 The elution profiles for extracted xylan fractions were referred to the elution profile for the monomeric sugars (arabinose, raminose, galactose, glucose, xylose and mannose), xylitol sugar, xylan from birch, xylan from oat split and H2O2-Black. The HPAEC-PAD chromatogram (Dinoex) showed that the monomeric sugars including xylitol were eluted in the p5 CarboPac PA 100 column with a retention time of 5 min (Figure 16A and B). Between 0 and 3 minutes, the elution profile of xylan from oat cleft showed a high intensity peak with a detection response> 300 nC which corresponds to the retention time of xylitol (Figure 16D). Otherwise, only intensity peaks (detection response of <20 nC) were shown between 3 and 6 min on the chromatogram for both xylan from birch and xylan from oat column. In contrast, the chromatogram of H 2 O 2 bleached bagassexylan (Bag B) showed multiple high intensity peaks with detection responses above 100 nC, occurring at retention times between 2 and 30 min (Figure 17A). The chromatograms for both Hoije and Lopeze extracted xylan showed light intensity peaks (<20 nC) within 25 minutes of retention time (Figures 17B and C). Among the extracted xylan samples, peaks corresponding to xylitol were present in the chromatograms of xylan from E.grandis (Figure 18A), bamboo (Figure 18C) and P.patula (Figure 18D), which were extracted by the Hoije method.

Xylose content xylan from E.grandis extracted by the Hoije method (EU H), bamboo, bagasse extracted by the Hoije method (Bag H) and P. patula were 92.00, 79.50, 71.00 and 61.30%, respectively ( figure 15). On the other hand, the xylose content of xylan from birch and oat column was 80.00 and 87.20%, respectively (Figure 15). The proportions of arabinose in xylan fractions from Bag H, P.patula and bamboo were 17.45, 15.50 and 10.50% respectively (Figure 15). Even am commercial xylan Irk] oat cleft has been reported to have 10% arabinose (Sigma), this study showed arabinose content of 7.4% (Figure 15). About 2.30% glucose was present in xylan fraction from EU H, while 13.20% glucose was present in xylan fractions from P.patula (Figure 15). In addition, EU-H contained 4.45% galactose and traces of raminose and arabinose (Figure 15). Total uronic acid content of uronic acid content in xylan from EU H, bamboo and P.patula was 12.83, 11.20 and 11.54% while xylan from bagasse contained 8.5% (Figure 15).

Xylan fractions extracted by the Hoije method, clA they were subjected to mild acid hydrolysis (72% H 2 O 2), gay between 16 and 55% insoluble residues (Figure 19). The highest proportion of acid-insoluble residues, 55%, is obtained from xylan extracted from P. patula using the Hoije method. The acid-insoluble residues from xylan from bagasse extracted by the Lopez method (Bag L) were 16%, while the reference material, xylan from birch, had 3.5% (Figure 19).

Structural ktin signs of extracted xylan Spectrum for 11-1-NMR and 13 C-NMR for reference xylan from birch, H 2 O 2 -bleached bagasse and xylan from oat clef showed characteristic signals for proton and carbon resonance (Figure 20A-D). The 1 H-NMR spectrum of the extracted xylan showed characteristic proton signals from xylose, 4-O-methylglucuronic acid and arabinose units at chemical shifts (5) between 3.3 and 5.7 ppm (Figure 20-22). In the proton signals of the 1-1-1 NMR for xylose in the xylan fractions from bagasse extracted by the Hoije (Bag H) and Lopez methods (Bag L) were shown at δ 4.44 / 4.45, 3.50, 3 , 67 and 4.01 ppm (Figure 21A). According to Vignon and Gey [1998, Carbohydrate Research 307: 107-111], such signals correspond to H1, H3, H4 and H5 p5 xylose units substituted with 4-O-methylglucuronic acid linked at 0-2. In addition, the proton spectra of Lopee-extracted bagasse (Bag L) and Hoije-extracted bagasse (Bag H) showed proton resonances from D-xylopyranosyl moiety substituted with 4-0-methylglucuronic acid at 0-2 and acetyl group at 0-3 at 5.72, 3 , 76 / 3.75, 3.97 / 3.96 ppm corresponding to H1, H2 and H4, respectively, of the PD-xylopyranosyl units (Figure 21A).

Proton spectra of xylan extracted from E.grandis by the Lopez method (EU L) and the Hoije method (EU H) showed signals at 4.48, 3.96 / 3.99, 3.63 / 3.68 and 3.50 / 3.52 ppm arising from xylose units substituted with 4-O-methylglucuronic acid (Figure 21C). According to Sims and Newman (2006), Adana chemical shifts may be derived from D-xylopyranosyl moiety residues substituted with 4-O-methylglucuronic acid at 0-2 and acetyl group at 03. In the same spectra, proton signals were derived from H1 and H3 for 4-0-methylglucuronic acid residues visible at 5.48 / 5.49 / 5.46 ppm and between 1.06 and 1.54 ppm 5.16 and 3.63 ppm (Figure 21C). In the proton spectrum of bamboo and P. patula, signals for 4-O-methylglucuronic acid residues were shown at 5.47 / 5.48 ppm (Figures 21A and C). SAclana signals can occur at 5.16 and 3.63 ppm [Sun et al., 2004, Carbohydrate Research 339: 291-300, Polym. Degrad. and Stability 84: 331-337, Carbohydrate Polymers 56: 195-204; Ebringrova et al., 1998, Carbohydrate Polymers 37: 231-239]. The proton spectrum of extracted xylan showed additional characteristic signals derived from C-2-linked arabinos to xylose units [Hoije et al., 2005, Carbohydr. Polym. 61: 266-275; Ebringerova et al., 1998, Carbohydrate Polymers 37: 231-239]. In the proton spectrum of Bag H and Bag L, C2-linked arabions were identified at 5.58, 5.60, 4.29 / 4.30 ppm (Figures 21A and C). The presence of arabinose in the proton spectrum of xylan from bamboo and P. patula was in accordance with Ebringerova et al. (1998) and Vignon and Gey (1998) identified inter alia, at, 58 / 5.59 ppm and 5.47 / 5.48 ppm, respectively (Figures 22A and C). Arabino signals were present in the proton spectrum of EU L and EU H at between 3.83 and 3.85 ppm (Figure 21C) while the presence of 0-2-linked acetyl groups which, based on p5 Shao et al. [2008, Wood Science Technology 42: 439-451]; Hoije et al. (2005) Sun et al. (2004); Ebringerova et al. (1998); Vig non and Gey (1998) supra were identified at 4-2.4 ppm (Figure 20-22). In addition, broad signals associated with aromatic and phenolic compounds derived from lignin residues were exhibited [Hoije et al., 200 supra; Xu et al., 2006, Carbohydrate Research and Oliveira et al., 2008, Chemical composition and lignin structural features of banana plant leaf sheath and rachis. In Hu, T.O. (ed.), Chapter 10: 171-188] between 6.5 and 7.9 ppm in the proton spectrum of the extracted xylan (Figures 21 and 22).

Characteristic carbon resonances of the five carbons in (14) linked [3-D-xylopyranosyl residues between 5,103 and 62 ppm were reflected in the 1-3 C-NMR spectrum of extracted xylan fractions (Figures 21 and 22). In the carbon spectrum of xylan fractions from E.grandis, resonances derived from C1 occur from xylose units with C2-linked arabino groups at 5 z 102.33 ppm while those from C1, C2, C3, C4 and C5 for arabinofuranosyl residues showed at 5 z 108, 81.7 , 78, 85.5 and 62 ppm (Figure 21D). In the spectrum, arabinose-associated carbon signals in EU H and EU L are said at 5 61.81 / 61.59 ppm (Figure 21D). In Bag L and Bag H, the identified arabino signals based on p5 were shown. C1, C2 and C4 resonances belonging to arabinofuranosyl residues monosubstituted xylose units at 0-3 (Ebringerove et al., 1998, supra) were present in the 13 C-NMR spectrum of both bamboo and P. patula at 108.60 / 108. 33 ppm, 81.71 / 81.42 ppm and 85.72 / 85.43 ppm, respectively (Figures 22B and D). The presence of 4-O-methylglucuronic acid residues in the extracted xylan fractions was evident from characteristic carbon signals derived from C1, C4, C6 and C5 at between 97 and 100 ppm, 83 and 84 ppm, 179-172 ppm and 59 and 61 ppm, respectively. (Haibibi and Vignon, 2005, Carbohydrate Research 34 537 4 340: 1431-1436; Xu et al., 2000, supra; Ebringerova et al., 1998, supra) (Figures 21 and 22). The presence of acetyl, phenol and aromatic groups arising from lignin compounds, and hexose sugar were identified in the 1-3 C NMR spectra from the extracted xylan fractions. In addition, acetyl groups were identified in all xylan fractions between 21-24 ppm (Figures 21 and 22). However, the presence of acetyl groups was evident in the 1-3 C NMR spectrum of xylan from birch (Figure 20B) and H 2 O 2 -bleached bagasse (Bag B) (Figure 20D). The methoxyl groups suggesting the narvaron of lignin compounds [Ebringerova et al., 1998, supra, Sun et al., 2004, supra, Xu et al., 2006 supra; maunu, 2008,13C CPMAS NMR Studies of wood, cellulose fibers and derivatives. In Hu, T.Q. (red)] was identified in the 1-3 C NMR spectrum at 56.62 / 56.58 ppm (Figures 21 and 22). The lignin compounds, especially those linked to arabinosyl side chains by ferulic acid bridges, were reflected by carbon signals at 140-160 ppm and 116.6-117.08 ppm (Figures 21 and 22). Other lignin compounds associated with ferulic or β-coumaric acid groups and of -CH3- in ArCOCH3 (Maunu, 2002, Progress in Nuclear Magnetic Response Spectroscopy 40: 151-174; Sun et al., 2004, supra) were found at 26-49 ppm , at 5,115.38 and 17.55 / 17.69 ppm in Bag L (Figure 21B, Spectrum 1) xylan fractions from bamboo and P.patula (Figures 22B and D). The presence of hexose sugars such as galactose or glucose [Sun et al., 2004 supra] was evident in the 1-3 C NMR spectrum, especially for EU H and EU Lvid 5 between 69 and 71 ppm (Figure 21B).

FTIR spectra of extracted xylan fractions showed characteristic bands for xylan residues, which included [3-glycoside linkers reflected at .-. 897 cm-1 (Figure 23). However, such a signal was present in the FTIR spectrum of extracted xylan Iran P.patula. In addition, the spectrum of extracted xylan signals in the band region between 1600 and 1200 cm -1 (Figure 23), which according to Fengel and Wegener [1989, Wood Chemistry, Ultrastructure, Reactions, Walter de Gruyter, Berlin, Germany] Jr. showed a region associated with aromatic associations arising from equation fractions. Bands arising from Iran oxygen ring living with CAr-OCH3 and methoxyl groups in lignin were reflected at 1329 cm-1 and 15911595, and 1460-1461 cm-1 in the spectrum of E.grandis and bamboo (Figure 23). Xylan fractions from bagasse, Bag H and Bag L contained signals of varying intensities in the range region 1600-1200 cm and spectra for Bag L reflected a relatively strong intensity band for C-H stretching vibrations at 2919 cm 1 (Figure 23). The FTIR spectrum for EU L showed strong signals related to lignin compounds at 1591 and 1379 cm-1, while in the EU 537 4 H spectrum multiple bands of lower strength were seen in the range 1600-1200 cm-1, especially at 1595, 1461 and 1329 cm-1 (Figure 23).

Controlled enzymatic removal of side chains from lignocellulosic products The purified α-glucuronidase (α-glu) from Schizophyllum commune removed 1.2 mg g-14-0-MegicA (1.3% available uronic acid) from xylan from birch, while about 1.6 mg g-1 4-0- MegIcA (2% available uronic acids) was released from BH-xylan fractions (Figure 24). The proportion of 4-0-MegIcA derived from Eucalyptus grandis-xylan, extracted by the Hoijem method (EH), and from Eucalyptus grandis-xylangel (ES) was about 1.3 mg g of substrate-1 (Figure 24). The lowest α-glu deposition rate from 4-0-MegIcA was <0.6 mg g of substrate-1 from H 2 O 2 bleached bagasse (BB) (Figure 24).

Determination of Optimal Conditions for Side Chain Removal Surface Response Diagrams for Depletion of Glucuronic Acid (4-0-MegIcA) from xylan from birch by α-glu were similarly reflected in both linear and quadratic ratios of hydrolysis time, temperature and specific dosage of α-glu-xylan. A maximum of 350 kg of g-1 substrate of 4-0-MegIcA was removed from birch by α-glu at xylan-specific dosage between 16500 and 18000 nkat g of substrate-1 d5 hydrolysis is challenged for a duration of between 9 and 10.2 hours at temperatures between 33, 5 and 42 ° C (Figure 25A-C).

The hydrolysis parameters showed significant effects on removal of 4-0-MegIcA from xylan from birch by a-glu and were in decreasing magnitude, from linear effects from a-glu - xylan-specific dosage [a-glu, nKatig (L)], temperature [Temp (L)] and quadratic effect on temperature [Temp (Q)] (Figure 26, Pareto diagram] The only significant interaction effect on the removal of 4-0-MegIcA from xylan from birch by a-glu was & An the linear effect of hydrolysis time and the quadratic effect of temperature [time (L) through temperature (Q)] (Figure 26, Pareto diagram) The optimal installation points for α-glu deposition of 4-0-MegIcA were between 9 h and 10.2 h, 33 , 5 and 42 ° C and 16500 and 18000 nCat g substrati The regression coefficients of the variable in the second degree polynomial adapted to the response surface diagram for 4-0 MegIcA deposition as a function of time, temperature and enzyme dose gay a regression coefficient R2 of 0, 90 (R2 adjusted = 0.81) (Figure 27)

Claims (8)

41

1. An isolated polypeptide having oi-glucuronidase activity and that can degrade a glucuronoxylan molecule by hydrolysis of a glycosidic Iinkage between a l\/leGlcA 5 residue and a non-terminal xylopyranosyl residue.

2. An isolated polypeptide having an amino acid sequence selected from the following group: the amino acid sequence of SEQ ID NO 1; an amino acid sequence at least 95% homologous to SEQ ID NO 1 or part 10 thereof;iii. an amino acid sequence at least 85% homologous to SEQ ID NO 1 or partthereof;iv. an amino acid sequence at least 75% homologous to SEQ ID NO 1 or partthereof; and15 v. a functional variant of any one of amino acid sequences listed in i-iv.

3. An isolated polypeptide having an amino acid sequence selected from thefollowing group:i. an amino acid sequence at least 65% homologous to SEQ ID NO 1 or partthereof;20 ii. an amino acid sequence at least 50% homologous to SEQ ID NO 1 or part thereof; and a functional variant of any one of amino acid sequences listed in i and ii.

4. The isolated polypeptide according to either claim 2 or claim 3, wherein the polypeptide is a biologically active fragment of the polypeptide. P1928PCOO(An enzyme with a-glucuronidase activity) 42

5. An isolated polynucleotide encoding a polypeptide according to any one of claims1 to 4, the polynucleotide having a nucleotide sequence selected from thefollowing group: i. the nucleotide sequence of SEQ ID NO 2; 5 ii. a nucleotide sequence at least 95% homologous to SEQ ID NO 2 or part thereof; iii. a nucleotide sequence at least 85% homologous to SEQ ID NO 2 or part thereof; and iv. a nucleotide sequence at least 75% homologous to SEQ ID NO 2 or part 10 thereof.

6. A method of isolating a polypeptide according to any one of claims 1 to 4, themethod including the steps of i. culturing a microbe capable of expressing the polypeptide in induction medium; and15 ii. isolating the polypeptide from the induction medium.

7. The method of isolating a polypeptide according to claim 6, wherein the step ofisolating the polypeptide from the induction medium is carried out using one ormore of anion-exchange chromatography, hydrophobic chromatography, andanion-exchange chromatography. 20

8. The method of isolating a polypeptide according to either claim 6 or claim 7,wherein the microbe is selected from the group including Pichia stipitis,Schizophyllum commune, Aspergillus clavatus, Neosartorya fischeri, Aspergillusfumigatus, Aspergillus terreus, Aspergillus oryzae, Sclerotinia sclerotiorum,Botryotinia fuckeliana, Pyrenophora tritici-repentis, Neurospora crassa, Gibberella 25 zeae, Podospora anserina, Coprinopsis cinerea okayama, Magnaporthe grisea, Fusarium sporotrichioides, Cryptococcus neoformans var. neoformans, Cellvibrio P1928PCOO(An enzyme with a-glucuronidase activity) 10. 11. 12. 43 japonicus, Saccharophagus degradans, Opitutus terrae, Phaeosphaeria nodorum, Bacteroides ovatus, and Streptomyces pristinaespiralis. The method of isolating the polypeptide according to claim 8, wherein the microbeis Pichia stipitis CBS 6054. A substantially enriched preparation of a polypeptide according to any one of claims 1 to 4. A substantially enriched preparation according to claim 10, wherein thepolypeptide is obtained from a culture of a microbe selected from the groupincluding Schizophyllum commune, Aspergillus clavatus, Neosartorya fischeri,Aspergillus fumigatus, Aspergillus terreus, Aspergillus oryzae, Sclerotiniasclerotiorum, Botryotinia fuckeliana, Pyrenophora tritici-repentis, Neurosporacrassa, Gibberella zeae, Podospora anserina, Coprinopsis cinerea okayama,Magnaporthe grisea, Fusarium sporotrichioides, Cryptococcus neoformans var.neoformans, Cellvibrio japonicus, Saccharophagus degradans, Opitutus terrae, Phaeosphaeria nodorum, Bacteroides ovatus, and Streptomyces pristinaespiralis. A substantially enriched preparation according to claim 11, wherein the polypeptide is obtained from a culture of Pichia stipitis CBS 6054. P1928PCOO(An enzyme with a-glucuronidase activity)