NEW XYLANASE WITH IMPROVED THERMOSTABILITY AND INCREASED ENZYME ACTIVITY ON ARABINOXYLAN

Abstract

The present invention relates to novel polypeptides with xylanase activity, especially xylanase variants, such as genetically engineered xylanase variants, which show improved thermostability, improved resistance against acid treatment and increased enzyme activity on arabinoxylan. The invention includes the use of said polypeptides in applications, such as for food or feed, for brewing or malting, for the treatment of xylan containing raw materials like grain-based materials, e.g. for the production of biofuels or other fermentation products, including biochemicals, and/or for the wheat gluten-starch separation industry, and methods using these polypeptides, as well as compositions (such as feed additive compositions) comprising said polypeptides.

Claims

1. A polypeptide, comprising or consisting of a polypeptide which has at least 75% amino acid sequence identity to the polypeptide according to SEQ ID NOs: 2 or 3, preferably of SEQ ID NO: 3, wherein said polypeptide has xylanase activity, and with the proviso that the polypeptide is not the polypeptide of SEQ ID NO: 1; characterized in that said polypeptide shows improved thermostability and/or resistance against acid treatment and/or an increased enzyme activity compared to the polypeptide of SEQ ID NO: 1, wherein improved thermostability means that said polypeptide displays a Tm.sub.on of 70 C. or higher and/or displays a Tm50 of 80 C. or higher and/or displays a Tm of 80 C. or higher; improved resistance against acid treatment means that an enzyme activity of at least 70% is retained after treatment at low pH, such as a pH at 2.5 to 5.5; and increased enzyme activity means that the specific enzyme activity is increased at least 1.6 fold.

2. The polypeptide according to claim 1 , wherein said polypeptide comprises or consists of a polypeptide having at least 75% amino acid sequence identity to a polypeptide of SEQ ID NO: 3, and wherein said polypeptide of SEQ ID NO: 3 shows a 1.6 fold increase of the specific enzyme activity and a Tm.sub.on of 81 .2 C. and/or a Tm50 of 85.7 C.

3. The polypeptide according to claim 1, wherein said polypeptide displays at least 40% enzyme activity, in particular xylanase activity, over a pH range from 5.5 to 9.5, and/or over a temperature range from 37 C. to 80 C.

4. The polypeptide according to claim Jany one of claims 1, wherein the enzyme activity is retained after acidic treatment of the polypeptide in the pH range from 2.0 to 5.5.

5. A nucleic acid molecule consisting of a nucleic acid sequence of SEQ ID NO: 9 or 10 encoding the polypeptide according to SEQ ID NO: 2 or 3.

6. An expression vector comprising the nucleic acid molecule as claimed in claim 5.

7. A host cell comprising the nucleic acid sequence of SEQ ID NO: 9 or 10 as claimed in claim 5, wherein said host cell expresses the polypeptide according to SEQ ID NO: 2 or 3.

8. A method for producing the polypeptide of SEQ ID NOs: 2 or 3. preferably of SEQ ID NO: 3, the method comprising culturing a host cell as claimed in claim 7 under conditions permitting the production of the polypeptide, and recovering the polypeptide from the culture.

9. A composition for addition to biomass or hemicellulose containing material, said composition comprising a polypeptide as claimed in claim 1, and optionally at least one formulating agent, excipient, stabilizer and/or a preservative.

10. The composition according to claim 9, wherein said composition is a liquid formulation, such as a solution or suspension, or a dry formulation, such as a powder or granulate.

11. The composition according to claim 9, wherein said composition comprises a sugar as heat stabilizing agent, which is eselected from sucrose, trehalose, sorbose, melezitose, verbascose, melibiose, sucralose and raffinose, or, when said composition is a liquid formulation, comprises a solvent, such as glycerol or water.

12. The polypeptide according to claim 1, wherein said polypeptide modifies the content of hemicellulose components, in particular the xylan content, to loosen compact structure or to reduce high viscosity of biomass or hemicellulose containing material.

13. Use of the polypeptide according to claim 1 during the production of animal feed, pulp and paper, bioenergy and in brewery or malting.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0171] Specific embodiments of the present invention are described in the working examples with reference to the accompanying drawings.

[0172] Herein,

[0173] FIG. 1 shows an SDS-PAGE of recombinantly produced enzymes of SEQ ID NOs: 1 to 3 respectively. PageRuler Prestained Protein Ladder 10 to 180 kDa (#26616, ThermoFisher Scientific) was used as protein standard.

[0174] FIG. 2 shows the relative enzyme activity increase (%) of the polypeptides of SEQ ID

[0175] NOs: 3 and 2 in relation to the enzyme activity of the wildtype enzyme of SEQ ID NO 1. Surprisingly, the enzyme of SEQ ID NO: 3 displays a 1,6 fold higher activity in comparison to the enzyme activity of the wildtype enzyme of SEQ ID NO: 1. Enzyme activity: the release of the amount (micromole) of reducing sugars from arabinoxylan per mg of enzyme per minute was determined with the DNSA assay compared to the activity of the polypeptide of SEQ ID NO: 1. Enzyme activity was measured in triplicates.

[0176] FIG. 3 shows the relative enzyme activity of the polypeptide SEQ ID NO: 3 tested under different pH and temperature conditions Enzyme activity is defined as the release of the amount (micromole) of reducing sugars from arabinoxylan per mg of enzyme per minute as was determined with the DNSA assay and compared to the activity at 70 C. and pH 7.0. Enzyme activity was measured in triplicates.

[0177] FIG. 4 shows the resistance of the polypeptide of SEQ ID NO: 3 against high temperatures simulating the pelleting process. Enzyme activity is defined as the release of the amount (micromole) of reducing sugars from arabinoxylan per mg of enzyme per minute as was determined with the DNSA assay and compared to the activity of the untreated enzyme. The activity was measured in triplicates at 60 C. at pH 6.5.

[0178] FIG. 5 shows the stability of the polypeptide of SEQ ID NO: 1 and SEQ ID NO: 3 against acid treatment. To simulate the gastric tract in vitro, the polypeptides of SEQ ID NO: 1 and SEQ ID NO: 3 were incubated at pH 3.5 at 40 C. for 30, 60 and 120 min. The remaining activity was determined by DNSA assay and compared to the activity of the untreated protein. Enzyme activity is defined as the release of the amount (micromole) of reducing sugars from arabinoxylan per mg of enzyme per minute was determined with the DNSA assay and compared to the activity of the untreated enzyme. The activity was measured in triplicates at pH 6.5 at 70 C.

[0179] FIG. 6 shows the residual activity of the polypeptide given in SEQ ID NO: 3 against proteolytic digestion by pepsin at 40 C. and pH 3.5. Residual activity was determined by measuring the release of reducing sugar equivalents (micromole per minute per mg enzyme) on arabinoxylan at 60 C. and pH 6.5 using the DNSA assay.

[0180] FIG. 7 shows the reduction of viscosity by the polypeptide of SEQ ID NO: 3 in comparison to Danisco Xylanase in an in vitro chicken intestine model.

[0181] FIG. 8 shows the time for liquefaction of wheat slurry without enzyme or with addition of different enzymes: A: alpha-amylase 25 mg/kg of Teramyl 3000L (Novozymes), B: alpha-amylase 25 mg/kg and SEQ ID NO 3, C: alpha-amylase 25 mg/kg, SEQ ID NO 3 and an endoglucanase SEQ ID NO 11 (EP17203087), D: alpha-amylase 25 mg/kg) cellulase/hemicellulase mixture Cellic CTec2 (Novozymes). Without an alpha-amylase no liquefaction was observed (asterisk). For SEQ ID NOs: 3 and 11 as well as Cellic CTec2 equal amounts of protein were used (0.4 mg/kg). All measurements were done in triplicate.

WORKING EXAMPLES

Example 1: Cloning of the polynucleic acids according to SEQ ID NOs: 8 to 10 encoding the GH11 xylanases of SEQ ID NOs: 1-3

[0182] Materials

[0183] Chemicals used as buffers and substrates were commercial products of at least reagent grade. Escherichia coli DH10B was used for routine cloning and E. coli BL21 Star (DE3) for expression of Clostridium stercorarium DSM8532 of SEQ ID NO 8.

[0184] DNA modification

[0185] Preparation of chromosomal and plasmid DNA, endonuclease digestion, and ligation were carried out by standard procedures (Sambrook J and Russell D W. 2001).

[0186] Cloning of genes encoding GH11 xylanases

[0187] The genes with sequences of SEQ ID NOs: 8 to 10 were amplified from chromosomal DNA from C. stercorarium DSM8532 in accordance with manufacturer's instructions (Phusion High-Fidelity DNA Polymerase, F530S, ThermoFisher Scientific) using primer set of SEQ ID NOs: 4 and 5. With the GH 11 xylanases of SEQ ID NO: 2 and 3, the effect of different protein module (i.e. CBM) deletions on enzyme activity and function was investigated. Two carbohydrate binding domains were deleted in the polypeptide of SEQ ID NO: 2 enzyme and all three carbohydrate binding modules were deleted in the polypeptide of SEQ ID NO: 3. The polynucleic acid of SEQ ID NO: 9 was amplified using primer 4 and 6, and the polynucleic acid of SEQ ID NO: 10 was amplified by employing primer 4 and 7. All PCR products were subsequently cloned by Gibson assembly (NEB, Cat. Nr. E2611S) in Ndel/Xhol digested pET24c(+) vector (Novagen, MerckMillipore) and sequenced by Eurofins to confirm the correct sequence.

[0188] Example 2: Protein production of the enzymes of SEQ ID NOs 1 to 3

[0189] Growth of cells

[0190] Fed-batch fermentations of recombinant E. coli strains harbouring the GH11 Xylanase genes from C. stercorarium DSM8532 of SEQ ID NO: 8 and the newly designed genes SEQ ID Nos: 9 and 10 were carried out in a 10 L Uni-Vessel controlled and equipped with a Biostat B Twin DCU (Sartorius AG, Gottingen, Germany). Temperature, pH, foam, turbidity, weight and dissolved oxygen were monitored online during fermentation. The dissolved oxygen (DO %) was set to 25% (vol/vol) and maintained with increasing agitation at constant air flow. The formation of foam was controlled by the addition of Antifoam 206 (Sigma Aldrich, St. Louis, Missouri, USA). A pH of 6.9 was maintained by addition of a 25% (vol/vol) ammonium hydroxide solution or 25% (vol/vol) HPO4 solution. E. coli strains were cultivated in Riesenberg medium (Korz et al., 1995) at the 10 L scale, the feeding solution consists of 1021 g/L glycerol, 20 g/L MgSO.sub.4.7 H.sub.2O, 13 mg/L EDTA, 4 mg/L CoCl.sub.2.6 H.sub.2O, 23.5 mg/L MnCl.sub.2.4 H.sub.2O, 2.5 mg/L CuC.sub.2.2 H.sub.2O, 5 mg/L H.sub.3BO.sub.3, 4 mg/L Na.sub.2MoO.sub.4 2 H.sub.2O, 16 mg/L Zn(CH.sub.3COO).sub.2.2 H.sub.2O, 40 mg/L Fe(III)citrate (Korz et al., 1995). After the consumption of the initial carbohydrate substrate, growth rate was controlled according to EQUATION 1, whereby m.sub.s, is the mass flow of substrate (g h.sup.l), set the desired specific growth rate (h.sup.l), Y.sub.xs the biomass/substrate yield coefficient (g g.sup.1), m the specific maintenance coefficient (g g.sup.1 h.sup.l), V the cultivation volume (L), and X the biomass concentration (g L.sup.1):

[00001]$\begin{array}{cc}{m}_s=\left(\frac{{}_{s.Math.e.Math.t}}{Y_{X/S}}+m\right).Math.V\left(t\right).Math.X\left(t\right).Math.e^{{}_{s.Math.e.Math.t}\left(t-t_F\right)}& \mathrm{EQUATION}.Math..Math.1\end{array}$

[0191] The inoculation procedure was the following: Based on a cryo-stock, a fresh agar plate containing adequate antibiotics was prepared. With one colony an Erlenmeyer flask containing 30 mL Lysogeny Broth (Sambrook et al. 1989) was inoculated and incubated for 12 to 15 h at 30 C. 30 mL of this first preculture was used to inoculate 500 mL of the fermentation medium in a 5 L Erlenmeyer flask and incubated for further 14 h. The 10 L fermenter was filled with 6 L fermentation medium and inoculated with 500 mL of the second preculture. Kanamycin was added at 50 pg/mL. Protein production was induced by changing the glycerol feed to lactose feed. Cells were harvested after 48 h by centrifugation for 1 h at 9000 rpm and 22 C. Portions of 300 g cells were solved in 3 L lysis buffer (50 mM MOPS pH 7.3, 0.1 M NaCI, 20 mM imidazol). Cell lysis was achieved by ultrasonic treatment in an ultrasonic flow-through chamber. Cell debris was separated by centrifugation (9000 rpm, 22 C.). Supernatant was clarified from residual cells or debris by tangential filtration applying a 0.2 m filter cassette and three volumes washing with lysis buffer. The enzyme solution was concentrated employing tangential filtration with a 30 kDa filter cassette followed by dialysis with three volumes lysis buffer. GH11 xylanases were purified by immobilized metal ion affinity chromatography (IMAC). Pure enzymes were eluted with elution buffer containing 50 mM MOPS, pH 7.3, 0.25 M imidazole, 0.1 M NaCI, and 20 mM CaCl2.

[0192] Another possibility for producing variants of the SEQ ID NO: 1 enzyme, such as the enzymes of SEQ ID NOs: 2 or 3 of the invention is to transform a competent B. subtilis strain with an appropriate vector comprising the mutated DNA and cultivating the recombinant strain in accordance with Park et al., 1991.

Example 3: Electrophoretic Methods

[0193] Sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis was performed in accordance with Laemmli (1970). Proteins were resuspended in denaturating buffer and heated for 15 min at 95 C. The PageRuler Prestained Protein Ladder 10 to 180 kDa (#26616, ThermoFischer Scientific) was used as molecular weight standard. The proteins were stained with Coomassie brilliant blue R-250 (Weber and Osbourne, 1969).

Example 4: Protein Quantification

[0194] The protein amount was determined by using Pierce BCA Protein Assay Kit (#23225, ThermoFisher Scientific) in accordance with the instructions of the manufacturer.

Example 5: Activity Test, Arabinoxylan Degradation

[0195] Xylanase activity

[0196] For the purpose of the present invention, any of the commercially available xylanase activity measurement kits is suitable to determine xylanase activity. One suitable way of measuring the xylanase activity is as follows:

[0197] Xylanase activity measurement was with a final concentration of 0.86% (wt/vol) arabinoxylan (wheat arabinoxylan, medium viscosity, Megazyme, Ireland), reaction buffer (100 mM MOPS, pH 6.5, 50 mM NaCl, 10 mM CaCl2) and the appropriate amount of enzyme. Quantification of reducing sugars was performed as described by Wood and Bhat (1988) using 3,5-dinitrosalicylic acid (DNSA). The amount of liberated reducing sugar ends was determined based on a calibration curve with glucose. One unit (U) is defined as the amount of enzyme required to liberate one mole reducing sugar equivalents in one minute. All assays were performed at least in triplicates.

[0198] The Clostridium stercorarium xylanase of SEQ ID NO: 1 and the variants of SEQ ID NOs: 2 and 3 were produced, purified and quantified as described in Examples 1-4. The specific activity of the enzymes of SEQ ID NOs: 1 to 3 was calculated as U/mmol. The results were calculated as ratio to the parent enzyme SEQ ID NO: 1. FIG. 1 shows the size of the enzymes of SEQ ID NOs: 2 and 3 compared to the size of the parent protein of SEQ ID NO: 1. The variants enzymes SEQ ID NOs: 2 and 3 have a reduced size of 57% and 35% respectively compared to the parent enzyme SEQ ID NO: 1 (FIG. 1). The calculation of the specific enzyme activity in respect to the molar mass of the individual enzymes revealed that the specific enzyme activity of SEQ ID NO: 2 compared to that of SEQ ID NO: 1 is reflected by the size difference perfectly. However, surprisingly the variant of SEQ ID NO: 3 displays an at least 1.6 fold specific enzyme activity of compared to the specific enzyme activity of the polypeptides of SEQ ID NOs: 1 and 2 (FIG. 2), which does not correspond to the size changes (FIG. 1). Surprisingly, the deletion of all carbohydrate binding modules leads to an increase of enzyme activity.

[0199] The activity assay was used to define the activity profile of SEQ ID NO: 3 over a broad range of temperatures (25 C. to 90 C.) and pH 4.0-10.0. The enzyme activity measured at 70 C. and pH 7.0 was set to 100%. We identified a sufficient enzyme activity (40% or higher) from 35 C. to 85 C. in a broad pH range from pH 5.0 to 9.5 (FIG. 3) indicating a suitable profile for different industrial applications.

Example 6: Thermostability (Tm.SUB.on .and Tm.SUB.50.) of the Polypeptides of SEQ ID NOs: 1-3

[0200] To determine the physical stability of the SEQ ID NOs 1-3 variants, differential scanning fluorimetry (DSF) was applied. Proteins were diluted for DSF to a concentration of 0.1 mg/ml in 20 l 50 mM K-phosphate buffer (pH 7.0, 50 mM NaCI and 5 Sypro Orange (Thermo Fisher Scientific)). Measurements were performed in a Bio-Rad Real-Time PCR CF X96 Touch Real-Time PCR Detection System. For this purpose the samples were equilibrated for 3 min at 25 C. and then heated with a ramp of 2 C/min to 98 C. The emitted fluorescence was detected in the HEX channel (Ex.: 52510 nm; Em.: 57515 nm). To determine the Tm.sub.50, experimental data were analyzed by non-linear regression via the Boltzmann equation as described by Niesen et al. (2007) using GraphPad Prism v6.

[00002]$y=L.Math.L+\frac{\mathrm{UL}-\mathrm{LL}}{1+\frac{\exp \left(T.Math.m_{5.Math.0}-X\right)}{\mathrm{slope}}}$

[0201] The Tm.sub.50 describes the temperature of 50% protein denaturation. This corresponds to the X-axis intercept with maximal slope. The onset of protein denaturation, when 1% of the total protein is denatured is described by Tm.sub.on, and was determined as described by Menzen and Friess (2013) with values obtained from the Boltzmann equation.

TABLE-US-00001 TABLE 1 Determination of the melting point of the polypeptides SEQ ID NOs: 1-3 (SEQ ID NO: 1) (SEQ ID NO: 2) (SEQ ID NO: 3) Tm.sub.50 [ C.] 82.9 0.57 82.8 0.8 85.7 0.21 Tm.sub.on [ C.] 67.0 0.04 63.5 0.25 81.2 0.27

[0202] The Tm.sub.50 and Tm.sub.on both reflect the thermostability of an enzyme. With the deletion of all carbohydrate binding modules, the Tm.sub.on of the polypeptide of SEQ ID NO: 3 was increased to 81.2 C., which is an increase of more than 10 C. compared to the wildtype enzyme of SEQ ID NO: 1. In addition an increase of Tm.sub.50 to 85.7 C. could be achieved by the deletion of all CBMs making SEQ ID NO: 3 more thermostable than SEQ ID NO: 1.

Example 7: Thermostability (TM) of the Polypeptides of SEQ ID NO: 3

[0203] By indirectly measuring thermostability enzyme inactivation can be measured as function of temperature. Here enzyme samples are incubated without substrate for a defined period of time e.g. 5 min at various temperatures and following incubation assayed for residual activity at the permissive temperature. Residual activity at each temperature is calculated as relative to a sample of the enzyme that has not been incubated at the elevated temperature. The resulting thermal denaturation profile (temperature versus residual activity) can be used to calculate the temperature at which 50% residual activity is obtained. This value is defined as the Tm value. The Tm value is the temperature at which 50% residual activity is obtained after 5 min incubation. The polypeptide SEQ ID NO: 3 was preincubated for 30, 60, and 300 s at pH 6.5 at 80, 85, 90, and 95 C. Afterwards, enzyme activity was determined at standard conditions as described in example 5 and compared to the untreated polypeptide of SEQ ID NO 3, respectively. For SEQ ID NO: 3 the Tm value is between 85 C. and 90 C. (FIG. 4). During pelleting for feed pellets high temperature is applied for a relatively short time period (e.g. 30 sec at about 80 C., WO2008063309). As shown in FIG. 4 the enzyme SEQ ID NO: 3 exhibits 80% or higher enzyme activity, even when the temperature exposure is up to 60 s and up to 95 C. Thus, the polypeptide of SEQ ID NO: 3 completely fulfills the requirements of the pelleting process.

Example 8: Resistence Against Acid Treatment

[0204] To simulate conditions occurring in the gastric tract of poultry in vitro, the polypeptides of SEQ ID NO: 1 and SEQ ID NO: 3 were incubated at pH 3.5 at 40 C. for 30, 60 and 120 min. The remaining activity was determined by DNSA assay and compared to the activity of the untreated protein in accordance with Example 5 unless the temperature was changed to 70 C. The parent enzyme SEQ ID NO: 1 shows a decrease in enzyme activity along with the prolonged acidic treatment. Surprisingly, this effect is diminished drastically for the enzyme SEQ ID NO: 3 lacking the carbon binding motives (FIG. 5). Even at conditions occurring in the chicken intestine, more than 70% of the initial activity can be restored after the simulated passage of the gastric tract.

Example 9: Resistence Against Proteolysis

[0205] Proteolytic stability of SEQ ID NO: 3 against pepsin was tested by incubating the enzyme for 30 and 60 minutes at 40 C. with 50 U/ml pepsin from gastric mucosa (Sigma-Aldrich) in incubation buffer (pH 3.5; 50 mM NaCI). Residual activity was determined as described in Example 5 and is shown in FIG. 6. Prolonged pepsin digestion didn't affect enzymatic activity and more than 85% residual activity could be recovered after 2 h of pepsin digestion of the enzyme.

Example 10: Reduction of Viscosity in the In Vitro Chicken Intestine Model

[0206] To demonstrate experimentally the production of animal feed pellets and the gastrointestinal passage of animal feed pellets, the following procedure was chosen: Enzyme was mixed with milled feed and heated at 95 C. for 2 minutes in a water bath. The simulation of the gastric passage was performed as described by Bedford and Classen (1993). Protease digestion was tested by incubating the feed enzyme mixture for 45 min at 40 C. and pH 3.0 with pepsin. Passage of the feed enzyme mixture through the small intestine environment was simulated for 2 h at 40 C. and pH 6.8 and then the viscosity was determined in a spherical viscometer. The same number of units was used for SEQ ID NO: 3 and the Danisco xylanase, which can be purchased from distributors such as Biochem Zusatzstoffe Handels- and Produktionsgesellschaft mbH, was used for comparison. The SEQ ID NO: 3 achieved an equivalent result and thus shows its effectiveness in this in vitro assay. (see also FIG. 7)

Example 11: Viscosity Reduction of Wheat Slurry

[0207] In first generation ethanol production, wheat slurry is an industrial important source for sugars. At the beginning mashing is performed at high temperature (>70 C.) to avoid microbial contamination. To reduce undesirable high viscosities and enable starch accessibility xylanase and cellulase are added early in the process together with starch hydrolysing alpha amylase. To keep the slurry in a pumpable and mixable state a rapid decrease in viscosity is an urgent need and a xylanase with robust activity at high temperature is needed.

[0208] To demonstrate the ability of the polypeptides of SEQ ID NOs: 1 to 3 to reduce viscosity at high temperature, 250 g shredded wheat were mashed with 500 mL of boiling water. After gaining a homogeneous slurry different enzymes were added: 25 mg/kg Teramyl 300L, an alpha-amylase from Bacillus licheniformis (Sigma-Aldrich, A4862), 0.4 mg/kg SEQ ID NO: 3, 0.4 mg/kg of a hemicellulase/cellulase blend (80% SEQ ID NO: 3, and 20% of a modified endoglucanase from Clostridium thermocellum (SEQ ID NO: 11, EP17203087)) or 0.4 mg/kg Cellic CTec2 (Sigma-Aldrich, SAE0020 SIGMA). Viscosity reduction was monitored as time to complete fluidity in a stirred tank reactor equipped with one Rushton impeller (BIOSTAT Single, 2 L Univessel) at 70 C. jacket temperature and 1000 rpm. Without any enzyme no liquefaction could be observed. For calculation reason, the time to complete liquefaction using Teramyl 300L was set to 100%. A combination of Teramyl 300L and the polypeptide of SEQ ID NO: 3 leads to a 40% decrease in liquefaction time. A 60% decrease could be achieved with Teramyl 300L, the polypeptide of SEQ ID NO: 3 and an additional endoglucanase from C. thermocellum (SEQ ID NO: 11, EP17203087), whereas the commercial available cellulose/hemicellulose mix Cellic CTec2, added in the same dosage, shows no effect (FIG. 8).

REFERENCES

[0209] Adelsberger, H., Hertel, C., Glawischnig, E., Zverlov, V. V., Schwarz, W. H., 2004. Enzyme system of Clostridium stercorarium for hydrolysis of arabinoxylan: reconstitution of the in vivo system from recombinant enzymes. Microbiology 150 (7), 2257-2266.

[0210] Bedford M. R. and Classen H. L. 1993. An In Vitro Assay for Prediction of Broiler Intestinal Viscosity and Growth When Fed Rye-Based Diets in the Presence of Exogenous Enzymes Poultry Science, Volume 72, Issue 1, 1 Jan. 1993, Pages 137-143.

[0211] Biely, P., Vrsansk, M., Tenkanen, M., Kluepfel, D., 1997. Endo-beta-1,4-xylanase families: differences in catalytic properties. Journal of biotechnology 57 (1-3), 151-166.

[0212] Bodie, E. A., Cuevas, W. A., Koljonen, M. Five thermostable xylanases from microtetraspora flexuosa for use in delignification and/or bleaching of pulp.. WO1995029997A1, WO1995029998A1 1994

[0213] Menzen, T., Friess, W., 2013.Temperature-Ramped Studies on the Aggregation, Unfolding, and Interaction of a Therapeutic Monoclonal Antibody. Journal of Pharmaceutical Sciences , Volume 103 , Issue 2 , 445-455.

[0214] Bouraoui, H., Desrousseaux, M.-L., loannou, E., Alvira, P., Manai, M., Remond, C., Dumon, C., Fernandez-Fuentes, N., O'Donohue, M. J., 2016. The GH51 alpha-L-arabinofuranosidase from Paenibacillus sp. THS1 is multifunctional, hydrolyzing main-chain and side-chain glycosidic bonds in heteroxylans. Biotechnology for biofuels 9, 140.

[0215] Brockmeier, U., Caspers, M., Freud!, R., Jockwer A., Noll, T. and Eggert T., 2006, Systematic Screening of All Signal Peptides from Bacillus subtilis: A Powerful Strategy in Optimizing Heterologous Protein Secretion in Gram-positive Bacteria, Journal of Molecular Biology. Volume 362, Issue 3, Pages 393-402.

[0216] Carre, B., Escartin, R., Melcion, J. P., Champ, M., Roux, G., Leclercq, B. Effect of pelleting and associations with maize or wheat on the nutritive value of smooth pea (Pisum satiuum) seeds in adult cockerels. Br. Poultry Sci. 1987,28, 219-229.

[0217] Chi, W J., Park, D. Y., Chang, Y K., Hng S K. (2012). A Novel Alkaliphilic Xylanase from the Newly Isolated Mesophilic Bacillus sp. MX47: Production, Purification, and Characterization. Appl Biochem Biotechnol 168: 899.

[0218] Collins, T., Gerday, C., Feller, G., 2005. Xylanases, xylanase families and extremphilic xylanases. FEMS Microbiology Reviews, 29: 3-23.

[0219] Evans, G. I., Lewis, G. K., Ramsay, G. and Bishop, J. M., 1985. Isolation of monoclonal antibodies specific for human c-myc proto-oncogene product. Mol Cell Biol. Dec; 5 (12):3610-6.

[0220] Fukunaga, N., Iwasaki, Y., Kono, S., Kita, Y., Izumi, Y., 1998. Thermostable xylanase. U.S. Pat. No. 5,916,795. 1994.

[0221] Gems, D., Johnstone, I. L. and Clutterbuck, A. J., 1991. An autonomously replicating plasmid transforms Aspergillus nidulans at high frequency. Gene. Feb 1; 98 (1):61-7.

[0222] Guo and Sherman, 1995. Molecular Cellular Biology 15: 5983-5990.

[0223] Dutta, T., Sengupta, R., Sahoo, R., Sinha Ray, S., Bhattacharjee, A., Ghosh, S. (2007). A novel cellulase free alkaliphilic xylanase from alkali tolerant Penicillium citrinum: production, purification and characterization. Let. Appl. Microbiol. 44:206-211.

[0224] Kolenov, K., Vransk, M., Biely, P., 2006. Mode of endo-, 4-xylanases of families 10 and 11 on acidic xylooligosaccharides. Journal of Biotechnology, 121:338-345.

[0225] Korz, D. J., Rinas, U., Hellmuth K., Sanders, E. A., Deckwer, W. D., 1995. Simple fed-batch technique for high cell density cultivation of Escherichia coli. J. Biotechnol. 39:59-65.

[0226] Kroll, J., Steinle, A., Reichelt, R., Ewering, E., and Steinbchel, A., 2009. Establishment of a novel anabolism based addiction system with an artificially introduced mevalonate pathway: complete stabilization of plasmids as universal application in white biotechnology. Metab Eng 11:168-177.

[0227] Laemmli, U. K., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 880-685.

[0228] Lombard V., Golaconda R. H., Drula E., Coutinho P. M., Henrissat B., 2014. The Carbohydrate-active enzymes database (CAZy) in 2013. Nucleic Acids Res 42:D490-D495.[PMID: 24270786].

[0229] Park, Y. S., Kai, K., lijima, S., Kobayashi, T., 1991. Enhanced 13-Galactosidase Production by High Cell-Density Culture of Recombinant Bacillus subtilis with Glucose Concentration control). Biotechnol Bioeng. 40(6), 686-696.

[0230] Prade, R. A. 1996. Xylanases: from Biology to BioTechnology, Biotechnology and Genetic Engineering Reviews, 13:1, 101-132.

[0231] Prakash, P., Jayalakshmi, S. K., Prakash, B. Rubul. M., Sreeramulu, K., 2012. Production of alkaliphilic, halotolerent, thermostable cellulase free xylanase by Bacillus halodurans PPKS-2 using agro waste: single step purification and characterization World J Microbiol Biotechnol 28: 183-192.

[0232] Niesen, F. H., Berglund, H., & Vedadi, M. (2007). The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nature protocols, 2(9), 2212-2221

[0233] Romanos, M. A., Scorer, C. A. and Jeffrey, J. C., 1992. Foreign Gene Expression in Yeast: a Review. YEAST VOL. 8: 423-488.

[0234] Sambrook, J, Russell, D. W., 2001.Molecular cloning: a laboratory manual 3rd edition. Coldspring-Harbour Laboratory Press, UK.

[0235] Sambrook, J., Fritsch, E. F., Maniatis. T., 1989. Molecular cloning: a laboratory manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

[0236] Simonen, M. and Palva, I., 1993. Protein secretion in Bacillus species. Microbiological Reviews 57: 109-137.

[0237] Schwarz, W. H., Adelsberger, H., Jauris, S., Hertel, C., Funk, B., Staudenbauer, W. L. 1990. Xylan degradation by the thermophile Clostridium stercorarium: cloning and expression of xylanase, beta-D-xylosidase, and alpha-L-arabinofuranosidase genes in Escherichia coli. Biochem Biophys Res Commun 170(1): 368-374.

[0238] Weber, K., Osborn, M., 1969.The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406-12.

[0239] Wilson, I. A., Niman, H. L., Houghten, R. A., Cherenson, A. R., Conolly, M. L., Lerner, R. A., 1984. The structure of an antigenic determinant in a protein. Cell. ; 37(3):767-78.

[0240] Wood T M, Bhat K M. Methods for measuring cellulase activities. Methods in enzymology, Vol. 160. 1988. p. 87-112.