CA2139001A1 - Combined action of endoglucanases and cellobiohydrolases - Google Patents

Abstract

The present invention discloses the use of combinations of cell wall degrading enzymes in the degredation of cell wall derived substrates. Specifically, the use of combinations of endoglucanases and cellobiohydrolases, comprising xyloglucanase activity is demonstrated.

Description

2~3~a~
W094/2~80 PCT~4/01531 Combined action of endoqlucanases and cellobiohydrolases Technical field The present invention is in the field of cell wall degradation. The present invention discloses the use of combinations of enzymes in the degradation of plant cell walls.
The advantage of the use of xyloglucanase activity is disclosed. Specific optimized ratios of cell wall degrading enzymes are also disclosed. Specifically, the combined action of endoglucanases and cellobiohydrolases in the degradation of a xyloglucan/cellulose complex is disclosed.
Furthermore it is demonstrated that these enzymes have a synergistic effect.

Backqround of the invention In the study of enzymatic plant cell wall degradation two 2s structural components of plant cell walls have been widely investigated, these are cellulose and pectin.
Apart from cellulose and pectic substances, a third structural element of the cell wall should be mentioned: the xyloglucans (Hayashi et al. (1984) Plant Physiol. 75 : 605-610, 30 Hayashi T. (1989) Ann. Rev. Plant Physiol. Plant Mol. Biol. 40 : 139-168). Xyloglucans consist of a cellulosic backbone about 70% of the glucose residues of this backbone are substituted with side chains. These side chains primarily contain xylose, galactose and fucose. Further, they hydrogen bond tightly to 35 cellulose (Hayashi et al. Plant Physiol. 83 : 384-389, Talbott L.D. and P.M.Ray (1992) Plant Physiol. 98 : 357-368) and are also bound to other cell wall components (Fry S.C. and J.G.

SUBSmUTE SHEET ~RULE 26) W094/26880 ~13 9 ~ O 1 PCT~4/01531 Miller (1989). Toward a working model of the growing plant cell wall : phenolic cross-linking reactions in the primary cell walls of dicotyledons. In : Lewis N.G., Paice M.G. Eds. The biosynthesis and biodegradation of plant cell wall polymers, 5 ACS, Washington DC, pp 33-46 and Talbott L.D. and P.M.Ray (1992), cited above).
Although they are present in relatively large amounts (about 20% in dicots according to T. Hayashi (1989, cited above) no attention has been paid to xyloglucans with respect 10 to the enzymatic liquefaction process of plant material (Voragen et al.(1992) Fluss. Obst.59 404-410). The activities screened for during the purification of glucanases are CMCase (often referred to as Cx or endoglucanase), Avicelase (often referred to as C1, cellobiohydrolase or exoglucanase) and 15 sometimes cellobiase or B-glucosidase. Xyloglllc~nAce (XGase) activity is generally not included. This means that to date no systematic use has been made of xyloglucanase activity in cell wall degradation.
Relatively little is known about the xyloglucans in the 20 process of fruit juice preparation. Just like pectin, they are known to be good gelling agents (M. Glicksman. (1986) In Glicksman M. ed.- Food Hydrocolloids 3, CRC Press, Boca Raton, Florida, pp. 191-202) and are used as such in Japan. For the liquefaction of apple tissue, efficient degradation of the bulk 25 component of the primary cell wall, cellulose, is essential.
The accessibility of cell wall embedded cellulose is reduced by the presence of other cell wall constituents such as pectin and xyloglucan.
It has been demonstrated that the removal of pectin 30 facilitates cellulose degradation. Whereas xyloglucans received little attention.
The present invention discloses the advantages of the use of xyloglucanase activity in the degradation of cell wall cellulose.

SUBSrltllTE SHEE~ (RlJLE 26) W094/~80 213 9 ~ D 1 PCT~4/01531 -SummarY of the invention The present invention discloses mixtures of endoglucanases and cellobiohydrolases comprising xyloglllc~Ace activity, which 5 are adapted for optimal cellulose degradation. One specific mixture comprises three enzymes; endogl~lc~nA.se I, endoglucanase IV and cellobiohydrolase, another specific mixture comprises two enzymes; endoglucanase V and cellobiohydrolase.
The ratio of xylogl~lc~nA~ to avicelase activity in the 10 mixture of endoglucanases and cellobiohydrolases is chosen to provide optimal cellulose hydrolysis.
In another embodiment of the invention, the mass ratios of the enzymes of the specific EndoI/EndoIV/CBH mixture are chosen such that at a constant (Endol + Endolv)/CBH mass ratio of 0.5-15 20 (g/g), an optimal molar Endol/Endolv ratio of 0.1-3 is used.
Preferably the mass ratio of endoglucanase/cellobio-hydrolase is 0.96 and the EndoI/EndoIV molar ratio is 0.33.
In addition, methods for preparation of the desired mixtures are described.
20 The present invention further discloses a method for the degradation of a cell wall embedded xyloglucan/cellulose complex by the combined action of endoglucanases (Endo), cellobiohydrolases (CBH) and xyloglucanase activity.
Specifically, apple cell wall cellulose is degraded.
25 The method makes use of a combination of endoglucanases and cellobiohydrolases. The endoglucanase is preferably selected on the basis of xyloglucanase activity. Any species producing enzymes having the described activities can in principle be used as a source for the enzymes. Preferably, the enzymes are 30 obtainable from Trichoderma, As~ergillus or Disporotrichum.
The present invention describes the use of xyloglucanase activity in the degradation of cell wall materials.
Specifically in the degradation of a xyloglucan/cellulose complex.
35 The present invention further discloses products obtained after treatment of plant material containing a xyloglucan/cellulose complex with cellulose-degrading enzyme SVB~TITUTE SHEET (RVLE 26) W094~0 ~ PCT~4/01531 mixtures containing xyloglucanase. Specifically, the product originates from apple. More specifically, the product is apple juice.
s Brief description of the drawinas Figure 1. Typical chromatograms showing the degradation products of apple-WUS solubilised by (combinations of) 10 glucanases, analysed on an Aminex HPX 22H column: A, CBH;
B, EndoI+CBH; C, EndoIV+CBH. The elution profile of a maltodextrin syrup is indicated by arrows with the corresponding degree of polymerisation. Typical degradation products of EndoIV are indicated by the shaded peaks. The 15 sum of these areas was used for quantification of the xyloglucan oligosaccharides.

Figure 2. The influence of the endoglllcAnAse/cellobiohydrolase-ratio on the release of cellobiose and oligomeric 20 xyloglucan fragments from apple-WUS; 0 cellobiose release by EndoI+CBH; 0 cellobiose release by EndoIV+CBH; release of xyloglucan fragments by EndoIV+CBH; FXG = fucosylated xyloglucan.

25 Figure 3. Release of cellobiose (~) and xyloglucan oligosaccharides (-) in a three enzyme system at a fixed (EndoI+EndoIV)/CBH mass ratio of 0.96~g/~g and varying amounts of EndoI and EndoIV.

30 Figure ~. Liquefaction of raw apple fruit tissue by PL and (upgraded, see Example 3) commercial enzyme preparations.
Raw apple fruit tissue was incubated in 200 mM succinate buffer pH 4.0 for 16 hrs at 40C, and 150 rpm. 1, no enzyme added; 2, Maxazyme (10 mU toward Avicel, 200 mu XGase); 3, 50 mU PL; 4, combination of 2 and 3; 5, Xyl 5000 (10 mU
toward Avicel, 100 mU XGase); 6, combination of 3 and 5; 7, as 6 plus 100 mU EndoV.

SUBSTITUTE S~EET (RlJLE 26) ~139~
W094/26880 PCT~4/01531 Figure 5. Release of cellobiose from apple fruit tissue by mixtures of purified glucanases. Blanched apple fruit v tissue was incubated (200 mM succinate buffer pH 4.0, 16 hrs, 40C, 150 rpm) with PL in combination with various 5 glucanase mixtures which were equal in Avicel degrading potential (0.26 mU) but different in their XGase activity (5 to 40 mU). Cellobiose was quantified by HPAEC.

Detailed description of the invention The present invention discloses the use of enzymes having xyloglucanase activity in the degradation of biological material containing xyloglucans.
15 Based on the finding that screening glucanases for CMCase and Avicelase activity is not an adequeate measure for finding enzymes with xyloglucanase activity, the glucanases are screened for activity on xyloglucans. It was found that there is a difference between the activities of the different 20 endoglucanases.
The present invention further describes the advantages of using mixtures of cellulose degrading enzymes comprising endoglucanases, cellobiohydrolases and xyloglucanase activity.
Preferably the mixture is obtained from Trichoderma, 25 Asperqillus or Disporotrichum. The mixtures are isolated from the growth medium of these microorganisms without further purification. It should be noted that the activities and ratios of the different enzymes in the mixtures depend on the substrate, the growth conditions and the strains used in 30 fermentation.
If the mixture so isolated contains the desired ratio of enzymatic activity the mixture is used as such. The desired ratio depends on the substrate which has to be degraded and is preferably determined for every substrate.
35 If the mixture that is isolated without further treatment does not contain the desired ratio of enzymatic activities, mixtures from different cultures are used to improve the ratio.

SUBSllTUrE SREET (RULE 26) W094~0 ~13 ~ O O 1 PCT~4/01531 It is also possible to mix culture fluids from growth of different strains or species. Alternatively, in order to obtain the desired ratio of enzymatic activity, the enzymes are purified. Purification can be performed with any method known 5 to a person skilled in the art. Preferably, the enzymes are purified to a level of purity of 70%. More preferably, the level of purity is 90%, especially preferably, it exceeds 95%.
Purification according to Beldman et al. (Eur. J. Biochem.
(1985) 146 : 301-308) yielded six endogluc~nAcec (EndoI to VI) 10 and two cellobiohydrolases (CBH). The nomenclature of the enzymes used in the present specification is in accordance with Beldman et al. (Biotech. Bioeng.(1988) 31 160-167), and therefore differs from other publications. Other procedures and sources than those described in the present specification can 15 also be used for obtaining the said enzymes. One alternative approach would be the cloning and expression of the gene(s) encoding the desired enzyme(s) in heterologous microorganisms, which would avoid contamination with other enzymes. It is however also possible to use a homologous microorganism for 20 cloning and overexpression of the desired gene(s). In that case the ratio of the different enzymes is influenced without external addition of enzyme. Inactivation of specific enzymes belonging to the mixture is also possible. Differential inactivation may be obt~i n~ by e.g. heating, pH alteration or 25 addition of specific inhibitors.
Preferably the mixtures of cellulose degrading enzymes comprising endoglucanases, cellobiohydrolase and xyloglucanase activity are obtained by mixing the purified enzymes in predetermined amounts or by combining mixtures with 30 predetermined activity giving the desired final enzymatic activity ratios.
The present invention discloses that the different enzymes have a synergistic effect when they are used in the degradation of cell wall material. This effect is especially clear when a 35 mixture is made of endoglucanases, comprising xyloglucanase activity, and cellobiohydrolases in specific ratios. T h e preferred mass ratios of the enzymes will depend on the SUBSTITU~E SH~ET (RULE 26) WOg4/26880 ~13 9 0 31 PCT~4/01531 substrates used.
The cellulase complex of Trichoderma viride (presently known as Trichoderma reesei) has been shown to contain six distinct endoglur~n~cec (Beldman et al (1985) Eur. J. Biochem.
5 146 : 301-308),. which can be subdivided into two classes: a specific (Endo I, II and III) and a non-specific (Endo IV, V
and VI) class, based on their ability to degrade xylans. In Example 1 it is demonstrated that the endoglucanases could be divided in three groups of enzymes based on their ability to 10 degrade xyloglucans: one group active on cellulose (Avicel), e.g. EndoI, one group active on xyloglucans, e.g. EndoIV, and one group active on both substrates, e.g. EndoV (see Table III).
The present invention discloses the advantage of the use of 15 endoglucanases IV, V and VI, comprising xyloglucanase activity, over endoglucanases I, II and III (Table III).
The present invention discloses the advantage of the combined action of EndoIV, which is specific for xyloglucan, and EndoI + CBH, specific for cellulose degradation, and the 20 advantage of the combined action of EndoV, which is specific for both xyloglucan and cellulose, and CBH, in the degradation of cell-wall embedded cellulose. It is further recognized that both aspects of the EndoI-EndoIV combination are represented in EndoV.
25 Furthermore, the use of combinations of these enzymes is demonstrated. In general, an enzyme comprising xyloglucanase activity, e.g. an enzyme belonging to the Endo IV to VI group is chosen to provide release of the xyloglucan from the surface of the xyloglucan/cellulose complex, whereafter (an) enzyme(s) 30 comprising cellulase activity, e.g. an enzyme of the Endo I to Endo III group together with CBH can attack the cellulose. It is recognized that the relative amounts of endoglucanases, cellobiohydrolases and xyloglucanase activity depend on the substrate. Upscaling of the process of cellulose degradation, 35 e.g. to industrial scale, may also affect the relative amounts of these enzymes. The present invention is demonstrated by the use of so-called water-unextractable solids (WUS) and by the SUBSTITUTE SHEET (RUL~ 26) W094/26880 ~13 9 0 ~1 PCT/~195~01531 use of apple tissue.
Example 2 demonstrates that for efficient degradation of cell wall cellulose three activities are important:
endogl~lcAnA~e (P~c~ssing activity towards Avicel), 5 cellobiohydrolase and xyloglu~AnAce. Optimal cellulose degradation is achieved when the cellobiose release is optimal.
Cellobiose release, on its turn, is optimal when the release of xyloglucan oligomers is maximal. For degradation of apple WUS, an optimal Endo/CBH molar ratio has been observed which varies 10 from 1.1 in case of EndoI/CBH to 14.9 in case of EndoIV/CBH.
However, twice as much cellobiose is released with optimal EndoIV/CBH as compared to optimal EndoI/CBH ratios. Relatively large amounts of EndoIV are required because of its low adsorbance to cellulose. Using the three enzyme system, 15 comparable cellulose degradation can be obtained, however, with much lower amounts of protein.
Example 2 further demonstrates that the endoglucanase/
cellobiohydrolase mass ratio is optimal between 0.5 and 20 (g/g). Using a constant mass ratio of 0.96 (EndoI + EndoIV/CBH) 20 it is demonstrated that an optimal ratio of EndoI/EndoIV ranges from 0.1 to 3. Preferably the ratio is between 0.20 and 0.60, more preferably the ratio is 0.33.
Specifically, the endoglucanases are EndoI and EndoIV and the mixture is characterized in that at a constant (Endo~ +
25 Endolv)/CBH mass ratio of 0.96 (g/g) the mixture contains a molar Endol/Endolv ratio of 0.33.
In Example 3, the importance of xyloglucanase activity for the degradation and liquefaction of apple tissue is demonstrated. A cellulase preparation with low xyloglucanase 30 activity is improved by addition of xyloglucanase, preferably by addition of an endogl~cAnAse selected from the group of EndoIV to EndoVI, more preferably by addition of EndoV.
Example 4 demonstrates that mixtures of purified enzymes produce similar effects. Increased cellobiose release from 35 apple tissue is observed upon increasing the amount of xyloglucanase activity, keeping the Avicel-degrading potential of the mixtures constant. A minimal amount of 25mU of SUBSrIME SHEET ~RULE 26) W094t26880 2 1 3 ~ PCT~4/01531 xyloglucanase activity, in the form of EndoV, is n~ceCc~ry upon 0.26mU of Avicel-degrading activity.

The mixtures as defined by the present invention can 5 advantageously be used on substrates having xyloglucans as components. Such substrates occur in food, feed, paper, pulp and textile raw materials. The mixtures are therefore advantageously employed in degradation processes in food, feed, paper, pulp and textile treatment.
The mixtures find particular use in the preparation of fruit juices. Specifically, in the preparation of apple juice.

The present invention describes the specificity of the six fungal endogluc~n~s~c for four different glucans (Example 1).
15 Further, the degradation of the glucan complex by the combined action of endoglucanases and cellobiohydrolase is disclosed, using model cell walls from apple (Example 2) or complete apple tissue (Examples 3, 4) as a substrate.
The synergistic effect of the use of specific combinations of 20 enzymes is disclosed.

ExPerimental 25 Materials Apples (Malus malus L., Rosaceae, var. Golden Delicious) were harvested in mid-October, 1989, and stored in controlled atmosphere (2C, 2% 2 and 5% CO2) for 4 months.
30 SiX endoglucanases (EndoI to VI) tE.C.3.2.1.4] and two cellobiohydrolase (CBH) [E.C.3.2.1.91] were purified from a commercial preparation from Trichoderma viride (Maxazyme Cl, Gist-Brocades, Delft, The Netherlands) as described by Beldman et al. (1985). Eur.J.Biochem. 146: 301-308.
35 Avicel crystalline cellulose (type SF) was obtained from Serva (Heidelberg, Germany), CMC (Akucell AF type 0305) from Akzo (Arnhem, The Netherlands) and tamarind seed xyloglucan SUBSJIME SHEET tRULE 26) W094/~880 213 9 0 ~1 PCT~4/01531 from Dainippon Pharmaceutical (Osaka, Japan).

Isolation of water-unextractable solids (WUS) 5 After remov~ng the core, the apples (5kg) were microwaved (Philips AKB 276/PH, 4kW, Switzerland) in portions of one kilogram in order to inactivate endogenous enzymes (polygalacturonase and pectin-esterase). The resulting material was peeled, ground and extracted extensively with warm 10 distilled water (50C) until the wash water contA~ minor amounts of sugars. Solids were collected by centrifugation (20min, 50,000~). The residual material was freeze-dried and ground in a Fritsch pulverisette (sieve l.Omm, Germany) and designated as WUS.
Preparation of xyloglucans Extraction of WUS. WUS (2g) was extracted with 0.05N NaOH
(200ml) containing 5mM 1,2-cyclohexylene-dinitrilotetraacetic 20 acid (CDTA) for 16h at 4C under continuous stirring. After centrifugation (20min, 50,000g) the residue was resuspended in lN KOH (320ml) containing 1% (w/w) NaBH4, extracted for 16h at 20C and centrifuged (20min, 50,000~). Using this residue, the latter procedure was repeated with 4N KOH containing 1% (w/w) 25 NaBH4. Between sequential extraction steps, the residue was washed twice with distilled water. The final residue was acidified to pH5 with HCl and freeze dried. The corresponding supernatants were collected, acidified to pH5 with HCl and dialysed extensively against distilled water.
30 Purification of the 4N KOH extract. The 4N KOH extract was depectinized on a DEAE Sepharose CL-6B column (40x440mm, Pharmacia , Uppsala , Sweden), equilibrated with 50mM sodium acetate buffer (pH5.0). After application of the sample (200ml), the column was washed with 400ml of buffer. The 35 fraction retained on the column was released by elution with 500ml lM sodium acetate buffer (pH5.0). Fractions (16.5ml) were assayed for both uronic acids and neutral sugar, pooled, SUBSTITU~E SHEET (RULE 26~

2133~1 WOg4/26880 ~ PCT~4101531 dialysed and freeze-dried. The neutral fraction consisted of xyloglucan (APFXG).
Mild acidic hydrolysis of APFXG. Purified apple xyloglucan (40mg) was treated with 25mN TFA (5mg/ml) at 60-C during 100h.
5 After dialysis the defucosylated XG (APXG) was freeze-dried.

Determination of enzyme activities.

Activities of endogl~lç~nAces and cellobiohydrolases on CMC
10 (CMCase), Avicel (avicelase) and tamarind seed xyloglucan (xyloglucanase) were determined by measuring the increase in reducing endgroups according to Somogyi (J.Biol. Chem. 195: 19-23). Incubations (40C, 0.1M succinate buffer pH 4.0) occured under non-substrate-limiting conditions. One milli-unit (mU) of 15 enzyme corresponds to the amount of reducing endgroups in nanomoles formed in one minute.

Analytical methods 20 Uronic acid content. Uronic acids (AUA) were estimated colorimetrically with an automated m-hydroxydiphenyl test (Thibault JF (1979) Lebensm.Wiss.Technol. 12: 247-251) using concentrated sulphuric acid containing 0.0125M Na2B4O7. Water-insoluble material was pretreated with 72% (w/w) H2SO4 (lh at 25 30C) and, after dilution with water, 2N H2SO4 (3h at 100C) prior to analysis.
Total neutral sugar content. The total neutral sugar content was determined colorimetrically with an automated orcinol/sulphuric acid assay (Tollier MT, Robin JP (1979) Ann.
30 Technol. Agric. 28: l-lS).
Neutral suaar com~osition. Water-insoluble material was subjected to a 7296 (w/w) H2S04 prehydrolysis (lh at 30OC) followed, after dilution with water, by a 2N H2SO4 hydrolysis (3h at 100C). Water solubles were hydrolysed with 2N TFA (lh 35 at 121C). Next, the released neutral sugars were converted to their alditol acetates (Englyst HN, Cummings JH (1984) Analyst 109: 937-942) and separated on a 3mx2mm i.d. glass column SU~STITUrE SHÉET (RUEE 26) W094/2~0 ~13 9 ~ ~1 PCT~4/01531 (packed with Chrom WAW 80-lOOmesh, coated with 3% OV275, Chrompack, Middelburg, The Netherlands) in a Carlo Erba Fractovap 2300 GC (Milan, Italy) operated at 200-C and equipped with a F.I.D. detector set at 270 C. Inositol was used as 5 internal st~n~
G1YCOSY1 linkaqe composition. The purified xyloglucan was methylated according to a modification of the Hakomori method (Sandford PA, Conrad HE (1966) Biochemistry 5: 1508-1517) and subsequently dialysed against water and dried by evaporation o (airstream, room temperature). This procedure was repeated once. Next, the methylated xyloglucan was hydrolysed using 2N
TFA (lh ar 121-C) which was removed by evaporation (airstream, room temperature). Sugars were reduced by adding 0.2ml of a freshly prepared 1.5N ammonia solution containing 75mg 15 NaBD~ml, and converted to alditol acetates (Englyst et al.
(1984). Analyst 109 : 937-942). The partially methylated alditol acetates (1~1) were analysed by on-column injections on a fused silica capillary column (30mxO.32mm i.d.; wall coated with DB 1701; 0.25~m film thickness; J & W Scientific, Folsom, 20 California, USA) in a Carlo-Erba Fractovap 4160 gas chromatograph equipped with a flame ionization detector (FID) set at 280 ~C. The temperature program was 80~180~C at 20-C/min, 180-230-C at 2-C/min, 230C for 3min. Identification of the compounds was confirmed by gas chromatography-mass 25 spectrometry (GC-MS) using a CP Sil 19 CB capillary column (26mxO.22mm i.d., 0.18~m film thickness; Chrompack Nederland B.V., Middelburg, The Netherlands) in a HP 5890 GC coupled to a Hewlett-Packard mass selective detector 5970-B and using a PAW-HP 300 Chem Station (Hewlett-Packard). The temperature program 30 was 160-185-C at 0.5-C/min, 185-230C at 10C/min, 230-C for 5.5min. Derivatives were quantified according to their effective carbon response (Sweet DP, Shapiro R~, Albersheim P
(1975) Carbohydr Res 40: 217-225).
Protein content. Protein content of enzyme preparations was 35 determined according to Sedmak J.J. and Grossberg S.E. (1977) (Anal.Biochem. 79: 544-552).

SUBSTITUTE SHEET (RULE 26) 21390~

Analysis of x~rloglucan and ce~ ose dearadation Droducts.
High-performance liquid chromatography was conducted at 85C
with a SP 8800 HPLC pump system (Spectra Physics, San Jose, CA
U.S.A.) fitted with a Shodex SE-61 refractometer (Showa Denko 5 K.K., Tokyo, Japan) and a Aminex HPX-22H column (7.8x300mm;
Bio-Rad Labs, Richmond, CA, U.S.A.) combined with a AG 50W X4 guard column (7.8x50mm, Bio-Rad Labs). The solvent was 0.01N
sulphuric acid pumped at a flow rate of 0.2ml/min. The injection volume was 20~1. Maltodextrins were used for o calibration. Quantitative analysis of cellobiose after degradation of apple tissue was performed with high-performance anion-exchange chromatography (HPAEC) using a Dionex Bio-LC
GPM-II quaternary gradient module equipped with a Dionex CarboPac PA100 column (250x4mm, 20C, Dionex, Sunnyvale, CA).
Samples (20 ~L) were injected using a SP8780 autosampler (Spectra Physics, San José, CA) equipped with a Tefzel rotor seal in a 7010 Rheodyne injector valve. Solvents were degassed and stored under helium using a Dionex EDM module. The eluate (1 mL/min) was monitored using a Dionex PED detector in the 20 pulsed-amperometric detection (PAD) mode. A reference Ag/AgCl electrode was used with a working gold electrode with the following pulse potentials and durations: E1 0.lV and 0.5s, E2 0.6V and 0.1s, E3 0.6V and 0.1s. Cellobiose in the incubation mixtures was quantified by application of the following 25 gradient: 0--12min, linear gradient of 25--85mM NaOH, 12--25 min, linear gradient of 0--100mM NaOAc in 85 mM NaOH. After each analysis the column was rinsed for 5 min with 1 M NaOAc in 100 mM NaOH, and equilibrated in 25 mM NaOH for 15 min.

ExamPle 1 Substrate specificitY of six endoqlucanases from Trichoderma viride The bulk of the xyloglucans was extracted with 4N KOH
(Table I). This extract also contained some pectic material which could effectively be removed with anion exchange SUBSrIME SI~EE~ LE 26) W094l26~0 ~13 9 ~ 31 PCT~4/01531 chromatography. A methylation analysis was carried out to characterize this purified xyloglucan. Based on Table II and literature (York WS, van Halbeek H, Darvill AG, Albersheim P
(1990) Carbohydr.Res. 200: 9-310), this polysaccharide consists s of a (1-4)-B-glucose backbone of which c. 42% contains a (6-1)-~-xylose terminus, c. 10% is substituted with (6-1)-~-Xyl-(2-1)-B-Gal and c. 13% has a (6-1)-~-Xyl-(2-1)-B-Gal-(2-1)-~-Fuc sidechain. Also some terminal arabinose is found which is most likely connected to xylose residues. The sugar composition 10 of the purified apple xyloglucan is consistent with the findings of others (Renard C.M.G.C. et al. (1991) Carbohydr.
Polymers 15: 387-403 and Ruperez P. et al.(1985) Carbohydr.Res.
142: 107-113). Undermethylation occurred to a limited extent, esp. of the hexose residues (c. 5%). It should be noted that 15 the purified xyloglucan still contains a slight contamination (c. 5%), most likely a galacto-(gluco)-mannan with a galactose attached to every nine of the mannose residues.
The purified xyloglucan (250~g) was dissolved in lOO~L
buffer and incubated for 1 or 20h with EndoIV, EndoV and EndoVI
20 (30ng) or EndoI, EndoII and EndoIII (400ng) respectively.
Enzyme dosage was such that no substrate limitation occurred.
The samples were then diluted twice and the increase in reducing sugars was determined according to Somogyi (1952) using glucose for calibration. Similar experiments were done 25 using defucosylated apple xyloglucan and CMC as a substrate.
The purified apple xyloglucan (APFXG), a defucosylated equivalent (APXG) and potato arabino xyloglucan (PoAXG) were used to determine the specificity of the endoglucanases. Table III summarizes the turnover numbers of the different 30 endoglucanases for the various glucans. Their activity towards CMC is in the same order of magnitude, except for EndoIII.
Activity measurements on xyloglucans clearly subdivide the endoglucanases into two classes. EndoI, II and III show a relatively low activity towards XG compared to EndoIV, V and 35 VI. In general, removal of the fucose residue slightly enhanced the degradation of xyloglucan by endoglllcAn~ses. However, the increased activity of, for instance, EndoI is not so dramatic SUBSTlTUrE SHEEI (RULE 26~

W094/~ 213 9 ~ O 1 PCT~P94/01531 _ 15 that a combination of EndoI and a fucosidase might prove an ~lternative for EndoIV in applications.
This example shows that characterisation of endoglucAnAcPc should not be solely based on CMCase and Avicelase activity 5 because the XGa-se potential of a preparation could easily be overestimated.

Table I. Composition of WUS and fractions obtained bY
10 seauential extractions thereof (mole%~.

Rha Fuc Ara Xyl Man Gal Glc GalA

15O. OSN NaOH 6 047 4 1 18 1 23 Residue 1 113 8 3 8 60 6 SUBSTllU~E SHEET ~RULE 26) W094/2~0 PCT~4/01531 ~13~0~1 16 Table II. GlycosYl linkaqe com~osition of the purified apple xYloglucan (APFXG) and potato xyloglucan fPOAXG) Glycosyl Methylated Deduced APFXG POAXG
5 residue position linkage (Molar ratio) Man 2,3,6 4-Man 4.8 5.3 Man 2,3 4,6-Man 0.5 1.7 Man unmeth. unident. 0.7 1.0 Ara 2,3,5 T-ara 1.3 7.9 Fuc 2,3,4 T-Fuc 6.3 Xyl 2,3,4 T-Xyl 17.7 9.1 Xyl 3,4 2-Xyl 9.8 10.5 Xyl 2,3 4-Xyl 1.0 1.6 5 Xyl unmeth. unident. 0.6 0.6 Gal 2,3,4,6 T-Gal 4.8 8.1 Gal 3,4,6 2-Gal 8.2 Gal unmeth. unident.
Glc 2,3,4 6-Glc 0.5 0.4 zo Glc 2,3,6 4-Glc 15.2 34.0 Glc 2,3 4,6-Glc 28.6 19.8 Glc unmeth. unident. a a 25 a) The amount of unmethylated glucose was not quantified due to a bad separation from inositol.

SUBSTIME SJ IEET (RULE 26) ~13~0û1 W094t26880 PCT~4/01531 -Table III. Turnover number of CMC, APFXG, APXG , POAXG and Avicel determined for the six endoglucanases and CBH of Trichoderma viride.
5 Enzyme CMC APFXG APXG POAXG Avicel~ Padsb (min~1) (min1) (min~1) (min~1) (min~1) (mg/mg) Endo I1518 9 32 36 0.6S 0.126 Endo II1363 4 17 22 0.31 0.090 10 Endo III212 4 8 15 0.91 0.026 Endo IV1139 2017 2237 2390 0.06 0.003 Endo V1049 600 781 1149 0.42 0.105 Endo VI974 807 1150 1183 0.23 0.004 CBH nd' nd' nd' - 0.48 0.063 a) Values according to Beldman et al. Eur.J.Biochem.(1985) 146 : 301-308.
b) Values according to Beldman et al.Biotech.Bioeng.30 : 251-257.
c) nd = not determined.

Exam~le 2 Deqradation of model cell walls from aD~le fruit by the combined action of endo~lucanases and cellobiohvdrolase From the sugar composition of the apple-WUS (Table I) 30 information on the constituent polysaccharides can be deduced as follows. Assuming all xylose is present in xyloglucan (Aspinall and Famous (1984) Carbohydr. Res. 4 : 193-214) which has a Glc/Xyl/Gal/Fuc-ratio of c. 7:4:2:1 (based on the methylation analysis), the xyloglucan fraction comprises about 35 25% (w/w) of the total amount of sugar residues, a value generally found for dicotyledons (Hayashi T. (1989) Ann.Rev.Plant Physiol.Plant.Mol.Biol.40 :139-168). The remaining part of the glucose (32% w/w) is assumed to be SUBSTlTUrE SHEET (RULE 26) W094/2~80 213 9 ~ O 1 18 PCT~4/01531 present as cellulose. This em~hAcizes the importance of the cellulose-xyloglucan complex accounting for about 57% of the apple cell-wall matrix.
For the time course studies, 20mg of WUS were susp~n~e~ in 5 a total volume of 1.5ml buffer containing 6.4~g of CBH and l~g of EndoI or EndoIV (Endo/CBH ratio of 0.16~g/~g) and incubated for defined time intervals up to 24h. A second series differed from the above in incubation time (3h) and a varying endoglucanase (I or IV) amount (Endo/CBH ratios ranging from 10 0.02 to 30~g/~g). In a last series the amount of EndoI+EndoIV
(6.1~g) and CBH (6.4~g) was kept constant at varying EndoI/EndoIV ratio and also incubated for 3h. The course of cellulose degradation was chec~ed for linearity at a Endo/CBH
ratio of 5~g/~g. After heating the incubation mixtures for 15 lOmin at lOO C and centrifugation (2min, 20,000~), the degradation products were analysed on HPLC. Generally, the synergistic action between Endo's and CBH is visualized by determining the increase in reducing end ~ r O~a upon degradation of Avicel. In a complex matrix like WUS, however, 20 this is not a proper approach since both cellobiose and oligomeric xyloglucan fragments contribute to the amount of reducing end groups. Therefore a HPLC method (Animex column) was used to determine both cellobiose (as a measure for cellulose hydrolysis) and the total of xyloglucan 25 oligosaccharides quantitatively. The isolated cell wall material was incubated with EndoI, EndoIV, CBH and various combinations thereof. Fig. lA shows that cellobiose is the only product formed by CBH whereas both endoglucanases did not release any cellobiose from WUS (data not shown). Fig. lC shows 30 a separation of cellobiose and other compounds (shaded area in Fig. lC). The latter were characterized as xyloglucan oligosaccharides after fractionation on BioGel P2 and semi-preparative CarboPac PA1 (data not shown).
Degradation of cellulose involves the concerted action of 35 endoglucanase and cellobiohydrolase, whereby the former creates new chain ends for the latter to work on. At low Endo/CBH
ratios the amount of endoglucanase is limiting whereas at high SUBSTITUTE SHEET (RUI E 26~

213~0i~
W094~6880 PCT~4101531 ratios Endo starts competing with CBH for binding sites to cellulose. This implies that there is an optimal ratio which is observed in Fig. 2 for both EndoI+CBH (l.lmole/mole) and EndoIV+CBH (14.9mole/mole). The difference in optimal molar 5 ratios with respect to cellulose hydrolysis can partly be explained by a different adsorption behaviour of both endoglucanases (Table III). A low adsorption on cellulose requires a larger amount of enzyme to reach the optimum.
Apart from the different optimal Endo/CBH ratios, the total 10 amount of cellobiose that was solubilised was particularly noteworthy. EndoIV has the ability to stimulate CBH to such an extent that the amount of cellobiose released from WUS after 3h reaction time was twice as high compared to incubations with EndoI+CBH. Cellulose degradation seems to relate to the 15 solubilisation of xyloglucan (Fig. 2), the former being optimal when the latter is maximal. It can be calculated that, at optimal EndoI/CBH, the cellulose turnover eYcee~-c the xyloglucan turnover. For optimal EndoIV/CBH the contrary is the case.
20 These data showed that optimal synergism with CBH was obtained with relatively small amounts of EndoI although cellulose hydrolysis proceeded less favourably than with EndoIV+CBH. The endoglucanases seemed to act differently on the cell wall material. Therefore, EndoI and EndoIV were added to a 25 fixed amount of CBH at various molar EndoI/EndoIV ratios, but at a constant Endo/CBH mass ratio of 0.96~g/~g, identical to the optimal EndoI/CBH ratio (as shown in Fig. 2).
All combinations tested, using the two different Endo's in combination with CBH, increased the degree of cellulose 30 hydrolysis (Fig. 3). At the optimal EndoI/EndoIV ratio for cellulose degradation of 0.33, the degree of hydrolysis was increased a factor 2 and l.25 when compared to digestion by only EndoI+CBH and EndoIV+CBH respectively. Cellobiose formation at this point equaled the amount reached at optimal 35 EndoIV/CBH ratio (Fig. 2), however, in this case a 7 to 8 fold smaller endoglucanase dosis was needed. Further, it should be mentioned that the optimum is rather broad indicating that a SUBSTITUTE SHEET (RULE 26) W094/26880 ~13 9 ~ ~1 PCT~4/01531 portion of only 20 mole% of either endogl~ n~ce almost gave a maximal effect. The sharp drop in the cellobiose formation at mole% EndoI is accompanied by a strong decrease in xyloglucan hydrolysis.
s The results- indicate that the removal of the xyloglucan coating is essential for efficient hydrolysis of cell wall embedded cellulose. The potential of EndoI+CBH to degrade cellulose is not fully used because the surface area of uncoated cellulose for the enzyme complex to bind to is 10 limiting. Due to the low turnover of xyloglucan by EndoI (Table III) no fast improvement in this situation is to be expected.
Despite the higher activity of EndoI towards cellulose (Avicel, Table III) compared to EndoIV, cellulose hydrolysis stays behind. For a combination of EndoIV+CBH, the turnover of 15 xyloglucan exceeds the turnover of cellulose and 'bare' cellulose microfibrils are readily degraded. Full cellulose degrading potential is used although relatively large amounts of EndoIV are needed due to its low adsorption onto the cellulose (Table III). A three enzyme system combines the 20 positive aspects of the previous two. By removing the xyloglucan coating, EndoIV enhances the accessibility of the cellulose for EndoI and CBH which display a larger activity towards this substrate than EndoIV. In this case, cellulose turnover almost equals xyloglucan turnover. An advantage of 2s this three enzyme system is that much smaller quantities of enzyme (at least 5 times on a molar basis) are needed to reach a similar cellobiose release as with EndoIV+CBH.
This example clearly demonstrates the importance of at least two different endoglucanase activities in the degradation 30 of model cell walls: one directed towards the cellulose and the other towards the xyloglucans.

SUBSTITUTE SHE~T (RULE ~

213~1)û1 W094/26880 PCT~4/01531 ExamDle 3 Degradation of aDple tissue bv ~xvlo~lucanase-u~qraded) commercial cellulase pre~arations s Two grams of raw apple fruit tissue were incubated in 3 mL
200 mM succinate buffer pH 4.0 containing 0.1% ascorbic acid at 40C for 16 hrs under continuous shaking (150 rpm). The buffer contained appropriate amounts of enzymes from two commercial 10 enzyme preparations from Gist-brocades: Maxazyme CL
(Trichoderma viride) and Xyl 5000 (Disporotrichum). The Avicel degrading potential was 10 mU and the concomitant XGase activities were 200 and lO0 mU for respectively Maxazyme and Xyl 5000. Pectin lyase (PL) (50 mU) (see for PL purification:
15 van Houdenhoven (Studies on pectin lyase, Ph.D. thesis (1975), Agricultural University Wageningen, The Netherlands) was added to appropriate incubations to degrade pectic material and increase the accessibility of the cellulose-xyloglucan network.
After treatment and visual evaluation, l mL was removed from 20 the incubation mixture, heated for lO min at 100C to inactivate the enzymes, centrifuged (2 min, 20.000 g), and the degradation products in the supernatant were analyzed by HPAEC.
Maxazyme alone is unable to degrade raw apple fruit tissue to a large extent within 16 hrs (Fig. 4). A minimum amount of 25 50 mU pectin lyase (PL) was required to enable Maxazyme to completely liquefy the material under the same circumstances.
Therefore, PL was added in this concentration to all incubations to ensure that the cellulose-XG network was accessible to the glucanases. No complete liquefaction could be 30 achieved by a combination of PL and Xyl 5000, although the cellulose degrading potential was similar to the combination PL
plus Maxazyme. However, the XGase activity was a factor 2 lower, and this might indicate that XGase activity is important in liquefaction. Therefore, the XGase activity of Xyl 5000 was 35 increased to the level of Maxazyme by addition of an appropriate amount of EndoV. Figure 4 shows that the extent of degradation is comparable to that obtained by a combination of SUBSTITUTE SHEFr (Rl)LE`.~6) W094/26880 213 9 ~ ~1 PCT~4/01531 PL plus Maxazyme. The cellobiose release upon addition of EndoV
increases 1.6 times but is still approximately 5 times lower when compared to an incubation with PL plus Maxazyme. Addition of a similar amount of EndoI protein had no effect on the s cellobiose release (data not shown). It should be noted that different apple batches gave a similar cellobiose release for the enzyme combinations outlined above. However, the extent of liquefaction as determined visually greatly differed, although similar patterns as in figure 4 were obtained. The commercial 10 preparations contain both endoglucanase (CMCase activity) as well as CBH (release of cellobiose from tissue). Although the cellulose degrading potentials of Maxazyme and Xyl 5000 were similar (lO mU), the performance of both preparations was very different (fig. 4). The CMCase activity in these preparations 15 is in the same order of magnitude, however, the activity toward Avicel of Maxazyme is ten fold higher. This indicates that Maxazyme contains much more CBH than Xyl 5000, and that endoglucanases are responsible for a relatively large part of the activity toward Avicel of the latter. This example further 20 shows the importance of a proper endoglucanase to CBH ratio, which has been demonstrated before. With respect to this ratio, the addition of EndoI or EndoV to Xyl 5000 might seem strange but the main objective here was to remove the xyloglucan coating from cell-wall-embedded cellulose.
25 This example shows that XGase activity is important in the liquefaction of apples.

ExamPle 4 Deqradation of a~le tissue usinq mixtures of ~urified enzYmes By combining EndoI and CBH at a mass ratio of approximately 3.5, a "low XGase" mixture was obtained. A "high XGase" mixture 35 was made using EndoV instead of EndoI. By combining these two mixtures in an appropriate way, five more mixtures with intermediate XGase activity were made. Avicelase activity in SUBSTITUTE SttEET (RULE 26) WOg4/~880 ~ 1 3 9 ~ ~1 PCT~4/01531 _ 23 the seven mixtures of purified enzymes was defined as the difference in activity toward Avicel of EndoI plus EndoV plus CBH and the corresponding mixture without CBH. Apple material was degraded with one of the seven mixtures described above, in s such a way that every incubation contained an equal amount of Avicelase activity (0.26 mU), but a different amount of XGase activity (4 to 40 mU).
Beldman et al. (1987) showed that degradation of Avicel crystalline cellulose by the different endoglucanases of o Trichoderma viride gave different (ratios of) products (Glucose, cellobiose, cellotriose). Upon incubation of apple WUS with EndoI or EndoIV plus CBH, however, cellobiose was the only product released. This discrepancy presumably also holds for the degradation of apple tissue because no cellotriose was 5 found in any of the incubation mixtures containing EndoI.
Glucose could not be analyzed since it naturally occurs in apple in large quantities. Avicelase activity of mixtures of purified glucanases was therefore defined as the difference in activity of endoglucanase plus CBH (endoglucanase/CBH-mass 20 ratio is approximately 3.5) and endoglucanase. It is hereby assumed that both enzymes (EndoI and EndoV) behave similar toward Avicel and apple cellulose.
Figure 5 shows that more cellobiose is released from blanched apple tissue when XGase activity is increasing.
25 'Naked' surface area is the limiting factor in cellulose degradation until the XGase activity reached a level of approximately 25 mU. After this point cellobiose release is probably limited by the amount of CBH. The performance of the glucanase mixtures was also evaluated visually. The extent of 30 liquefaction was comparable to sample "3" in Figure 4 (data not shown), indicating that cellobiose release was determined in the initial stage of tissue degradation. These experiments were repeated with a doubled dose of glucanases as well as an increased incubation time (40 hrs). Incubations with low XGase 35 activity still showed some pieces of tissue, whereas the apple material in incubations with high XGase activity was completely liquefied (data not shown).

SUBSTITUTE SWE~T ~ULE-26) W094/26880 ~ 24 PCT~P94/01531 Also with purified enzymes we have been able to demonstrate the importance of XGase activity for degradation of apple tissue. Furthermore, results on apple WUS can be translated to complete tissue.

SUBSTITUTE SHEET tRULE 26)

Claims (17)

Claims

1. An isolated mixture of cellulose degrading enzymes comprising endoglucanases, cellobiohydrolases and xyloglucanase activity.

2. A mixture of cellulose degrading enzymes comprising endoglucanases, cellobiohydrolases and xyloglucanase activity obtained by mixing the enzymes in predetermined amounts.

3. A mixture according to claims 1 or 2, characterized in that the xyloglucanase activity resides in an endoglucanase.

4. A mixture according to claim 3, characterized in that the xyloglucanase activity resides in an endoglucanase selected from the group consisting of the Trichoderma reesei endoglucanases EndoIV to EndoVI.

5. A mixture according to any one of the claims 2 to 4 wherein at least one of the enzymes added was in a purified state.

6. A mixture according to any one of the previous claims, characterized in that the mixture contains a sufficient amount of xyloglucanase activity to provide optimal cellulose degradation.

7. A mixture according to any one of the previous claims characterized in that the enzymes are obtainable from Trichoderma, Asperqillus, or Disporotrichum.

8. A mixture according to any one of the claims 1 to 7, characterized in that the endoglucanases are EndoI and EndoIV
from Trichoderma reesei and further characterized in that at a constant (Endoi + Endoiv)/CBH mass ratio of 0.5-20 (g/g) the mixture contains a molar Endoi/Endoiv ratio of 0.1-3.

9. A mixture according to claim 8, characterized in that the endoglucanases are EndoI and EndoIV from Trichoderma reesei and further characterized in that at a constant (Endoi + Endoiv)/CBH
mass ratio of 0.96 (g/g) the mixture contains a molar Endoi/Endoiv ratio-of 0.33.

10. A method for the preparation of a mixture of cellulose degrading enzymes comprising endoglucanases, cellobiohydrolases and xyloglucanase activity, characterized in that said enzymes are mixed in predetermined amounts.

11. A method for the degradation of material containing a xyloglucan/cellulose complex comprising the combined action of endoglucanaces, cellobiohydrolases and xyloglucanase activity.

12. A method according to claim 11, characterized by the use of a combination of endoglucanases, cellobiohydrolases and xyloglucanase activity obtainable from Trichoderma, Aspergillus or Disporotrichum.

13. A method according to claim 11, characterized by the use of a mixture according to any one of the claims 1 to 9.

14. A method according to claim 11, characterized in that the action of pectin lyase is included.

15. A method according to any one of the claims 11 to 14 wherein the material is selected from the group comprising:
food, feed, paper, pulp and textile raw materials.

16. A product obtained after treatment of material containing a xyloglucan/cellulose complex with an enzyme mixture according to any one of the claims 1 to 9.

17. A product according to claim 16, characterized in that said product originates from apple.