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)
  • C12Y302/01037—Xylan 1,4-beta-xylosidase (3.2.1.37)
• • A—HUMAN NECESSITIES
  • A21—BAKING; EDIBLE DOUGHS
  • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
  • A21D8/00—Methods for preparing or baking dough
  • A21D8/02—Methods for preparing dough; Treating dough prior to baking
  • A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
  • A21D8/042—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes with enzymes
• • 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
  • A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof
  • A23L7/10—Cereal-derived products
  • A23L7/104—Fermentation of farinaceous cereal or cereal material; Addition of enzymes or microorganisms
  • A23L7/107—Addition or treatment with enzymes not combined with fermentation with microorganisms
• • 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)
• • 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)
  • C12N9/2477—Hemicellulases not provided in a preceding group
  • C12N9/248—Xylanases
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/02—Monosaccharides
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P19/00—Preparation of compounds containing saccharide radicals
  • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
• • 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)
  • C12Y302/01008—Endo-1,4-beta-xylanase (3.2.1.8)
• • 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)
  • C12Y302/01055—Alpha-N-arabinofuranosidase (3.2.1.55)

Definitions

• the invention relates to non-starch polysaccharide hydrolysing enzymes, in particular xylan-degrading enzymes which are endogenously present in wheat malt, wheat kernel or fractions thereof, to a method for obtaining such enzymes, as well as to the use of these enzymes in food, feed and paper and pulp technology.
• NSPs non-starch polysaccharides
• NSP hydro ⁇ lysing enzymes are increasingly employed in the breadmaking industry (McCleary 1986; Ter Haseborg and Himmelstein 1988; Maat et al 1992). Such enzymes, when added at an optimal dosage, clearly improve the machinability of doughs and the volume and appearance of breads (Maat et al 1992; yet et al 1992). NSPs also influence the digestibility of feeds rich in cereals and legumes (in particular rye or wheat). Production of sticky droppings and poor growth and feed conversion, especially in younger broilers are often related with these compounds (Moran et al 1969; Campbell et al 1983; Fengler et al 1988; Pettersson et al 1988).
• NSP attacking enzymes in the pulp and paper industry has become more and more important (Wong et al 1988) due to their bleach boosting properties helping to replace the use of damaging bleaching agents (Farrell et al 1992, Nissen et al 1992; Wong and Saddler 1992).
• Other applications of these NSP hydrolysing enzymes are clarifying juices, beers and wines, extracting coffee, plant oils and starch and producing food thickeners (Wong and Saddler 1992).
• the NSP hydrolysing enzymes encompass a large group of enzymes, which are classified according to their substrate specificity and to their mode of action (Dekker 1979).
• the common sources of industrially available NSP-hydrolases are the culture media of microorganisms.
• NSP hydrolysing enzymes from microorganisms have received a great deal of attention due to their applications as described above, the relative importance of endogenous enzymes present in wheat malt, wheat kernel or fractions thereof is described in only a limited number of reports (Preece and McDougall 1958; Kulp 1968b; Lee and Ronalds 1972; Schmitz et al 1974; Bremen 1981; Adlung 1985, Beldman et al 1995).
• the NSP hydrolysing activity was mostly found in the outer fractions of the kernel, mainly the bran and short fractions.
• the invention relates to enzymes having xylan-degrading activity, obtainable by extraction of wheat or fractions thereof, such as wheat flour, which enzymes have an apparent molecular weight between 25,000 and 68,000 Da.
• the enzymes have endo-xylanase, ⁇ -xylosidase and arabinofuranosidase activities.
• the invention also relates to a method for obtaining non-starch poly ⁇ saccharide hydrolysing enzymes from wheat malt, wheat kernel or fraction thereof by subjecting such preparations to fractionation steps, obtaining enzymes having enzymatic activity toward p-nitrophenyl- ⁇ -D-xylopyranoside, p-nitrophenyl- ⁇ -L- arabinofuranoside, xylan from oat spelts and arabinoxylan from wheat.
• Some of these enzymes from wheat flour were found to have a very specific mode of action, differ ⁇ ent from most microbial enzymes, and are therefore of major importance for particu ⁇ lar industrial applications.
• the invention furthermore relates to the use of these xylan-degrading enzymes, especially of the endo-xylanases, as bread improver, for the treatment of cereals, such as for animal feedstuffs, for the production of xylose, and for gluten- starch separation or syrup processing.
• these xylan-degrading enzymes especially of the endo-xylanases, as bread improver, for the treatment of cereals, such as for animal feedstuffs, for the production of xylose, and for gluten- starch separation or syrup processing.
• NSP hydrolysing enzymes are a group of enzymes degrading non-starch polysaccharides. Due to the complexity and heterogeneity of non-starch polysacchar ⁇ ides, several types of endo- and exo-acting enzymes are known to be involved in the hydrolysis of these polysaccharides. The discussion of these enzymes will be easier with a previous description of their substrates.
• One of the most abundant non- starch polysaccharides in cereals is (arabino)-xylan. This polysaccharide consists of a homopolymeric backbone of 1,4-linked ⁇ -D-xylopyranose units. Depending on its origin, the backbone may be substituted.
• Xylans from cereals have been shown to be highly arabinosylated.
• the arabinofuranosyl residues are attached to the main chain by ⁇ -1,3 and/or ⁇ -1,2 glycosidic linkages.
• These arabinofuranosyl residues can be esterified with phenolic acids such as -coumaric and ferulic acids (4-hydroxycinna- mic acid and 4-hydroxy-3-methoxycinnamic acid, respectively).
• Glucuronic acid residues or their 4-O-methyl ethers are also present.
• the crucial enzyme for de- polymerisation of (arabino)xylan is endo-xylanase (EC 3.2.1.8), which attacks the main chain, generating non-substituted or branched xylo-oligosaccharides.
• endo-xylanase EC 3.2.1.8
• the mode of action of different endo-xylanases and the hydrolysis products vary according to the source of the enzymes. Most of the endo-xylanases are generally found to hydrolyse main chain linkages at regions of the substrate not substituted with arabinose.
• the main chain substituents are liberated by the corresponding (exo-acting) glycosidases and esterases, as follows: ⁇ -L-arabinosyl residues by ⁇ -L-arabino- furanosidase (EC 3.2.1.55), 4-O-methyl-D-glucuronosyl residues or D-glucuronosyl residues by ⁇ -glucuronidase, -coumaric acid and ferulic acid residues by the corresponding esterases.
• ⁇ -Xylosidase (EC 3.2.1.37) is the enzyme component that attacks xylo-oligosaccharides generated by the action of ⁇ -xylanase and other hydrolases from the non-reducing end, liberating D-xylose as the only product of hydrolysis.
• ⁇ -Xylosidase is important when complete hydrolysis of (arabino)-xylan is required.
• the enzymes liberating (arabino)-xylan substituents act synergistically with the depolymerising ⁇ -xylanases.
• Debranching enzymes create new sites on the main chain for productive complex formation with ⁇ -xylanases.
• ⁇ -xylanase facilitates the interaction of debranching enzymes with the substrate. Synergism between ⁇ -xylanase and ⁇ -L-arabinofuranosidase has been demonstrated.
• the present invention provides a method for isolation and purification of these xylan-degrading enzymes, although the invention is not restricted to these methods.
• a crude lyophilised aqueous enzyme extract from wheat flour is dissolved in phosphate buffer and subjected to gradual ammonium sulphate precipitation.
• the fraction that precipitates between 30 and 70 % ammonium sulphate saturation shows activity towards p-nitrophenyl- ⁇ -D-xylopyranoside, p-nitrophenyl- ⁇ -L-arabino- furanoside and xylan from oat spelt, indicating xylosidase, arabinofuranosidase and endo-xylanase activity respectively.
• Preparative anion exchange chromatography fractionates the exo-acting enzymes (xylosidase and arabinofuranosidase) from the endo-acting enzyme.
• the protein fraction that precipitates between 80 and 100 % ammonium sulphate saturation shows hydrolysing activity against arabinoxylan.
• a protein with MW of 30,000 Da and pi > 9.0 is obtained, further referred to as P-30,000 or endoxylanase B.
• the N-terminal aminoacid sequence of this protein is given in SEQ ID NO. 1 (VAIACSASGFENCEEEQPK), wherein the identity of the cysteine residues at position 5 and 13 could not be un ⁇ ambiguously confirmed.
• the N-terminal protein sequence data shows 89 and 84% homology (assuming the cysteines are confirmed) with the internal aminoacid sequence 32-50 of two clones of friabilin, a 15,000 Da grain softness protein (GSP), as deduced from the DNA sequences found by Rahman et al, 1994. These two GSP clones have the aminoacid sequences VAIAPSASGSENCEEEQPK and VAIAPSAS- GFEDCEEEHPK, respectively. This indicates that the isolated protein represents an endogenous wheat enzyme.
• GSP grain softness protein
• the fraction that remains in solution after 100 % ammonium sulphate saturation of the crude wheat extract shows one clear protein band after SDS-poly ⁇ acrylamide gel electrophoresis under non-reducing conditions with a MW of approximately 6,500.
• the N-terminal protein sequence data of this protein are IDCGHVDSLVRPCLSYVQGG and show 100 % homology with the N-terminal aminoacid sequence of a phospholipid transfer protein of wheat with a MW of about 8-9,000.
• the invention thus provides four isolated xylan-degrading enzymes derived from wheat flour, denoted as ⁇ -xylosidase, arabinofuranosidase, endoxylanase A and endoxylanase B.
• These enzymes are defined by their physical properties and their activity pattern as described in the examples. They may be produced by separation from wheat flour extracts as described herein, but they may also be produced by cloning their gene into a suitable host organism, e.g. a mould (especially an jispergillus species) or a yeast, using an endogenous or exogenous promoter, cultur ⁇ ing the host organism under suitable, commonly used conditions, and isolating the enzymes produced by these organisms.
• a suitable host organism e.g. a mould (especially an jispergillus species) or a yeast
• xylan-degrad ⁇ ing enzymes this term should be understood to include enzymes degrading arabino- xylans and other polysaccharides containing arabinose and/or xylose units.
• novel endoxylanase endoxylanase B
• This enzyme is further defined by reference to its N-terminal aminoacid sequence.
• the invention relates in particular to an endoxylanase which in its protein sequence contains at least 8 aminoacids which are in the same relative position as the amino acid sequence of SEQ ID NO. 1 and contains the sequence Phe-Glu-Asn.
• aminoacid sequence contains a contiguous series of at least 8 aminoacids corresponding to the sequence of SEQ ID NO. 1.
• aminoacid sequence contains a contiguous series of at least 8 aminoacids corresponding to the sequence of SEQ ID NO. 1.
• the invention also pertains to the use of these enzymes, alone or in combi ⁇ nation, including the use in bread making and in the treatment of cereals, e.g. for the production of animal feedstuffs.
• Other desired uses include the production of xylose
• ⁇ -xylosidase optionally together with one or more of the other enzymes
• arabinose arabinose
• arabinose arabinose
• xylo-oligosaccharides endoxylanase A and/or B
• Wheat flour (Camp Remy, 3300 g), was suspended in 9900 ml of 0.1 M sodium phosphate buffer (pH 7.0) and stirred for 30 min. The supernatant (10,000 g, 30 min, 4°C) was dialysed (MW cut-off 3500 Da, 48 h, 4°C) against deionised water and lyophilised to produce a crude enzyme extract. Crude enzyme extract (60 g) was dissolved in 1700 ml phosphate buffer (pH
• fraction AS 0-30 The supernatant was successively adjusted to 70%, 80% and 100% AS saturation and the precipitates separated in a similar manner (fractions AS 30-70, AS 70-80 and AS 80-100, respectively). The final supernatant was dialysed and lyophilised in the same way resulting in fraction AS >100.
• Fraction AS 30-70 was further fractionated by anion exchange chromato ⁇ graphy (Q-Sepharose HP 35/100, Pharmacia, SE; 20 mM Tris buffer pH 8.0). The column was washed with buffer and bound proteins were then eluted with a stepwise gradient of the buffer + 1.0 M NaCl. Fractions containing glycosidase ( ⁇ -xylosidase and arabinofuranosidase) activity were pooled, dialysed and lyophilised to yield fraction AS 30-70 I and fractions with endo (xylanase) activity were pooled, dialysed and lyophilised to yield fraction AS 30-70 II.
• anion exchange chromato ⁇ graphy Q-Sepharose HP 35/100, Pharmacia, SE; 20 mM Tris buffer pH 8.0. The column was washed with buffer and bound proteins were then eluted with a stepwise gradient of the buffer + 1.0 M NaCl.
• Fraction AS 30-70 I was further fractionated by hydrophobic interaction chromatography (Phenyl Superose HR 5/5, Pharmacia, SE; 50 mM sodium phosphate buffer pH 7.0 containing 1.2 M AS). A linear gradient from 1.2 to 0 M AS was used. Fractions were dialysed against 50 mM sodium acetate (pH 5.5) yielding fractions AS 30-70 Ia, exhibiting ⁇ -L-arabinofuranosidase activity, and AS 30-70 Ib, exhib ⁇ iting ⁇ -D-xylosidase and arabinofuranosidase activity.
• Fractions Ia and Ib were subjected to cation exchange chromatography (Mono S column, HR 5/5, Pharmacia, SE; 50 mM sodium acetate, pH 5.5) to remove contaminating proteins.
• the eluted fractions were pooled and dialysed (pH 8.5, 20 h).
• Final purification was performed by anion exchange chromatography (Mono Q, HR 5/5; 20 mM Tris pH 8.5); the absorbed proteins were eluted with linear gradient of 0 to 0.5 M NaCl, followed by a linear gradient from 0.5 to 1.0 M NaCl.
• the final fractions contained xylosidase and arabino ⁇ furanosidase activity, respectively.
• Fraction AS 30-70 II was further fractionated by hydrophobic interaction in the same way as fraction I, except that an AS gradient from 0.6 M to 0.24 M was used.
• the pooled, dialysed fractions were subjected to a final anion exchange chromatography (Mono Q) (fraction AS 30-70 II Q) and contained endoxylanase (referred to herein as endoxylanase A) activity.
• Fraction AS 80-100 was further purified on a Bio-gel P-10 column (Bio-
• fractions were pooled, dialysed (3500 Da) and lyophilised to yield fraction AS 80- 100 GS containing endoxylanase (referred to herein as endoxylanase B) activity.
• the molecular weights of the purified enzymes were determined by SDS- PAGE on 12.5% or 20.0% polyacrylamide gels under non-reducing conditions with the PhastSystem unit (Pharmacia). The isoelectric points were determined with the
• Fraction AS 30-70 Ia SQ ( ⁇ -xylosidase, example 1) is capable of hydro ⁇ lysing xylose- oligomers from dimers up to at least pentamers. It can also release xylose form wheat arabinoxylan and oat spelts xylan, but more efficiently from the latter. This shows that it cannot debranch branched xylans.
• Fraction AS 30-70 Ib SQ (arabinofuranosidase, example 1) is capable of hydrolysing arabinoxylans having different substitution patterns, containing 1,3- ⁇ - and/or 1,2- ⁇ - ⁇ -linked arabinose residues.
• the major hydrolysis product is presum- ably a substituted arabinose (HPAEC retention time between xylose and arabinose), together with arabinose.
• Endoxylanase B is also capable of hydrolysing oat spelts xylan, although the differ ⁇ ence in yield of oligosaccharide production is smaller than with the arabinoxylans.
• the degradation of (arabino)-xylan without the need to use further xylano- lytic enzymes and without release of major amounts of arabinose and xylose may in particular be important for the feed industry since pentose sugars are poorly utilised and may even be detrimental in high concentrations e.g. to fowl.
• Viscosity measurements of an incubation mixture of wheat arabinoxylan with endoxylanase B show a clear decrease in viscosity in time, indicat ⁇ ing the presence of an endo-acting enzyme, thus supporting our findings in example 3.
• the soluble arabinoxylans form highly viscous solutions and in this way influence dough rheology.
• Addition of endoxylanase B to wheat flour decreases the viscosity of arabinoxylans present in the flour during breadmaking and influence the bread-making quality of the flour.
• the enzyme can e.g. be added to the process water containing starch and gluten. Transport problems with highly viscous solutions (syrups) can be resolved using this enzyme. Again the enzyme can simply be added to the syrup and, if appropriate, the mixture can be pasteurised.

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• 1996
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