Classifications

• • 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
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23K—FODDER
  • A23K20/00—Accessory food factors for animal feeding-stuffs
  • A23K20/10—Organic substances
  • A23K20/163—Sugars; Polysaccharides
• • 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
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/20—Reducing nutritive value; Dietetic products with reduced nutritive value
  • A23L33/21—Addition of substantially indigestible substances, e.g. dietary fibres
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/7016—Disaccharides, e.g. lactose, lactulose
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H1/00—Processes for the preparation of sugar derivatives
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
  • C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
  • C07H3/04—Disaccharides
• • 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
  • C12P19/12—Disaccharides
• • 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
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
  • C12P7/58—Aldonic, ketoaldonic or saccharic acids
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
  • C12Y101/03—Oxidoreductases acting on the CH-OH group of donors (1.1) with a oxygen as acceptor (1.1.3)
  • C12Y101/03004—Glucose oxidase (1.1.3.4)
• • 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)
• • C—CHEMISTRY; METALLURGY
  • C13—SUGAR INDUSTRY
  • C13K—SACCHARIDES OBTAINED FROM NATURAL SOURCES OR BY HYDROLYSIS OF NATURALLY OCCURRING DISACCHARIDES, OLIGOSACCHARIDES OR POLYSACCHARIDES
  • C13K1/00—Glucose; Glucose-containing syrups
  • C13K1/02—Glucose; Glucose-containing syrups obtained by saccharification of cellulosic materials
• • C—CHEMISTRY; METALLURGY
  • C13—SUGAR INDUSTRY
  • C13K—SACCHARIDES OBTAINED FROM NATURAL SOURCES OR BY HYDROLYSIS OF NATURALLY OCCURRING DISACCHARIDES, OLIGOSACCHARIDES OR POLYSACCHARIDES
  • C13K13/00—Sugars not otherwise provided for in this class
  • C13K13/007—Separation of sugars provided for in subclass C13K
• • 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
  • C12P2203/00—Fermentation products obtained from optionally pretreated or hydrolyzed cellulosic or lignocellulosic material as the carbon source

Definitions

• the invention is directed to a method for the enzymatic production of cellobiose from sugar beet and other cellulose- containing biomass.
• Cellobiose is a disaccharide which
• Cellobiose is a valuable ingredient in the food, animal feed, chemicals, cosmetics and pharmaceutical industries.
• Cellobiose can be produced by chemical and biochemical methods from polymeric cellulose or from disaccharides such as sucrose.
• cellobiose When using cellulosic feedstocks, cellobiose can be prepared either by chemical acidolysis or by enzymatic hydrolysis.
• cellulose is subjected to acids, such as sulfuric acid or glacial acetic acid to produce cellobiose octaacetate which is subsequently converted to cellobiose by chemical deacetylation (Pereira at al . , (1988): Methods Enzymol . , 160, 26) .
• acids such as sulfuric acid or glacial acetic acid
• cellobiose octaacetate which is subsequently converted to cellobiose by chemical deacetylation
• cellobiose is produced from a cellulose containing feedstock by treatment with cellulose-hydrolyzing enzymes (Taniguchi (1989, Nippon Nogeikagaku Kaishi, 63, 1133).
• the enzymatic method can lead to higher yields and in contrast to the chemical methodology does not require toxic reagents nor is it associated with waste disposal problems.
• a physico-chemical pre- treatment which may encompass a hydrothermal treatment in the absence or presence of a base or acid catalyst, such sulfuric acid (US 4908311, JP2G06204294 ⁇ can be applied.
• inhibitory compounds that reduce enzyme hydrolysis activity and limit product yields (Palmqvist and Hahn-Hagerdal (2000) , Bioresource Technology, 74, pp. 25-33) .
• pretreatments can be applied to remove unwanted biomass components, such as pectins and hemicelluloses (Bonnin, E. et al. (2000) Carb.Poly., 41, pp. 143-151, Micard, V. et al . , (1997) Lebensmitt. Wiss. U. Technol . 30, pp. 284-291) from complex biomass such as sugar beet pulp.
• pectinase preparations with very low cellulose side-activities are applied to selectively solubilise and remove non-cellulose components. Subsequently, the remaining cellulose enriched solid phase can be contacted with cellulase enzyme
• endo- cellulases e.g. endoglucanases
• exo-cellulases e.g.
• cellobiohydrolases and di- and oligosaccharide hydrolizing enzymes (e.g. /3-glucosidases , also often referred to as cellobiase) .
• endo- and exo-cellulases release oligo- and disaccharides (cellobiose) from polymeric cellulose chains
• ⁇ - glucosidases beta-giucosidases , BGL
• BGL activities are undesirable.
• Most cellulase preparations contain /5-glucosidase activity, which will degrade cellobiose to glucose. This ⁇ -glucosidase activity can be inhibited by the addition of an inhibitor such as D- gluconolactone (Reese et al . , 1971, Carbohydrate Research, 18, 381-388) .
• BGL activities can be accomplished in-situ by addition of inhibitors, such as D-glucono-lactone (Bonnin et al., (2000), Carbohydr. Polymers 41, pp. 143-151), removal of BGL from the enzyme complex is commonly achieved by inhibitors, such as D-glucono-lactone (Bonnin et al., (2000), Carbohydr. Polymers 41, pp. 143-151), removal of BGL from the enzyme complex is commonly achieved by
• Bioengineer. 41, pp. 405-410) can be used as chromatographic resins which allow selective retention of endo- and exo- cellulase enzymes.
• Glucose oxidase has been applied for the removal of glucose from egg white (Sisak et al., 2006, Enzyme and Microbial
• cellobiose is released from cellulosic substrates by adding a cellulase enzyme at an enzyme to substrate ratio of 1 - 20 % w/w dry matter at 45 - 55 °C for 2 - 600 hours in a 50 - 200 mM, pH: 4.5 ⁇ 5.5 buffer (Micard et al., (1997), Lebensm. -Wiss.u. -Technol. 30, pp. 284- 291, Micard et al . 2000, Vanderghemet al . (2009), Appl .
• the cellobiose yield is limited by the catalytic efficiency of cellobiohydrolase (CBH) which is significantly inhibited by the end product cellobiose (Ki ⁇ 0,3 raM) .
• CBH cellobiohydrolase
• Ki ⁇ 0,3 raM end product cellobiose
• the by-product glucose needs to be separated from the cellobiose besides other impurities.
• Glucose can be removed by passing the solution through an immobilized yeast column whereby glucose is metabolized selectively by the yeast. Cellobiose is recovered afterwards (US 4, 908, 311) .
• Cellobiose can also be recovered by cooling the solution to 0 °C or below to precipitate cellobiose.
• an organic solvent can be added to precipitate the cellobiose. Desalting and decoloring of cellobiose can be done by
• the cellobiose can be used as a building block in polymers, as food ingredient, such as animal feed additive, for example as pre-biotic additive, as cosmetics additive or ingredient, or in an anti-inflammatory pharmaceutical preparations (Briton et al. (2003) Ann. New York .Acad . Sci . pp. 750-54; Briton et al . (2003) Hemijsk Industrija 57, pp. 622-625, US 5219842; Kurita et al., (2003), Poly . Chem. Ed . 18, pp. 365-370).
• Enzyme costs and cellobiose product yields are critical parameters of enzymatic cellobiose production methods from an economic point of view.
• the problem to be solved by the present invention is therefore the provision of an economic method for production of
• cellobiose comprising an enzymatic degradation of cellulose from a cellulose-containing material.
• the problem to be solved by the present invention also includes the provision of a method for producing cellobiose at high concentrations.
• the present invention provides an economically efficient process for producing cellobiose from a cellulose containing material, whereby high substrate yields can be achieved.
• the present invention also provides an economically efficient process for producing cellobiose from a cellulose containing material, whereby low enzyme/substrate are used.
• the present invention covers a process for producing cellobiose, comprising the following steps: c) subjecting a cellulose containing material to a
• cellulase treatment at a dry matter content between 7.5 and 50 % (w/w) , preferably between 10 and 25% (w/w) , and more preferably between 12 and 20% ⁇ w/w) , and
• the substrate used for the production of cellobiose is a cellulose containing material.
• Many cellulose containing materials are suitable substrates for the process according to the present invention, in particular microcrystalline
• the cellulose containing material used as substrate in the process of the present invention is or comprises sugar beet pulp.
• the process above is preceeded by the follwong steps: subjecting a cellulose containing material to a treatment that produces a liquid phase
• the enzymatic treatment above is carried out in the presence of an enzyme preparation
• the cellulase composition used exhibits reduced or minimal ⁇ -glucosidase activity.
• Various ways for obtraining such a cellularase composition are possible, including, but by no means limited to: obtaining the cellulase composition from an organism devoid of enzymes exhibiting ⁇ - glucosidase activity, removing any polypeptides exhibiting ⁇ - glucosidase activity, inhibiting any polypeptides exhibiting ⁇ -glucosidase activity.
• the cellobiose cellobiose containing liquid phase obtained by the process above is subjected to a further work-up step, which comprises:
• cellobiose optionally concentrating the cellobiose solution, and further purifying the cellobiose from the (optionally concentrated) cellobiose solution.
• the cellobiose may be purified for example by one or more of the following: cellobiose
• crystallization chromatography, nanofiltration, precipitation by organic solvents, or the use of microorganisms, whereby crystallization is preferred.
• glucose removal is its oxidation to gluconic acid.
• Gluconic acid may then be removed by one or several of the following: ion exchange chromatography, precipitation, electrodialysis , whereby ion exchange chromatography is preferred.
• the present invention provides an economically efficient process for producing cellobiose from a cellulose containing material, whereby high substrate yields can be achieved.
• the present invention also provides an economically efficient process for producing cellobiose from a cellulose containing material, whereby low enzyme/substrate are used.
• the substrate used for the production of cellobiose is a cellulose containing material.
• Many cellulose containing materials are suitable substrates for the process according to the present invention, in particular microcrystalline
• the relevant substrate contains a significantly larger ratio of amorphous cellulose versus crystalline
• amorphous cellulose at 50 % or more relative to the total amount of cellulose, and which is or comprises preferably fruit pulp or vegetable residue, or is sugar beet biomass or is or comprises even more preferably sugar beet pulp. Preferred is a content of 50 % or more amorphous cellulose relative to the total amount of
• cellulose (w/w) , and particularly preferred is a content of 60 - 99% amorphous cellulose (w/w) in the cellulose containing material subjected to the enzymatic hydrolysis step.
• Physicochemical treatment can be acid treatment or heat treatment or any combination thereof such that part or the entire crystalline cellulose portion of the substrate is rendered amorphous .
• the cellulose containing substrate is any form of sugar beet, vegetable or fruit biomass.
• the substrate is sugar beet pulp, or vegetable residues, or fruit pulp residues.
• the cellulose containing material used as substrate in the process of the present invention is or comprises sugar beet pulp.
• Sugar beet pulp means the residual biomass material after extraction of part or all sucrose from sugar beet (Beta vulgaris) .
• Sugar beet pulp can be used freshly after extraction, with or without pressing.
• Fresh sugar beet pulp typically has a dry matter content between 19 and 29 % w/w.
• Sugar beet pulp can also be used after drying, with or without pelleting.
• Dried sugar beet pulp typically has a dry matter content of 85 - 90 % w/w.
• the substrate can be cellulosic or lignocellolosic material.
• the material can either be fresh (e.g. fresh sugar beet pulp, wet cellulose from a paper mil), or dried (e.g. dried straw, dried sugar beet pulp, wood, paper) or in any other form, including any form of preserved materials.
• the substrate is fresh sugar beet pulp, ensiled sugar beet pulp, dried sugar beet pulp, or dried and pelleted sugar beet pulp.
• sugar beet pulp is the substrate and is treated with acid at elevated temperature at high dry matter content.
• the transportation of such material into and out of the pretreatment reactor is achieved by using a
• the mixing can be carried out by using special kinds of stirring equipment such as anchor agitators or segmented spiral stirrers, which guarantee a uniform power input as well as a homogeneous distribution of temperature and added substances.
• the present invention covers a process for producing ceilobiose, comprising the following steps: c) subjecting a cellulose containing material to a cellulase treatment at a dry matter content between 7.5 and 50 % (w/w) , preferably between 10 and 25% (w/w) , and more preferably between 12 and 20% (w/w) , and d) separating the ceilobiose containing liquid phase from the solid phase obtained in c) .
• the hydrolysis can be performed in various ways, e.g. as batch, repeated batch, continuous, semi-continuous , using a membrane based process, or in a counter- flow setup, or in any
• the steps c) and d) of the process above are repeated multiple times, whereby fresh aqueous phase, and optionally no cellulase or no enzyme preparation comprising a polypeptide exhibiting cellulase activity is added to the solid phase in subsequent cycles.
• the enzyme composition and various preferred embodiments thereof are given in detail below .
• steps c ⁇ and d) of the process above are carried out simultaneously.
• a technical way of putting this embodiment into practice is the removal of the ceilobiose via the liquid phase, e.g. by using a membrane reactor configuration. It was found that by using a membrane reactor configuration the space time yield was significantly improved.
• membranes are used that retain the solid phase in the reactor but permit the solubilized components, like ceilobiose and others, to pass through the membrane.
• micro- or ultrafiltration membranes are used.
• Ultrafiltration may generally be used to retain particles of a size of a few nanometres or more, whereas micro-filtration, which employs porous membranes with pore diameters between 0.05 and 10 micrometres ( ⁇ m) is able to separate particles in the ⁇ size range.
• Ultrafiltration membranes are typically- able to retain oils, fats, waxes, resins, as well as solids such as sand, grinding residues and metal hydroxides.
• soluble components such as salts and low molecular organic substances usually are not retained with ultra-filtration membranes.
• a typical ultrafiltration membrane has a cut-off between 500 D and 50 kDa, or between 30 kDa and 3.5 kDa.
• both micro- or ultrafiltration membranes are suitable for this separation process. An example thereof is given in example 10 below.
• additional aqueous phase such as water or buffer
• the volume in the membrane reactor is kept at a constant level.
• additional dry matter i.e. additional cellulose containing solids, is added during the hydrolysis. Two or three of these particularly preferred embodiments may be combined with each other .
• the dry matter content in the hydrolysis step is kept constant or increased over time during the hydrolysis.
• the cellobiose concentration on average is increased, which is considered beneficial for the downstream processing of the cellobiose.
• the ultrafiltration membrane has a cut-off between 3.5 kD and 30 kDa.
• the nanofiltration step is used to concentrate the
• steps c) and d ⁇ of the process above are carried out simultaneously. This may be done by continuously replacing the cellobiose containing liquid phase by fresh aqueous phase.
• a repeated batch process is used, dry matter content between 7.5 and 50 % (w/w) , preferably between 10 and 25% (w/w) , and more preferably between 12 and 20% (w/w), and an enzyme-to- substrate ratio of 0.05 - 1 % enzyme d.m substrate (w/w) . This allows for short cyling times and a high overall yield.
• a membrane reactor is used, which enables the continuous removal of the cellobiose
• the membrane is preferably a
• the ultrafiltration membrane has a cut-off between 3.5 and 30 kD .
• the nanofiltration step is used to concentrate the cellobiose.
• a microfiltration membrane is used besides an ultra- and a nanofiltration membrane in order to improve the clarification of the product stream.
• step b) The cellulase treatment of step b) above for cellobiose
• This enzyme preparation preferably comprises at least one polypeptide having cellobiohydrolase activity (EC 3.2.1.91, CBH) .
• the enzyme/substrate ratio in step c) of the process above is 0.01 to 0.5 %, preferably 0.05 to 0.1 % .
• the cellulase preparation also contains at least one polypeptide with beta- endo-glucanases activity ⁇ EC 3.2.1.4, EG ⁇ to break down the polymeric substrate cellulose into smaller fragments which are more amendable to hydrolysis by a cellobiohydrolase activity.
• the cellulase preparation is derived from a fungal fermentation, e.g. from the
• the cellulase preparation used for degradation of the cellulose containing material in the process above exhibits reduced ⁇ - glucosidase activity.
• the cellulase in an even more preferred embodiment, the cellulase
• preparation used in the process above originates from an organism devoid of enzymes that exhibit significant ⁇ - glucosidase activity, and preferably from an organism wherein at least one gene, more preferably all genes encoding
• polypeptides exhibiting ⁇ -glucosidase activity have been deleted or rendered dysfunctional.
• the original cellulase preparation contains ⁇ -glucosidase activity, but the ⁇ -glucosidase activity is minimized or eliminated by adding the original cellulase preparationto an adsorption material selected from cellulose-, chitin- and chitosan-containing materials, followed by washing the adsorption material, which yields a cellulase
• the adsorption material is the cellulose-contianing solid phase obtained in step a) of the process described below.
• said step a) comprises subjecting a cellulose containing material to a treatment that produces a liquid phase containing pectin, hemicellulose and/or their degradation products, and a solid phase
• ⁇ -glucosidase activity is removed or reduced by passing the cellulase preparation over sugar beet pulp, which leads to selective retention of cellulase (CBH, EG) activites on the designated substrate, whereas ⁇ -glucosidase activity is removed or reduced,
• a strain is used for the production of the cellulase preparation that is deficient in ⁇ -glucosidase activity.
• the ⁇ -glucosidase deficient expression strain can be generated by conventional mutagenesis and selection, or by selective molecular biology and generation of specific knock out strains.
• the enzyme preparation comprising a polypeptide exhibiting cellulase activity in the process above comprises at least one cellobiohydrolase from the following group of cellobiohydrolases (EC 3.2.1.91): a glycosylhydrolase belonging to family 6 or family 7 of glycosylhydrolases .
• the families of glycosylhydrolases are well known to the person skilled in the art, and have been classified by Henrissat et al . (1998, FEBS Lett. 425(2) :352- 354) .
• polypeptides exhibiting cellobiohydrolases from the following group of cellobiohydrolases (EC 3.2.1.91): a glycosylhydrolase belonging to family 6 or family 7 of glycosylhydrolases .
• the families of glycosylhydrolases are well known to the person skilled in the art, and have been classified by Henrissat et al . (1998, FEBS Lett. 425(2) :35
• Cellobiohydrolase activity CBHI from Trichoderma reesei (SEQ ID NO. 1) , CBHII from Trichoderma reesei (SEQ ID No, 2) , a polypeptide exhibiting cellobiohydrolase activity and having 70 % or more, preferably 80 % or more, and even more
• polypeptide exhibiting cellobiohydrolase activity is produced by secretion from a recombinant yeast or fungal expression host that is devoid of measurable ⁇ -glucosidase activity.
• the cellulase preparation is produced from an organism devoid of enzymes that exhibit any measurable ⁇ -glucosidase activity.
• an organism is used wherein at least one gene, more preferably all genes encoding for polypeptides exhibiting measurable ⁇ - glucosidase activity have been deleted or rendered
• Trichoderma reesei strain in which one or more genes encoding
• polypeptides exhibiting ⁇ -glucosidase activity have been deleted or rendered dysfunctional.
• a Trichoderma reesei strain is used in which at least the gene bgll (E.C. 3.2.1.21 ⁇ has been deleted or rendered dysfunctional .
• a recombinant cellulase or recombinant cellulases are used, which are heterologously expressed by an expression host which does not exhibit any detectable BGL activity.
• at least one gene encoding one of the following cellobiohydrolase enzymes is cloned by recombinant DNA techniques into an expression host : a
• glycosylhydrolase belonging to family 6 or family 7 of glycosylhydrolases (Henrissat et al . 1998, FEBS Lett.
• polypeptides exhibiting cellobiohydrolase activity that belong to one of the following: Cellobiohydrolase I, Cellobiohydrolase II. Even more preferred are the following polypeptides exhibiting
• Cellobiohydrolase activity CBHI from Trichoderma reesei ⁇ SEQ ID NO. 1), CBHII from Trichoderma reesei (SEQ ID No. 2), a polypeptide exhibiting cellobiohydrolase activity and having 70 % or more, preferably 80 % or more, and even more preferably 90 % or more identity with any of SEQ ID NO . 1 or SEQ ID NO. 2.
• the cellobiohydrolases are preferably produced by secretion from a recombinant yeast or fungal expression host that is devoid of measurable ⁇ -glucosidase activity.
• a non-limiting list of such organisms includes Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, Kluyveromyces lactis.
• the cellulase in another embodiment of the invention, the cellulase
• D-gluconolactone can be added to the reaction or can be produced in situ, from glucose by the enzyme glucose oxidase and oxygen. D-gluconolactone may decompose spontaneously and form gluconic acid (Guiseppi-Elie et al . , 2009, Journal of Molecular Catalysis B: Enzymatic, 58, 118-123) .
• D-gluconolactone is continuously formed in- situ and thus it is present during the complete time of reaction despite its possible
• the concentration of the formed by-product hydrogen peroxide, which has a damaging effect on glucose oxidase is minimized by immobilization of glucose oxidase on activated carbon, which has a strong catalytic action, for hydrogen peroxide decomposition. This has the additional advantage that the half amount of the consumed molecular oxygen is recovered.
• catalase or activated carbon is added to the reactor.
• Catalase or activated carbon is added to the reactor.
• activated carbon catalyzes the decay of hydrogen peroxide to water and molecular oxygen. For this reason, it is possible to produce the molecular oxygen also in-situ by feeding hydrogen peroxide to the hydrolysis reactor avoiding complicated ventilation with air or oxygen.
• the by-product glucose is removed by the use of glucose oxidase (GOX) from cellobiose produced by alternative enzymatic methods.
• GOX glucose oxidase
• Alternative methods are the enzymatic production of cellobiose from starch
• Other alternative methods utilize the transferase activity of the ⁇ -glucosidase at high concentrations of glucose (Murray P, Aro N, Collins C, Grassick A, Penttila. M, Saloheimo M, Tuohy M. , Protein Expr Purif. 2004 Dec ; 38 (2 ) : 248 -57 ) .
• the enzyme mixture comprising a polypeptide exhibiting
• cellulase activity can be of different bacterial, plant, yeast or fungal origin, e.g. from bacterial origin such as Bacillus sp., Streptomyces sp . , Pseudomonas sp., Escherichia sp., or from yeast origin, e.g. from Pichia sp . , Saccharomyces sp., Candida sp., Kluyveromyces sp., Yarrowia sp . ,
• Schizosaccharomyces sp. or from other fungal origin, e.g. from Acremonium sp., Aspergillus sp . , Aureobasidiurn sp . , Cryptococcus sp . , Filibasidium sp., Fusarium sp., Humicola sp . , Penecillium sp. , Talaromyces sp . , Thermoascus sp . , or Trichoderma sp .
• the enzyme preparation comprising a polypeptide exhibiting cellulase activity originates from a Trichoderma sp . , more specifically from Trichoderma reesei , Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiaturn or Trichoderma viride.
• Trichoderma sp Trichoderma sp .
• Trichoderma reesei Trichoderma harzianum
• Trichoderma koningii Trichoderma longibrachiaturn or Trichoderma viride.
• the cellulase preparation is either produced in its native host, e.g. originating from bacteria, plant, yeast or fungi, or the enzymes are expressed heterologously by a host organism.
• a host organism e.g. a bacterium, a yeast or a fungal strain, e.g. Escherichia coll, Bacillus subtilis, Bacillus brevis, Bacillus coagulans, Bacillus megaterium, Bacillus stearothermophilus, Streptomyces sp., Pseudomonas sp . , Pichia pastoris, Pichia angusta,
• Saccharomyces cerevisiae Kluyveromyces sp . , Hansenula sp . , Yarrowia sp., Trichoderma reesei, Aspergillus niger,
• Penicillium sp Aspergillus oryzae, or Penicillium sp .
• the dry mass of the respective biomass is determined
• the enzyme concentration is determined by measuring total protein content of the respective enzyme stock solution using the colorimetic Bradford (Bradford, M. M.
• the solid, cellulose containing substrate optionally after solid-liquid separation, is incubated with the cellulase preparation at one or more of the following conditions:
• the cellulosic material is incubated with the cellulase preparation for 0.1 - 24 hours at 10 - 80 °C and a pH between 3.0 and 9.0 and an enzyme-to-substrate ratio of 0.01 - 3 % w / w d.m. substrate.
• the protein content of the cellulase preparation and the dry matter content of the substrate are used to adjust the enzyme- to-substrate w/w ratio.
• the cellulose containing material is incubated with the cellulase preparation for 0.5 - 6 hours, at 40 - 60 °C at a pH between 4.5 and 5.5 with a dry matter content of 2 - 20 % and an enzyme-to-substrate ratio of 0.05 - 1 % w/w d.m substrate.
• the cellulose containing material is incubated with the cellulase preparation for 2 - 6 hours at 45 - 55 °C at a pH between 4.5 - 5.0 with a dry matter (d.m.) content of 7.5 - 15 % and an enzyme-to-substrate ratio of 0.05 - 0.1 % w/w d.m. substrate.
• the substrate is further treated before enzymatic hydrolysis.
• the aim of the treatment is to provide better availability of the cellulose in the subsequent hydrolysis step.
• the treatment can be a chemical, physical, or enzymatic processing, or a combination thereof.
• the treatment can be either of the following, an acid treatment, by addition of an inorganic or organic acid, a base treatment, by addition of an inorganic or organic base, application of temperature, or pressure, or application of an organosolv process, by addition of an organic solvent, the use of ionic liquids, or a
• hydrothermal pretreatment with or without application of pressure, or freeze-thaw cycles, or addition of any catalyst, homogeneous or heterogeneous, or addition of one or more enzymtic activities, or any combination of the above.
• the treatment combines physical and chemical (i.e. physicochemical ) processing.
• the substrate is heated in combination with acid treatment.
• sugar beet pulp is the substrate and is treated with acid at elevated temperature at high dry matter content.
• hydrolysis can be performed in various ways, e.g. as batch, repeated batch, continuous, semi -continuous , using a membrane based process, or in a counter-flow setup, or in any combination thereof.
• the dry matter content is between 7.5 and 50 % w/w. In a more preferred embodiment the dry matter content is between 10 and 25% w/w. In an even more preferred embodiment, the dry matter content is between 12 and 20% w/w.
• the cellulose containing substrate is treated with an acid in combination with temperature and pressure, whereas the acid can be any inorganic or organic acid, the temperature is within the range of 80 - 210 °C and the pressure is between 0.5 and 100 bar. In more preferred embodiment the temperature is between 95 and 135 °C.
• the cellulose containing substrate is treated with an acid in combination with
• the acid is either sulfuric acid (H2SO4) , sulfurous acid (H2SO3) , hydrochloric acid (HCl) , carbonic acid ⁇ H2CO3) or carbon dioxide (CO2 ⁇ , nitric acid
• HNO3 nitrous acid
• HNO2 nitrous acid
• the temperature is within the range of 80 - 210 °C and the applied pressure is within 0.5 - 100 bar. In a more preferred embodiment the temperature is between 95 and 135 °C.
• sulfuric acid is added to the substrate and the substrate is treated at a dry matter content between 20 and 25 % w/w at a temperature between 95 and 135 °C,
• the sulfuric acid dosage is preferably between 0.1 and 1% w/w (sulfuric acid/substrate) .
• the treatment renders part or all of the non-cellulose
• part or all of the soluble non-cellulose material is removed before the cellobiose- producing hydrolysis step.
• Non-cellulose material can comprise hemicellulose , pectin, hydrolysis products from hemicellulose and pectin, lignin, mineral salts, organic acids, alcohols, oligosaccharides, sucrose, and/or other soluble sugars.
• the present invention also covers a process for
• producing cellobiose comprising the following steps: a) subjecting a cellulose containing material to a
• the dry matter content of the solid phase obtained in a) is 7.5 to 25 % (w/w) , preferably 10 to 20% (w/w) , thereby providing a cellobiose containing liquid phase and a solid phase;
• the dry matter content during the treatment in step a) is between 7.5 and 50 % (w/w), preferably between 10 and 30 %.
• the non-cellulose material of sugar beet pulp comprises pectin (polygalacturonic acid) and hemicellulose (galactoarabinan) , comprising the monomers galacturonic acid, arabinose, galactose, rhamnose and/or xylose.
• pectin polygalacturonic acid
• hemicellulose galactoarabinan
• at least 80 % w/w of the pectin and hemicellulose fraction is renedered soluble by the described treatment whereas at least 80 % w/w of the cellulose remains in the solid phase.
• the portion of the substrate that is rendered soluble by the treatment step is removed by solid-liquid-separation.
• the solid-liquid separation can be performed by continuous or discontinuous centrifugation, filter pressing, or band
• pectinases and arabinases are added prior to the separation step.
• incubation with these enzymes is carried out for a duration of 10 min to 12 hours at a temperature between 20 and 60 °C.
• pectinases and arabinases are added prior to the separation step, and incubated for a duration of 10 min to 4 hours at temperatures between 30 and 50 °C.
• the pectinase and arabinase enzyme is produced by culturing one of the following microorganisms: Aspergillus niger, Penicillium foetidus .
• the liquid phase after separation is used as a source for value adding products. It contains polymers like pectin, arabinan, galactan,
• the polymers can be used in purified form or as a mixture (e.g. as thickener, surfactant, emulsifier, in feed or food applications) .
• the liquid phase contains monomeric sugars and sugar derivatives of the above mentioned polmers in various amounts (0-100% total sugar and sugar acid contents), e.g. galacturonic acid, galactose, arabinose, xylose, rhamnose, glucose and fructose.
• the monomeric sugars and sugar acids can be used directly, after purification as pure substances, or in various mixtures. They can be used as source for value adding products, after chemical or biochemical conversion to yield sugar acids (e.g. gluconic acid, galactonic acid, arabonic acid, xylonic acid, rhamnonic acid), sugar diacids (e.g. galactaric acid, glucaric acid, arabaric acid, xylaric acid, rhamnaric acid) , sugar alcohols (e.g. xylitol, arabitol, galactitol, rhamnitol) , furfural, hydroxymethylfurfual , furandicarboxylic acid and deriviatives thereof.
• the conversion step is done by enzymatic treatment, by homogeneous and/or by heterogeneous catalysis.
• the present invention relates to a process for producing cellobiose from a cellulose containing material, wherein the liquid phase is removed after the hydrolysis step.
• the cellobiose containing liquid phase obtained by the process above can be collected and concentrated.
• the cellobiose contained in the cellobiose containing liquid phase of step d) of the process above is subjected to a further step e ⁇ , which
• the cellobiose solution comprises: optionally concentrating the cellobiose solution, and further purifying the cellobiose from the (optionally concentrated) cellobiose solution, preferably to a purity of at least 50 %, more preferably of at least 60 %, more
• step e) comprises one or more of the following: cellobiose crystallization, chromatography, nanofiltration, precipitation by organic solvents, or the use of
• Concentration can by achieved by processes well known in the art, e.g. by evaporating water, by membrane techniques, or by removing water via extraction into another liquid phase.
• the water is removed by a membrane technique using ultra-, micro- and nano-filtration,
• This can be followed by a further concentration step using a thin film evaporizer, to concentrate the cellobiose from 10% ⁇ w/w) up to 40% (w/w) .
• the water is removed by a membrane technique using ultra-, micro- and nano-filtration, concentrating the cellobiose from a concentration between 0.1 % and 1.5 % (w/w) up to 10% (w/w), followed by a further concentration step, using a thin film evaporizer to
• the cellobiose containing liquid is concentrated by a thin film evaporizer
• the cellobiose containing liquid is concentrated by a thin film evaporizer
• the concentrated liquid contains 50 - 95 % w/w cellobiose (relative to the total amount of saccharides) , 1 - 40 % w/w glucose, and minor quantities of e.g. arabinose, xylose, galactose, rhamnose, galacturonic acid, fructose and other impurities .
• crystallization chromatography, nanofiltration, precipitation by organic solvents, or the use of microorganisms, whereby crystallization is preferred.
• the resulting liquid can have a brownish color. Therefore, optionally a decoloration step is added to the process which removes part or all of the colored substances. This is preferably done by adsorption of the impurities to an adsorber material, preferably to charcoal, ion exchange resins or bentonites. In a preferred embodiment, this is done by a chromatography step using an adsorption to an adsorption material which is or comprises bentonite.
• the present invention also comprises a process for purifying cellobiose from a composition comprising cellobiose and glucose, comprising a step of converting glucose to gluconic acid by glucose oxidation in the presence of glucose oxidase (GOX) and oxygen.
• a process for purifying cellobiose from a composition comprising cellobiose and glucose comprising a step of converting glucose to gluconic acid by glucose oxidation in the presence of glucose oxidase (GOX) and oxygen.
• GOX glucose oxidase
• the by-product glucose is removed from the cellobiose by the use of glucose oxidase (GOX) (and optionally catalase or activated carbon) to oxidize the glucose to gluconic acid.
• GOX glucose oxidase
• Catalase or activated carbon is added for the purpose to remove the product ⁇ 02 and to avoid unspecific oxidation of the product cellobiose.
• glucose in the liquid phase containing cellobiose obtained by the process above is converted to gluconic acid by glucose oxidation, preferably in the presence of glucose oxidase (GOX) and oxygen.
• glucose oxidase GOX
• the gluconic acid obtained by the process above is removed from the liquid phase containing cellobiose by one or several of the following: ion exchange chromatography, precipitation, electrodialysis , whereby ion exchange
• gluconic acid may be removed from the reaction mixture by ion exchange chromatography.
• the glucose present in the liquid phase may be converted to D-gluconolactone in situ.
• the D-gluconoiactone may then be used to inhibit the activity of any polypeptides exhibiting ⁇ -glucosidase activity.
• the process for obtaining cellobiose according to the present invention may be carried out in such a way, that the enzymatic activity of any polypeptides with ⁇ -glucosidase activity is inhibited by an inhibitor, preferably D-gluconolactone , whereby the D-gluconolactone is preferably generated in situ from glucose comprised in the liquid phase, according to the process for glucose oxidation described above.
• an inhibitor preferably D-gluconolactone
• immobilized, thermostable glucose oxidase, catalase or activated carbon and oxygen are used to oxidize glucose to gluconic acid, at 30 - 60 °C, and substrate concentrations of 5 - 50 g/1 glucose and other
• the gluconic acid is removed by standard ion exchange chromatography.
• the gluconic acid can be recovered from the ion exchange resin, e.g. by elution with an acid.
• gluconic acid is removed by precipitation, or by electrodialysis making use of an anion-selective membrane.
• microorganisms e.g. bacteria, yeast, fungi
• microorganisms e.g. bacteria, yeast, fungi
• the used microorganisms are yeasts (e.g. Saccharomyces cerevisiae, Klyveromyces sp.,
• Saccharomyces cerevisiae is used for this process step.
• Another option for purification of cellobiose involves selective crystallization of the cellobiose, in order to purify the product and to produce an easily storable product.
• the method for final preparation and storage of the product is not restricted to any particular one, and can involve
• the product can be used as amorphous powder, in a crystalline form or as a cellobiose syrup.
• the cellobiose obtained by the process above is further purified to a purity of more than 99% (w/w) .
• the cellobiose preparation obtainable by the process according to the present invention can be applied as food ingredient, animal feed additive, cosmetics ingredient.
• the cellobiose preparation can be used in an anti-inflammatory pharmaceutical preparation.
• the produced cellobiose can be used in many applications, e.g. as pre-biotic substance for feed and food applications, as a low caloric ingredient in food, as a building block in
• polymers in organic synthesis, as additive in polymers, as an active pharmaceutical ingredient in therapeutic applications, as building block in polymers, or in cosmetic applications.
• the cellobiose is used as a feed additive that improves the animal feed product .
• the feed can be used for feeding pigs, piglets, calves, cattle, and/or poultry such as chicken and turkey.
• the cellobiose is typically used in a concentration of 0.01 - 1 % w/w of cellobiose in the dry matter content of the animal feed composition.
• the cellobiose is used in a concentration of 0.05 - 0.5 % w/w of cellobiose. In a more preferred variant of this embodiment the cellobiose is used in a concentration of 0.05 - 0.2 % w/w of cellobiose.
• the combination product containing 50 - 90 % cellobiose (w/w) and 5 - 50 % gluconic acid (w/w) is used as an animal feed ingredient, with a concentration of 0.05 - 1.0 % w/w, more preferably 0.05 - 0.5 % w/w, and even more preferably 0.05 - 0.2 % w/w of cellobiose.
• the cellobiose preparation is added to an animal feed preparation at a dosage of 0.01 - 1% (w/w), preferably at a dosage of 0.05 - 0.2% (w/w) .
• the cellobiose preparation can have a prebiotic effect on the intestinal flora of the animal.
• the addition of cellobiose can improve the overall health status of the animal. Furthermore, the addition can lead to an increased feed uptake and/or weight gain.
• the animal feed is preferably used as a feed for pig, fish, cattle and/or
• poultry more preferably for pig and poultry.
• the cellobiose in another embodiment of the invention, the cellobiose
• preparation is used as a food ingredient.
• Applications in food comprise ingredients in baking, beverages, as well as
• sucrose dosage in the dough is reduced.
• sweeteners such as xylitol, maltitol, and/or stevia
• Such dough characteristics are one or more of the following: browning, aroma formation, mouth feel .
• the invention relates to the cellobiose preparation described above for use in a food composition providing a prebiotic effect to the food thereby- improving the overall health status of the person consuming the food, in particular by improving the intestinal flora.
• the cellobiose in another embodiment of the invention, the cellobiose
• This cellobiose composition is intended for use in an oral pharmaceutical composition effective in the treatment of inflammatory bowel diseases, such as Colitis ulcerosa, Crohn's disease, Collagenous colitis, Lymphocytic colitis, Ischaemic colitis, Diversion colitis and/or Behget ' s syndrome.
• inflammatory bowel diseases such as Colitis ulcerosa, Crohn's disease, Collagenous colitis, Lymphocytic colitis, Ischaemic colitis, Diversion colitis and/or Behget ' s syndrome.
• the gluconic acid obtainable by the process according to this invention may be applied, e.g. in the food or feed industry, as preservative, as cosmetic additive, in organic syntheses.
• the mixture comprising gluconic acid and cellobiose obtainable by the process according to this invention is used as a combination product in the feed industry, combining the beneficial effects of cellobiose with the benefits of an organic acid.
• the present invention comprises process for producing
• cellobiose which may be carried out comprising the following steps: a) subjecting a cellulose containing material to a
• the dry matter content of the solid phase obtained in b) is 7.5 to 25 % (w/w) , preferably 10 to 20% (w/w) , thereby providing a cellobiose containing liquid phase and a solid phase ;
• the treatment a) can be a chemical, physical, or enzymatic processing, or a combination thereof.
• the treatment renders part or all of the non-cellulose material in the substrate soluble. Thereby a portion of the non-cellulose material after the treatment is in the liquid phase whereas the majority of the cellulose is in the solid phase.
• the separation step b) can be carried out by any method suitable for solid-liquid separation, such as sieving, filtration etc.
• the enzymatic hydrolysis step c) can be carried out efficiently when the dry matter content of the solid phase obtained in b) is 7.5 to 25 % (w/w) , preferably 10 to 20% (w/w) .
• the cellulase treatment of step c) can be a treatment with a single type of polypeptide or an enzyme composition comprising at least one type of cellulase. Enzymes or enzyme compositions comprising Cellobiohydrolase activity are preferred.
• the enzyme or enzyme composition used in step c) may be obtained from a native or recombinant source.
• the enzyme or enzyme composition of step c) exhibits reduced or minimal ⁇ -glucosidase activity.
• Methods for obtaining such an enzyme (composition) comprise obtaining it from an organism that exhibits minimal ⁇ -glucosidase activity, inhibiting any measurable ⁇ -glucosidase activity by addition of inhibitors, such as such as D-gluconolactone , or removing any polypeptides exhibiting measurable ⁇ -glucosidase activity by passing the enzyme composition over an adsorption material selected from cellulose-, chitin- and chitosan- containing materials.
• any polypeptides exhibiting measurable ⁇ -glucosidase activity do not bind strongly to such materials and can therefore be eluted, while the desired enzyme (s) remain bound to the adsorption material.
• the adsorption material used in this step is the cellulose-containing material to be hydrolyzed. Especially preferred is that the adsorption material is the cellulose containing solid phase obtained in step a) below. Typically, the adsorption process is much quicker than the hydrolysis process .
• the cellobiose-containing liquid phase may be separated from the solid phase by any method suitable for solid-liquid separation, such as any type of filtration, preferably nano— ultra- or microfiltration .
• the cellobiose containing liquid phase may be substituted by fresh or recycled aqueous phase. It is possible to remove the cellobiose-containing liquid phase simultaneously to addition of new aqueous phase, but it is alternatively possible to carry out these steps
• Steps c) and d) of the process above may be carried out simultaneously. This may be done by continuously replacing the cellobiose containing liquid phase by fresh aqueous phase, such as using a membrane reactor configuration as described above.
• the present invention also relates to a process for removal of glucose from an aqueous phase comprising the use of glucose oxidase (GOX) (and optionally catalase or activated carbon ⁇ in the presence of oxygen to oxidize the glucose to D- gluconolactone .
• Catalase is necessary to remove the product H2O2 and to avoid unspecific oxidation of the product
• Gluconic acid originating from hydrolysis of D- gluconolactone may be removed from the reaction mixture by precipitation or ion exchange chromatography.
• D-gluconolactone may be formed in situ; the D-gluconolactone may then be used as an inhibitor of any polypeptides exhibiting measurable ⁇ - glucosidase activity, as described above.
• the product obtained by the process of this invention is also an inherent component of the invention.
• the product may be used for example as food additive, e.g. as pre-biotic food additive.
• the invention also relates to a product for use in the treatment of diseases such as inflammatory diseases.
• sugar beet biomass refers to the complete and unprocessed root tissue of Beta vulgaris including the outer peel and the internal pulp. Dry tissue typically contains 80% (w/w) soluble sucrose, while beet pulp is mainly composed of polymeric sugars and pectin including typically 7 % pectin (mainly polygalacturan) , typically 7 % cellulose and typically 7 % hemicellulose , mainly in the form of
• pectin rich biomass refers to the unprocessed or processed plant tissues of fruits and vegetables tissue, which are composed of polymeric sugars and pectin, including 1 - 50 % pectin, 1 - 80 % cellulose and 1 - 80 % hemicellulose.
• dry matter refers to the mass to biomass ratio determined after removal of water from fresh tissue, e.g. by using an IR-balance.
• polymeric substrate means substances composed of either a specific monomeric constituent or a limited variety of defined monomeric constituents covalently linked in a linear or partially branched molecular structure.
• An example is the insoluble fraction of raw lignocellulosic feedstocks, which can contain significant amounts of such polymeric substrates such as cellulose, xylan, mannan, galactan, arabinoxylan, glucoronoxylan, glucomannan, and xyloglucan.
• enzyme activity refers to the catalytic activity under appropriate conditions under which the enzyme serves as a protein catalyst.
• enzyme activity is the activity at which the enzyme converts specific polymeric or artificial substrates to specific oligomeric or monomeric products.
• cellulase refers to a polypeptide exhibiting cellulase activity.
• cellulase preparation refers to a cellulase or a composition comprising at least one polypeptide exhibiting cellulase activity.
• enzyme preparation is used synonymously with “cellulase preparation” .
• the cellulase preparation preferably comprises at least one polypeptide having cellobiohydrolase activity (EC 3.2.1.91, CBH) .
• cellobiohydrolase or "CBH” (E.C. 3.2.1.91) refers to an enzyme, which catalyses the hydrolysis of polymeric cellulose to cellobiose and other beta-D-glucose oligomers.
• ⁇ -glucosidase or "beta-glucosidase” or "BGL”
• E.C, 3.2.1.21 refers to an enzyme, which catalyses the hydrolysis of cellobiose, cellotriose, cellotetraose , and other beta-D-glucose oligomers to the corresponding glucose monomer .
• membrane reactor configuration refers either to the combination of a reactor and one or more external membrane modules which are connected to the reactor, or to a reactor with one or more submerged membrane modules.
• Example 3 Concentration of cellobiose in liquid phases at Different Dry Matter Factors
• Cellobiose degrading activities were removed according the procedure described in example 2.
• 50 mM acetate buffer pH 5.0 was added to the solid fractions to reach a dry matter content w/w of (i) 15, (ii) 7.5, and (iii) 2.5 %, respectively.
• the mixtures were incubated for 1 h at 50 °C followed by a phase separation step.
• the amount of glucose and cellobiose in the liquid phases was determined by HPLC as described in Example 2. Data for cellobiose concentrations in relation to the substrate dry matter content are shown in Fig. 2.
• Example 1 460 g pretreated sugar beet pulp (see Example 1) was contacted with (i) 75 mg, (ii) 37.5 mg, and (iii) 7.5 mg cellulase preparation from T. reesei for 5 min at room temperature.
• Example 2 460 g pretreated (see example 1) or untreated sugar beet pulp and Avicel were treated as shown in Example 2 to remove cellobiose degrading activities.
• Acetate buffer was added to the solid fraction to reach a dry matter content of 2.5% and the mixture was incubated for 1 h at 50 °C.
• the amount of glucose and cellobiose in the liquid phases was determined by HPLC as described in Example 2.
• Example 2 460 g pretreated sugar beet pulp (see Example 1) was treated as shown in Example 2.
• 50 mM acetate buffer pH 5.0 was added to the solid fraction to reach a dry matter content of 7.5 % w/w.
• the mixture was incubated for 1 h at 50 °C followed by a liquid/solid phase separation step. The separation of the liquid phase, addition of buffer to the same dry matter content followed by incubation were repeated six times.
• the amount of glucose and cellobiose were determined in the particular liquid fractions of each step by HPLC as described in Example 2. Glucose and Cellobiose production from sugar beet pulp is shown in Fig. 5.
• Example 7 GOX Substrate specificity
• the substrate specificity of glucose oxidase was assessed using the following assay: Each tested saccharide was
• Glucose oxidase was immobilized on glass beads coated with polyethylenimine and glutaraldehyde . 20 mg of these beads with an overall activity of 100 U were added to 100 mL of a 100 mM Na-acetate buffer containing 25 mM glucose, 150 mM cellobiose and 70,000 U catalase. The reaction was performed in a reactor which was continuously vented with air with a ventilation rate of 1 L/min. Aliquots were taken and the glucose concentration was quantified using HPLC. The data for the conversion of glucose to gluconic acid by glucose oxidase are shown in Fig. 7.
• Example 9 Separation of Glucose and Gluconic Acid by Ion Exchange Chromatography
• ultrafiltration membrane unit with a cut-off of 10.000 Dalton. Enzymes and water were added in order to adjust the dry matter content to 9% . The temperature was kept constant at
• transmembrane pressure was between 0.7 and 1.5 bar.
• the retentate was pumped back into the reactor, the permeate was collected.
• the permeate mass was continuously measured and its cellobiose content was analyzed by HPLC.
• water was added. After nine hours the hydrolysis was stopped by switching off the pump and by cooling the suspension to 4 °C.
• Fig. 1 Yield of Cellobiose and Glucose in % of Cellulose Substrate in the mixture after Removal of Cellobiose Degrading Enzymatic Activities in the Hydrolysis Step
• Fig. 2 Concentration of Cellobiose after Hydrolysis depending on the Dry Matter content
• Fig. 3 Cellobiose Production from Sugar Beet Pulp at
• Fig. 4 Production of Cellobiose from different Cellulosic Materials
• Fig. 5 Yield of Cellobiose after Hydrolysis of Sugar Beet Pulp
• Fig. 7 Decrease of Glucose Concentration due to the Activity of Glucose Oxidase
• Fig. 8 Separation of Gluconic acid from Cellobiose by Ion Exchange Chromatography

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