Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
  • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
  • C12Y302/01091—Cellulose 1,4-beta-cellobiosidase (3.2.1.91)
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K10/00—Animal feeding-stuffs
  • A23K10/10—Animal feeding-stuffs obtained by microbiological or biochemical processes
  • A23K10/14—Pretreatment of feeding-stuffs with enzymes
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
  • A23L2/70—Clarifying or fining of non-alcoholic beverages; Removing unwanted matter
  • A23L2/84—Clarifying or fining of non-alcoholic beverages; Removing unwanted matter using microorganisms or biological material, e.g. enzymes
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
  • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
  • A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
  • A23L5/25—Removal of unwanted matter, e.g. deodorisation or detoxification using enzymes
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
  • C08B37/0006—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid
  • C08B37/0057—Homoglycans, i.e. polysaccharides having a main chain consisting of one single sugar, e.g. colominic acid beta-D-Xylans, i.e. xylosaccharide, e.g. arabinoxylan, arabinofuronan, pentosans; (beta-1,3)(beta-1,4)-D-Xylans, e.g. rhodymenans; Hemicellulose; Derivatives thereof
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
  • C12N9/2405—Glucanases
  • C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
  • C12N9/2437—Cellulases (3.2.1.4; 3.2.1.74; 3.2.1.91; 3.2.1.150)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
  • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
  • C12Y302/01004—Cellulase (3.2.1.4), i.e. endo-1,4-beta-glucanase
• • D—TEXTILES; PAPER
  • D06—TREATMENT OF TEXTILES OR THE LIKE; LAUNDERING; FLEXIBLE MATERIALS NOT OTHERWISE PROVIDED FOR
  • D06M—TREATMENT, NOT PROVIDED FOR ELSEWHERE IN CLASS D06, OF FIBRES, THREADS, YARNS, FABRICS, FEATHERS OR FIBROUS GOODS MADE FROM SUCH MATERIALS
  • D06M16/00—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic
  • D06M16/003—Biochemical treatment of fibres, threads, yarns, fabrics, or fibrous goods made from such materials, e.g. enzymatic with enzymes or microorganisms
• • D—TEXTILES; PAPER
  • D21—PAPER-MAKING; PRODUCTION OF CELLULOSE
  • D21C—PRODUCTION OF CELLULOSE BY REMOVING NON-CELLULOSE SUBSTANCES FROM CELLULOSE-CONTAINING MATERIALS; REGENERATION OF PULPING LIQUORS; APPARATUS THEREFOR
  • D21C5/00—Other processes for obtaining cellulose, e.g. cooking cotton linters ; Processes characterised by the choice of cellulose-containing starting materials
  • D21C5/005—Treatment of cellulose-containing material with microorganisms or enzymes

Definitions

• 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.
• the combined action of endoglucanases and cellobiohvdrolases in the degradation of a xyloglucan/cellulose complex is disclosed.
• xyloglucans 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. 5 '• 605-610, 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 cellulose (Hayashi et al. Plant Physiol. 8_3 : 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.
• C x or endoglucanase a group consisting of C x or endoglucanase and Avicelase (often referred to as C,, cellobiohydrolase or exoglucanase) and sometimes cellobiase or ⁇ -glucosidase.
• Xyloglucanase (XGase) activity is generally not included. This means that to date no systematic use has been made of xyloglucanase activity in cell wall degradation.
• the present invention discloses the advantages of the use of xyloglucanase activity in the degradation of cell wall cellulose. Summary of the invention
• the present invention discloses mixtures of endoglucanases and cellobiohydrolases comprising xyloglucanase activity, which 5 are adapted for optimal cellulose degradation.
• One specific mixture comprises three enzymes; endoglucanase I, endoglucanase IV and cellobiohydrolase, another specific mixture comprises two enzymes; endoglucanase V and cellobiohydrolase.
• the ratio of xyloglucanase to avicelase activity in the 10 mixture of endoglucanases and cellobiohydrolases is chosen to provide optimal cellulose hydrolysis.
• the mass ratios of the enzymes of the specific EndoI/EndoIV/CBH mixture are chosen such that at a constant (Endoi + Endo ⁇ v)/CBH mass ratio of 0.5- 15 20 (g/g), an optimal molar Endoi/Endoiv ratio of 0.1-3 is used.
• the mass ratio of endoglucanase/cellobio- hydrolase is 0.96 and the EndoI/EndoIV molar ratio is 0.33.
• 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. 5
• 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.
• the enzymes are 0 obtainable from Trichoderma. Asper ⁇ illus 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. 5
• the present invention further discloses products obtained after treatment of plant material containing a xyloglucan/cellulose complex with cellulose-degrading enzyme mixtures containing xyloglucanase. Specifically, the product originates from apple. More specifically, the product is apple juice.
• Figure 1 Typical chromatograms showing the degradation products of apple-WUS solubilised by (combinations of) 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 sum of these areas was used for quantification of the xyloglucan oligosaccharides.
• FIG. 1 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 40°C, and 150 rp . 1, no enzyme added; 2, Maxazyme (10 mU toward Avicel, 200 m ⁇ 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.
• Figure 5 Release of cellobiose from apple fruit tissue by mixtures of purified glucanases.
• Blanched apple fruit tissue was incubated (200 mM succinate buffer pH 4.0, 16 hrs, 40°C, 150 rpm) with PL in combination with various 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.
• the present invention discloses the use of enzymes having xyloglucanase activity in the degradation of biological material containing xyloglucans. 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 endoglucanases.
• the present invention further describes the advantages of using mixtures of cellulose degrading enzymes comprising endoglucanases, cellobiohydrolases and xyloglucanase activity.
• the mixture is obtained from Trichoderma. Asper ⁇ illus 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 fermentation.
• 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. 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. It is also possible to mix culture fluids from growth of different strains or species.
• the enzymes are purified. Purification can be performed with any method known 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.
• 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 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 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 substrates used.
• Trichoderma viride presently known as Trichoderma reesei
• Trichoderma reesei The cellulase complex of Trichoderma viride has been shown to contain six distinct endoglucanases (Beldman et al (1985) Eur. J. Biochem. 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.
• the endoglucanases could be divided in three groups of enzymes based on their ability to degrade xyloglucans: one group active on cellulose (Avicel) , e.g. Endol, 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 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 Endol + CBH, specific for cellulose degradation, and the 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. 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) comprising cellulase activity, e.g. an enzyme of the Endo I to Endo III group together with CBH can attack the cellulose.
• an enzyme(s) comprising cellulase activity e.g. an enzyme of the Endo I to Endo III group together with CBH can attack the cellulose.
• cellulase activity e.g. an enzyme of the Endo I to Endo III group together with CBH can attack the cellulose.
• the relative amounts of endoglucanases, cellobiohydrolases and xyloglucanase activity depend on the substrate. Upscaling of the process of cellulose degradation, 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
• Example 2 demonstrates that for efficient degradation of cell wall cellulose three activities are important: endoglucanase (possessing activity towards Avicel) , cellobiohydrolase and xyloglucanase. Optimal cellulose degradation is achieved when the cellobiose release is 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, 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
• the endoglucanases are Endol and EndoIV and the mixture is characterized in that at a constant (Endoi + Endo ⁇ v)/CBH mass ratio of 0.96 (g/g) the mixture contains a molar Endoi/Endoiv ratio of 0.33.
• Example 3 the importance of xyloglucanase activity for the degradation and liquefaction of apple tissue is demonstrated.
• a cellulase preparation with low xyloglucanase activity is improved by addition of xyloglucanase, preferably by addition of an endoglucanase 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 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 xyloglucanase activity, in the form of EndoV, is necessary upon 0.26mU of Avicel-degrading activity.
• the mixtures as defined by the present invention can 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 endoglucanases for four different glucans (Example 1) . 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 enzymes is disclosed.
• Apples (Malus malus L. , Rosaceae, var. Golden Delicious) were harvested in mid-October, 1989, and stored in controlled atmosphere (2 ⁇ C, 2% 0 2 and 5% C0 2 ) for 4 months.
• Six endoglucanases (Endol to VI) [E.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.
• 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 from Dainippon Pharmaceutical (Osaka, Japan) .
• the apples After removing 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 distilled water (50°C) until the wash water contained minor amounts of sugars. Solids were collected by centrifugation (20min, 50,000g). The residual material was freeze-dried and ground in a Fritsch pulverisette (sieve 1.0mm, Germany) and designated as WUS.
• WUS (2g) was extracted with 0.05N NaOH (200ml) containing 5mM 1,2-cyclohexylene-dinitrilotetraacetic acid (CDTA) for 16h at 4°C under continuous stirring. After centrifugation (20min, 50,000g) the residue was resuspended in IN KOH (320ml) containing 1% (w/w) NaBH 4 , extracted for 16h at 20°C and centrifuged (20min, 50,000g). Using this residue, the latter procedure was repeated with 4N KOH containing 1% (w/w) NaBH 4 . Between sequential extraction steps, the residue was washed twice with distilled water. The final residue was acidified to pH5 with HC1 and freeze dried.
• CDTA 1,2-cyclohexylene-dinitrilotetraacetic acid
• the corresponding supernatants were collected, acidified to pH5 with HC1 and dialysed extensively against distilled water.
• 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 fraction retained on the column was released by elution with 500ml 1M sodium acetate buffer (pH5.0). Fractions (16.5ml) were assayed for both uronic acids and neutral sugar, pooled. dialysed and freeze-dried. The neutral fraction consisted of xyloglucan (APFXG) .
• Uronic acid content 20 Uronic acid content.
• Uronic acids were estimated colorimetrically with an automated m-hydrox ⁇ diphenyl test (Thibault JF (1979) Lebensm.Wiss.Technol. 12.: 247-251) using concentrated sulphuric acid containing 0.0125M Na 2 B 4 0 7 . Water- insoluble material was pretreated with 72% (w/w) H 2 S0 4 (lh at
• Total neutral su ⁇ ar content The total neutral sugar content was determined colorimetrically with an automated orcinol/sulphuric acid assay (Tollier MT, Robin JP (1979) Ann.
• Neutral su ⁇ ar composition Water-insoluble material was subjected to a 72% (w/w) H 2 S0 4 prehydrolysis (lh at 30°C) followed, after dilution with water, by a 2N H 2 S0 4 hydrolysis (3h at 100°C) . Water solubles were hydrolysed with 2N TFA (lh 5 at 121°C) . 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.
• 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 (airstream, room temperature) . This procedure was repeated once.
• 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 NaBD ⁇ ml, and converted to alditol acetates (Englyst et al.
• Protein content Protein content of enzyme preparations was determined according to Sedmak J.J. and Grossberg S.E. (1977) (Anal.Biochem. 7_9: 544-552). Analysis of xvlo ⁇ lucan and cellulose degradation products. High-performance liquid chromatography was conducted at 85°C with a SP 8800 HPLC pump system (Spectra Physics, San Jose, CA U.S.A.) fitted with a Shodex SE-61 refractometer (Showa Denko 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).
• SP 8800 HPLC pump system Spectra Physics, San Jose, CA U.S.A.
• Shodex SE-61 refractometer Showa Denko K.K., Tokyo, Japan
• Aminex HPX-22H column
• the solvent was 0.01N sulphuric acid pumped at a flow rate of 0.2ml/min.
• the injection volume was 20 ⁇ l.
• Maltodextrins were used for 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, 20°C, Dionex, Sunnyvale, CA) .
• HPAEC high-performance anion-exchange chromatography
• Samples (20 ⁇ L) were injected using a SP8780 autosampler (Spectra Physics, San Jose, 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 pulsed-amperometric detection (PAD) mode. A reference Ag/AgCl electrode was used with a working gold electrode with the following pulse potentials and durations: E., 0.1V and 0.5s, E 2 0.6V and 0.1s, E 3 0.6V and 0.1s.
• Cellobiose in the incubation mixtures was quantified by application of the following gradient: 0-+12 min, linear gradient of 25 ⁇ 85 mM NaOH, 12 ⁇ 25 min, linear gradient of O ⁇ lOO mM 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.
• the purified xyloglucan (250 ⁇ g) was dissolved in lOO ⁇ L buffer and incubated for 1 or 2Oh with EndoIV, EndoV and EndoVI (30ng) or Endol, 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 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 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. Endol, II and III show a relatively low activity towards XG compared to EndoIV, V and VI.
• removal of the fucose residue slightly enhanced the degradation of xyloglucan by endoglucanases.
• the increased activity of, for instance, Endol is not so dramatic that a combination of Endol and a fucosidase might prove an alternative for EndoIV in applications.
• Table I Composition of WUS and fractions obtained bv se ⁇ uential extractions thereof fmole%..
• the constituent polysaccharides From the sugar composition of the apple-WUS (Table I) 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 25% (w/w) of the total amount of sugar residues, a value generally found for dicotyledons (Hayashi T.
• a HPLC method (Animex column) was used to determine both cellobiose (as a measure for cellulose hydrolysis) and the total of xyloglucan oligosaccharides quantitatively.
• the isolated cell wall material was incubated with Endol, EndoIV, CBH and various combinations thereof.
• Fig. 1A shows that cellobiose is the only product formed by CBH whereas both endoglucanases did not release any cellobiose from WUS (data not shown) .
• Fig. 1C shows a separation of cellobiose and other compounds (shaded area in Fig. 1C) . The latter were characterized as xyloglucan oligosaccharides after fractionation on BioGel P2 and semi- preparative CarboPac PA1 (data not shown) .
• 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 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 exceeds the xyloglucan turnover. For optimal EndoIV/CBH the contrary is the case.
• EndoIV+CBH For a combination of EndoIV+CBH, the turnover of 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 positive aspects of the previous two. By removing the xyloglucan coating, EndoIV enhances the accessibility of the cellulose for Endol and CBH which display a larger activity towards this substrate than EndoIV. In this case, cellulose turnover almost equals xyloglucan turnover.
• An advantage of 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.
• Pectin lyase (PL) 50 mU) (see for PL purification: 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, 1 mL was removed from the incubation mixture, heated for 10 min at 100°C to inactivate the enzymes, centrifuged (2 min, 20.000 g) , and the degradation proclucts 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 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 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.
• the commercial preparations contain both endoglucanase (CMCase activity) as well as CBH (release of cellobiose from tissue) .
• CMCase activity endoglucanase
• CBH release of cellobiose from tissue
• the CMCase activity in these preparations 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.
• Avicelase activity of mixtures of purified glucanases was therefore defined as the difference in activity of endoglucanase plus CBH (endoglucanase/CBH-mass ratio is approximately 3.5) and endoglucanase. It is hereby assumed that both enzymes (Endol 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. '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 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.

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