HUT70470A - Combined action of endoglucanases and cellobiohydrolases - Google Patents

Abstract

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

Description

The present invention relates to the destruction of the cell wall. The invention discloses the use of enzyme combinations in the degradation of plant cell walls. It discloses the advantage of using xyloglucanase activity. It also provides the most favorable ratios of enzymes that break down the cell wall. Specifically, it reports the combined effect of endoglucanases and cellobiohydrolases on the degradation of a xyloglucan / cellulose complex.

It further demonstrates that these enzymes are synergistic.

To date, two structural components of the plant cell wall, cellulose and pectin, have been extensively studied in the study of enzymatic degradation of the plant cell wall.

Apart from cellulose and pectin materials, a third cell wall structural element should be mentioned: the xyloglucans [Hayashi et al .; Plant Physiol. 75: 605-610 (1984); Hayashi T .; Ann. Port. Plant Physiol. Plant Mole. Bioi., 1989, 40, 139-168. Xyloglucans contain a cellulose backbone in which about 70% of the glucose groups are substituted by side chains. These side chains contain primarily xylose, galactose and fucose. Further, they are strongly bound to cellulose by hydrogen bonding [Hayashi et al .; Plant Physiol. 83, 384-389; Talbott L.D. and P.M.Ray; Plant Physiol., 98, 357-368 (1992)] and other cell wall components [Fry, S.C. and J.G. Miller; (1989). For a working model of a growing plant cell wall: phenolic cross-linking reactions in the primary cell wall of dicotyledons. Lewis N.G.; Paice M.G.; Eds. The Biosynthesis and Biodegradation of Plant Cell Wall Polymers, ÁCS, Washington, DC, 33-36.

page and Talbott L.D. and P.M.Ray; (1992), supra.

Although xyloglucans are present in relatively large amounts (about 20% in dicotyledons according to T. Hayashi (1989, cited above)), no attention has been paid to xyloglucans in the enzymatic liquefaction process of plant material [Voragen et al .; Fiüss. Obst., 5, 9, 404-410 (1992)]. The activities screened during the purification of glucanases are (carboxymethyl) cellulase (often identified as C _x or endoglucanase) (CMCase), avicellase (often identified as C 4, cellobiol hydrolase or exoglucanase) and sometimes cellobiase or β-glucosidase. Xyloglucanase (XGase) activity is generally absent. This means that xyloglucanase activity is not regularly used in cell wall degradation.

Relatively little is known about the role of xyloglucans in fruit production. Xyloglucans, like pectin, are known to be good gel-forming agents [M. Glicksman; edited by Glicksman M., Food Hydrocolloids 3, CRC Press Boca Raton, Florida (1986) 191-202] and used as such in Japan. Effective decomposition of the bulky component of the primary cell wall, cellulose, is essential for the flow of apple tissues. The accessibility of cellulose embedded in the cell wall is reduced by the presence of other cell wall components such as pectin and xyloglucan.

Removal of pectin has been shown to promote cellulose degradation. However, little attention was paid to xyloglucans.

The present invention discloses the advantages of using xyloglucanase activity in cellulosic cell wall degradation.

The present invention relates to mixtures of endoglucanases with xyloglucanase activity and cellobiohydrolases which are adapted for optimal cellulose degradation. A typical mixture contains three enzymes; endoglucanase I, endoglucanase IV and cellobiohydrolase, another typical mixture containing two enzymes; endoglucanase V and cellobiohydrolase.

In the mixture of endoglucanases and cellobiohydrolases, the ratio of xyloglucanase to avicellase activity is chosen to provide optimum cellulose hydrolysis.

In another embodiment of the invention, the weight ratios of typical EndoI / Endo-IV / CBH blend enzymes are chosen such that a constant weight ratio of (Endol + EndoIV) / CBH of 0.5-20 (g / g) and 0.1-3 an optimal EndoI / EndoIV molar ratio is used.

Preferably, the weight ratio of endoglucanase / cellobiohydrolase is 0.96 and the molar ratio EndoI to EndoIV is 0.33.

In addition, processes for preparing the claimed mixtures are described.

It is a further object of the present invention to provide a process for the degradation of xyloglucan / cellulose complex endoglucanases (Endo), cellobiohydrolases (CBH) and xyloglucanase activity embedded in a cell wall. Typically, apple cell wall cellulose is degraded.

The process utilizes a combination of endoglucanases and cellobiohydrolases. The endoglucanase is preferably selected on the basis of xyloglucose activity. In principle, any strain producing enzymes having the indicated activities can be used as an enzyme source. The enzymes are preferably obtained from Trichoderma, Aspergillus or Disporotrichum.

The invention discloses the use of xyloglucanase activities in the degradation of cell wall materials. Typically in the degradation of a xyloglucan / cellulose complex.

The present invention further provides products obtained by treating plant material containing xyloglucan / cellulose complex with xyloglucanase-containing cellulosic enzyme mixtures. The product is typically derived from apples. Even more typically, the product belongs to apples.

Brief Description of the Drawings

Figure 1. The analysis of the characteristic chromatograms of the degradation products of apple water-non-extractable water (WUS) solubilized with glucanases (combinations thereof) on an Aminex HPX 22H column shows: The curve is CHB; Curve B Endol + CBH; Curve C Endo-IV + CBH. The elution profile of a maltodextrin syrup is indicated by arrows, corresponding to the degree of polymerization. Representative degradation products of EndoIV are indicated by the coated peaks. The sum of these areas is used to quantify xyloglucan oligosaccharides.

Figure 2. Effect of endoglucanase / cellobiohydrolase ratio on release of cellobiose and oligomeric xyloglucan fragments from apple-WUS; □ release of cellobiose by EndoI + CBH; o release of cellobiose with EndoIV + CBH; · Release of xyloglucan fragments with EndoIV + CBH; FXG = fucosylated xyloglucan.

Figure 3. Release of cellobiose (Δ) and xyloglucan oligosaccharides (1) in a three enzyme system, with a fixed weight ratio (EndoI + EndoIV) / CBH of 0.96 pg / g and varying amounts of Endol and EndoIV.

Figure 4. Liquidation of crude apple fruit tissue with pectin lyase (PL) and (see Example 3, improved) commercial enzyme preparations. Raw apple fruit tissue was incubated in 200 mM succinate pH 4.0 for 16 hours at 40 ° C and 150 rpm. 1, without addition of enzyme; 2, Maxazyme (10 mU activity against Avicel, 200 mU XGase); 3.50 mU PL; A combination of 4, 2 and 3; 5, Xyl 5000 (10 mU activity against Avicel, 100 mU against XGase); A combination of 6, 3 and 5; 7 than 6 plus 100 mU EndoV.

Figure 5. Release of cellobiose by a mixture of glucanases purified from apple fruit tissue. Pre-cooked apple fruit tissue (200 mM succinate pH 4.0 in buffer for 16 hours at 40 ° C, 150 rpm) was incubated with the same Avicel degradation potential (0.26 mU) but with different XGase activity (5-40mU). PL with different glucanase mixtures. Quantification of cellobiose was performed by high performance affinity elution chromatography (HPAEC).

Detailed Description of the Invention

The present invention relates to the use of enzymes having xyloglucanase activity in the degradation of biological materials containing xyloglucans.

Based on the finding that glucanase screening for CMCase and Avicellase activity is not a suitable measure for finding enzymes with xyloglucan activity, glucanases were screened for xyloglucan activity. We found that there is a difference in the activity of different endoglucanases.

The invention further provides the advantages of using cellulose-degrading enzyme mixtures containing endoglucanases, cellobiohydrolases and xyloglucanase activities. Preferably the mixture is obtained from Trichoderma, Aspergillus or Disporotrichum. The mixtures are isolated without further purification from the growth medium of these microorganisms. It should be noted that the activity and ratio of different enzymes in the mixture will depend on the substrate, the growth conditions and the strains used in the fermentation.

If the mixture so isolated contains the desired ratio of enzyme activity, the mixture itself is used. The desired ratio depends on the substrate to be degraded and is preferably determined for each substrate.

If the isolated mixture does not contain the desired ratio of enzyme activities without further treatment, mixtures from different cultures are used to improve the ratio. It is also possible to mix the culture fluids from the propagation of different strains or varieties. Alternatively, the enzymes are purified to obtain the desired enzyme activity ratio. Purification can be carried out by any method known to those skilled in the art. Preferably, the enzymes are purified to a purity level of 70%. More preferably, the purity level is 90%, more preferably greater than 95%. Beldman et al. [Eur. J.

Biochem., 146, 301-308 (1985)] yields six endoglucanases (EndoI-VI) and two cellobiohydrolases (CBH). The enzyme nomenclature used in the present invention is described in Beldman et al. Biotech. Bioeng., 31, 160-167 (1988)] and is therefore different from other publications. In addition to those described herein, methods and sources for obtaining the disclosed enzymes may be used. An alternative approach may be cloning and production of the gene (s) encoding the desired enzyme (s) in heterologous microorganisms that cannot be infected with other enzymes. However, a homologous microorganism may also be used for cloning and production of the desired gene (s) in excess. In this case, the ratio of different enzymes can be influenced without external enzyme addition. It is also possible to inactivate typical enzymes of the mixture. Differential inactivation can be achieved, for example, by heating, changing the pH, or by adding specific inhibitors.

Preferred cellulose-degrading enzyme mixtures containing the activities of endoglucanases, cellobiohydrolases and xyloglucanase are obtained by mixing predetermined amounts of purified enzymes or by combining predefined activity mixtures to obtain desired final enzyme activity ratios.

The invention discloses that when used to degrade cell wall material, various enzymes have a synergistic effect. This effect is particularly clear when the mixture is prepared from endoglucanases and cellobiohydrolases having specific proportions of xyloglucanase activity. Preferred weight ratios of enzymes depend on the substrate used.

The Trichoderma viride cellulase complex (currently known as Trichoderma reesei) has been shown to contain six distinct endoglucanases [Beldman et al; Eur. J. Biochem., 146, 301-308 (1985)], which can be divided into two classes based on their ability to degrade xylans: one specific (Endo I, II and III) and one non-specific (Endo IV, V and VI). Example 1 illustrates that, by virtue of their ability to degrade xyloglucan, endoglucanases can be divided into three groups of enzymes: one group for active cellulose (Avicel), e.g. Endol, one group for active xyloglucans, such as EndoIV, and in Table 3).

The invention discloses the advantageous use of endoglucanase IV, V and V1 containing xyloglucanase activity over endoglucanase I, II and III (Table 3).

The invention discloses that the combined action of xyloglucan-specific EndoIV and cellulose-specific endol + CBH is beneficial in the degradation of cellulose embedded in the cell wall, and the combined effect of both xyloglucan and cellulose-specific EndoV and CBH is preferred. Further, it has been recognized that both characteristics of the EndoI-EndoIV combination are present in EndoV.

Further, the use of these enzymes in combinations is shown. Generally, an enzyme containing xyloglucanase activity, such as the Endo Group IV-VI enzyme, is chosen to liberate xyloglucan from the surface of the xyloglucan / cellulose complex, followed by (one) cellulase activity enzyme (s), e.g. can attack cellulose with. It has been recognized that the relative amount of endoglucanases, cellobiohydrolases and xyloglucanase activities depends on the substrate. Increasing the size of the cellulose degradation process, for example to industrial size, can also affect the relative amount of these enzymes. The invention is illustrated by the use of so-called water-non-extractable solids (WUS) and apple tissue.

Example 2 demonstrates that three activities are important for the efficient degradation of cell wall cellulose: endoglucanase (having activity against Avicel), cellobiohydrolase, and xyloglucanase. Optimal cellulose degradation is achieved if cellobiose lysis is optimal. The implementation of cellobiose lysis is optimal when xyloglucan lysis is maximal. According to our observations, the optimal molar ratio of Endo / CBH for apple WUS degradation varies from 1.1 for EndoI / CBH to 14.9 for EndoIV / CBH. However, twice the amount of cellobiose is dissolved with optimal EndoIV / CBH ratios compared to optimal EndoI / CBH ratios. Due to its low absorbance against cellulose, relatively large amounts of EndoIV are required. Using the three enzyme systems, similar cellulose degradations can be obtained, but with much smaller amounts of protein.

Further, Example 2 demonstrates that the optimal endoglucanase / cellobiohydrolase ratio is between 0.5 and 20 (g / g). Using a constant weight ratio of 0.96 to (EndoI + EndoIV / CBH), we show that the optimal EndoI / EndoIV ratio is between 0.1 and 3. Preferably the ratio is between 0.20 and 0.60, more preferably the ratio is 0.33.

Endoglucanases are typically Endol and EndoIV, and the mixture is characterized by a 0.96 (g / g) constant weight ratio (EndoI + EndoIV) to CBH of 0.33 EndoI / EndoIV.

Example 3 illustrates the important role of xyloglucanase activity in apple tissue degradation and fluidity. The cellulase composition with low xyloglucanase activity is improved by the addition of xyloglucanase, preferably by the addition of endoglucanase selected from the group EndoIV-EndoVI, more preferably by addition of EndoV.

Example 4 shows that mixtures of purified enzymes produce similar effects. Keeping the Avicelt degradation potential of the mixtures constant at increasing the amount of xyloglucanase activity increases the cellobiose leaching from apple tissue. In the form of EndoV, a minimum amount of 25mU xyloglucanase activity is required for 0.26mU Avicel degradation activity.

Mixtures as defined in the present invention may advantageously be used on substrates containing xyloglucans as constituents. Such substrates are found in food, feed, paper, pulp and textile raw materials. Therefore, in the degradation processes, the mixtures can be advantageously used in the treatment of food, feed, paper, pulp and textiles.

Mixtures are particularly useful in the preparation of fruit juices. Typically useful in the production of apple juice.

The present invention demonstrates the specificity of six fungal endoglucanases against four different glucans (Example 1). Furthermore, the degradation of the glucan complex by the combined action of endoglucanases and cellobiohydrolases is demonstrated using apple cell wall sample (Example 2) or whole apple tissue (Example 3, 4).

The synergistic effect of using typical enzyme combinations is also shown.

Experimental part

Materials

Apples (Malus malus L., Rosaceae, var. Golden Delicious) were harvested in mid-October 1989 and stored for four months in a controlled atmosphere (2 ° C, 2% O2 and 5% CO2).

Six endoglucanases (EndoI-EndoVI) [E.C.3.2.1.4] and two cellobiohydrolases (CBH) [E.C.3.2.1.91] were purified from a commercial preparation of Trichoderma viride (Maxazyme Cl, Grist-Brocades, Delft, The Netherlands) according to Beldman et al. Eur. J. Biochem. 146: 301-308 (1985).

Avicel crystalline cellulose was obtained from Serva (Heidelberg, Germany), CMC (type Akucell Af 0305) from Akzo (Arnhen, The Netherlands) and tamarind core xyloglucan from Dainippon Pharmaceutical (Osaka, Japan). ).

Non-Water Extractable Solids (WUS)

To inactivate endogenous enzymes (polygalacturonases and pectin esterases), apples (5 kg) were treated with microwave (Philips AKB 276 / PH, 4kW, Switzerland) in kilograms after removal of the nucleus. The resulting material was peeled, ground and thoroughly extracted with warm distilled water (50 ° C) until the washing water contained only a small amount of sugar. The solid was collected by centrifugation (20 min, 50000g). The residue was freeze-dried and ground on a Fritsch pulverisette (1.0 mm sieve, Germany) and labeled as WUS.

Preparation of Xyloglucans

Extraction of WUS. 2 g of WUS was extracted with 200 ml of 0.05 M sodium hydroxide solution containing 5 mM 1,2-cyclohexylene dinitrilotetraacetic acid (CDTA) with stirring for 16 hours at 4 ° C. After centrifugation (20 min, 50,000 g), the residue was resuspended in 320 ml of 1M potassium hydroxide solution containing 1% by weight sodium [tetrahydroborate] (1), and extracted for 16 hours at 20 [deg.] C. min, 50000g). The latter procedure was repeated using this residue with 4M potassium hydroxide solution containing 1% by weight sodium [tetrahydroborate] (1). Between successive extractions, the residue was washed twice with distilled water. The final residue is acidified to pH 5 with hydrochloric acid and freeze-dried. The appropriate supernatants were collected, acidified to pH 5 with hydrochloric acid and thoroughly dialyzed against distilled water.

Purification of the 4M potassium hydroxide extract. The 4M potassium hydroxide extract was pectinated on a DEAE Sepharose CL-6B column (40x440mm, Pharmacia, Uppsala, Sweden) equilibrated with 50mM sodium acetate buffer (pH5.0). After applying 200 ml of sample, the column is washed with 400 ml of buffer. The fraction retained on the column was dissolved by elution with 500 mL of 1M sodium acetate buffer (pH 5.0). The fractions (16.5 ml) were assayed for both uronic acids and neutral sugars, pooled, dialyzed and freeze-dried. The neutral fraction consists of xyloglucan (APFXG).

Slight acid hydrolysis of APFXG. 40 mg of purified apple xyloglucan was treated with 25 mM trifluoroacetic acid (TFA) (5 mg / ml) at 60 ° C for 100 hours. After dialysis, the deprotected XG (APXG) is freeze-dried.

Determination of enzyme activities.

The activities of endoglucanases and cellobiohydrolases on CMC (CMCase), Avicel (avicellase) and tamarind core xyloglucanase (xyloglucanase), increased the number of reducing end groups according to Somogyi [J. Biol. Chem., 195, 19-23]. Incubations (40 ° C, 0.1M succinate buffer, pH 4.0) were performed under non-substrate limited conditions. One unit of enzyme (mU) corresponds to the amount of reducing end group expressed in nanomoles per minute.

Analytical procedures for uric acid content. An automated m-hydroxydiphenyl assay using concentrated sulfuric acid containing 0.0125M disodium [heptaoxotetetraborate] uronic acid (AUA) [Thibault JF; Lebensm.Wiss.Technol., 12, 247-251 (1979)] is determined colorimetrically. The water-insoluble material is pre-treated with 72% sulfuric acid (1 hour at 30 ° C) and after dilution with water, 1M sulfuric acid (3 hours at 100 ° C) prior to analysis.

Total neutral sugar content. All neutral sugars were colorimetrically determined by an automated orcinol / sulfuric acid test [Tollier MT, Robin JP; Ann. Technol. Agric. , 28, 115 (1979)].

♦ ··

Neutral sugar composition. The water-insoluble material was subjected to a 72 wt% pre-hydrolysis of sulfuric acid (1 hour at 30 ° C) followed by dilution with water followed by hydrolysis with 1M sulfuric acid (3 hours at 100 ° C). The water-soluble materials were hydrolyzed with 2M TFA (1 h at 121 ° C). In the next step, the dissolved neutral sugars are converted to their alditol acetates [Englyst HN, Cummings JH; Analyst, 109, 937-942 (1984)] and separated on a (3% OV275 coated Chrom WAW 80-100mesh filling, Chrompack, Middelburg, The Netherlands) 3m x 2mm internal glass column operating at 200 ° C and 270 ° C. on a Carlo Erba Fractovap 2300 gas chromatograph (GC) equipped with a flame ionization (FID) detector (Milan, Italy). Inositol is used as the internal standard.

Glycosyl bond composition. Purified xyloglucan according to the modified Hakomori procedure [Sandfort PA, Conrad HE; Biochemistry, 5, 1508-1517 (1966)], followed by dialysis against water and drying by evaporation (air flow, room temperature). This procedure is repeated once. In the next step, the methylated xyloglucan is hydrolyzed using 2M trifluoroacetic acid (TFA) (1 hour at 121 ° C) which is removed by evaporation (air flow, room temperature). The sugars were reduced by addition of 0.2 ml of freshly prepared 1.5M ammonia solution containing 75 mg of sodium [tetradeuterium borate] (1) / ml and converted to alditol acetate [Englyst et al .; Analyst, 10 9, 937-942 (1984)]. Partially methylated alditol acetates (Ιμΐ) were analyzed by column injection on a silica gel capillary column (30mx0.32mm internal diameter; DB 1701 wall plated; 0.25pm film thickness; J & W Scientific, Folsom, California, USA) at 280 ° C. on a Carlo-Erba Fractovap 4160 gas chromatograph equipped with an adjusted flame ionization detector (FID). The temperature program is 80 to 180 ° C at 20 ° C / min, 180 to 230 ° C at 2 ° C / min, 230 ° C for 3 minutes. Identification of compounds using a CP Sil 19 CB capillary column (26mx0.22mm internal diameter, Ο, ΐδμιη film thickness; Chrompack Nederland BV, Middelburg, The Netherlands) in an HP 5890 gas chromatograph (GC) coupled to a Hewlett-Packard 5970-B mass-selective detector Confirmed by gas chromatography mass spectrometry using -HP 300 Chem Station (Hewlett-Packard). The temperature program is 160 to 185 ° C at 0.5 ° C / min, 185 to 230 ° C at 10 ° C / min, 230 ° C for 5.5 minutes. Quantification of derivatives according to their effective carbon reaction [Sweet DP, Shapiro RH, Albersheim P; Carbohydr. Sl., 40, 217-225 (1975)].

Protein content. The protein content of the enzyme preparations was determined by Sedmak J.J. and Grossberg, S.E. [Anal. Biochem. 7, 9, 544-552 (1977).

Analysis of xyloglucan and cellulose degradation products. Combined with an Aminex HPX-22H column (7.8x300mm) combined with a Shodex SE-61 refractometer (Showa Denko KK, Tokyo, Japan) and an AG 50W X4 protective column (7.8x50mm, Bio-Rad Labs), Bio-Rad Labs, Richmond, CA , USA) was analyzed by high performance liquid chromatography at 85 ° C using an SP 8800 HPLC pump system (Spectra Physics, San Jose, CA USA). The solvent was 0.005M sulfuric acid pumped at a flow rate of 0.2ml / min. The injected volume is 20μ1. Maltodextrins are used for calibration. After degradation of apple tissue, quantitative analysis of cellobiose was performed on a Diomex Bio-LC GPM-II Quaternary Gradient Module (HPAEC) equipped with a Diomex CarboPack PA100 column (250x4mm, 20 ° C, Diomex, Sunnyvale, CA). Samples (20 μΐ) were injected with a SP8780 automatic sampler (Spectra Physics, San Jósé, CA) equipped with a Tefzel rotor seal in a 7010 Rheodyne injector valve. The solvents were degassed and stored under helium gas using a Dionex EDM module. The eluate (1 ml / min) was monitored using a Dionex PED detector in pulsed amperometric detection (PAD) mode. Silver / silver chloride reference electrode was used as an active in the following pulse potentials and durations aranyelektróddal E] _ O, 1V, and o, 5s, E ₂ 0, 6 V and 0, ls, E3 0,6V and 0, ls. The cellobiose in the incubation mixtures is quantitated using a gradient of 0 to 12 minutes, a linear gradient of 25 to 85 mM sodium hydroxide, 0 to 100 mM sodium in 85 mM sodium hydroxide, 12 to 25 minutes. -acetate linear gradient. After each analysis, the column was rinsed with 1 M sodium acetate in 100 mM sodium hydroxide solution for 5 minutes and equilibrated with 25 mM sodium hydroxide solution for 15 minutes.

Example 1;

Specificity of the six endoglucanase substrates from Trichoderma viride.

Most of the xyloglucans were extracted with 4M potassium hydroxide solution (Table 1). This extract also contains all pectic substances which can be effectively removed by anion exchange chromatography. Methylation analysis was performed to characterize this purified xyloglucan. Table 2 and literature [York WS, van Halbeek H, Darvill AG, Albersheim P; Carbohydr. Gap. 200: 9-310 (1990)], this polysaccharide is composed of (1-4) -β-glucose units, of which about 42% (6-> 1) -CC-

contains about 10% (6-> 1) -a-Xyl- (2->) of the end group xylose

1) -β-Gal substituted and about 13% is one (6->

1) It has a side chain -d-Xyl- (2-> 1) -β-Gal- (2-> 1) -α-Fuc. We also found some arabinose end groups most likely to be linked to xylose groups. The sugar composition of purified apple xyloglucan is the same as described by other experts [Renard C.M.G.C. et al. ; Carbohydr. Polymers, 15, 387-403 (1991) and

Ruperez P. et al. 142: 107-113 (1985)]. Undermethylation, especially of hexose groups, occurs only to a limited extent (about 5%). It should be noted that the purified xyloglucan still contains a small amount of contamination (about 5%), most likely a galacto (gluco) mannan, with one galactose attached to every ninth mannose group.

250µg of purified xyloglucan was dissolved in 100µl buffer and incubated for 1 or 20 hours with EndoIV, EndoV and EndoVI (30ng) and Endol, EndoII and EndoIII (400ng). The enzyme core is such that no substrate restriction occurs. The samples are then diluted twice and the increase in reducing sugars is determined using glucose for calibration according to Somogyi (1952). Similar experiments were performed using de-fucose apple xyloglucan and CMC substrate.

Purified apple xyloglucan (APFXG), a fucose-free equivalent (APXG) and potato arabinoxyloglucan (PoAXG) were used to determine the specificity of endoglucanases. Table 3 summarizes the cycle numbers of the different endoglucanases for the different glucans. Their activity against CMC, except for EndoII, is of the same order of magnitude. The activity measurements for xyloglucans clearly divide endoglucanases into two classes. Endol, II and III exhibit relatively low activity against XG compared to Endol, V and V1. Removal of the fucose group generally slightly increases the degradation of xyloglucan by xyloglucanases. However, for example, the increase in Endol activity is not so dramatic that the combination of Endol and a fucosidase would prove to be an alternative solution to EndoIV in the applications of the invention.

In this example, it is shown that the characterization of endoglucanases cannot be based solely on CMCase and Avicellase activities, since the XGase potential of a formulation can easily be overestimated.

Table 1

WUS composition and fractions obtained by batch extraction (mole%).

Rha fuc Price xyl Mán Gal GLC Ga 1A WUS 1 1 13 10 2 9 42 22 0.05M NaOH 6 0 47 4 1 18 1 23 1M KOH 1 3 15 21 4 13 31 12 4M KOH 1 5 5 29 3 12 40 5 the rest 1 1 13 8 3 8 60 6

Table 2.

The glycosyl bond composition of purified apple xyloglucan (APFXG) and potato xyloglucan (PoAXG).

glycosyltransferase methylated Derived APFXG POAXG group position binding (Molar gold) Mán 2,3,6 4-Man 4.8 5, 3 Mán 2.3 4.6-Man 0.5 1.7 Mán not meth- not identifiable 0.7 1.0 acylated to Price 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 xyl not meth- not identifiable 0, 6 0, 6 acylated to Gal 2,3,4,6 T-Gal 4.8 8.1 Gal 3,4,6 2-Gal 8.2 - Gal not annual not identifiable - - acylated to Glc 2,3,4 6-Glc 0.5 0.4 Glc 2,3,6 4-Glc 15.2 34.0 Glc 2.3 4,6-Glc 28, 6 19, 8 Glc not annual not identifiable the the acylated to

the)

Due to poor separation from an inositol, the amount of non-methylated glucose was not determined.

Table 3

Cycle counts for CMC, APFXG, APXG, POXG, and Avicel for six endoglucanases and CHB from Trichoderma viride

Enzyme CMC APFXG APXG POAXG Avicel ³ Pad ^b (min 1) (minutes ^- · '·) (min 1) (min ^- 1) (minutes ^- · '-) (Mg / mg) Endol 1518 9 32 36 0, 65 0, 126 EndoII 1363 4 17 22 0.31 0, 090 endothelia 212 4 8 15 0, 91 0.26 EndoIV 1139 2017 2237 2390 0.06 0,003 EndoV 1049 600 781 1149 0.42 0, 105 EndoVI 974 807 1150 1183 o, 23 0,063 CBH nd ^c nd ^c nd ^c - 0.48 0,063

a) Values Beldman et al .; Eur. J. Biochem., 146, 301-308 (1985),

b) Values Beldman et al. ; Biotech.Bioeng. , 30, 251-257.

c) nd = not determined.

Example 2;

Degradation of apple fruit cell wall model by combined action of endoglucanases and cellobiohydrolases

The following conclusions can be drawn for the polysaccharide components of the apple-WUS sugar composition (Table 1). Assuming all xylose is present in xyloglucan in which the ratio of Glc / Xyl / Gal / Fuc base units is 7: 4: 2: 1 (based on methylation analysis) [Aspinall and Famous; Carbohydr. Gap. 4, 193-214 (1984)], containing about 25% by weight of total sugar residues around the xyloglucan fraction, which is generally found in dicotyledonous plants [Hayashi T; Ann.Rev.Plants Physiol.Plants.Mol.Bioi., 40, 139-168 (1989)]. The rest of the glucose (32% by weight) is presumed to be present as cellulose. This underlines the importance of the cellulose xyloglucan complex, which represents about 57% of the apple cell wall matrix.

For duration studies, 20 mg of WUS was suspended in 1.5 ml total buffer containing 6.4 µg of CBH and 1 µg of Endol or EndoIV (Endo / CBH ratio 0.16 µg / µg) and incubated for 24 hours at specified time intervals. . The second series differs from the above by varying amounts of endoglucanase (I or IV) during the incubation time (3 hours) and (with Endo / CBH ratios in the range of 0.02 µg / pg to 30 µg / µg). In the final series, the amounts of EndoI + EndoIV (6, 1pg) and CBH (6.4pg) were kept constant with variable Endol / EndoIV ratios and also incubated for 3 hours. The linearity of the cellulose degradation is checked at a 5µg / pg Endo / CBH ratio. After incubation mixtures are heated at 100 ° C for 10 min and centrifuged (2 min, 20000 g), the degradation products are analyzed by HPLC. The synergistic activity between the Endo's and the CBH is usually detected by quantifying the increase in reducing end groups upon Avicel degradation. However, this is not an appropriate approach in a complex matrix such as WUS, since both cellobiose and oligomeric xyloglucan fragments contribute to the amount of reducing end groups. Therefore, an HPLC method is used to quantify both cellobiose (as a measure of cellulose hydrolysis) and total xyloglucan oligosaccharides. The isolated cell wall material was incubated with Endol, Endo-IV, CBH and various combinations thereof. 1A. Figure 1B shows that no product produced by CBH is cellobiose, whereas no endoglucanase loses any cellobiose from WUS (data not shown). IC. Figure 1B shows the separation of cellobiose and other components (coated area in Figure IC). The latter were characterized as xyloglucan oligosaccharides after fractionation on BioGel P2 and semi-preparative CarboPac PA1 (data not shown).

The breakdown of cellulose occurs by the concerted action of endoglucanase and cellobiohydrolase, the former forming new ends for the latter to function. At low Endo / CBH ratios, the amount of endoglucanase is limiting, while at high ratios Endo competes for cellulose binding sites with CBH. This implies that there is an optimum ratio, as shown in Figure 2, to both EndoI + CBH (1.1 mol / mol) and EndoIV + CBH (14.9 mol / mol). The difference in optimal molar ratios for cellulose hydrolysis is partly explained by the different adsorption behavior of the two endoglucanases (Table 3). In the case of low adsorption on cellulose, a greater amount of enzyme is required to reach the optimum.

Apart from the different optimal Endo / CBH ratios, the total amount of cellobiose dissolved is particularly noteworthy. EndoIV stimulates CBH to such an extent that after 3 hours of reaction, the amount of cellobiose lysed from WUS is twice that of incubation with EndoI + CBH. The cellulose degradation apparently depends on the solubilization of xyloglucan (Figure 2).

optimal if optimal EndoI / CBH xyloglucan cycle, position.

These data are maximum for the latter.

cellulose

Optimal EndoIV / CBH show that

We can calculate that cycles exceed the ratio of inverse to small amounts of relatively

Endol provides optimum synergism with CBH, although cellulose hydrolysis is less favorable than EndoIV + CBH. Endoglucanases appear to act in a different way on cell wall material. Therefore, Endol and EndoIV (as shown in Figure 2) were added to a constant amount of CBH with different molar ratios of EndoI / EndoIV and with a constant weight ratio of EndoI / CBH of 0.96 pg / g constant EndoI / CBH.

All of the tested combinations using two different Endo's in combination with CBH increase the degree of cellulose hydrolysis (Figure 3). The optimum EndoI / EndoIV ratio for cellulose degradation

0.33, and the degree of hydrolysis increases by a factor of 2 and 1.25, respectively, when compared to degradation with EndoI + CBH and EndoIV + CBH only. At this point, cellobiose formation is equivalent to that achieved with the optimal EndoIV / CBH ratio (Figure 2), but in this case a 7-8 fold lower dose of endoglucanase is required. Furthermore, it should be noted that the optimum is quite wide, indicating that a single dose of only 20 mole% of any endoglucanase produces maximum effect. The large drop in cellobiose formation at 80 mol% is associated with a strong reduction in xyloglucan hydrolysis.

The results indicate that removal of the xyloglucan coating is essential for efficient hydrolysis of cellulose embedded in the cell wall. The cellulose-degrading potential of EndoI + CBH is not fully exploited because it is limited by the uncoated cellulosic surface area capable of binding the enzyme complex. Due to the small number of cycles of xyloglucan with Endol (Table 3), no rapid improvement is expected in this situation. Despite the higher activity of Endol compared to EndoIV against cellulose (Avicel, Table 3), cellulose hydrolysis is lagging behind. For an EndoIV + CBH combination, the xyloglucan cycle exceeds the cellulose cycle and the "bare cellulose microfibers are rapidly degraded. The total cellulose-degrading potential is exploited, although relatively high amounts of EndoIV are required due to its low adsorption to cellulose (Table 3). The three enzymatic systems combine the positive aspects of the previous two. By removing the xyloglucan coating, EndoIV improves the availability of cellulose to Endol and CBH, which show greater activity against this substrate than EndoIV. In this case, the cellulose cycle is almost equal to the xyloglucan cycle. One advantage of these three enzyme systems is that much smaller amounts of enzyme (at least five times lower on a molar basis) are required for cellobiose lysis similar to EndoIV + CBH.

In this example, it is clearly demonstrated that at least two different endoglucanase activities are important in the degradation of the cell wall model, one targeting cellulose directly and the other targeting xyloglucans.

Example 3;

Degradation of apple tissue with commercial cellulase preparations (improved by xyloglucanase)

Two grams of crude apple fruit tissue was incubated at 40 ° C for 16 hours with continuous shaking (150 rpm) in 3 ml of 200 mM succinate pH 4.0 with 0.1% ascorbic acid. The buffer contains appropriate amounts of enzymes from two commercial enzyme preparations available from Gist-Brocades, Maxazyme CL (Trichoderma viride) and Xyl 5000 (Disporotrichum). Avicelt has a degradation potential of 10 mU and the accompanying X-Gase activities of 200 and 300 mU for Maxazyme and Xyl 5000 respectively. For appropriate incubation, pectin lyase (PL) (50 mU) is used to degrade the pectin material and increase the accessibility of the cellulose xyloglucan mesh [see PL Purification: van Houdenhoven; Studies on pectin lyase, Ph. D. thesis, Agricultural University Wageningen, The Netherlands (1975)]. After treatment and visual evaluation, 1 ml of the incubation mixture is withdrawn, heated to 100 ° C for 10 minutes to inactivate the enzymes, centrifuged (2 min, 20000g) and the supernatant degradation products analyzed by HPAEC.

Maxazyme alone is not capable of major degradation of raw apple fruit tissue within 16 hours (Figure 4). A minimum amount of 50 pU of pectin lyase (PL) is required to allow Maxazyme to fully fluidize under the same conditions. Therefore, PL at this concentration is added to all incubations to ensure accessibility of the cellulose XG network to the gluconases. A combination of PL and Xyl 5000 does not provide complete liquefaction, though

the cellulose-degrading potential is similar to that of the PL plus Maxazyme combination. However, XGase activity is reduced by a factor of 2, which may indicate that XGase activity is important for fluidity. Therefore, the activity of Xyl 5000 XGase is increased to the level of Maxazyme by addition of sufficient amount of EndoV. Figure 4 shows that the degree of degradation is similar to that obtained with PL plus Maxazyme. Addition of EndoV increases the cellobiotic lysis to 1.6-fold, but is still approximately 5-fold lower than a PL plus incubation with Maxazyme. Addition of a similar amount of Endol protein does not affect cellobiose lysis (data not shown). It should be noted that the various apple batches give a similar cellobiotic lysis to the enzyme combinations outlined above. However, the visually determined degree of fluidity is very different, although a diagram similar to Figure 4 is obtained. Commercial formulations also contain endoglucanase (CMCase activity) and CBH (tissue lysis of cellobiose). The performance of Maxazyme and Xyl 5000 is very different, although the cellulose-degrading potential of the two formulations is similar (10 mU) (Figure 4). In these formulations, CMCase activity is of the same order of magnitude, but with an activity of up to ten times that of Maxazyme Avicel. This indicates that Maxazyme contains much more CBH than Xyl 5000 and that the latter is responsible for a relatively large part of its activity against Avicel. This example further illustrates the importance of the appropriate endoglucanase CBH ratio, which has been described previously. Given this ratio, it might seem strange to add Endol or EndoV to Xyl 5000,

however, the main task here is to remove the xyloglucan coating from cellulose embedded in the cell wall.

This example demonstrates the importance of XGase activity in apple liquefaction.

Example 4;

Degradation of apple tissue using mixtures of purified enzymes

By mixing Endol and CBH at a ratio of about 3.5% by weight, a mixture of "low XGase activity" is obtained. “A mixture of high XGase activity is obtained using EndoV instead of Endol. By mixing the two mixtures appropriately, five additional mixtures of intermediate XGase activity are prepared. Avicellase activity in seven mixtures of purified enzymes is defined as the difference between the activity of Endol plus EndoV plus CBH and the corresponding CBH-free mixture against Avicel. The apple material was digested with one of the seven mixtures described above such that each incubation contained the same amount of Avicellase activity (0.26 mU) and different XGase activity (4-40 mU).

Beldman et al. (1987) showed that the degradation of Avicel crystalline cellulose with different endoglucanases of Trichoderma viride gives different (proportional) products (glucose, cellobiose, cellotriose). However, after incubating apple WUS with Endol or EndoIV plus CBH, the only dissolved product is cellobiose. This contradiction is presumably also true for apple tissue, since none of the incubation mixtures containing Endol contain cellotriose. Glucose cannot be analyzed because naturally high levels of glucose are present in al ··· today. Avicellase activity of mixtures of purified glucanases is therefore defined as the difference between endoglucanase plus CBH (endoglucanase / CBH weight ratio of about 3.5) and endoglucanase activity. This assumes that both enzymes (Endol and EndoV) behave similarly to Avicel and apple cellulose.

Figure 5 shows that with increasing XGase activity, more cellobiose is leached from blanched apple tissue. In cellulose degradation, “bare surface area is a limiting factor until XGase activity reaches about 25 mU. After this point, cellobiose lysis is likely to be limited by the amount of CBH. The performance of glucanase mixtures is also visually evaluated. The degree of liquefaction is similar to that of Example 3 in Figure 4 (data not shown), indicating that cellobiose lysis is determined by the initial stage of tissue degradation. These experiments were repeated with double-dose glucanases and increased incubation times (40 hours). At low XGase activity incubations, a few pieces of tissue are still visible, while at high XGase activity incubations the apple material is completely liquefied (data not shown).

Purified enzymes can also demonstrate the importance of XGase activity in the degradation of apple tissue. In addition, results for apple WUS can be applied to whole apple tissue.

Claims (14)

Claims An isolated mixture of cellulose-degrading enzymes containing endoglucanases, cellobiohydrolases and xyloglucanase activities. A mixture of cellulose-degrading enzymes containing the activities of endoglucanases, cellobiohydrolases and xyloglucanase, characterized in that the mixture is obtained by mixing a predetermined amount of the enzymes. The mixture according to claim 1 or 2, characterized in that the xyloglucanase activity is present in an endoglucanase. The mixture according to claim 3, wherein the xyloglucanase activity is present in an endoglucanase selected from Trichoderma reesei EndoIV-EndoVI endoglucanases. The mixture according to any one of claims 2 to 4, characterized in that at least one of the enzymes is added in a purified state. The mixture according to any one of the preceding claims, characterized in that the mixture contains the amount of xyloglucanase activity required for optimal cellulose degradation. Ί. Mixture according to any one of the preceding claims, characterized in that the enzymes are obtained from Trichoderma, Aspergillus or Disporotrichum. A mixture according to any one of claims 1 to 7, characterized in that the endoglucanases are Endol and EndoIV from Trichoderma reesei and further characterized in that at a constant weight ratio (Endol + EndoIV) / CBH of 0.520 (g / g) mix Contains a molar ratio of 0.1-3 EndoI / EndoIV. The mixture according to claim 8, characterized in that the endoglucanases are Endol and EndoIV from Trichoderma reesei, and further that at a constant weight ratio (Endol + EndoIV) / CBH of 0.96 (g / g), the mixture is 0 , 33 molar ratio of EndoI / EndoIV. 10. A process for preparing a mixture of cellulose-degrading enzymes containing endoglucanases, cellobiohydrolases and xyloglucanase activities, wherein the enzymes are mixed in predetermined amounts. 11. A process for the degradation of a material comprising a xyloglucan / cellulose complex, characterized in that the process comprises the combined action of endoglucanases, cellobiohydrolases and xyloglucanase activities. 12. The process according to claim 1, wherein the activities of endoglucanases, cellobiohydrolases and xyloglucanases from Trichoderma, Aspergillus or Disporotrichum are used in combination. 13. The method of claim 11, wherein the method according to claims 1-9. A mixture according to any one of claims 1 to 6. 14. The method of claim 11, further comprising the action of pectin lyase. Method according to any one of claims 11 to 14, characterized in that the material is selected from the group consisting of food, feed, paper, pulp and textile raw materials.