Temperature-stable β-pyranosidase

Abstract

The invention relates to temperature-stable polypeptides with -pyranosidase activity. The polypeptide substrates include -glucopyranosides and -xylopyranosides. The polypeptides can be expressed alone or as fusion proteins for example in yeast or bacteria and subsequently purified. The polypeptides according to the invention can be used alone or in a mixture with other enzymes for the degradation of plant raw materials, among others for the enzymatic degradation of biomass containing lignocellulose, in particular hemicellulose and the hemicellulose component xylan. The enzymes are suitable for use in textile processing, as an additive of detergents, or in the food or feed industry.

Claims

1. A fusion protein, said fusion protein comprising: a first polypeptide and a second polypeptide, said first polypeptide comprising: an amino acid sequence having at least 90% sequence identity with SEQ ID NO. 2, and a catalytic domain capable of hydrolysing a -glycosidic bond on a pyranose; wherein said first polypeptide is a recombinant polypeptide having -pyranosidase activity; wherein said -pyranosidase activity is thermostable in the temperature range of 60-100 C.; and wherein said second polypeptide is at least one of a signal peptide, an affinity tag, or a protease cleavage site.

2. The fusion protein according to claim 1, wherein the -pyranosidase activity is -xylopyranosidase activity, -glucopyranosidase activity or a combination of these two activities.

3. The fusion protein according to claim 1, wherein said first polypeptide is capable of hydrolysing a -glycosidic bond on a xylose.

4. The fusion protein according to claim 1, wherein said first polypeptide is capable of hydrolysing at least one of the -glycoside bonds contained in a substrate, wherein said substrate is at least one of xylobiose, xylotriose, xylotetraose, or xylan.

5. The fusion protein of claim 1 wherein the pNP--cellobioside activity of said first polypeptide is 5% or less compared with the pNP--xylobioside activity of said first polypeptide wherein such activity is determined for each of pNP--cellobioside and pNP--xylobioside by incubating 0.01-6.0 mM at 60-90 C., in about 50 mM universal buffer at a pH of 5-7 for 5 to 30 minutes.

6. A vector containing a nucleic acid, said nucleic acid encoding a polypeptide said polypeptide comprising: an amino acid sequence having at least 90% sequence identity with SEQ ID NO. 2, and a catalytic domain capable of hydrolysing a -glycosidic bond on a pyranose; wherein said polypeptide is a recombinant polypeptide having -pyranosidase activity; wherein said -pyranosidase activity is thermostable in the temperature range of 60-100 C.

7. A host cell transformed with a vector according to claim 6.

8. The host cell according to claim 7, said host cell being an eukaryote selected from the group consisting of Saccharomyces cerevisiae, Yarrowia lipolytica, Schizosaccharomyces lactis, Kluyveromyces lactis, Pichia methanolytica, Pichia pastoris, Pichia angusta, Hansenula polymorpha, Aspergillus niger, Chrysosporium lucknowense, -Trichoderma reesei, and Penicillum sp.

9. The host cell according to claim 7, said host cell being a methylotrophic yeast selected from the group consisting of Pichia methanolytica, Pichia pastoris, Pichia angusta, and Hansenula polymorpha.

10. The host cell according to claim 7, said host cell being a prokaryote selected from the group consisting of Bacillus sp., Bacillus subtilis, Bacillus licheniformis; Bacillus megaterium, Thermus thermophilus, Pseudomonas fluorescens, Fervidobacterium sp., and Escherichia coli.

11. The host cell according to claim 7, said host cell being a Bacillus subtilis.

Description

DETAILED DESCRIPTION OF THE INVENTION

(1) Polypeptides with -Pyranosidase Activity

(2) The present invention relates to polypeptides with -pyranosidase activity comprising an amino acid sequence having at least 71%, if possible at least 75%, if possible at least 80%, and preferably at least 85% sequence identity with SEQ ID NO 2. The sequence identity is determined as described in the section Definitions.

(3) SEQ ID NO. 2 is contained in SEQ ID NO. 1. SEQ ID NO. 1 contains the amino acid sequence of the polypeptide FgXyl3a. The DNA underlying the SEQ ID NO. 1, represented by SEQ ID NO. 3, originates from the gene bank clone Bgl13 which contains a section of genomic DNA from Fervidobacterium gondwanense (F. gondwanense). Finding the SEQ ID NO. 3 and determining the domain structure are described in detail in example 3.

(4) In short, SEQ ID NO. 2 comprises that region of SEQ ID NO. 1 which contains both structural domains of the glycoside hydrolase and was determined by a sequence comparison with SEQ ID NO. 4 (enzyme BglB of Thermotoga neapolitana, Pozzo et al., 2010, J. Mol. Biol., 2: 397 (3), 724-739). SEQ ID NO. 2 thus comprises amino acid residues 13 to 638 of SEQ ID NO. 1.

(5) The present invention also relates to polypeptides comprising an amino acid sequence which has at least 70%, if possible at least 75%, if possible at least 80%, and preferably at least 85% sequence identity with SEQ ID NO. 1.

(6) The invention further comprises polypeptides comprising a structural domain 1 of a glycoside hydrolase of family 3 (GHF3) and a structural domain 2 of a glycoside hydrolase of family 3 (GHF3), wherein at least one of these two domains has at least 70%, if possible at least 75%, if possible at least 80%, and preferably at least 85% sequence identity with the corresponding domain FgXyl3a, hat. Structural domain 1 of FgXyl3a consists of the region comprising amino acid residues 13 to 381 and 586 to 383 of SEQ ID NO. 1; the structural domain 2 of FgXyl3a consists of the region comprising the amino acid residues 382 to 585 of SEQ ID NO. 1. The method for determining structural domains is described in example 1. It may be applied generally to polypeptides the sequence identity of which satisfies at least the criteria described herein.

(7) The cloning (example 2), expression (example 3), purification from a cell extract (examples 4 and 5) and determination of the molecular weight (example 6) of a polypeptide with -pyranosidase activity according to the present invention are described in the examples.

(8) The -pyranosidase activity of the polypeptide according to the present invention is selected from -xylopyranosidase activity, -glucopyranosidase activity and a combination of these two activities, the -xylopyranosidase activity being preferred. The detection of activity and the determination of the substrate specificity of a polypeptide according to the present invention are described in detail in examples 7 and 8.

(9) In a preferred embodiment, the present invention relates to a polypeptide with -pyranosidase activity which satisfies at least one of the above criteria and which is capable of hydrolysing at least one of the -glycoside bonds contained in the following substrates: xylobiose, xylotriose, xylotetraose, xylan. The determination of the hydrolysis products of a polypeptide with -pyranosidase activity is described in detail in example 13.

(10) In more preferred embodiment, the present invention relates to a polypeptide with -pyranosidase activity which satisfies at least one of the above criteria and the activity of which vis-vis pNP--cellobioside is markedly lower than vis-vis pNP--xylobioside, namely 5% or less vis-vis pNP--cellobioside compared with the activity vis-vis pNP--xylobioside. A method for determining substrate specificity is described in example 8.

(11) In a yet more preferred embodiment, the present invention relates to a polypeptide with -pyranosidase activity which satisfies at least one of the above criteria and which has maximum activity vis-vis pNP--xylopyranoside in the acidic, neutral or slightly basic range, i.e. in a range of pH 4.0 to pH 8.0, therein, if possible, in the slightly acidic to neutral range, i.e. in a range of pH 5.5 to 7.0, and preferably in a range of pH 6.2 to 6.8. A method for determining the influence of the pH value on the activity of a polypeptide with -pyranosidase activity is described in more detail in example 11.

(12) In a yet more preferred embodiment, the present invention relates to a pH-stable polypeptide with -pyranosidase activity which satisfies at least one of the above criteria and the maximum activity of which vis-vis pNP--xylopyranoside after 48 hours of incubation at a pH of 9.0 is at least 50%, if possible at least 60%, if possible at least 70%, and preferably at least 80%. A method for determining the influence of the pH value on the activity of a polypeptide with -pyranosidase activity is described in more detail in example 11.

(13) In a yet more preferred embodiment, the present invention relates to a polypeptide with -pyranosidase activity which is active at high temperature and which satisfies at least one of the above criteria and which has maximum activity vis-vis pNP--xylopyranoside in a range between 60 C. and 100 C., if possible between 70 C. and 95 C., and preferably between 80 C. and 90 C. A method for determining the influence of the temperature on the activity of a polypeptide with -pyranosidase activity is described in more detail in example 9.

(14) In an yet more preferred embodiment, the present invention relates to a temperature-stable polypeptide with -pyranosidase activity which satisfies at least one of the above criteria and the maximum activity of which vis-vis pNP--xylopyranoside after 3 hours of incubation at 60 C. is at least 40%, preferably at least 50%. A method for determining the influence of the temperature stability on the activity of a polypeptide with -pyranosidase activity is described in more detail in example 10.

(15) Fusion Protein

(16) The polypeptide according to the present invention may either occur in the isolated form or it may be fused to one or more additional oligo- or polypeptides. That is, in a further preferred embodiment the polypeptide satisfying at least one of the criteria described above may be present as a fusion protein, wherein it is preferably fused to one of the following: a carbohydrate binding domain of another protein, a signal peptide, an affinity tag or a protease cleavage site.

(17) The fusion protein according to the present invention is not limited to the method by which it has been obtained but comprises fusion proteins of all kinds as long as they contain a component satisfying at least one of the above criteria. One option is to obtain the fusion protein according to the present invention by methods of molecular biology. As a person skilled in the art will know, methods for preparing nucleic acids encoding fusion proteins are standard methods of molecular biology which are described in greater detail, for example, in Sambrook et al. (Molecular cloning, a laboratory manual, 2.sup.nd edition, 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). In a preferred embodiment, the fusion protein besides the polypeptide with -xylosidase activity according to the present invention may be fused to a carbohydrate binding domain of another protein. In a preferred embodiment, the polypeptide with -xylosidase activity according to the present invention may be fused to a polypeptide selected from one or more of the following group: signal peptide, affinity tags, protease cleavage site. Translational coupling may be used to direct the expressed polypeptide according to the present invention to cellular compartments or organelles or to export it from the host cell. Signal peptides which are also called signal sequences are well known to a person skilled in the art and include the lead sequences of the periplasmatic proteins OmpA, OmpT, PelB, PhoA. Signal sequences for the export of proteins are found, for example, in naturally occurring secreted proteins, for example proteins with carbohydrate-modifying characteristics such as cellobiohydrolase I or II, endoglucanasae, AmyE, and in S. cerevisiae Mf or chicken egg lysozyme. Cleavage sites for proteases which are suitable as tags for recombinantly expressed proteins are well known to a person skilled in the art. Protease cleavage site means a poly- or oligopeptide comprising a peptide bond which may be specifically cleaved by a certain protease, and a recognition sequence which is usually near the cleavage site and is recognised by the corresponding protease. The protease cleavage sites that may be used in accordance with the present invention are not subject to any limitation. They expressly include the cleavage site of the tobacco etch virus (TEV) protease, the cleavage sites of the blood coagulation factors from mammals such as factor Xa or thrombin. The affinity tags that may be used according to the present invention are not subject to any limitations. As a person skilled in the art knows, affinity tags that have proved to be advantageous for the purification of many polypeptides are described in detail, for example, in Sambrook et al. (Molecular cloning, a laboratory manual, 2nd edition, 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). Expressly included in the definition of the present invention is an oligohistidine tag as described in examples 1 and 5.

(18) Enzyme Mixture

(19) A mixture of enzymes may also be used in accordance with the invention. This mixture contains the polypeptide with -pyranosidase activity which satisfies at least one of the above criteria and comprises one, two or more additional enzymes. These additional enzymes may be selected from the group comprising pectinases, endoxylanases, -glucosidases, -glucanases, cellobiohydrolases, -xylosidases, -arabinofuranosidases, -glucuronidases, acetyl xylan esterases. The enzyme mixture according to the invention thus comprises the polypeptide with -pyranosidase activity which satisfies at least one of the criteria described above and one or more pectinases and/or one or more endoxylanases and/or one or more -glucosidases and/or one or more -glucanases and/or one or more cellobiohydrolases and/or one or more -xylosidases and/or one or more -arabinofuranosidases and/or one or more -glucuronidases and/or one or more acetyl xylan esterases.

(20) In a preferred embodiment, the enzyme mixture according to the invention comprises the polypeptide with -pyranosidase activity which satisfies at least one of the criteria described above and one or more of -arabinofuranosidases and/or one or more -glucuronidases and/or one or more acetyl xylan esterases.

(21) Unless specifically stated otherwise, the term comprises in the present application is used to indicate that further components may optionally be present in addition to the components listed under comprises. However, it is considered a special embodiment that the term comprises includes the possibility that no other components are present, i.e. the term comprises under this special embodiment is understood to mean the same as consists of.

(22) Nucleic Acid and Vector

(23) The present invention also relates to a nucleic acid encoding the polypeptides described above. One example for such a nucleic acid is shown by SEQ ID NO. 3. The nucleic acid according to the present invention may be a component of an expression cassette. A person skilled in the art will be well aware of the typical components of an expression cassette; for example, they are described in more detail in Sambrook et al. (Molecular cloning, a laboratory manual, 2.sup.nd edition, 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). In addition to the coding sequence, the expression cassette typically contains at least: one promoter and one terminator. The expression level of a gene encoding a polypeptide according to the present invention may be adjusted by the number of copies of the gene introduced into the host cell; preferably more than one copy is present. The promoter may be adjusted for optimised expression so that it will, for example, respond to the addition of a chemical or the change of one or more physical parameters in such a manner that the induction of a gene may be switched on or off. Examples for inducible promoters include the tetracycline repressor system, the Lac-repressor system (Beneyx, 1999, Curr. Opin. Biotechnol. 10: 411: 422), systems inducible by copper ions (Hottinger et al., 2004, Yeast, 10: 283-296), systems inducible by methanol (Cereghino et al., 2000, FEMS Microbiol. Reviews 24: 45-66) or the temperature-inducible PL promoter. Alternatively, derepression of the promoter by reaching a favourable physiological state of the culture may be a useful strategy (promoters PhoA, Trp, Adh2, Fmdh, CBH1 (Price et al., 1990, Methods Enzymol., 185-308-318; Hollenberg, 1995, U.S. Pat. No. 3,589,585). Methods to further increase the yield include the co-expression of one or more proteins involved in the translation, proteins involved in target control, proteins involved in folding (for example chaperones of the Hsp70-family, protein disulfide isomerases), or of proteases assisting correct processing.

(24) The expression cassette may be integrated in a vector which is either propagated episomally in the host cell or is integrated in its genome. Examples of typical vectors are bacterial plasmids, yeast plasmids, centromer-containing linear DNA, constructs of viral origin such as SV40, phage DNA, bacculovirus, vaccinia, adenovirus, chicken pox virus, pseudo-rabies and combinations of vectors of bacterial, eukaryotic and viral origin. Integration may be achieved by methods well known to a person skilled in the art of molecular biology, such as homologous recombination, transposition, or by using viral transfection systems. Also included are episomal systems for expression, one or more copies of which are integrated into the genome of the host cell either in a planned or unplanned manner. Moreover all vector systems are included which permit the heterologous expression of a polypeptide according to the invention in a host cell.

(25) Host Cell

(26) The present invention also relates to a host cell transformed with the vector described above. Preferred methods for introducing the vector construct into the host cell include transformation, transfection, conjugation and hybridisation. Transformation may be effected by electroporation, protoplast fusion, lipofection, ballistic bombardment, chemical transformation based on calcium chloride, polyethylene glycol (PEG) or manganese chloride. Other strategies include the application of viral particles. Another option is the use of naturally competent organisms as host cells.

(27) The host cell according to the present invention may be a prokaryote or a eukaryote. A eukaryotic host cell is preferably selected from the groups consisting of Saccharomyces cerevisiae, Yarrowia lipolytica, Schizosaccharomyces lactis, Kluyveromyces lactis, Pichia methanolytica, Pichia pastoris, Pichia angusta, Hansenula polymorpha, Aspergillus niger, Chrysosporium lucknowense, Trichoderma reesei, Penicillum sp.

(28) In a particularly preferred embodiment, the eukaryotic host cell is a methylotrophic yeast, preferably from the group including Pichia methanolytica, Pichia pastoria, Pichia angusta, Hansenula polymorpha.

(29) A prokaryotic host cell according to the present invention is preferably selected from the group comprising Bacillus sp., Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium, Thermus thermophilus, Pseudomonas fluorescens, Fervidobacterium sp., Escherichia coli.

(30) After transformation of the host cell and growth to the desired cell density, the selected inducible promoter may be induced by a chemical activator or by changing one or more physical parameters so as to induce the cultivated host cells to produce the desired polypeptide.

(31) Purification Method

(32) The present invention also relates to a method for purifying the above-described polypeptide with -pyranosidase activity. In a particular embodiment, the polypeptide is not secreted by the host cell. After culturing, the host cells are then isolated and the polypeptide according to the present invention contained therein is isolated. Such a process is described in examples 3 to 5. In a particular embodiment, the polypeptide according to the present invention is produced as a fusion protein with at least one signal peptide which directs the recombinant protein to secretion from the host cell.

(33) In detail, the process comprises the following steps: a) obtaining the host cell transformed with a vector as described above; b) cultivating the host cell under conditions where the polypeptide with -pyranosidase activity is expressed, c) purifying the polypeptide with -pyranosidase activity.

(34) The host cell according to a) may be recovered by methods well known to a person skilled in the art, for example as described in the chapter host cell.

(35) The manner in which step b) is performed is influenced by many factors which are basically known to a person skilled in the art. Thus the growth medium, the growth temperature and other conditions of growth are selected depending on the kind of host cell used. The conditions under which the polypeptide is expressed further depend especially on the selection of the promoter used as described above. Different methods for harvesting host cells from a protein-expressing cell culture are well known to a person skilled in the art and include, for example, centrifugation of the medium containing the host cells and filtration of the medium containing the host cells. Then the cells are typically subjected to lysis, either by chemical or mechanical methods or by a combination of both.

(36) The manner of performing step c) is selected depending on the nature of the expressed polypeptide. In particular embodiments, in which a fusion protein containing the polypeptide according to the present invention and another domain such as an affinity tag is expressed, the characteristics of the affinity tag may also be used to purify the fusion protein. One example for such an affinity tag is a tag comprising several, typically at least six, histidine residues as described in examples 3 to 5.

(37) In a preferred embodiment, step c) of the process for purifying the polypeptide described above comprises heat precipitation. For this purpose, a mixture of different polypeptides comprising the desired polypeptide is brought to a certain temperature which is generally higher than the temperature at which the host cell grew during expression. This mixture may comprise two or more different polypeptides. Such a mixture may also be a lysate which is produced when the cells expressing the desired polypeptides are subjected to lysis, for example with mechanical or chemical methods or a combination of both, or an extract obtained or enriched from said lysate.

(38) Depending on their properties, different proteins precipitate at different elevated temperatures. In this way, an enrichment in the in the cell lysate or the extract recovered therefrom can be achieved. By further increasing the temperature, the desired protein may optionally be precipitated.

(39) Use

(40) The present invention also relates to the use of the polypeptide with -pyranosidase activity or the mixture containing the polypeptide with -pyranosidase activity described above for any purpose for which such enzyme activity is required or desired.

(41) In particular, the present invention describes the use of the polypeptide with -pyranosidase activity or the mixture containing the polypeptide with -pyranosidase activity for degrading one or more substrates containing one or more -xylopyranosidic and/or one or more -glucopyranosidic bonds, hereinafter called -xylopyranoside or -glucopyranoside.

(42) In a preferred embodiment, the present invention relates to the above-described use for the enzymatic degradation of biomass containing lignocellulose.

(43) In a more preferred embodiment, the present invention relates to the above-described use in textile processing. In a particularly preferred embodiment, the present invention relates to the above-described use as an additive to detergents. In a further particularly preferred embodiment, the present invention relates to the above-described use in the food and/or feed industry. Finally, in a further particularly preferred embodiment, the present invention relates to a combination of two or more of these applications.

(44) In a particularly preferred embodiment, the present invention relates to the above-described use for the production of fruit juices. In a more preferred embodiment, the present invention relates to the above-described use for the production of wine or beverages containing wine, and in a further more preferred embodiment, the present invention relates to the above-described use for the production of beer or beverages containing beer. In a further more preferred embodiment, the present invention relates to the above-described use for the production of oil, preferably olive oil, rapeseed oil or sunflower oil. Finally, in a further more preferred embodiment, the present invention relates to the above-described use for the production of baked goods.

EXAMPLES

Example 1

Identification and Characterisation of -Pyranosidase FgXyl3A of Fervidobacterium Gondwanense

(45) For identification of cellulolytic and xylanolytic activity, a gene bank of F. gondwanense was screened. The gene bank was prepared according to the following method: Genomic DNA was purified from Fervidobacterium gondwanense cells using the Qiagen genomic DNA isolation kit (Qiagen GmbH, Hilden). The DNA obtained was degraded into clonable fragments of several kilo bases in length by partial digestion with Sau3Al; these fragments were ligated with lambda-ZAP-Express-Predigested (Stratagene) vector arms and the ligation products then packed into phage particles as instructed by the manufacturer. The primary phage library was amplified in E. coli XL1-Blue MRF cells and the phagemide library cut out by means of helper phages ExAssist and stably established after transfection in E. coli XLOLR.

(46) For sample taking, bacteria of the E. coli strain XLOLR containing parts of the genome of F. gondwanense in the vector pBK-CMV (kanamycin resistance, phagemide vector with Lac-Promoter, Agilent Technologies, Waldbronn) were plated on an LB selection tray and incubated at 37 C. over night. The colonies that had grown were transferred to a fresh medium and reincubated at 37 C. over night.

(47) For the identification of colonies with -glucosidase activity, esculin (Sigma-Aldrich, Munich) was used as a substrate. For this purpose, the colonies were detected coated with esculin agar (0.1% (w/v) esculin, 0.01% (w/v) ammonium iron(III) citrate, 50 mM Na-acetate, 1% (w/v) agarose, pH 6.0). It was possible to detect activity from the formation of a brown halo around the colonies which is due to the decomposition of the esculin into glucose and esculetin. Esculetin forms a complex with iron ions which becomes visible by the brown colouring. Clones with enzymatic activity were isolated and cultivated.

(48) Screening with the substrate esculin by means of colorimetric detection methods resulted in the identification of the gene bank clones Bgl13.

(49) In order to determine the open reading frames (ORFs) encoding the active protein, the plasmid of clone Bgl13 was sequenced by means of primer walking using the standard primers T3 and T7.

(50) TABLE-US-00001 T3 5-ATTAACCCTCACTAAAGGGA-3 T7 5-TAATACGACTCACTATAGGG-3

(51) This standard method is described in more detail in Sambrook et al. (Molecular cloning, a laboratory manual, 2.sup.nd edition, 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

(52) The overall size of the inserts was 8479 base pairs (bp). For identification of open reading frames on the insert, the ORF Finder from the NCBI data base was used; five open reading frames were determined. So as to be able to allocate a possible function to the proteins encoded by the ORFs, their amino acid sequences were compared with known sequences by means of blastp from the NCBI-data base. BLAST represents the Basic Local Alignment Search Tool (Altschul et al., 1997, Nucleic Acids Res 25 (17): 3389-3402). The amino acid sequence of ORF 1 displayed similarities with -xylosidases of the glycoside hydrolase family 3 and, according to the respective nomenclature for glycoside hydrolases, ORF 1 was designated xyl3A and the corresponding protein FgXyl3A (Henrissat et al., 1998, FEBS Lett. 425 (2): 352-354).

(53) The gene xyl3A encoding the -pyranosidase consists of 2328 bp and encodes the protein FgXyl3A with of a size of 775 amino acids. FgXyl3A has a calculated molecular weight of 85.9 kilodalton (kDa) and a theoretical isoelectric point (pI) of 5.55. A signal sequence could not be determined by SignalP 3.0.

(54) The amino acid sequence of FgXyl3A is shown in SEQ ID NO. 1.

(55) A sequence comparison of FgXyl3A with sequences of known -xylosidases was performed with the aid of blastp from the NCBI data base. The amino acid sequence of FgXyl3A showed the highest congruence with a putative -xylosidase of the glycoside hydrolase family 3 of Thermotoga neapolitana with an identity of 68%. The sequence of FgXyl3A contains the preserved amino acids which are characteristic for proteins of the glycoside hydrolase family 3 (Zverlov et al., 1997, Microbiology 143 (Pt 11): 3537-3542).

(56) The domain structure of FgXyl3A was determined with the aid of InterProScan. Accordingly, the protein FgXyl3A consists of two domains, namely of an N-terminal catalytic domain and of a C-terminal domain, both domains showing similarity to the glycoside hydrolase family 3 (FIG. 1).

(57) SEQ ID NO. 1 contains a region which comprises the two structural domains determined by a sequence comparison with the protein BgIB of Thermotoga neapolitana (Pozzo et al., 2010, J. Mol. Biol., 2:397 (3), 724-739). Structural domain 1 contains the amino acid residues Leu13 to Ile381 and Gln586 to Tyr638 of SEQ ID NO. 1 and was determined by a sequence comparison by means of InterProScan with SEQ ID NO. 4. A conserved aspartate residue is present there (Asp281) which was identified as a catalytic nucleophil in glycoside hydrolases of the family 3 (Zverlov et al., 1997, Microbiology 143 (Pt 11): 3537-3542; Wulff-Strobel & Wilson, 1995, J. Bacteriol. 177 (20): 5884-5890).

(58) Structural domain 2 contains the amino acid residues Val382 to Val585 of SEQ ID NO. 1 and was determined by a sequence comparison by means of InterProScan with SEQ SD NO. 4. SEQ ID NO. 2 is the region of SEQ ID NO. 1 which includes both structural domains and hence comprises the amino acid residues 13 to 638 of SEQ ID NO. 1.

(59) The underlying nucleotide sequence of the full-length protein FgXyl3a is given by SEQ ID NO. 3.

Example 2

Cloning the -Pyranosidase FgXyl3A of Fervidobacterium Gondwanense

(60) For cloning xyl3A, the gene was amplified by means of the following oligonucleotides by a polymerase chain reaction (PCR) according to Sambrook et al. (Molecular cloning, a laboratory manual, 2.sup.nd edition, 1989. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.):

(61) TABLE-US-00002 xyl3A_BamHIF 5-GGGGATCCGAGATATATAAGGATTCTTC-3 xyl3A_HindIII.R 5-GGAAGCTTTTAGAAAGTGTAAACTTTTG-3

(62) The oligonucleotides contained a BamHI cleavage site at the 5 terminus and a HindIII cleavage site at the 3 terminus. The PCR was performed in a thermal cycler (Gene Amp PGR System 2400, Perkin-Elmer, Massachusetts, USA) and the PCR products purified with the High Pure PCR Product Purification Kit (Roche Diagnostics, Mannheim) as instructed by the manufacturer. The amplified gene was ligated into the vector pJET1.2 (CloneJET PCR Cloning Kit (Fermentas, St. Leon-Rot)) with the aid of the 3A overhangs formed by high fidelity polymerase in accordance with the instructions of the manufacturer. By restriction with the endonucleases BamHI and HindIII, the gene xyl3A was cut from the vector and ligated into the vector pQE-30 cut with the same restriction enzymes (ampicillin resistance, expression vector with a sequence for an N-terminal 6 histidine tag with T5-Promotor, Qiagen, Hilden). Correct cloning of xyl3A into the vector pQE-30 was verified by sequencing. For gene expression, the recombinant plasmid pQE-30::xyl3A (FIG. 2) was transformed into the E. coli strain M15[pREP4] (Nal.sup.s Str.sup.s Rif.sup.s Thi.sup. Lac.sup. Ara.sup.+ Gal.sup.+ Mtl.sup. F.sup. RecA.sup.+, Uvr.sup.+ Lon.sup.+ pREP4, Qiagen Hilden). By cloning xyl3A into the pQE-30 vector a sequence encoding a His tag was attached to the 5 terminus of the gene. This results in a size of 786 amino acids and a calculated molecular weight of 87.2 kDa for the recombinant protein FgXyl3A.

Example 3

Expression of Fgxyl3A in Escherichia coli (E. coli)

(63) The recombinant clone of Escherichia coli (E. coli) M15/pQ E-30::xyl3A (example 2) was incubated at 37 C. Cell cultivation of the E. coli strains was performed in LB-Medium (10 g/l of trypton, 5 g/l of yeast extract, 10 g/of 1 NaCl, pH 7.0) according to Sambrook et al. (Molecular cloning, a laboratory manual, 2.sup.nd edition, 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

(64) The cultivation of strains with plasmid- or genome-coded resistance against antibiotics was performed under selection pressure by adding the appropriate antibiotic. The concentration of antibiotics used corresponded to the recommendations of Sambrook et al. (Molecular cloning, a laboratory manual, 2.sup.nd edition, 1989, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.). The cultivation of the cells was carried out aerobically at 37 C. on a shaker (Certomat R, B. Braun Biotech International, Melsungen) at 160 rpm. The growth of the cells was determined on the basis of the optical density (OD) at a wavelength of 600 nm in a spectrophotometer (UV-1602, Shimadzu Deutschland, Duisburg). The induction of the gene expression of recombinant E. coli strains was performed by adding 1 mM of isopropyl--D-thiogalactopyranoside (IPTG) at an OD.sub.600 nm of 0.6-0.8. This was followed by the incubation of the cells at 37 C. and 160 rpm for 4.5 hrs and subsequent centrifugation of the cells in a Sorvall RC 5C Plus centrifuge (Thermo Fisher Scientific, Langenselbold) for 20 minutes at 4221g and 4 C., obtaining about 2.0 g of cells (humid weight) of 500 ml culture volume. The pellet was used directly for the preparation of cell-free crude extract (example 4) or stored at 20 C.

Example 4

Preparation of a Crude Extract of the Recombinant E. coli Clone M15/pQE-30::xyl3A

(65) For obtaining a crude extract, the cell pellet (example 3) was washed twice in lysis buffer (50 mM of NaH.sub.2PO.sub.4, 300 mM of NaCl, 10 mM of imidazole, pH 8.0), with 20 minutes of centrifugation at 4500g and 4 C. each time. The pellet was then taken up in a cell lysis buffer, using 5 ml of lysis buffer per 1 g of cells. This was followed by cell lysis by means of ultrasound (Branson Sonifier, Danbury, Conn., USA), wherein 3 cycles were performed for 5 minutes each on ice (output control: 50%, duty cycle: 5). Cell debris was then separated by centrifugation for 20 minutes at 20200g and 4 C. and the supernatant transferred into a new vessel. Optionally, heat precipitation of the crude extract was performed at 60 C. for 15 minutes and the precipitated protein separated by centrifugation for 20 minutes at 20200g and 4 C. The crude extract was stored at 4 C.

(66) The crude extract was tested for -xylosidase activity by a standard test (example 7) and used for purification (example 5).

Example 5

Purification of FgXyl3A

(67) The multi-step purification of FgXyl3A by (1) heat precipitation, (2) affinity chromatography and (3) gel filtration resulted in the almost complete purification of the protein. (1) Heat precipitation: After the first purification step by means of heat precipitation (example 4), a yield of 41.7% was obtained (Table 1). (2) Affinity chromatography: The further purification of FgXyl3A with an attached His tag (examples 2, 3 and 4) was achieved through affinity chromatography by means of Ni2.sup.+ nitrilotriacetic acid (Ni2.sup.+-NTA, Qiagen, Hilden). An AKTA purifier system (GE Healthcare, Munich) with the following components was used for performing the affinity chromatography: monitor UV-900, pump P-900, in-line mixer M-925, motor valve INV-907 and fraction collector Frac-950. The buffers used were filtered before use (0.45 m cellulose mixed ester filter, Whatman, Dassel) and the protein samples centrifuged for 10 minutes at 13000g and 4 C. After equilibration of the column matrix with 5 ml column volumes of cell lysis buffer, the crude extract or heat-precipitated crude extract was loaded onto the column at a flow rate of 1 ml/minute. Unbound proteins were removed by washing of the column matrix with 3 column volumes of washing buffer (50 mM of NaH.sub.2PO.sub.4, 300 mM of NaCl, 20 mM imidazole, pH 8.0) at a flow rate of 2 ml/minutes. The elution of bound proteins was performed by rinsing the column with 5 column volumes of elution buffer (50 mM NaH.sub.2PO.sub.4, 300 mM of NaCl, 250 mM imidazole, pH 8.0) at a flow rate of 2 ml/minute. The fraction size of the samples collected was 2 ml. The fractions containing the target protein were combined, concentrated with the aid of micro-concentrators (Amicon Ultra-15 centrifugal filter unit, 10 kDa cut-off size, Milipore, Schwalbach) and used for further purifications steps or, respectively, subjected to dialysis and used for biochemical characterisation. This further purification by means of Ni2.sup.+-NTA agarose resulted in a marked loss in yield which was 1.13%. (3) Gel filtration: For the further purification of FgXyl3A with an attached His tag (examples 2, 3 and 4) by means of gel filtration, an AKTA Fast Protein Liquid Chromatography (FPLC) plant (GE Healthcare, Munich) with the following components was used: monitor UPC-900, pump P-920, in-line mixer M-925, engine valve INV-907 and fraction collector Frac-950. The purification by means of gel filtration was performed by a HiLoad 16/60 Superdex 200 prep grade column (GE Healthcare, Munich). The protein samples were centrifuged for 10 minutes at 13000g and 4 C. before use and the used buffers filtered (0.45 m cellulose mixed ester filter, Whatman, Dassel). At a flow rate of 1 ml/minute the column was loaded with 1 ml of the protein sample maximum and rinsed with 1.5 column volumes of 50 mM Na.sub.2HPO.sub.4 and 150 mM of NaCl (pH 7.0). Fractions of a size of 2 ml were collected. The fractions containing the target protein were combined and concentrated with the aid of micro-concentrators (Amicon Ultra-15 centrifugal filter unit, 10 kDa cut-off size, Milipore, Schwalbach). Subsequent desalting and the change of buffers of protein solutions was achieved by dialysis. For this purpose, dialysis tubing (MembraCel MWCO 3500, Serva, Heidelberg) was boiled in the buffer used and then loaded with the protein sample. Dialysis was performed in 50 mM Na-acetate, pH 6.0 and, respectively, pH 6.5 over night at 4 C. with stirring against the 100-fold volume of the protein solution. After dialysis, the samples were used for biochemical characterisation. As a result of the last purification step by gel filtration a yield of 0.18% and a purification factor of 95 was obtained. The specific activity was 20.9 U/mg vis-vis pNP--xylopyranoside (Table 1).

(68) The purification of histidine-tagged FgXyl3a is shown in FIG. 3 and Table 1.

(69) TABLE-US-00003 Total Overall Specific Purification protein activity activity Yield factor Purification step [mg] [U] [U/mg] (%) [x-fold] Crude extract 1263.4 273.7 0.22 100 1 Heat precipitation 345.9 114.3 0.33 41.7 1.5 Ni.sup.2+-NTA 3.1 3.1 0.38 1.13 1.7 Gel filtration 0.015 0.32 20.9 0.18 95

(70) The determination of protein concentrations was performed according to Bradford (1976, Anal Biochem 72: 248-254). For this purpose, 1 ml of Bradford reagent (0.007% (w/v) Serva Blau G-250, 5% (v/v) of ethanol, 8.5% (v/v) of H.sub.3PO.sub.4) was mixed with 10 l of the protein sample and incubated at room temperature for 5 minutes. The absorbance was determined at the wavelength =595 nm by spectrophotometry. Bovine serum albumin (BSA) in concentrations of 0.1-1.0 mg/ml was used for preparing calibration lines.

Example 6

Determination of the Molecular Weight of FgXyl3A

(71) The determination of the molecular weight of FgXyl3A was performed by means of denaturing and native polyacryl amide gel electrophoresis.

(72) The denaturing separation of proteins was performed by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) according to Laemmli (1970, Nature 227 (5259): 680-685). Unstained Protein Molecular Weight Marker or, respectively, PageRuler Unstained Protein Ladder by Fermentas (St. Leon-Rot) was used as the protein standard. The gel run was carried out in a Minuteni-Protean Tetra system (Bio-Rad, Munich) in 25 mM of tris, 192 mM of glycine, 0.1% (w/v) SDS at constant 200 V. The gels were incubated for 30 minutes in Coomassie dye solution (0.25% (w/v) Serva Blau G-250, 45% (v/v) ethanol, 10% (v/v) acetic acid) and then decoloured in a decolouring solution (30% (v/v) ethanol, 10% (v/v) acetic acid). The gels were incubated in a drying solution (20% (v/v) ethanol, 2% (v/v) glycerine) for 10 minutes for the purpose of preservation and dried with the aid of the DryEase Minuteni-Gel Drying System (Invitrogen, Karlsruhe).

(73) The determination of the molecular weight of native proteins and native protein complexes was performed by native polyacryl amide gel electrophoresis. 4-12% (w/v) tris-glycine gradient gels (Anamed Electrophorese, Gro-Bieberau) were used for this purpose. Before the run, native sample buffer (7.5% (v/v) glycerine, 0.04% (w/v) of bromophenol blue, 33 mM tris, pH 6.8) was added to the protein sample. High Molecular Weight-Marker (GE Healthcare, Munich) served as the protein standard. The gel run was performed in a Novex XCell II Minuteni-Cell facility (Invitrogen, Karlsruhe) for 3 hrs at constant 120 V in 25 mM tris and 192 mM glycine. The gels were incubated in Coomassie dye solution, decoloured and dried for preservation.

(74) Under denaturing conditions, the recombinant protein had a molecular weight of 87 kDa (FIG. 4 A). Under native conditions, a molecular weight of 529.7 kDa was determined (FIG. 4 B). This indicates that the protein FgXyl3A might be present as a homohexamer.

Example 7

Detection of the Activity of FgXyl3A in SDS Gels

(75) The detection of enzymatic activity with the aid of zymograms was performed after the electrophoretic separation of the proteins by SDS-PAGE (example 6). After the run, the gels were incubated in 1% (v/v) Triton X 100 at room temperature (RT) for 1 hr. The -xylosidase activity of FgXyl3A was detected by incubation of the gels in 0.1% (w/v) esculin, 0.01% (w/v) ammonium iron(III) citrate and 50 mM Na-acetate (pH 6.0) for 30-60 minutes at 60 C.

Example 8

Examination of the Enzymatic Activity of FgXyl3A

(76) For the determination of the substrate spectrum of FgXyl3A, the activity of the purified recombinant proteins (example 5) vis-vis pNP--D-galactopyranoside, pNP--D-glucopyranoside, pNP--D-glucopyranoside, pNP--D-cellobioside and pNP--D-xylopyranoside was examined. The substrates were used in a final concentration of 2 mM and the activities were measured by the following standard tests. (1) Determination of the -glucosidase activity: The activity of the -glucosidase was determined in a modified manner according to Park et al. (2005, Appl. Microbiol. Biotechnol. 69 (4): 411-422) using pNP--D-glucopyranoside (Sigma-Aldrich, Munich) as the substrate. Unless otherwise indicated, a reaction sample contained 2 mM pNP--D-glucopyranoside and 50 mM Na-acetate (pH 6.0) in an overall volume of 1 ml. Before adding 0.02 U of the enzyme, the reaction samples were incubated at 80 C. for 5 minutes. The reactions were performed at 80 C. for 10 minutes and then stopped by adding 100 l of 0.1 M Na.sub.2CO.sub.3 and placing the sample on ice. All the measurements were taken in the form of triple determinations. The enzyme was added to the control batches only after Na.sub.2CO.sub.3 had been added and incubation on ice performed. The determination of the released p-nitrophenol was performed at a wavelength of =410 nm. The enzymatic activity was calculated by the following formula:
Enzymatic activity (U/ml)=(E.Math.V.sub.Tot)/(d.Math.e.Math.t.Math.V.sub.E) E is defined as the change in extinction, V.sub.Tot as the reaction volume (ml), d as the thickness of the cuvette (cm), e as the molar extinction coefficient (M.sup.1 cm.sup.1), t as the reaction time (minutes) and V.sub.E as the volume of the enzyme sample (ml). 16.56 M-1 cm-1 is used as the molar extinction coefficient. 1 U -glucosidase activity was defined as the amount of enzyme which releases 1 mol of p-nitrophenol per minute under standard conditions. (2) Determination of the -xylosidase activity: pNP--D-Xylopyranoside (Sigma-Aldrich, Munich) was used as the substrate for determining the activity of -xylosidase in the modified form according to Park et al. (2005, Appl. Microbiol. Biotechnol. 69 (4): 411-422). Unless otherwise described, the standard reaction to determine the -xylosidase activity was carried out in the presence of 2 mM pNP--D-xylopyranoside and 50 mM Na-acetate (pH 6.5) in a total volume of 1 ml. The reactions were started by adding 0.016 U enzyme to the batches pre-incubated at 85 C. for five minutes and then carried out at a temperature of 85 C. for 10 minutes. The reactions were stopped by adding 100 l 0.1 M Na.sub.2CO.sub.3 and placing the samples on ice. All the measurements were taken in the form of triple determinations. Reaction samples to which the enzyme was added only after addition of Na.sub.2CO.sub.3 and incubation on ice served as controls. The spectrophotometric measurement of the released p-nitrophenols was performed at a wavelength of =410 nm. The enzymatic activity was calculated by the following formula:
Enzymatic activity (U/ml)=(E.Math.V.sub.tot)/(d.Math.e.Math.t.Math.V.sub.E) E is defined as the change in extinction, V.sub.Tot as the reaction volume (ml), d as the thickness of the cuvette (cm), e as the molar extinction coefficient (M.sup.1 cm.sup.1), t as the reaction time (minutes) and V.sub.E as the volume of the enzyme sample (ml). 16.56 M-1 cm-1 was used as the molar extinction coefficient. 1 U -xylosidase activity was defined as the amount of enzyme which led to a release of 1 mol of p-nitrophenol per minute under standard conditions.

(77) FgXyl3A showed the highest specific activity with 20.9 U/mg vis-vis pNP--xylopyranoside (Table 2). In addition, a specific activity of 1.3 and, respectively 9.3 U/mg vis-vis pNP-- and pNP--glucopyranoside was determined. The substrate pNP--galactopyranoside was also hydrolysed by the -xylosidase, the specific activity being 2.7 U/mg. Hydrolysis of the disaccharide pNP--cellobioside by Xyl3A was not detected (Table 2).

(78) TABLE-US-00004 TABLE 2 Substrate specificity of FgXyl3A Substrate Specific activity [U/mg] pNP--xylopyranoside 20.9 pNP--glucopyranoside 9.3 pNP--glucopyranoside 1.3 pNP--galactopyranoside 2.7 pNP--cellobioside 0

Example 9

Influence of the Temperature on the Activity of FgXyl3A

(79) For determination of the temperature profile of FgXyl3A, the enzymatic activity of the recombinant proteins (example 5) was measured in a temperature range of 10-115 C. with pNP--xylopyranoside as the substrate as described in example 8. For this purpose, the reaction batches were pre-incubated at the relevant temperature for 5 minutes before adding the enzyme. Activity was determined by the standard test (example 8).

(80) FgXyl3A showed -xylosidase activity in a wide temperature range (FIG. 5). More than 50% of the relative activity was measured at temperatures of 70-95 C., maximum activity being measured at 85 C. A pronounced decrease in activity was observed at temperatures above 95 C.; for example, the activity of FgXyl3A at 100 C. was 23% of the maximum activity. 18-38% of the relative activity was measured in a temperature range of 50-65 C.

Example 10

Temperature Stability of FgXyl3A

(81) For the determination of temperature stability, recombinant FgXyl3A (example 5) was incubated at temperatures of 60-90 C. for up to 24 hrs. The residual activity was determined under standard conditions (example 8).

(82) Measured by the residual -xylosidase activity, FgXyl3A was stable for a period of 3 hours at a temperature of 60 C. Incubation at 70-90 C., on the other hand, caused deactivation of the enzyme after a few minutes (FIG. 6). By pre-incubation of FgXyl3A at 60 C., 65% of residual activity was determined after 2 hrs; after 6 hrs of incubation the activity was 30%. After a longer incubation of 24 hrs, the enzyme exhibited a residual activity of 25%. At a temperature of 70 C., FgXyl3A exhibited 30% of residual activity after 5 minutes of incubation; and 15% residual activity after 10 minutes of pre-incubation. The half-life of FgXyl3A was 215 minutes at 60 C. and 6 minutes at 70 C.

Example 11

Influence of the pH-Value on the Activity of FgXyl3A

(83) For the determination of the pH profile of FgXyl3A, the activity of the recombinant enzyme (example 5) in a pH range of 2.0-11.0 with pNP--xylopyranoside as the substrate was measured (example 8). The reactions were carried out in the presence of 50 mM of universal buffer (16.7 mM H.sub.3PO.sub.4, 16.7 mM of acetic acid, 16.7 mM of H.sub.3BO.sub.4, Britton & Robinson, 1931) under standard conditions (example 8). The maximum activity of FgXyl3A vis-vis -xylosidase was measured at a pH value of 6.5 (FIG. 7). In the pH range of 5.5-7.0, the -xylosidase exhibited more than 45% of the relative activity, a pronounced decrease in activity being observed at pH values above 7.0. In the acidic pH range, the enzyme did not exhibit any activity at pH 2.0-3.0 and at pH 4.0 exhibited a maximum activity of 6%. A relative activity of 36% was measured at pH 5.0.

Example 12

Determination of the pH Stability of FgXyl3A

(84) For determination of the pH stability, recombinant FgXyl3A (example 5) was incubated in 50 mM of universal buffer with pH-values of 3.0-10.0 for 48 hrs at room temperature. This was followed by the determination of the residual activity under standard conditions (example 8), but in the presence of 50 mM of universal buffer having a pH-value of 6.5.

(85) In the slightly acidic and neutral pH range of 5.0-7.0, FgXyl3A exhibited high stability with a residual activity vis-vis -xylosidase of 90-95% (FIG. 8). In the acidic pH range, on the other hand, a loss in activity was observed. For example, the residual activity was 66% at pH 3.0 and 4.0. A small loss in activity was observed in the alkaline pH range, where residual activity of 87% was measured at pH 8.0, 81% at pH 9.0 and 80% at pH 10.0.

Example 13

Product Analysis by High-Performance Liquid Chromatography (HPLC)

(86) For detection of the hydrolysis products formed, 0.5% (w/v) of beech wood xylan, xylobiose, xylotriose and xylotetraose were incubated in 50 mM NaH.sub.2PO.sub.4 (pH 6.5) with 0.1 U -xylosidase for up to 3 hrs at 80 C. The samples were boiled for 10 minutes to stop the reaction and then centrifuged for 10 minutes at 13000g and 4 C. The supernatants of the samples were filtered (0.2 m pore size, Pall, Darmstadt) and transferred into sample bottles with micro-inserts (CS Chromatographie Service, Langerwehe). 20 l of each sample was injected. The analysis of the products was performed using an Aminex HPX 42-A column (Bio-Rad, Munich), using degassed H.sub.2O (LiCrosolv, Merck, Darmstadt) as the mobile phase and selecting a flow rate of 0.6 ml/minute at a temperature of 70 C.

(87) After incubation of the enzyme for 3 hrs in the presence of xylobiose, a major part of the substrate was hydrolysed to form xylose (FIG. 9). The incubation of FgXyl3A with xylotriose resulted in the formation of xylose and xylobiose, a large proportion part of the substrates being hydrolysed (FIG. 10). If FgXyl3A was incubated with the substrate xylotetraose, the hydrolysis products xylose, xylobiose and xylotriose were detected, xylose being the main product (FIG. 11). Longer incubation did not result in an increased degradation of xylotetraose. If the FgXyl3A was incubated in the presence of beech wood xylan, small amounts of xylose were detected as the hydrolysis product.

Example 14

Kinetics of FgXyl3A

(88) The determination of the kinetic parameters of the -xylosidase activity of FgXyl3A was performed at substrate concentrations of 0.01-6.0 mM pNP--D-xylopyranoside. The reaction was performed under standard conditions (example 8). The determination of v.sub.max and K.sub.M was done by non-linear regression according to Michaelis-Menten and yielded a v.sub.max value of 0.53 mol/min and a K.sub.M value of 0.06 mM (FIG. 12).

(89) Overview of the Sequences in the Sequence Listing:

(90) SEQ ID NO. 1: FgXyl3A full-length protein

(91) SEQ ID NO. 2: catalytic domain GHF 3 from SEQ ID NO. 1 (corresponds to amino acid residues 13 to 638 of SEQ ID NO. 1)

(92) SEQ ID NO. 3: DNA sequence encoding SEQ ID NO. 1

(93) SEQ ID NO. 4: barley -D-glucan-exohydrolase isoenzyme Exo I, full length

SHORT DESCRIPTION OF THE DRAWINGS

(94) FIG. 1: Domain structure of FgXyl3A

(95) FIG. 1 shows the domain structure -xylosidase FgXyl3A determined by means of InterProScan. GHF: glycoside hydrolase family.

(96) FIG. 2: Vector map of the recombinant plasmid pQE-30::xyl3A

(97) The xyl3A gene encoding the -xylosidase FgXyl3A gene was ligated into the expression vector pQE-30 using the restriction cleavage sites BamHI and HindIII. T5: T5 promoter, bla: -lactamase, ColE1: replication origin.

(98) FIG. 3: Purification and detection of the activity of FgXyl3A

(99) The purification and activity of FgXyl3A was detected by means of SDS-PAGE. The proteins were dyed with Coomassie-Blau; the activity of FgXyl3A was detected by esculin. Track 1: Protein standard PageRuler Unstained Protein Ladder (Fermentas, St. Leon-Rot), Track 2: Crude extract of E. coli M15/pQE-30 (control, 35 g), track 3: crude extract of E. coli M15/pQE-30::xyl3A (37 g), track 4: heat-precipitated crude extract of E. coli M15/pQE-30::xy/3A (35 f), track 5: Ni.sup.2+-NTA fraction of FgXyl3A (40 g), track 6: gel filtration fraction of FgXyl3A (1.1 g), track 7: activity gel of the gel filtration fraction of FgXyl3A (1.1 g).

(100) FIG. 4: Determination of the molecular weight of FgXyl3A

(101) The molecular weight of FgXyl3A was determined by means of denaturing and native polyacryl amide gel electrophoresis. In the Figure the logarithm of the molecular weight is plotted against the running distance of the proteins in the gel (Rf value). Molecular weights of the standard proteins in (A): Recombinant proteins having a size of 200, 150, 120, 100, 85, 70 and 60 kDa. Molecular weights of the standard proteins in (B): thyroglobulin (669 kDa), ferritin (440 kDa), catalase (232 kDa), lactate dehydrogenase (140 kDa), albumin (66 kDa).

(102) FIG. 5: Temperature profile of FgXyl3A

(103) The activity of FgXyl3A was determined in a temperature range of 10-115 C. under standard conditions. Reaction parameters: 2 mM pNP--xylopyranoside, 50 mM Na-acetate, pH 6.5; 10 mM incubation time.

(104) FIG. 6: Temperature stability of FgXyl3A

(105) The temperature stability of FgXyl3A was determined after pre-incubation of the enzyme at temperatures of 60-100 C. Reaction parameters: 2 mM pNP--xylopyranoside, 50 mM Na-acetate, pH 6.5, 85 C.; 10 min incubation time.

(106) FIG. 7: pH-Profile of FgXyl3A

(107) The activity of FgXyl3A was determined in a pH range of 2.0-11.0 under standard conditions. Reaction parameters: 2 mM pNP--xylopyranoside, 50 mM universal buffer, 85 C.; 10 mM incubation time.

(108) FIG. 8: pH Stability of FgXyl3A

(109) The pH stability of FgXyl3A was determined after pre-incubation of the enzyme at pH values of 3.0-10.0 under standard conditions. Reaction parameters: 2 mM pNP--xylopyranoside, 50 mM universal buffer, 85 C.; 10 minutes incubation time.

(110) FIG. 9: Hydrolysis of xylobiose by FgXyl3A

(111) The hydrolysis products were analysed after incubation of FgXyl3A for 3 hrs in the presence of 0.5% (w/v) xylobiose, 20 mM Na-phosphate buffer, pH 6.5, 80 C. by HPLC.

(112) FIG. 10: Hydrolysis of xylotriose by FgXyl3A

(113) The hydrolysis products were analysed after incubation of FgXyl3A for 3 hrs in the presence of 0.5% (w/v) xylotriose, 20 mM Na-phosphate buffer, pH 6.5, 80 C. by HPLC.

(114) FIG. 11: Hydrolysis of xylotetraose by FgXyl3A

(115) The hydrolysis products were analysed after incubation of FgXyl3A for 3 hrs in the presence of 0.5% (w/v) xylotetraose, 20 mM Na-phosphate buffer, pH 6.5, 80 C. by HPLC.

(116) FIG. 12: Kinetics of FgXyl3A

(117) The figure shows the Michaelis-Menten plot of FgXyl3A. The activity of FgXyl3A was determined under standard conditions. Reaction parameters: 0.01-6.0 mM of pNP--xylopyranoside, 50 mM of Na-acetate, pH 6.5, 85 C.; 10 minutes incubation time.