WO2010113020A1 - An enzyme with alpha-glucuronidase activity - Google Patents

Abstract

The present invention relates to an isolated polypeptide having α-glucuronidase activity and that can degrade glucuronoxylan molecules by hydrolysis of a glycosidic linkage between a MeGIcA residue (glucuronic side chain) and a non- terminal xylopyranosyl residue. The isolated polypeptide is capable of cleaving glycosidic linkages within glucuronoxylans from plant biomass, thereby removing internal glucuronic side chains. The invention also relates to amino acid sequences of the isolated polypeptide, and homologous thereto, as well as isolated polynucleotides having nucleic acid sequences encoding the polypeptides. The invention further relates to methods of isolating the polypeptides from microbial cultures, such as that of Pichia stipitis, using chromatographic techniques, and to methods of producing substantially enriched preparations of the polypeptides.

Description

AN ENZYME WITH σ-GLUCURONIDASE ACTIVITY

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, to a method of isolating the polypeptide, and to a substantially enriched preparation thereof.

BACKGROUND TO THE INVENTION

The pulp and paper industry processes large quantities of plant biomass or wood. This biomass comprises complex carbohydrates, such as cellulose, hemicellulose and lignin, which must be partially degraded and processed to produce a paper product. Wood contains about 20% hemicelluloses, of which xylans form an essential part in both hardwoods and softwoods. The major group of hemicelluloses found in hardwoods are the glucuronoxylans. These comprise a /M ,4-linked D-xylopyranose backbone with 4-O-methyl D-glucuronic acid substituents linked σ-1 ,2. In addition, the 2,3 positions of the xylose backbone may be partially acetylated. The glucuronoxylan content of hardwood is typically between 15 and 30% by weight of the wood.

During pulping processes, hemicelluloses undergo various changes. Hemicelluloses such as xylan do not form tightly packed crystalline structures like those of cellulose, due to the presence of side chains in the xylan structure. This attribute of hemicelluloses means that these polysaccharides are more easily degraded than cellulose. While some hemicelluloses and hemicellulose degradation products are dissolved in the cooking liquors, such as the readily alkali-soluble hemicelluloses, some are degraded into lower molecular weight products that may either remain in an insoluble form within the fiber matrix or be dissolved into the cooking liquors. The fraction of hemicelluloses and hemicellulose degradation products that is dissolved in the cooking liquors is then lost to the process, resulting in a loss of yield of the wood. Hemicelluloses are believed to contribute to the swelling of the pulp and therefore the conformability of the wet fibers during sheet formation as a result of their noncrystalline hydrophilic nature. During the degradation of xylan, it is therefore desirable to minimize the loss of hemicellulose and hemicellulose degradation products in the cooking liquors, and to maximize the retention of the hemicellulose in the fiber matrix.

In order to produce desirable hemicellulose degradation products, selective degradation of hemicellulose is carried out by the use of enzymes. The enzymatic degradation of the hemicellulose xylan is a complex process requiring the action of various enzymes falling generally into two categories: i) enzymes degrading the polysaccharide main chain, such as endo-β-1 ,4-xylanase (EC 3.2.1.8) and β-xylosidase (EC 3.2.1.37); and ii) enzymes that liberate side chains, the main chain substituents, so called accessory xylanolytic enzymes, that include α-glucuronidase (EC 3.2.1.139), α-L-arabinofuranosidase (EC 3.2.1.55), acetylxylan esterase (EC 3.1.1.72) and feruloyl esterase ( 3.1.1.73). While the endo-xylanase attacks the main chains of xylans and /?-xylosidase hydrolyzes xylooligosaccharides to xylose, the σ-arabinofuranosidase and a- glucuronidase remove the arabinose and 4-O-methyl glucuronic acid substituents, respectively, from the xylan backbone. The esterases hydrolyse the ester linkages between xylose units of the xylan and acetic acid (acetylxylan esterase) or between arabinose side chain residues and phenolic acids, such as ferulic acid (ferulic acid esterase) and p-coumaric acid (p-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. Currently, only one GH family, GH67, exclusively harbours α-glucuronidases. The activity of these enzymes are, however, limited as they liberate MeGIcA or GIcA only from those fragments of glucuronoxylan (aldouronic acids), in which the uronic acid is linked to the non-reducing terminal xylopyranosyl residue. These α-glucuronidases do not cleave glycosidic linkages within polymeric substrates, such as within glucuronoxylans. The only α-glucuronidase described to date that is capable of liberating MeGIcA side chains from hardwood glucuronoxylan is the enzyme present in the cellulolytic system of the wood rotting fungus Schizophyllum commune. Formulas 1 to 4 show the glycosidic linkages in fragments of glucuronoxylan that are attacked (<-) or not attacked (x) by GH67 α- glucuronidases.

Xylβi -4Xylβ1 -4Xylβ1 -4XyI- Xylβ1-4Xylβ1-4Xyl* Xylβi -4Xylβ1 -4Xylβ1 -4XyI- 2 2 2 2 2 Aryl

I <- I x I <- l <- | ^χ l^x α1 α1 α1 α1 α1 α1 MeGIcA MeGIcA MeGIcA GIcA GIcA MeGIcA

Formula 1 Formula 2 Formula 3 Formula 4

These enzymes also do not hydrolyze aryl α-glucuronides, which serve as substrates of non-hemicellulolytic family 4 α-glucuronidases. Although an α- glucuronidase that hydrolyzes aryl α-D-glucuronosides is known in GH4, this enzyme does not recognize glucuronoxylan or its fragments as substrates. The types of glycosidic linkages cleaved by the α-glucuronidases to date are therefore limited.

There is a need for an improved method of enzymatically hydrolyzing glucuronoxylan to increase the retention of glucuronoxylan in the fiber matrix during plant biomass degradation, and to enzymes for use therein.

OBJECT OF THE INVENTION

It is an object of this invention to provide an alternative method of enzymatically hydrolyzing glucuronoxylan that may alleviate, at least to some extent, the abovementioned problems.

SUMMARY OF THE INVENTION

In accordance with the invention there is provided an isolated polypeptide having σ-glucuronidase activity and that can degrade a glucuronoxylan molecule by hydrolysis of a glycosidic linkage between a MeGIcA residue and a nonterminal 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 at least 95% homologous to SEQ ID NO 1 or part thereof;

iii. an amino acid sequence at least 85% homologous to SEQ ID NO 1 or part thereof;

iv. an amino acid sequence at least 75% homologous to SEQ ID NO 1 or part thereof;

v. an amino acid sequence at least 65% homologous to SEQ ID NO 1 or part thereof;

vi. an amino acid sequence at least 50% homologous to SEQ ID NO

1 or part thereof; and

vii. a functional variant of any one of amino acid sequences listed in i- vi. Further features of the invention provide for the polypeptide to have a molecular weight of about 120 kDa, and for the polypeptide to be a biologically active fragment of the polypeptide.

The invention extends to an isolated polynucleotide encoding a polypeptide according to the invention, the polynucleotide having a nucleotide sequence selected from the following group:

i. the nucleotide sequence of SEQ ID NO 2;

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 thereof.

The invention also provides a method of isolating a polypeptide according to the invention, the method including the steps of

i. culturing a microbe capable of expressing the polypeptide in induction medium; and

ii. isolating the polypeptide from the induction medium.

Further features of the invention provide for the step of isolating the polypeptide from the induction medium to be carried out using one or more of anion- exchange chromatography, hydrophobic chromatography, and anion-exchange chromatography. The invention is typically carried out in the following steps

i. culturing a microbe capable of expressing the polypeptide in induction medium and producing microbial biomass;

ii. separating the microbial biomass from the induction medium;

iii. fractionating the induction medium by anion-exchange chromatography to obtain a first eluant;

iv. fractionating the first eluant by hydrophobic interaction chromatography to obtain a second eluant;

v. fractionating the second eluant by anion-exchange chromatography to obtain a third eluant; and

vi. fractionating the third eluant by anion-exchange chromatography to obtain a fraction comprising the isolated polypeptide.

Further features of the invention provide for the microbe to be selected from the group including Pichia stipitis, Schizophyllum commune, Aspergillus clavatus, Neosartorya fischeri, Aspergillus fumigatus, Aspergillus terreus, Aspergillus oryzae, Sclerotinia sclerotiorum, Botryotinia fuckeliana, Pyrenophora tritici- repentis, Neurospora crassa, 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; and for the microbe to preferably be Pichia stipitis CBS 6054. Still further features of the invention provide for the method of isolating the polypeptide to further include the step of concentrating one or more of the induction medium; the first eluant, the second eluant, the third eluant, and the fraction comprising the isolated polypeptide.

The invention further provides a substantially enriched preparation of a polypeptide according to the invention.

Further features of the invention provide for the polypeptide to be purified from a culture of a microbe selected from the group including Schizophyllum commune, Aspergillus clavatus, Neosartorya fischeri, Aspergillus fumigatus, Aspergillus terreus, Aspergillus oryzae, Sclerotinia sclerotiorum, Botryotinia fuckeliana, Pyrenophora tritici-repentis, Neurospora crassa, 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; and for the microbe to preferably be Pichia stipitis CBS 6054.

Still further features of the invention provide for the induction medium to be glucose YNB medium supplemented with xylooligosaccharides and methyl- /?xylopyranoside; for the xylooligosaccharides to be at a concentration of 0.5 mg/ml; for the methyl-βxylopyranoside to be at a concentration of 0.33 mg/ml; for the fractionating the induction medium by anion-exchange chromatography to obtain a first eluant to be carried out using a HiTrap DEAE-FF column; for the first eluant to be obtained by the application of a first elution buffer to the HiTrap DEAE-FF column, the first elution buffer comprising a NaCI gradient of 0 to 1.0 M in approximately 50 mM sodium-phosphate buffer at approximately pH 7.0; for the fractionating the first eluant by hydrophobic interaction chromatography to obtain a second eluant to be carried out using a Butyl-FF column; for the second eluant to be obtained by the application of a second elution buffer to the Butyl- FF column, the second elution buffer comprising a decreasing (NH₄^SO₄ gradient of 1.1 M to 0.61 M in approximately 50 mM acetate buffer at approximately pH 4.0; for the fractionating the second eluant by anion-exchange chromatography to obtain a third eluant to be carried out using a Tricorn MonoQ 5/50GL column; for the third eluant to be obtained by the application of a third elution buffer to the Tricorn MonoQ 5/50GL column, the third elution buffer comprising an increasing NaCI gradient of 0 to 1.0 M in approximately 50 mM sodium-acetate buffer at approximately pH 4.0; fractionating the third eluant by anion-exchange chromatography to obtain a substantially pure enzyme fraction to be carried out using a Tricorn MonoQ 5/50GL column; for the substantially pure enzyme fraction to be obtained by the application of a fourth elution buffer to the Tricorn MonoQ 5/50GL column, the fourth elution buffer comprising an increasing NaCI gradient of 0 to 1.0 M in approximately 50 mM sodium- phosphate buffer at approximately pH 7.0.

Still further features of the invention provide for the polypeptide to be obtained from a culture of a microbe selected from the group including Schizophyllum commune, Aspergillus clavatus, Neosartorya fischeri, Aspergillus fumigatus, Aspergillus terreus, Aspergillus oryzae, Sclerotinia sclerotiorum, Botryotinia fuckeliana, Pyrenophora tritici-repentis, Neurospora crassa, 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 pristinaespirali; and for the microbe to preferably be Pichia stipitis CBS 6054. BRIEF DESCRIPTION OF THE DRAWINGS

Further features of the invention will now become apparent from the following description, by way of example only, with reference to the accompanying Figures and SEQ ID NOS.

In the drawings:-

Figure 1 is a summary of σ-glucuronidase purification from the induction medium of P. stipitis CBS 6054;

Figure 2 is an SDS-PAGE gel of purified P. stipitis σ-glucuronidase,

Lane 1 - protein markers (Fermentas #SM 0431), lane 2 - σ-glucuronidase, 10 μg protein, lane 3 - σ-glucuronidase, 20 A/g; lane 4 - protein marker (SERVA #39216);

Figure 3 is a chromatogram of the TLC analysis of products formed from aldopentauronic acid (XyI-XyI(MeGIcA)-XyI-XyI) (A) and glucuronoxylan (B) upon the action of purified P. stipitis σ-glucuronidase, A: samples: lane 1 and lane 8 - xylose and xylooligosaccharide standards, 2 - aldopentauronic acid (XyI-XyI(MeGIcA)-XyI-XyI), lane 3 - enzyme blank, lanes 4-7 - products of enzyme reaction after 1, 10, 60 min and 18 h, respectively, B: samples: lane 1 and lane 5 - xylose and xylooligosaccharide standards, lane 2 - glucuronoxylan control, lane 3 and lane 4 - products of enzyme reaction after 4 h and 18 h, respectively; Figure 4 is a summary of a BLAST search performed using a sequence of P. stipitis. The sequences of microorganisms used for the alignment and construction of the phylogenetic tree are shown in bold;

Figure 5 is a schematic representation of the homology alignment of the amino acid sequence of the P. stipitis CBS 6054 a- glucuronidase with the nine protein sequences from Aspergillus fumigatus Af293, Pyrenophora tritici-repentis Pt- 1 C-BFP, Neurospora crassa OR74A, Gibberella zeae PH-1 ,

Cellvibrio japonicus Ueda107, Coprinopsis cinerea okayama 7#130, Aspergillus oryzae RIB40, Bacteroides ovatus ATCC 8483, Streptomyces pristinaespiralis ATCC 25486 that showed more than 50% identity;

Figure 6 shows a summary of xylan substrates used for evaluating enzymatic substrate specificity and degree of removal of xylan side chains;

Figure 7 shows the Box-Behnken experimental set up for removal of

4-O-methyl-D-glucuronic acid from birch xylan by σ-glu;

Figure 8 shows the central composite design for effect of oatspelt xylan concentration and enzyme dosage on arabinose removal;

Figure 9 shows a bar graph of the content (% OD biomass) of extractives and ash of bagasse, pine (Pinus patula), and bamboo (Bambusidae balcooa); Figure 10 shows a bar graph of the Klason 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 pentosan of of bagasse, pine (Pinus patula), and bamboo (Bambusidae balcooa);

Figure 12 shows a bar graph of the xylan yield (% pentosan) extracted using ultrapurification and ethanol precipitation protocols from bagasse, pine (Pinus patula), and bamboo (Bambusidae balcooa);

Figure 13 shows solid state ¹³C-CPMAS NMR spectra showing the effect of mild alkali xylan extraction on the integrity of cellulosic fibres in (A) Pinus patula , (B) Bagasse, (C) Eucalyptus grandis and (D) giant bamboo. The spectra 1 , 2, and 3 denote: raw material, extractive free material, and post xylan extracted material and ^** denotes peaks for resonances of carbon in glucose units of less ordered cellulose;

Figure 14 shows a comparison of neutral sugar composition of lignocellulosic materials before (EF) and after xylan 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 of neutral sugars and uronic acid of pre-extracted xylan;

Figure 16 shows the elution profiles of xylan on HPAEC-PAD (Dionex) CarboPac P10 column from (A) monomeric sugars, (B) xylitol), (C) birch xylan (Roth, and (D) Oatspelt xylan;

Figure 17 shows the elution profiles of xylan on HPAEC-PAD (Dionex)

CarboPac column P10 from (A) mild alkali extracted H₂O₂ bleached bagasse (Bag B), (B) mild alkali extracted ultra purified bagasse (Bag H), and (C) mild alkali extracted ethanol precipitated bagasse (Bag L);

Figure 18 shows the elution profiles of xylan on HPAEC-PAD (Dionex) CarboPac column P10 from (A) Eucalyptus grandis H [EU

H) and (B) Eucalyptus grandis, L [EU L], (C) bamboo and (D) Pinus patula;

Figure 19 shows a bar graph of the insoluble fraction obtained after 72% acid hydrolysis of mild alkali extracted xylan H₂O₂ bleached bagasse (Bag B), ultrapurified bagasse (Bag H), ethanol precipitated bagasse (Bag L), bamboo, ultrapurified E. grandis (EU H), ethanol precipitated E. grandis (EU L), and P. patula (Pine) referenced to Birch xylan (Roth);

Figure 20 shows the characterisation of xylan by (A) ¹H-NMR and (B)

¹³C-NMR analyses of birch xylan, (C) ¹H-NMR and (D) ¹³C- NMR analyses of H₂O₂ bleached bagasse (Bag B), and (E) 1H-NMR and (F) ¹³C-NMR analyses of oatspelt xylan. Me denotes Methyl group from glucuronic acid and Ac = acetyl group, Ar = arabinose group;

Figure 21 shows the characterisation of xylan by (A) ¹H-NMR and (B) 1³C-NMR analyses of bagasse, (C) ¹H-NMR and (D) ¹³C-

NMR analyses of E. grandis xylan: In the spectra (1) = Lopez extracted xylan (Bag L or EU L), and (2) Hoije ultapurified xylan (Bag H or EU H);

Figure 22 shows the characterisation of xylan by (A) ¹H-NMR and (B)

¹³C-NMR analyses of bamboo, (C) ¹H-NM R and (D) ¹³C- NMR analyses of P. patula xylan;

Figure 23 shows the FTIR spectra of xylan extracted from different types of lignocellulosic materials from bottom (iv) birch^**, (F) ethanol precipitated bagasse [Bag L] (2), (E) ultrapurified bagasse [Bag H] (1 ), (D) oatspelt xylan*, (C) bamboo, (B) ethanol precipitated E. grandis [EU L] (2), (B) ultrapurified E. grandis [EU H] (1) and (A) P. patula;

Figure 24 shows the removal of 4-O-MeglcA by AbfB and σ-glu from oatspelt/birch, mild alkali pre-extracted bagasse Hόije (BH), H₂O₂ bleached bagasse (BB), bamboo (BM), and Pinus patula (PP) xylan, and by σ-glu from mild alkali pre- extracted Eucalyptus grandi, (EH), Eucalyptus grandis gel

(ES) extracted from pulp;

Figure 25 shows response surface plots response surface plots of glucuronic acid removal as a function of (A) time (h) and temperature (⁰C) at 16500 nKat g^'1 substrate, (B) temperature (⁰C) and enzyme dose (nKat g^'1 substrate) at 9 h, and (C) time (h) and enzyme dose (nKat g^'1 substrate) at 33.5⁰C;

Figure 26 shows interaction effects between time, temperature, and enzyme dose on glucuronic acid removal. First columns from top show the interaction between temperature (Temp) and time, enzyme dose (AbfB /σ-glu) and time, and enzyme dose (σ-glu) and temperature. In the second columns top right, the cell shows the size and significance of the treatment and interaction effects as measured by the size of bar graph. The t (i, i₄) values are indicated at the end of each bar graph in the respective Pareto chart. The vertical dotted line in the Pareto chart 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);

SEQ ID NO 1 is the deduced amino acid sequence of the α-glucuronidase gene of P. stipitis, as available in Genbank accession number XP 001385893; and

SEQ ID NO 2 is the DNA sequence of the α-glucuronidase gene of P. stipitis, as available in Genbank accession number XM 001385893 and the published P. stipitis genome sequence (Vrsanska et al 2007). DETAILED DESCRIPTION WITH REFERENCE TO THE DRAWINGS

The invention relates to an isolated polypeptide which has σ-glucuronidase activity and is capable of degrading glucuronoxylan molecules found in plant biomass. Unexpectedly, this polypeptide demonstrates advantages of enzymatic activity in degrading glucuronoxylan molecules that are not seen in other enzymes previously reported to exhibit σ-glucuronidase activity. Such other σ- glucuronidase are limited in their hydrolysis of glycosidic linkages of glucuronoxylan molecules as they are only capable of hydrolysing a glycosidic linkage 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 hydrolysing a glycosidic linkage between a MeGIcA residue and a non-terminal xylopyranosyl residue. Furthermore, the σ- glucuronidase activity of the isolated polypeptide of the invention is broadly applicable to a variety of glucuronoxylan molecules obtained from various plant biomass sources.

The present invention provides for an isolated polypeptide having an amino acid sequence that is the amino acid sequence of SEQ ID NO 1 ; or one that is substantially similar thereto, such as a sequence that is at least 95% homologous to SEQ ID NO 1 or part thereof; 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 thereof; a functional variant of any one of these amino acid sequences. The identity of the full-length isolated polypeptide may be confirmed with reference to its molecular weight of about 120 kDa, using techniques such as SDS-PAGE, or σ-glucuronidase activity assays for the identification of biologically active fragments of the polypeptide. The polypeptide 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. Such microbes include Schizophyllum commune, Aspergillus clavatus, Neosartorya fischeri, Aspergillus fumigatus, Aspergillus terreus, Aspergillus oryzae, Sclerotinia sclerotiorum, Botryotinia fuckeliana, Pyrenophora tritici-repentis, Neurospora crassa, 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.

Since the polypeptide is secreted into the induction medium of the microbial culture, various chromatographic techniques may be used 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 eluant; fractionating the first eluant by hydrophobic interaction chromatography to obtain a second eluant; fractionating the second eluant by anion-exchange chromatography to obtain a third eluant; and fractionating the third eluant by anion-exchange chromatography to obtain a fraction comprising the isolated polypeptide. Further steps of concentrating one or more of the induction medium; the first eluant, the second eluant, the third eluant, and the fraction comprising the isolated polypeptide are also carried out.

The isolated polypeptide of the invention may be provided in the form of a substantially enriched preparation of the polypeptide. A substantially enriched 1.8

preparation as produced according to the invention generally means that the predominant protein species or component of the preparation is the polypeptide of SEQ ID NO 1 , or one substantially similar thereto. However it will be appreciated that more purified forms of the substantially enriched preparation are included within the scope of the invention, such as preparations comprising at least 75% of the polypeptide; preparations comprising at least 80% of the polypeptide; preparations comprising at least 90% of the polypeptide; and preparations comprising at least 90% of the polypeptide. A substantially enriched preparation produced according to the invention also generally refers to the substantially absence of biologically active enzymes capable of hydrolysing the main chain of a glucuronoxylan molecule.

EXAMPLE 1

Materials and Methods

P. stipitis strain and its cultivation

P. stipitis CBS 6054 was grown in flasks in medium containing YNB (Difco, 6.7 g/l), L-asparagine (2 g/l), KH₂PO₄ (5 g/l) and carbon source (glucose or beechwood glucuronoxylan, 10 g/l) at a temperature 30⁰C and an agitation of 180 rpm. Exponential-grown cells were harvested at a cell density of 0.15-0.20 mg/ml (dry weight).

α-Glucuronidase substrates and products

Deacetylated glucuronoxylan was extracted from beech sawdust [Ebringerova et al 1967, Holzforschung 21 :74-77]. Aldotetraouronic acid XyI(MeGIcA)XyI-XyI, the shortest acidic product of glucuronoxylan hydrolysis by family 10 endoxylanases, and aldopentaouronic acid XyI-XyI(MeGIcA)XyI-XyI, the shortest acidic product of glucuronoxylan hydrolysis by family 11 endoxylanases were prepared as previously described [Biely et al, 1997, J. Biotechnol. 57:151-166]. 4-O-Methyl-D-glucuronic acid (MeGIcA) was prepared by de-esterification of its methyl ester by S. commune glucuronoyl esterases [Spanikova and Biely, 2006, FEBS Lett. 580:4597-4601].

P. stipitis α-glucuronidase production in the absence of xylan

The enzyme, which became a subject of purification was produced in induction experiments which were carried out as follows: Exponential-phase cells grown in a 1% glucose YNB medium were collected by centrifugation, washed twice with basal YNB medium (without carbon source) and suspended in the same medium supplied with 0.5 mg/ml of xylooligosaccharides mixture (XYLO-OLIGO 70, Suntory Limited, Japan) and 0.33 mg/ml of methyl-/?-xylopyranoside. The cell concentration was 0.6-0.8 mg/ml dry weight (105⁰C). After 24 h incubation on a shaker (180 rpm) at 30⁰C for 24 h the mixture was centrifuged and the clear supernatant used for purification of extracellular σ-glucuronidase which was coinduced with endo-β-1 ,4-xylanase.

Purification of P. stipitis α-glucuronidase

The clear induction medium (600 ml) was 300-fold concentrated on Amicon 10 kDa cut-off membranes. The secreted proteins were first fractionated by anion- exchange chromatography on a HiTrap DEAE-FF (GE Healthcare, Sweden) column using elution with NaCI gradient (0-1.0 M) in 50 mM sodium-phosphate buffer (pH 7.0). Fractions containing σ-glucuronidase, eluting as a peak between 0.2 - 0.26 M NaCI, were pooled, concentrated and desalted, then equilibrated in 50 mM acetate buffer (pH 4.0) containing 2M (NH₄)₂SO₄. The eluant obtained was resolved by hydrophobic interaction chromatography on a Butyl-FF column (5 ml) (GE Healthcare) eluted with a decreasing gradient of (NH₄)₂SO₄ in the same buffer. σ-Glucuronidase eluted at a concentration of between 1.1 and 0.61 M (NhU)₂SO₄. The fractions having σ-glucuronidase activity were pooled, desalted, concentrated and resolved by two additional anion-exchange chromatography steps using a Tricorn MonoQ 5/50GL (Amersham, UK) column (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 NaCI (0-1.0 M). In the second step the acetate buffer was replaced with 50 mM sodium phosphate buffer (pH 7.0). Fractions having σ-glucuronidase activity were desalted and concentrated by membrane filtration on Microcon (10 kDa cut-off, Millipore Co., USA).

Sequence analysis of purified protein

Purified α-glucuronidase was resolved by SDS PAGE on 10% acrylamide gels and electro-blotted onto a polyvinylidene difluoride membrane (Milipore 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).

α-Glucuronidase assay

Purification fractions having σ-glucuronidase activity were qualitatively identified by TLC analysis using a silicagel (Merck Silica gel 60 on aluminum plates) in ethyl acetate: acetic acid: 1-propanol: formic acid: water (26:10:5:1:15, by vol.). The presence of free MeGIcA derived from aldouronic acids (10 mg/ml) or glucuronoxylan (2%) dissolved in 0.05 M sodium acetate buffer (pH 4.4) was indicative of -glucuronidase activity. The enzyme was usually used at concentration 50 μg protein/ml. A quantitative assay based on determination of free MeGIcA liberated from aldopentauronic acid (XyI-XyI(MeGIcA)-XyI-XyI, 10 mg/ml) or beechwood glucuronoxylan (2%) according to the method of Milner and Avigad [Milner and Avigad, 1967, Carbohyd. Res. 4:359-361] was used to detect substrates and products of -glucuronidase activity. The substrate and products were detected using N-(naphtylethylenediamine)-dihydrochloride reagent [Buonias, 1980, Anal. Biochem. 106:291-295]. The reagent is brown in colour in the presence of MeGIcA and purple in colour in the presence of xylose-containing compounds. Protein samples (1-10 μg, depending on purity) were incubated for 10-60 min in 0.1 ml reaction mixture containing the substrate in 50 mM acetate buffer (pH 4.4). The reaction was stopped by adding 0.3 ml copper reagent and boiling for 10 min at 100⁰C, followed by addition of 0.2 ml Nelson reagent and 0.4 ml water. The absorbance was measured at 600 nm using calibration with GIcA. One unit of α-glucuronidase was defined as the amount of enzyme producing 1 μmol of uronic acid in 1 min from aldopentauronic acid (XyI-XyI(MeGIcA)-XyI- XyI.

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 227:680-685] and comparison with similarly resolved unstained protein molecular weight markers (FERMENTAS, Canada) and dyed protein markers (SERVA, GmbH). IEF was performed on Multiphor Il system (GE Healthcare, Sweden) using SERVALYT PRECOTES 3-6 precast gels and IEF markers 3-10 (SERVA, GmbH). Results

Isolation of α-glucuronidase

A new α-glucuronidase was observed during the growth of several P. stipitis strains on various types of xylan. The highest growth of the yeast was observed on glucuronoxylan. Although glucuronoxylan was utilized as the main carbon source to a limited extent when compared with the utilization of D-xylose, D- glucose or β-1 ,4-xylooligosaccharides, there was no accumulation of acidic oligosaccharides in the medium. All fragments released from glucuronoxylan were completely utilized which suggested that the yeast secreted an α- glucuronidase into the medium. Analysis of the growth medium of the yeast after growth on glucuronoxylan revealed strong α-glucuronidase activity. The partially purified enzyme was found to be capable of debranching glucuronoxylan and liberating MeGIcA from aldouronic acids in which the MeGIcA was linked to internal xylopyranosyl residues. The enzyme could not be purified from a partially-spent glucuronoxylan medium as the presence of xylan residues increased the viscosity of the concentrated medium and interfered with the enzyme purification.

The extracellular α-glucuronidase was, however, successfully purified from the medium when washed exponential phase glucose-grown cells were incubated in a synthetic medium supplied with a mixture of xylobiose, xylotriose and methyl β-D-xylopyranoside, which are inducers of xylanolytic enzymes in P. stipitis. The level of extracellular α-glucuronidase induced under these conditions was 0.015 U/ml which represented about 20% of the activity observed during incubation of cells with 1% glucuronoxylan. This level of α-glucuronidase was sufficient for its purification from the induction medium containing only dialyzable nutritional 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 resolved to a single band by SDS PAGE analysis, 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 endoxylanase or β-xylosidase activity. The only reaction exhibited by the enzyme with aldopentaouronic acid having the structure XyI-XyI(MeGIcA)-XyI-XyI* or with beech glucuronoxylan was the liberation of MeGIcA residues (Figure 3). The enzyme also liberated MeGIcA from aldotetraouronic acid XyI(MeGIcA)-XyI-XyI*

(not shown) which similarly serves as a substrate of family 67 α-glucuronidases (Biely, 2003, Xylanolytic enzymes. In: Handbook of Food Enzymology, pp. 879-

916. Marcel Dekker, Inc. NY). The initially clear glucuronoxylan solution showed increased opalescence and increased viscosity due to efficient debranching.

The amount of uronic acid released from beechwood glucuronoxylan by the enzyme after a long-term treatment was 0.35 μmol per 1 mg of glucuronoxylan, which represents 75% of total MeGIcA content in the polysaccharide.

Optimum conditions for enzyme activity

The pH optimum of enzyme activity was found to be 4.4. At pH 4.0 and pH 5.5 the activity represented 26.5% and 51.6% of the activity at the optimum pH, respectively. The temperature optimum for the enzyme activity was 60⁰C (18.43 U/mg at polymeric glucuronoxylan), but the protein was found to be unstable at this temperature, with 50% loss of activity in 30 min. The protein was found to be stable for at least 3h at 40⁰C, with specific activity on glucuronoxylan 3.01 U/mg. The pi of σ-glucuronidase calculated from sequence (ExPASy online ProtParam tool) was 4.64, but isoelectrofocusing data indicated the protein pi as being 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 corresponded exactly with the P. stipitis gene XP 0013855930 deduced from the available genome sequence of P. stipitis, starting with amino acid number 19, coding for a protein of unknown function but having a similar molecular mass as the isolated α-glucuronidase (SEQ ID NO 1). The part of the gene coding for the first 18 amino acid corresponds obviously to the enzyme secretion signal sequence, which is not preserved in the matured extracellular protein comprising of 957 amino acids.

A BLAST search was performed using the sequence of P. stipitis α- glucuronidase which uncovered similar genes in genomes of many other microorganisms, mostly fungi. The list of microorganisms with the orthologues of the P. stipitis α-glucuronidase gene having identity higher than 34% and similarity higher than 51% is shown in Figure 4. All orthologues correspond to proteins of around 1000 amino acids, which correspond to a molecular mass about 120 kDa, in all cases to proteins of unknown function. The highest identity (54%), and the highest similarity (69%) was exhibited by the gene sequence of Aspergillus clavatus.

The alignment of eight selected homologous sequences (shown in bold in Figure 4) with the P. stipitis α-glucuronidase sequence is shown in Figure 5. The alignment shows 6 conserved glutamic acid residues and 12 conserved aspartic acid residues, two of which may be the amino acids involved in catalyzing the reaction. Furthermore, 3 tyrosines and 6 tryptophans are also conserved. In case the aromatic amino acids are surface-exposed, they could play important role in recognition of the xylan main chain as one of the conditions of the enzyme to operate on the polymeric substrate.

The P. stipitis enzyme liberates MeGIcA residues linked to terminal or internal xylopyranosyl residues of glucuronoxylan and aldouronic acids generated from the polysaccharide on the action of endoxylanases. The P. stipitis CBS 6054 sequence is phylogenetically distant from both GH families 67 and 4.

The xylose-fermenting yeast Pichia stipitis is unique in that it has also a limited ability to utilize xylan as a carbon source. The yeast was found to preferably hydrolyze the hardwood glucuronoxylan possibly because the set of its xylanolytic enzymes is limited to the production of only three enzymes, endo-β- 1 ,4-xylanase, α-glucuronidase and β-xylosidase. This property of the xylanolytic system of the yeast corresponds to its natural habitat, that of the digestive gut of passalide beetles that feed on hard wood biomass rich in acetylglucuronoxylan. During growth on this deacetylated glucuronoxylan, the first two enzymes were secreted by the yeast into the growth medium and no accumulation of acidic oligosaccharides (aldouronic acids) therefore occurred during growth on this carbon source. The level of secreted endoxylanase by this yeast has previously been found to be so low as to discourage further studies of the xylanolytic system of this yeast. It is therefore surprising that this poor xylanase secretes a new and useful α-glucuronidase.

The newly described type of α-glucuronidase has the ability to liberate MeGIcA residues from polymeric substrate. Its action on aldotetraouronic acid XyI(MeGIcA)XyI-XyI, the shortest acidic product of glucuronoxylan hydrolysis by family 10 endoxylanases confirms that the new family α-glucuronidases exhibits the catalytic activity of GH67 enzymes. The N-terminal amino acid sequence of α-glucuronidase of S. commune did not match any sequences of the enzymes grouped in the new family.

The ability of this novel α-glucuronidase enzyme to debranch glucuronoxylan is believed to influence the physico-chemical properties of the polysaccharide. Deacetylation of acetylglucuronoxylan or the removal of α-L-arabinofuranosyl side chains in arabinoxylan is proposed to decrease the solubility of the polysaccharides and eventually result in precipitation of the debranched polymer. Treatment of plant biomass using the novel α-glucuronidase enzyme herein described is proposed to decrease the loss of glucuronoxylan in the cooking liquors produced during paper manufacture and to maximise the retention of the glucuronoxylan in the fiber matrix.

The removal of methyl-glucuronic acid and glucuronic acid from glucuronoxylan is expected to produce compositions having useful application in the paper and pulp industry and pharmaceutical field. Increased amounts of glucuroxylan in paper production not only contributes to conserving plantbiomass in paper making processes, but could provide unique properties in paper strength, paper coating and retaining printer ink. The re-precipitation of glucuroxylan can be used in the pharmaceutical field for coating of medicine to extend their storage and to assist in slow release of medicinal compounds. The hydrolysis of glucuronoxylan with through the synergistic activities of α-glucuronidase together with /?-xylanases and /?-xylosidases can release fermentable sugars from glucuronoxylan for conversion to commodity products, such as ethanol, lactate and other fine chemicals.

EXAMPLE 2 Materials and methods

Statistical analysis

Unless stated otherwise, samples were conducted in triplicates. Analysis of variance (ANOVA) including sample means and standard deviations were performed using Microsoft Excel and Statistica 2007.

Optimisation of xylan extraction for analysis of α-Glucuronidase activity

The selectivity and effectiveness of the two mild alkali xylan extraction methods which were adopted from Hoije et al. [2005, Carbohydr. Polym. 61: 266-275] and De Lopez et al. [1996, Biomass and Bioenergy, 10: (4) : 201-211] were assessed for potential use in integrated production of substantially pure xylan biopolymers and pulp production from the feedstock commonly found in South Africa. The assessment was based on five factors as follows: (1) xylan 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 the raw material post xylan extraction. A summary of the xylan substrates used for evaluating enzymatic substrate specificity and degree of removal of xylan side chains is shown in Figure 6.

Raw material characterisation

The feedstock used included Eucalyptus (Eucalyptus grandis), pine (Pinus patula), giant bamboo (Bambusa balcooa), and sugarcane (Saccharum officinarum L) bagasse). The E. grandis chips were supplied by The Transvaal Wattle Cooperatives from Piet Retief, Mpumalanga Province, whereas the P. patula trees were harvested from Stellenbosch University forest plantations in the Western Cape Province of South Africa. The giant bamboo stems (one and half year plant) were supplied from mature plantations located in Paarl in the Western Cape Province of South Africa. The bagasse was a by-product from the sugar processing industry which was donated by TBS Company located in Nkomazi region of the South-Eastem Lowveld of Mpumalanga province in South Africa. Oatspelt xylan (Sigma), birch xylan (Roth), and mild alkali extracted H₂O₂ bleached bagasse xylans (donated by Prof. A.M. F. Milagres, University of Sao Paulo, Brazil) were used as reference xylans.

The feedstock materials were prepared for analysis according to TAPPI test methods (TAPPI, T264 cm-97 (2002-2003)), and NREL Laboratory Analytical Procedures (NREL LAP) [Hammes et ai, 2005, Laboratory Analytical Procedure (LAP), version 2005, NREL Biomass Program. National Bioenergy Center]. Chips derived from the various feedstocks were dried to a moisture content (me) of «10%, and subsequently conditioned to a relative humidity of 55% at 20 ⁰C for at least 24 h prior to 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 μm/40 mesh size, and 250 μm/60 mesh size with a lid and pan. The particulates that passed through 425 μm/40 mesh size but were retained on a 250 μm/60 mesh sieve were collected for chemical composition analyses and those retained on the 425 μm/40 mesh were used for xylan extraction. The moisture content of the feedstock was determined using National Renewable Energy Laboratory Analytical Procedure (NREL LAP) for determination of total solids in biomass [Hammes et ai, 2005, Laboratory Analytical Procedure (LAP), NREL Biomass Program. National Bioenergy Center]. The percent moisture content was calculated as a % of oven dry (o.d) weight biomass. Extractives were determined in two sequential steps, starting with cyclohexane/ethanol (2:1) followed by hot water extraction, using soxhlet apparatus. Both extractions were done according to TAPPI Test Method T 264 om-88, and NREL LAP methods [Sluiter et al., 2005, Analytical Procedure (LAP), version 2006. NREL Biomass Program. National Bioenergy Center]. The extractives were quantified on a moisture free basis.

Klason lignin (acid insoluble) content of the feedstock was determined following a NREL LAP method for determination of structural carbohydrates and lignin in biomass [Sluiter et al., 2005, Analytical Procedure (LAP), version 2006. NREL Biomass Program. National Bioenergy Center] and TAPPI test procedures (T249 cm-85). The Klason lignin was calculated on o.d. mass.

Seifert cellulose content was determined according to the analytical methods outlined by Browning [1967, Methods of wood chemistry, VoI II. lnterscience publishers], and Fengel and Wegner [1989, Wood Chemistry, Ultrastucture, Reactions. Walter de Gruyter, Berlin, Germany]. Extractive free material weighing 1.1g oven dry was treated with a mixture of acetyl acetone (6 ml), dioxane (2 ml) and 32 % HCI (2 ml) in round bottom flasks followed by incubation in a boiling water bath for 30 min. The treated samples were transferred quantitatively into pre-weighed sinter glass crucibles for vacuum filtration and washing. The residues were successively washed with 100 mL each of methanol, cyclo-dioxane, warm water (8O⁰C), methanol, and diethyl ether and subsequently dried at 105⁰C for 2 h. The Seifert cellulose content was defined as the weight of the dried residue presented as a percentage of the extractive free material. Monomeric sugar composition of the acid hydrolysate was analysed after storage at -20^°C for at least 24 h. The analysis was performed in high performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD, (Dionex) that was equipped with a gradient pump GP 50, a Carbopac PA 10 (4 mm x 250 mm) column, and electrochemical detector (ED40). The data acquisition and analysis were performed using PEAKNET software package. The eluents were 250 mM NaOH and Milli-Q-water in the ratio of 1.5:98.5 at a flow rate of 1 ml_ min^"1. Sodium acetate (1M NaOAc) eluent was used when acid sugars (glucuronic/ methyl glucuronic acid) were analysed. The samples were filtered on 0.22 μm pore size filters before analysis on HPAEC-PAD. The quantity of the sugars was determined from standard plot of the respective analytical grade sugars (arabinose, rhaminose, 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 pentosan content in the feedstocks was determined according to TAPPI standard methods for measuring pentosans in wood and pulp (T223 cm-84). The xylan content was calculated from a standard plot prepared from xylose (analytical grade) using equation 3.1.1 as percentage of the o.d. biomass [Xyl = xylχ cf , Where: XyI = Xylan content (mg), xyl = Xylose content (mg), cf = Correction factor (0.88)].

The ash content was determined by a thermogravimetric method. Lignocellulosic samples (0.5 g) were incinerated in a Muffle furnace at 575 ± 25°C for 4 h or until a constant weight was obtained. Ash content was calculated as a percentage of the initial o.d. biomass.

Xylan extraction and characterisation Extraction of xylan from the feedstock was performed using the two mild alkali extraction methods described in section 2.9 above. The Hoije method involved post xylan extraction ultrapurification using membrane dialysis (MWCO 12-14 kDa) whereas the Lopez method involved fractionation of the hydrolysates by ethanol precipitation. In both methods, xylan extraction was performed without prior removal of solvent and hot water extractives. The extracts were concentrated before ultrapurification or fractionation to a third of the initial volume using a rotary evaporator (Rotavapor Bϋchi R-124, Switzerland) under vacuum at 40 ⁰C. The extraction efficiency was defined as the yield of xylan per theoretical content of pentosans in the material. The Lopez method was limited to extraction of xylan from E. grandis and bagasse only.

The structure and chemical composition of the feedstocks for xylan extraction were analysed pre- and post- xylan extraction by solid state ¹³C-Nuclear Magnetic Resonance Cross-Polarisation/Magic Angle Spinning (¹³C-NMR CP/MAS) on a Varian VNMRS 500 wide bore solid state NMR spectrometer with an operating frequency of 125 MHz for ¹³C, using a 6mm T3 probe with a probe temperature of 25 ⁰C. Dry samples were loaded into 6 mm zirconium oxide rotors for analysis. Spectra were recorded using cross-polarisation and magic angle spinning (CP/MAS). The speed of rotation was 5 kHz, the proton 90° pulse was 5μs, the contact pulse 1500μs and the delay between repetitions 5 sec. Chemical shifts were determined relative to TMS by setting the downfield peak of an external adamantane reference to 38.3 ppm. The carbon resonances in the solid state NMR spectra were assigned according to Larsson et a/. [1999, Solid State Nuclear Magnetic Resonance 15: 31-40]; Renard and Jarvis [1999, Plant Physiology 119: 1315-1322], Maunu [2002, Progress in Nuclear Magnetic Response Spectroscopy 40: 151-174], Lahaye et al. [2003, Carbohydrate Research 338: 1559-1569]; Atalla and lsogai [2005, Recent developments in spectroscopic and chemical characterisation of cellulose. In Dumitriu, S. (ed.) Marcel Dekker. New York, pp 123-157], Virkki et al. [2005, Carbohydrate Polymers 59: 357-366], Geng et al. [2006, International Journal of Polymer Characterization 11: 209-226], and 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].

The extracted xylan samples were analysed using solid state ¹³C-Nuclear Magnetic Resonance Cross-Polarisation/ Magic Angle Spinning (¹³C-NMR CP/MAS) and Liquid ¹³C and ¹H NMR and Fourier Transform Infrared (FTIR) spectroscopy. The xylan samples were subjected to a ¹³C and a ¹H NMR run either on a Varian Inova 400 or 600 NMR spectrometer. ¹³C NMR spectra were collected using a 1.3s acquisition time and 1s pulse delay at 25 ⁰C. The ¹³C spectra were collected overnight (minimum 19000 scans). ¹H NMR spectra were collected after filtration of the sample with a 4.8 s acquisition time at 50 ⁰C. ¹H spectra were collected with 64 scans and pre-saturation of the HDO peak. The ¹³C and ¹H NMR spectra were interpreted according to assignment of characteristic signals of related feedstock presented by Ebringerova et al. [1998, Carbohydrate Polymers 37: 231-239], 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], Grόndahl 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.Q. (ed)], Shao et al. [2008, Wood Science Technology 42: 439-451]. In FTIR spectroscopy, dry solid samples of the xylan were recorded on a Nexus 670 spectrometer from Thermo Nicolet with the Smart Golden Gate ATR accessory installed. This single-reflection 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 of 4000 to 650 cm^"1 using 16 scans at 6 cm^"1 resolution and were calibrated against a previously recorded background. Thermo Nicolet's OMNIC® Software was used for collecting and processing of the infrared spectra. The spectra signals for FTIR were interpreted according to characteristic 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 Carbopac™ PA100 column (4 x 250 mm) and a guard column, and electrochemical detector (ED40) for pulsed amperometric detection (PAD). The PA 100 column separates monomers and oligomers up to a degree of polymerisation (DP) 10 which usually elutes within a retention time of 25 min. The HPAEC PA100 column bases its separation on DP and degree of substitution, thus the longer the retention time, the higher the DP or degree of substitution (Combined CarboPac manual pp 52-56). Samples (10 //L) were injected into the column and were eluted with helium degassed 0.25M NaOH, MiIIi-Q H₂O, and 1 M NaOAc at a flow rate of 1 mLmin^"1. Elution profiles of the samples were referenced to elution profiles of monomeric sugars (arabinose, rhaminose, galactose, glucose, xylose and mannose), and polymeric xylan (birch, and oatspelt xylan) and H₂O₂ bleached bagasse. Samples with less intense peaks < 20 nC or no peaks eluting within the 25 min retention time were considered polymeric with DP>10 sugar units. The composition of neutral sugars in the extracted xylan samples were determined on HPAEC-PAD (Dionex) on 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 bottles (50 ml_) into which 1 ml_ 72% H₂SO₄ was added. The mixture was incubated at 3O⁰C in a water bath for 1 h. De-ionized water (30 ml_) was added followed by autoclaving at 121⁰C for 1 h. The samples were cooled to room temperature before filtering. The filter cake was dried at 105 ⁰C for residual Klason lignin determination. The liquid fraction was filtered through a 0.22 μm pore size filter before subjecting it to HPAEC— PAD (Dionex) on Carbopac PA 10 column. The monomeric sugars were quantified from standard plots of analytical grade arabinose, rhaminose, galactose, glucose, xylose, and mannose). The total neutral sugar content of the samples was presented relative to the initial xylan o.d mass.

Determination of uronic acid composition

Uronic acid content of the xylan samples and the feedstocks were quantified using chromatographic and colorimetric methods. In the chromatographic method, a two step acid hydrolysis method adopted from Prof. A.M. F. Milagres of University of Sao Paulo, Brazil was used. Xylan samples (150 mg o.d mass) were hydrolysed in 0.75 ml_ of 72% (w/w) H₂SO₄ in McCartney bottles. The mixture was incubated at 45⁰C for 7 min in a water bath after which 22.5 mL of distilled water were added. The bottles were loosely covered and autoclaved at 121⁰C for 30 min. After cooling to room temperature, the liquid fraction was separated by vacuum filtering through glass micro fibre filters (GF/A- Whatman). The liquid fraction was further filtered through a 0.22 μm filter and kept frozen overnight at -2O⁰C before analysing for glucuronic acid content using HPAEC— PAD (Dionex) on Carbopac PA 10 column. Quantification of uronic acid was based on standard plots for glucuronic acid (Sigma). Uronic acid losses during autoclaving were accounted for by autoclaved glucuronic acid at 121 ⁰C for 1 h in 4% H₂SO₄. In the colorimetric method, carbazole-sulfuric assay adopted from Li et al. [2007, Carbohydr. Res. 342 (11): 1442-1449] was used. Total uronic acid concentration was determined from standard curve plot for D-galacturonic acid (Merck) and in both methods uronic acid content was presented as percentage of the initial xylan amount.

Removal of xylan side chains

The degree of selective removal of 4-0- methyl glucuronic acid (4-O-MeglCA) side groups by σ-D-glucuronidase of Schizophyllum commune (σ-glu) was determined using xylan derived from Eucalyptus grandis, Pinus patula, Bambusa balcooa, and bagasse found in South Africa.

The σ-glu from S. Commune was assessed for selective removal of 4-O- MeglcA side chains from xylan derived from hardwood, softwood and grass (including cereals) sources with the aim of developing a controlled enzymatic technology for diversification of the xylan functional properties. Therefore, the effect of hydrolysis time, temperature and enzyme xylan specific dosage on the removal of 4-0- MeglcA side chains, and the subsequent modification of viscosity, solubility, precipitation and aggregation of the xylan was examined. Xylan samples substituted with arabinose and /or 4-0 methyl glucuronic acid (4- O MeglcA) side chains (Figure 7). Oatspelt xylan (Sigma) and birch xylan (Roth) were utilised as model xylans. Xylan solution (1% w/v) for each material was prepared in de-ionized water (dH₂O). The xylan that showed limited solubility in water was prepared by first dissolving in ethanol and subsequently heating according to de Wet et al. [2008, Appl. Microbiol. Biotechnol. 77: 975-983]. Xylan solutions were made in bulk and stored in vials at -2O⁰C. Oat spelt xylan (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-O-MeglcA: glucose, and xylose) [Kormelink and Voragen, 1993, Carbohydr. Res. 249: 345-353] made in similar way were used as model xylan. The enzyme used was σ-D- glucuronidase (σ-glu) with specific activity = 300 nKat mg^'1 purified from wild type Schizophyllum commune (VTT-D-88362- ATCC 38548 (donated by Prof. Matti Siika-aho of VTT Biotechnology institute in Finland), for the selective removal of 4-O-methyl-D-glucuronic acid/D-glucuronic acid side groups. The enzyme aliquots were stored at 4⁰C.

A xylan solution (1% w/v) prepared from 4-O-MeglcA substituted substrates was treated with σ-glu (9000 nKat g^"1) in 5 ml_ reaction volumes consisting of 2.5 ml_ of the substrate and made up to 5 ml_ with 0.05M acetate buffer, pH 4.8. The reactions proceeded for 16 h at 4O⁰C.

4-0- MeglcA side chain sugar release was analysed using (HPAEC-PAD) on Carbopac PA 10 column eluted with helium degassed MiII-Q H₂O, 25OmM NaOH and 1M NaOAc (for acid sugars only). D-glucuronic acid was used as a standard sugar. Insolubilization, precipitation and aggregation of the xylan hydrogels were confirmed by visual inspection (photographs taken) and quantified by measuring viscosity using Rheometer (MCR501). The degree of xylan precipitation was quantified by determining residual xylose in solution using phenol-sulphuric assay for total sugar [Dubois et al., 1956, Annal. Chem. 28 (3):350-356].

A three factor Box-Behnken statistical design experiment with 3 central points making a total of 15 runs was run in duplicates; Statistica 7.0 software programme, (StatSoft, Inc., 1984-2005) was used for designing and analysis using a response surface method (RSM) as shown in Figure 8. Regression and ANOVA analyses were performed to determine the size and significance of individual and interaction effects of the hydrolysis parameters on viscosity. Optimal conditions were determined using the desirability function. The response surface plots were fitted with a second order polynomial as follows: Z = βo + β\x\ + β\ iXi² + βixi + βnxi¹ + βϊXi + βaxi² + ε Where: Z = Viscosity (mPa.s), /?o + /?i βn = linear regression coefficient, /?n....

/?_nn= Quadratic regression coefficient, e= Error, and X-i, X₂, X₃= Hydrolysis time, temperature, and enzyme xylan specific dosage.

Optimal conditions for side chain removal

The optimal combination levels of hydrolysis parameters were determined: time, temperature, and dosage of σ-D-glucuronidase for removal of 4-O-methyl glucuronic acid side chain.

The effect of xylan loading, enzyme loading, hydrolysis time, and temperature, and their interaction on σ-glu removal of 4-O-MeglcA side chains from oatspelt and birch xylan, was determined by using Response surface Methodology (RSM). Time, temperature, and enzyme xylan specific dosage constituted the independent variable whereas degree of side chain removal and viscosity change formed the dependent variables. Statistica 7.0 software programme, (StatSoft, Inc., 1984-2005) was used for designing and analysing the experiments. Statistical analysis included regression and ANOVA analyses. Pareto Chart plots were used to present size and significance of effects while the desirability and profiling function was used to determine optimal set point for the hydrolysis parameters. The oatspelt xylan (1% w/v) was prepared according to de Wet et al. [2008, Appl. Microbiol. Biotechnol. 77: 975-983]. The solutions were made in bulk and stored in vials at -2O⁰C. σ-D-glucuronidase (σ-glu) 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 of VTT Biotechnology Institute in Finland), was used for the selective removal of 4-O- methyl-D-glucuronic acid/D-glucuronic acid side groups. The enzyme aliquots were stored at 4⁰C. D-Glucuronic acid (Sigma) was used as standard sugar.

Optimization of hydrolysis parameters

Optimal set points for time, temperature, and enzyme dosage for the σ-glu removal of 4-0- MeglcA from birch xylan were determined in a three factor Box- Behnken statistical design with 3 central points making a total of 15 runs in duplicates. The hydrolysis parameters were each tested at two levels and middle point with the highest, middle and lowest levels denoted as 1, 0, and -1 respectively. Temperature was tested at 3O⁰C and 5O⁰C, time at 1 h and 16 h, and enzyme dosage of σ-glu was 2 000 nKat g^"1 and 18 000 nKat g^'1. The central points for temperature and time were 40 ⁰C, and 8.5 h, while for σ-glu xylan specific dosage were 360000 nKat g^'1 and 11000 nKat g^"\ respectively.

The variables were coded according to the equation:[jc. Xj=

coded value for variable.Xi = natural value, ΔXj= scaling factor (half the range of the independent variables which constituted Time, temperature, and enzyme xylan specific dosage)]

4-O- MeglcA side chains were analysed using (HPAEC-PAD) on Carbopac PA 10 column eluted with helium degassed MiII-Q H₂O, 25OmM NaOH and 1M NaOAc (for acid sugars only). D-glucuronic acid was used as a standard sugar.

The response surface plot was fitted with a second order polynomial which included both linear and quadratic interactions as follows:

Z

Ai 22*^ +A₂₃₃*²*? +* 000724

39

Where: Z= Response (degree of side chain removal), β₀ + βi β_n.... = linear regression coefficient, /?n.... /W...= Quadratic regression coefficient, X-i, X2, X3 = Hydrolysis time, temperature, and enzyme xylan specific dosage or xylan and enzyme loading, e= Error.

RESULTS

Extracting and characterising xylan from lignocellulosic materials

The chemical composition of bagasse, pine (Pinus patula), and bamboo (Bambusidae balcooa) is presented in (Figures 9-11). Bagasse had the highest ash (8.6%) and solvent extractives (6.2%) (Figure 9), lignin (30.0%) (Figure 10), cellulose (53.80%) and pentosans (22.00%) (Figure 11). Both E.grandis and P. Patula had ash and extractive contents of less than 3% (Figure 9). However, P. patula displayed the lowest pentosan level (8.49%). The cellulose level in E.grandis and bamboo was in the range of 40-43% (Figure 11) where as the lignin content was about 23 % (Figure 10). The extraction of xylan from P. patula, bagasse, E. grandis, and bamboo by the Hoije method gave extraction efficiencies of 71.20, 65.50, 35.20, and 20.20%, respectively (Figure 12) whereas extraction of xylan from bagasse and E.grandis using the Lopez method gave extraction efficiencies of 28.00 and 12.00 %, respectively (Figure 12).

The solid state ¹³C-CP/MAS NMR spectra for unprocessed, extractive free, and xylan extracted residue of P. patula, bagasse, E.grandis, and bamboo materials displayed characteristic signals originated from the six carbon resonances of the anhydrous glucose ring in cellulose which were assigned according to Atalla and lsogai [2005, Recent developments in spectroscopic and chemical characterisation of cellulose. In Dumitriu, S. (ed.) Marcel Dekker. New York, pp 123-157] (Figures 13A-D spectra 1). Beginning with the upfield of the spectra, the C6 of the primary alcohol group emerged at chemical shift (δ) 60-70 ppm and the resonances for a cluster of C2, C3, and C5 of the ring carbons other than those anchoring the glycosidic linkage were displayed at δ 70-81 ppm, the C4 resonances at δ 81-93 ppm, and the C1 at δ 102-108 ppm. In addition, typical doublets were displayed in C4 and C6 resonances (upper field) that represent less ordered (armophous) cellulose and in the ordered (crystalline) cellulose (downfield) (Atalla and lsogai, 2005) in spectra of all the lignocellulosic feedstock materials (Figures 13A-D). However, the doublets for C6 in the spectra of P. patula (Figure 13A spectra 1) were more resolved than in bagasse (Figure 13B), E.grandis (Figure 13C) and bamboo (Figure 13D). Furthermore, characteristic signals for acetyl groups (at δ 20 - 22 ppm, aliphatic groups at δ 30 - 40 ppm, methyl (CH₃) arising from lignin residues at δ 50-60 ppm, C1 of arabinose residues at δ 110 -120 ppm, aromatic compounds from lignin residues at δ 140 - 160 ppm, and C6 of uronic acid residues or carbonyl groups at δ 170 - 190 ppm were in accordance with Liitia et al. [2001, Holzforschung 55: 503-510]; Maunu [2002, Progress in Nuclear Magnetic Response Spectroscopy 40: 151-174]; Lahaye et al. [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 spectra of the unprocessed raw materials (Figures 13A-D spectra 1). The ¹³C-CP/MAS NMR spectra of the feedstock in which extractives were removed showed changes in line and splitting pattern of signals in the upfield of C4 and C6 s and the resonances between δ 81-93; 60- 70 and 20-22 ppm respectively (Figures 13A-D spectra 2). Whereas the¹³C- CP/MAS NMR spectra for feedstock from which xylan was removed showed disappearance or reduction in intensity of signals emanating from acetyl, aliphatic, methyl, aromatic, C6 of uronic/carbonyl groups at δ 20 - 22 , 30 - 40, 50-60, 140 - 160 and 170 - 190 ppm, respectively (Figures 13A-D spectra 3). The¹³C-CP/MAS NMR spectra for bagasse from which xylan was removed displayed sharpened signals particularly in resonances between δ 30 and 40 ppm which originate from aliphatic groups (Oliveira, 2008) and complete disappearance of reasonances arising from methyl groups at δ 40-50 ppm (Figure 13B spectra 3). Whereas in ¹³C-CP/MAS NMR spectra of P. patula, E. grandis, and bamboo, a reduced intensity of the signal for methyl group was notable (Figure 13A, C, D spectra 3, respectively).

The initial glucose levels in extractive free bagasse, bamboo, P. patula and E. grandis were 68.0, 66.0, 61.0, and 59.0 %, respectively. Upon xylan extraction using the Hoije method the glucose proportion in the feedstocks increased to 75.0, 76.0, 65.0, and 79.0%, respectively while xylose concentration decreased from 14.0 to 10.0 %, 27.0 to 25 %, 35 to 19%, 30.0 to 22.0% in P. patula, bagasse, E. grandis and bamboo, respectively (Figures 14A-D). Furthermore the xylan extraction corresponded to a decrease in arabinose, and galactose content in all the feedstock (Figures 14A-D). The concentration of mannose (16.0%) which was detectable only in P. patula feedstock increased to 18% in the xylan extracted residue (Figure 14A). The presence of uronic acids was detectable in all the four lignocellulosic feedstock materials (Figure 15). The highest and lowest uronic acids content were found in E. grandis and bagasse feedstocks, respectively (Figure 15).

The elution profiles of the extracted xylan fractions were referenced to the elution profiles of the monomeric sugars (arabinose, rhaminose, galactose, glucose, xylose, and mannose), xylitol sugar, birch xylan, oatspelt xylan, and H₂O₂ bleached bagasse (Bag B). The HPAEC-PAD (Dionex) chromatogram showed that the monomeric sugars including the xylitol eluted on CarboPac PA 100 column within a retention time of 5 min (Figures 16A and B). Between 0 and 3 min, oatspelt xylan elution profile showed a high intensity peak with detector response > 300 nC which corresponded to retention time for xylitol (Figure 16D). Otherwise, between 3 and 6 min the chromatogram for both birch xylan and the oatspelt xylan displayed only low intensity peaks (detector response of < 20 nC) (Figures 16C and D respectively). Whereas, the chromatogram for H₂O₂ bleached bagasse xylan (Bag B) displayed multiple peaks of high intensities with detector response of over 100 nC appearing at retention time between 2 and 30 min (Figure 17A). The chromatograms of both Hoije and Lopez extracted xylan displayed low intensity peaks (<20 nC) within 25 min retention time (Figure 17B and C). Among the extrcated xyla samples, the peak corresponding to xylitol was present in the chromatogram of xylan from E. grandis (Figure 18A), bamboo (Figure 18C) and P. patula (Figure 18D) extracted by the Hoije method.

The xylose content of xylan from E. grandis extracted by Hoije method (EU H), bamboo, bagasse extracted by Hoije method (Bag H), and P. Patula was 92.00, 79.50, 71.00 and 61.30%, respectively (Figure 15). Whereas, the xylose content in birch and oatspelt xylan was 80.00 and 87.20 %, respectively (Figure 15). The proportion of arabinose in Bag H, P. patula, and bamboo xylan fractions was 17.45, 15.50, and 10.50%, respectively (Figure 15). Although, commercial oatspely xylan is reported to have 10% arabinose (Sigma), this study showed arabinose content of 7.4 % (Figure 15). About 2.30 % glucose was present in EU H xylan fraction whereas 13.20% glucose was present in P. patula xylan fractions (Figure 15). In addition, the EU-H contained 4.45 % galactose and traces of rhaminose and arabinose (Figure 15). Total uronic acid content of Uronic acid content of EU H, bamboo, and P. Patula xylan was 12.83, 11.20 and 11.54% whereas bagasse xylan contained 8.5% (Figure 15). The xylan fractions extracted by Hoije method when subjected to mild acid (72% H₂SO₄) hydrolysis yielded between 16 and 55% insoluble residues (Figure 19). The highest acid insoluble residues 55% were obtained from xylan extracted from P. Patula using the Hoije method. The acid insoluble residues from bagasse xylan extracted by the Lopez method (Bag L) was 16% whereas the reference material, birch xylan had 3.5% (Figure 19).

Structural characteristics of the extracted xylan

The ¹H NMR and ¹³C NMR spectra of the reference xylan from birch, H₂O₂ bleached bagasse, and oatspelt xylan displayed characteristic signals for proton and carbon resonances (Figure 20A-D). The ¹H NMR spectra of the extracted xylan displayed characteristic proton signals from xylose, 4-0- methylglucuronic acid, and arabinose units at chemical shifts (δ) between 3.3 to 5.7 ppm (Figures 20-22). In the ¹H NMR the proton signals for xylose in the xylan fractions from bagasse extracted by Hoije (Bag H) and by Lopez (Bag L) methods were displayed 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 of xylose unit substituted with 4-0- methylglucuronic acid linked at O-2, respectively. In addition, the proton spectra for Lopez extracted bagasse (Bag L) and Hoije extracted bagasse (Bag H) displayed proton resonances from D- xylopyranosyl units residues substituted with 4-O- methylglucuronic acid at O-2 and acetyl group at O-3 at δ 4.72., 3.76/3.75, 3.97/3.96 ppm that correspond to M ₁ H2 and H4 of the β-D- xylopyranosyl units, respectively (Figure 21 A).

The proton spectra of xylan extracted from E. grandis by Lopez method (EU-L) and Hoije method (EU-H) displayed signals at δ 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) such chemical shifts could originate from D-xylopyranosyl units residues substituted with 4-O- methylglucuronic acid at O-2 and acetyl group at 0-3. In the same spectra, proton signals originating from H1 and H3 of the 4-O- methylglucuronic acid residues were evident at δ 5.48/5.49/5.46 ppm, and between 1.06 and 1.54 ppm δ 5.16 and 3.63 ppm (Figure 21C). In the proton spectra of bamboo, and P. patula, signals for the 4-0- methylglucuronic acid residues were displayed at 5.47/5.48 ppm (Figure 21 A and C). Such signals could appear at δ 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; Ebringerova et al., 1998, Carbohydrate Polymers 37: 231-239]. The proton spectra of the extracted xylan further displayed characteristic signals originating from C-2 linked arabinose to xylose units [Hδije et al., 2005, Carbohydr. Polym. 61 : 266-275; Ebringerova et al., 1998, Carbohydrate Polymers 37: 231-239]. In the proton spectra for Bag H and Bag L, the C2 linked arabinose were identified at δ 5.58, 5.60, 4.29/4.30 ppm (Figure 21 A and C). The presence of arabinose in the proton spectra of bamboo and P. patula xylan were in accordance with Ebringerova et al. (1998) and Vignon and Gey, (1998) identified inter alia, at δ 5.58/5.59 ppm and 5.47/5.48 ppm, respectively (Figures 22A and C). Arabinose signals were present in the proton spectra of EU-L and EU-H at δ between 3.83 and 3.85 ppm (Figure 21C) whereas the presence of O- 2 linked acetyl groups which based on Shao et al. [2008, Wood Science Technology 42: 439-451]; Hόije et al.(2005) Sun et al. (2004); Ebringerova et al. (1998); Vignon and Gey (1998) supra were identified at δ 4-2.4 ppm (Figures 20-22). In addition, broad signals associated with aromatic or phenolic compounds originating from lignin residues (Hδije et al., 2005 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.Q. (ed). Chapter 10: 171-188] were displayed between δ 6.5 and 7.9 ppm in the proton spectra of the extracted xylan (Figures 21 and 22).

The characteristic carbon resonances from the five carbons of (1→ 4) linked β-D- xylopyranosyl residues between δ 103 and 62 ppm were reflected in the ¹³C NMR spectra of the extracted xylan fractions (Figures 21 and 22). In the carbon spectra of E. grandis xylan fractions, the resonance originating from C1 of xylose units with C2- linked arabinose groups appeared at δ «102.33 ppm while those from C1 , C2, C3, C4, and C5 of arabinofuranosyl residues displayed at δ =408, 81.7, 78, 85.5, and 62 ppm (Figure 21D). In the spectra the arabinose associated carbon signals in EU H and EU L were seen at δ 61.81/61.59 ppm (Figure 21D). In Bag L and Bag H the arabinose signals identified based on Vignon and Gey (1998) supra were displayed at δ 112.50/ 111.56 and 89.31 ppm which correspond to C1 and C2 of C-2 linked arabinose residues, respectively (Figure 21B). The C1, C2, C4 resonances belonging to arabinofuranosyl residues mono substituted xylose units at O-3 (Ebringerova et al., 1998, supra) were present in the ¹³C NMR spectra of both bamboo and P. patula at δ 108.60/108.33 ppm, 81.71/81.42 ppm, 85.72/85.43 ppm respectively (Figure 22B and D). The presence of 4-0- methyiglucuronic acid residues in the extracted xylan fractions was evident from characteristic carbon signals that originate from C1, C4, C6 and C5 at δ between 97 and 100 ppm, 83 and 84 ppm, 179 - 172 ppm, and 59 and 61 ppm, respectively (Habibi and Vignon, 2005, Carbohydrate Research 340: 1431-1436; Xu et al., 2000, supra; Ebringerova et al.,1998, supra) (Figures 21 and 22). The presence of acetyl, phenolic, and aromatic groups arising from lignin compounds, and hexose sugars were identified in the ¹³C NMR spectra of the extracted xylan fractions. In addition, acetyl groups in all the xylan fractions were identified between δ 21-24 ppm (Figures 21 and 22). However, absence of the acetyl signals was apparent in ¹³C NMR spectra of birch xylan (Figure 20B) and H₂O₂ bleached bagasse (Bag B) (Figure 20D). The methoxyl groups signifying presence 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. (ed)]; were identified in the ¹³C NMR spectra at δ 56.62/ 56.58 ppm (Figures 21 and 22). The lignin compounds particularly those linked to arabinosyl side chains through ferulic acid bridges were reflected by carbon signals at δ 140-160 ppm and 116.6- 117.08 ppm (Figures 21 and 22). Other lignin compounds associated with ferulic or p-coumaric acids groups and of the -CH₃- in Ar-COCH₃ (Maunu, 2002, Progress in Nuclear Magnetic Response Spectroscopy 40: 151-174; Sun et al. 2004, supra) were seen at δ 26-49 ppm at δ 115.38 and δλ 7.55/17.69 ppm in Bag L (Figure 21 B spectra 1) bamboo and P. Patula xylan fractions (Figures 22B and D). The presence hexose sugars such as galactose or glucose [Sun et al., 2004, supra] was evident in the ¹³C NMR spectra, in particular of EU H and EU L at δ between 69 and 71 ppm (Figure 21B).

The FTIR spectra of the extracted xylan fractions displayed characteristic bands for xylan residues which included /?-glycosidic linkages reflected at «897 cm^'1 (Figure 23). However, such signal was absent in the FTIR spectra of the extracted xylan from P. Patula. In addition the spectra of the extracted xylan displayed signals in the band region between 1600 and 1200 cm^'1 (Figure 23), which according to Fengel and Wegener [1989, Wood Chemistry, Ultrastucture, Reactions. Walter de Gruyter, Berlin, Germany] is a region associated with aromatic compounds that originate from lignin fractions. Bands arising from syringyl ring breathing with C_Ar-OCH₃, and methoxyl groups in lignin were reflected at 1329 cm^"1 and 1591-1595, and 1460-1461 cm^"1 in the spectra of E. grandis and bamboo (Figure 23). The bagasse xylan fractions, Bag H and Bag L contained signals of varying intensities in the 1600 - 1200 cm^"1 wavelength region and spectra for Bag L reflected a relatively strong intensity band for C-H stretching vibrations at 2919 cm^"1 (Figure 23). The FTIR spectra for EU L displayed two intense signals related to lignin compounds at 1591 and 1379 cm^{" 1} while in EU H spectra, multiple bands of lesser intensity in the 1600-1200 cm^"1 region in particular at 1595, 1461, and 1329 cm^"1 were seen (Figure 23).

Controlled enzymatic removal of side chains from lignocellulosic feedstocks

The purified σ-glucuronidase (cr-glu) from Schizophyllum commune removed 1.2 mg g^"1 4-O-MeglCA (1.3% available uronic acid) from birch xylan whereas about

1.6 mg g^'1 4-O-MeglCA (2 % available uronic acids)was released from the BH xylan fractions (Figure 24). The proportion of 4-O-MeglCA removed from

Eucalyptus grandis xylan extracted by the Hoije method (EH) and from

Eucalyptus grandis xylan gel (ES) was about 1.3 mg g substrate^"1 (Figure 24). The lowest σ-glu removal of 4-O-MeglCA was < 0.6 mg g substrate^"1 from H₂O₂ bleached bagasse (BB) (Figure 24).

Determination of optimal conditions for removal of side chains

Similarly, the response surface plots for removal of glucuronic acid (4-O- MeglcA) from birch xylan by σ-glu reflected both linear and quadratic relationships with hydrolysis time, temperature, and σ-glu xylan specific dosage. A maximum of 350 ug g^"1 substrate of 4-O-MeglcA was removed from birch by σ-glu at xylan specific dosage between 16500 and 18000 nkat g substrate^"1 when hydrolysis was performed for durations of between 9 and 10.2 h at temperatures between 33.5 and 42⁰C (Figure 25A-C).

The hydrolysis parameters showed significant effects on removal of 4-O-MeglcA from birch xylan by σ-glu and were in descending magnitude, from linear effects from σ-glu xylan specific dosage [σ-glu, nKat/g (L)], temperature [Temp (L)], and the quadratic effect of temperature [Temp (Q)] (Figure 26, Pareto chart). The only significant interaction effect on the removal of 4-O-MeglcA from birch xylan by σ-glu was from the linear effect of hydrolysis time and the quadratic effect of temperature [ time (L) by temperature (Q)] (Figure 26, Pareto chart). The optimal set points for σ-glu removal of 4-O-MeglcA were between 9 h and 10.2 h, 33.5 and 42 ⁰C, and 16500 and 18000 nKat g substrate^'1. The regression coefficients of the variable in the second-order polynomial model fitted to the response surface plots for 4-O-MeglcA removal as a function of time, temperature and enzyme dose gave a regression coefficient R² of 0.90 (R² adjusted = 0.81) (Figure 27).

Claims

1. An isolated polypeptide having σ-glucuronidase activity and that can degrade a glucuronoxylan molecule by hydrolysis of a glycosidic linkage between a MeGIcA residue and a non-terminal xylopyranosyl residue.

2. 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 at least 95% homologous to SEQ ID NO 1 or part thereof;

iii. an amino acid sequence at least 85% homologous to SEQ ID NO 1 or part thereof;

iv. an amino acid sequence at least 75% homologous to SEQ ID NO 1 or part thereof; and

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 the following group:

i. an amino acid sequence at least 65% homologous to SEQ ID NO 1 or part thereof;

ii. an amino acid sequence at least 50% homologous to SEQ ID NO 1 or part thereof; and iii. 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.

5. An isolated polynucleotide encoding a polypeptide according to any one of claims 1 to 4, the polynucleotide having a nucleotide sequence selected from the following group:

i. the nucleotide sequence of SEQ ID NO 2;

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 thereof.

6. A method of isolating a polypeptide according to any one of claims 1 to 4, the method including the steps of

i. culturing a microbe capable of expressing the polypeptide in induction medium; and

ii. isolating the polypeptide from the induction medium.

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

Diα9ftPrnrv4n e>n7vme> with a-alucuronidase activity)

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, Aspergillus fumigatus, Aspergillus terreus, Aspergillus oryzae, Sclerotinia sclerotiorum, Botryotinia fuckeliana, Pyrenophora tritici-repentis,

Neurospora crassa, Gibberella zeae, Podospora anserina, Coprinopsis cinerea okayama, Magnaporthe grisea, Fusarium sporothchioides, Cryptococcus neoformans var. neoformans, Cellvibrio japonicus, Saccharophagus degradans, Opitutus terrae, Phaeosphaeria nodorum, Bacteroides ovatus, and Streptomyces pristinaespiralis.

9. The method of isolating the polypeptide according to claim 8, wherein the microbe is Pichia stipitis CBS 6054.

10. A substantially enriched preparation of a polypeptide according to any one of claims 1 to 4.

11. A substantially enriched preparation according to claim 10, wherein the polypeptide is obtained from a culture of a microbe selected from the group including Schizophyllum commune, Aspergillus clavatus, Neosartorya fischeri, Aspergillus fumigatus, Aspergillus terreus, Aspergillus oryzae, Sclerotinia sclerotiorum, Botryotinia fuckeliana, Pyrenophora tritici-repentis, Neurospora crassa, 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.

12. A substantially enriched preparation according to claim 11 , wherein the polypeptide is obtained from a culture of Pichia stipitis CBS 6054.