The present invention relates to the provision of xcex1-glucuronidase in pure, isolated form or as a recombinantly produced enzyme and the use;hereof as a xylan side-group hydrolysing enzyme, in particular in food manufacturing, as a feed additive or in processing of cellulosic pulps.
Biodegradation of hemicellulose requires accessory enzyme activities that remove non-xylose substituents, in particular glucuronic acid substituents, from the xylan backbone in addition to endoxylanases and xcex2-xylosidases. The content of glucuronic acid substituents present in xylans varies with the sources of xylan such as hardwood xylans, softwood xylans and cereal xylans.
The configuration of hardwood xylan is presented in FIG. 1. The backbone of hardwood xylan consists of (1,4)-xcex2-D-linked xylopyranose residues. About every tenth xylose unit carries a single, terminal side chain consisting of 4-O-methylglucuronic acid attached directly to the 2-position of xylose. Seven out of 10 xylose residues contain an O-acetyl group at C-2, at C-3, or at both positions (Timell, 1967). Additionally, hardwood xylans contain minor amounts of rhamnose and galacturonic acid residues (Puls and Schuseil, 1993). O-acetyl-4-O-methyl-glucurono-xylan comprise between 10-35% of hardwoods (Timell, 1967).
Softwood xylans consist of a backbone of xcex2-1,4-linked D-xylopyranose residues. There is one 4-O-methylglucuronosyl residue attached to C-2 per 5-6 xylose units. Instead of acetyl-substituents as found in hardwoods, softwood xylans contain alfa-L-arabinofuranose residues directly linked to C-3 of the xylose. One, arabi-nose occurs per eight or nine xylose units, see FIG. 2 (Timell, 1967). Arabino-4-O-methyl-glucuronoxylan amounts to 10-15% of the softwood hemicelluloses (Puls and Schuseil, 1993; Timell, 1967).
Cereal xylans, which are commonly referred to as arabinoxylans consist of a linear backbone of (1,4)-xcex2-D-linked xylopyranose units to which alfa-L-arabinofuranosyl substituents are attached at O-2 or O-3 or at both of these positions, FIG. 3 (Voragen et al., 1992). There are two main types of arabinoxylans in annual plants. In the endosperms there is a highly branched form which basically is devoid of glucuronic acid. In the more lignified tissues, much less branched forms substituted with additional glucuronic acid residues and/or 4-O-methylether thereof as well as galactose units are found. Glucuronic acid residues are linked to O-2 on the xylan backbone (Viikari et al., 1993)
The amount of glucuronic acid in arabinoxylan varies considerably between cereals of different origins and between fractions of individual cereals. Glucuronoarabinoxylans are found in rice and sorghum and in the bran fraction of wheat, rye and barley and in a number of lignocellulosic by-products like e.g. sunflower meal. There are alsoacetyl groups present in cereal arabinoxylans, particularly in sorghum. They are ester-linked to O-2, O-3 or O-2,3 of xylose. In addition, phenolic acids like ferulic and coumaric acid have been found ester bound to the arabinofuranosyl residues of arabinoxylans (Voragen et al., 1992).
There is currently a considerable industrial interest in the enzymatic hydrolysis of xylans either with the aims of having complete hydrolysis to obtain xylose or with the objective of obtaining enzymatic pulping and bleaching in paper manufacturing.
xcex1-Glucuronidases are produced naturally by several micro-organisms, and up till now isolation of the enzyme from 7 different sources has been described. These source organisms are: Trichoderma reesei Rut C-30 (Siika-aho et al., 1994), Trichoderma viride (Ishihara et al., 1990), Aspergillus niger (Uchida et al., 1992b), Thermoascus aurantiacus (Khandke et al., 1989), Agaricus bisporus (Korte, 1990), Thermoanaerobacterium sp. strain JW/SL-YS485 (Shao et al., 1995) and Helix pomatia (snail) (Kawabata et al., 1995).
The xcex1-glucuronidases isolated from A.niger and Helix pomatia are intracellular enzymes whereas the others are extracellular enzymes. A molecular weight above 90 kDa are common for these isolated xcex1-glucuronidases. They all have an acidic isoelectric point and pH optimum. The fungal xcex1-glucuronidases, except the Agaricus enzyme are single polypeptide chains, while the bacterial xcex1-glucuronidase, the Agaricus and the snail enzyme are dimers. Isoenzymes have only been found in Aspergillus niger (Uchida et al., 1992b).
In general, the specific activity of xcex1-glucuronidase decreases with increasing chain length of the substrate. Only the Thermoascus aurantiacus enzyme has any activity on polymeric xylan. The xcex1-glucuronidase from Helix pomatia is the only isolated enzyme which had activity towards p-nitro-phenyl-xcex1-D-glucuronide.
Several reports have shown a synergistic effect between xylanase, xcex2-xylosidase and xcex1-glucuronidase in the breakdown of glucuronoarabinoxylans (Siika-aho et al., 1994). xcex1-Glucuronidase therefore has potential application in the total hydrolysis of hemicellulose to produce xylose (Uchida et al., 1992a).
In WO 93/11296 is disclosed a method for the enzymatic treatment of cellulosic pulps where an xcex1-glucuronidase preparation is used to remove metal ions (bound through the carboxylic groups) from the pulp. This improves the bleaching properties of the pulp. Through removal of the glucuronic acid groups the pulp is also-rendered more susceptible to xylanase treatment.
WO 94/21785 discloses xylanases which are useful in food production and which may enhance the utilization of animal feed. However, there is no specific teaching of the use of an xcex1-glucuronidase for such purposes.
Thus, the prior art is silent with respect to improvements which can be obtained specifically by use of xcex1-glucuronidases in the manufacturing of food products including bakery products, and as an additive to enhance the utilization and digestibility of animal feeds. However, as it is described herein, a highly advantageous effect of xcex1-glucuronidase was demonstrated in animal feed ingredients with respect to improving the nutritional value of animal feedstuffs e.g containing annual plants which will result in an enhanced utilization of feed and accordingly of conversion rate for such feedstuffs. Furthermore, nutritionally and quality improving effects of xcex1-glucuronidase alone or in combination with other enzyme activities in dietary fiber-containing food products, such as doughs and finished bakery products including in particular bakery products based on wholemeal, were demonstrated.
Whereas enzyme preparations having xcex1-glucuronidase activity presently can be obtained from microorganisms, in particular fungal species, which produce the enzyme naturally, such preparations are crude in the sense that they contain several other enzyme activities such as cellulase or protease activity. Such impure enzyme preparations are industrially less feasible, since the associated non-xcex1-glucuronidase enzyme activities may be undesirable in several application areas and since it is difficult or impossible to provide enzyme products having a well-defined and standardized xcex1-glucuronidase activity.
Evidently, enzyme preparations only having, or substantially only having xcex1-glucuronidase activity may be provided by extensive purification from media in which xcex1-glucuronidase producing organisms have been cultivated (when the enzyme is secreted out of the cell) or by isolating the enzyme from cells producing the enzyme naturally (when the enzyme is accumulated intracellularly). For specific application areas such as in the food industry such purified, naturally produced xcex1-glucuronidase preparations may be useful.
However, the provision of xcex1-glucuronidase in this manner in industrially needed amounts and at a feasible cost level is not possible due to the low amount of the enzyme being produced naturally in the above source organisms.
It is therefore an important aspect of the present invention to provide the means for producing xcex1-glucuronidase using recombinant DNA technology. Not only will it be possible to provide such recombinantly produced enzyme at a lower cost level, but it will also be possible to obtain xcex1-glucuronidase preparations which do not contain other, undesirable enzyme activities.
Accordingly, the invention relates in a first aspect to an isolated DNA fragment coding for an enzyme having xcex1-glucuronidase activity, wherein the isolated DNA fragment is obtainable from an Aspergillus species and in a further aspect the invention pertains to a recombinant vector into which such a DNA fragment has been inserted and to a host cell transformed with the vector according to invention.
In a still further aspect there is provided a method of producing xcex1-glucuronidase, the method comprising cultivating the above host cell under conditions where the xcex1-glucuronidase is expressed, and harvesting the enzyme from the cells and/or the cultivation medium.
It is also an objective of the invention to provide a method of improving a dough and/or a baked product comprising adding to the dough an amount of xcex1-glucuronidase which increases the specific volume of the baked product as determined by the rape seed displacement method by at least 5% such as at least 10% and a method of enhancing the nutritional value of an animal feed comprising xylans, the method comprising adding to the feed xcex1-glucuronidase in an amount which results in an increase of the amount of dialysable metal ions in the feed of at least 5%.
In other aspects the invention relates to an isolated, substantially pure enzyme having xcex1-glucuronidase activity, characterised in that it in its glycosylated form has a molecular weight which is about 116 kDa, it has its maximum activity at about 60xc2x0 C., it shows an isoelectric point of about 5.2 and its pH of optimal activity is in the range of 4.5 to 6, preferably about 5, the enzyme comprising at least one of the amino acid sequences:
(i) EDGYDGWLRYAPVHRDLH (SEQ ID NO:1)
(ii) XDGYDGWLRYAPVSCDLHCRQALPSHIVLLXSTK (SEQ ID NO:2)
(iii) AGFQSILSTXLTSHPFQXDSSASILVATLDXYRQK (SEQ ID NO: 3)
(iv) IXGEADGVEPAPVDYVV (SEQ ID NO: 4)
(v) APSGVYDIGVNYYDLYGGQSK (SEQ ID NO: 5)
(vi) YGPIDFQVREPTSPLFANLYQTNTAIELEVSQEYLGQQCHF (SEQ ID NO:6)
(vii) WTLSVGDK (SEQ ID NO:7)
(viii) TVLDFDLRVDHKPSMVRDIISGQRFXRTLGGWAAVVNVGTXR (SEQ ID NO:8)
where X can be any amino acid, and to a method of treating a cellulosic pulp which comprises that the pulp is contacted with the above enzyme under conditions where at least part of the glucuronic acid groups of the pulp is removed.
xcex1-Glucuronidase is, as it is mentioned above, produced naturally by several microorganisms, in particular by a number of fungal species. Thus, a DNA fragment coding for the enzyme can e.g. be derived from a Trichoderma species such as T. reesei, an Aspergillus species including A. niger, A. tubigensis (this species is also sometimes referred to as Aspergillus tubigensis, A. awamori, A. terreus, A. oryzae, an Thermoascus species, e.g. T. auranticus or an Agaricus species such as A. bisporus. Other potential fungal sources of DNA sequences coding for xcex1-glucuronidase include a Schizophyllum species, an Aureobasidium species, a Phanerochaete species, a Fusarium species, a Penicillium species, a Curvularia species, a Tyromyces species, a Cryptonectria species and a Myceliophtora species.
xcex1-Glucuronidase encoding DNA fragments may also be derived from certain bacterial species such as a Thermoanaerobacterium species, a Streptomyces species or a Bacillus species. Additionally, xcex1-glucuronidase encoding DNA sequences may be derived from an animal species such as a snail.
Among the above mentioned source organisms Aspergillus niger and Aspergillus tubigensis are genetically and physiologically very closely related and morphologically indistinguishable species which differ only in very few phenotype traits.
In a specific embodiment, the DNA fragment coding for xcex1-glucuronidase codes for an enzyme comprising at least one of the amino acid sequences:
(i) EDGYDGWLRYAPVHRDLH (SEQ ID NO:1)
(ii) XDGYDGWLRYAPVSCDLHCRQALPSHIVLLXSTK (SEQ ID NO:2)
(iii) AGFQSILSTXLTSHPFQXDSSASILVATLDXYRQK (SEQ ID NO:3)
(iv) IXGEADGVEPAPVDYVV (SEQ ID NO:4)
(v) APSGVYDIGVNYYDLYGGQSK (SEQ ID NO: 5)
(vi) YGPIDFQVREPTSPLFANLYQTNTAIELEVSQEYLGQQCH (SEQ ID NO:6)
(vii) WTLSVGDK (SEQ ID NO:7)
(viii) TVLDFDLRVDHKPSMVRDIISGQRFXRTLGGWAAVVNVGTXR (SEQ ID NO:8)
where X can be any amino acid.
An xcex1-glucuronidase which comprises at least one of the specifically mentioned amino acid sequences may be derived as a native enzyme from a natural source for the enzyme. However, such an enzyme may also in accordance with the invention be provided by means of recombinant DNA technology and thus be produced by a recombinant homologous species or a heterologous species. It has been found that the enzyme as produced naturally may be glycosylated. However, it is envisaged that a heterologous xcex1-glucuronidase producing species may produce the enzyme in a non-glycosylated form. It may be advantageous to provide the enzyme in the non-glycosylated form, since, as it is demonstrated herein, such a form may have a higher activity than the natively glycosylated form. A non-glycosylated form may be provided either by selecting a production microorganism which do not glycosylate the enzyme or by subjecting a glycosylated enzyme to a chemical or enzymatic treatment whereby bound sugars are removed.
In accordance with the invention, xcex1-glucuronidase may be provided as an isolated, substantially pure enzyme preparation, although it may also, for certain industrial purposes, be used as a crude preparation such as the cultivation medium in which the xcex1-glucuronidase producing organism has been cultivated. The purification of xcex1-glucuronidase can be carried out according to any known method for protein purification including the methods which are described in the following examples.
The xcex1-glucuronidase according to the invention may advantageously be-provided as a composition comprising the enzyme and at least one further component. In particular it may be useful to incorporate into such a composition at least one further enzyme, the type of which will depend on the intended use of the composition. Thus, additional enzyme components may be selected from an other hemicellulase such as a xylanase or a xcex2-xylosidase, a cellulase, a lipase, a starch degrading enzyme, a protease and an oxidoreductase. In this context, useful starch degrading enzymes include xcex1-1,4 exo-glucanases such as amyloglucosidases or xcex2-amylases, and xcex1-1,6-endoglucanases including pullulanases and isoamylases, and xcex1-amylase. Useful oxidoreductases include hexose oxidase and glucose oxidase.
With respect to combinations of xcex1-glucuronidase and a xylanyase, it was found that the effect of such combinations depend on the type of xylanase. Xylanases have different substrate affinities such that some types have a preference for water insoluble pentosans (WIP) and others for water soluble pentosans (WSP). As it is described in the following, it may for certain applications, such as in doughs for bread products be advantageous to combine the xcex1-glucuronidase with a xylanase having WIP substrate preference, whereas for other applications including the production of dry cereal bakery products such as crackers, crispbread and biscuits a combination with a xylanase having WSP substrate preference may be preferred.
In specific embodiments of the invention the DNA fragment is derived from an Aspergillus species including as examples Aspergillus niger, Aspergillus tubigensis and Aspergillus awamori. Specifically, the DNA fragment may comprise the coding sequence as shown in FIG. 10 attached hereto ora derivative or a mutant hereof. In the present context, xe2x80x9cderivative or mutantxe2x80x9d encompasses any modification of the fragment which implies that the coding sequence when expressed codes for an enzyme having xcex1-glucuronidase activity. Thus, the term includes homologous DNA sequences, the term xe2x80x9chomologousxe2x80x9d being intended to indicate a DNA sequence which hybridizes to the sequence of FIG. 10 under specified conditions such as e.g. the conditions described in the following Example 5 or. by presoaking in 5xc3x97SSC and prehybridizing for 1 h at about 40xc2x0 C. in solution of 5xc3x97SSC, 5xc3x97Denhardt""s solution, 50 mM sodium phosphate, pH 6.8 and 50 xcexcg of denatured sonicated calf thymus DNA, followed by hybridization in the same solution supplemented with 50 xcexcCi 32-P-dCTP labelled probe for 18 h at about 40xc2x0 C. followed by washing 3 times in 2xc3x97SSC, 0.2 SDS, at 40xc2x0 C. for 30 minutes.
More specifically, the term is intended to refer to a DNA sequence which is at least 70% homologous to the sequence of FIG. 10, such as at least 75%, at least 80%, at least 85%, at least 90% or event at least 95% homologous. The term is intended to include modifications of the above sequence such as nucleotide substitutions which do not give rise to another amino acid sequence of the xcex1-glucuronidase or nucleotide substitutions which give rise to a different amino acid sequence and therefore, possibly, a different protein structure which might give rise to an xcex1-glucuronidase mutant with different properties than the wild-type enzyme. Other examples of possible modifications are insertion of one or more nucleotides into the sequence, addition of one or more nucleotides at either end of the sequence, or deletions of one or more nucleotides at either end or within the sequence.
One specific example of a DNA fragment according to the invention is the fragment contained in lambda phage which is deposited under the accession No. NCIMB 40801.
In accordance with the invention there is in an important aspect hereof provided a method of producing xcex1-glucuronidase using a host cell which its transformed with a recombinant vector into which a DNA fragment comprising a sequence coding for xcex1-glucuronidase is inserted. Such a sequence can be derived from any cell naturally producing xcex1-glucuronidase including the above mentioned source organisms.
In the present context, the term xe2x80x9chost cellxe2x80x9d is used to designate any cell which is transformable with a gene coding for xcex1-glucuronidase. Thus, a useful transformable cell may be selected from a fungal species, including a yeast cell, a bacterial cell, a plant cell and an animal cell, the term xe2x80x9ctransformablexe2x80x9d being used to describe the ability of a cell to receive DNA by means of any conventional technique whereby a DNA sequence can be inserted into a cell in a manner allowing the gene to be expressed in the cell. Thus xe2x80x9ctransformationxe2x80x9d in this context encompasses introduction of DNA into a cell by conventional transformation methods, insertion by means of transposable elements or recombination. It will be understood that the DNA which is inserted into a cell may in addition to the sequence coding for xcex1-glucuronidase also comprise sequences regulating the expression of the gene including promoter sequences and initiation and stop sequences and sequences coding for a signal peptide. Specifically, the promoter regulating the transcription of the the coding sequence may be a native promoter for the sequence or it can be a promoter not naturally associated with the coding sequence, i.e. a foreign promoter. The promoter sequence may be inserted on replicon carrying the coding sequence or it may be inserted into a different replicon in such a manner that it is operatatively associated wiht the xcex1-glucuronidase encoding sequence. Advantageously, the promoter may be a promoter which is regulatable by substances in the growth medium or by physical conditions such as temperature.
A plant which is transformed with a gene coding for xcex1-glucuronidase is also contemplated.
The coding sequence and regulatory sequences may be inserted into a chromosome of the transformable recipient cell or may be introduced by means of extrachromosomal replicons such as plasmids. The gene may-be under the control of sequences already present in the cell being transformed or sequences which is introduced with the coding sequence.
The choice of host cell to be transformed will depend on the capacity of a given species or strain to express the gene effectively. In particular it may be advantageous to select a cell which excretes the enzyme out of the cell, i.e. produce the enzyme extracellularly. This evidently reduces the downstream processing considerably, since the enzyme may be harvested directly from the cultivation medium. However, it may be required to use a production strain in which all or part of the enzyme is accumulated intracellularly. In this case the harvesting of the enzyme includes steps whereby the enzyme is isolated from the cells.
In accordance with the invention there is also provided a recombinant host cell which is transformed with a gene coding for xcex1-glucuronidase and the host cell is capable of expressing said gene.
In useful embodiments the microorganism which is transformed with a gene coding for xcex1-glucuronidase is a fungal species selected from a yeast species, a Trichoderma species, an Aspergillus species, a Thermoascus species, an Agaricus species or a bacterial species including a Thermoanaerobacterium species.
Many food products comprise vegetable components which has a content of hemicellulose. The hemicellulose forms part of what is generally referred to as dietary fiber or non-starch polysaccharides. Dietary fiber is generally not degraded by the human or animal digestive enzymes, but the fiber may be at least partially degraded by the intestinal microbial flora. It is, based on the known enzymatic activities of xcex1-glucuronidase, contemplated that the nutritional value of food products can be enhanced by the addition of this enzyme to the products or components hereof, or by subjecting dietary fiber-containing food ingredients to a treatment with the enzyme according to the invention. Thus, in the context of food manufacturing the enzyme may be applied in the manufacturing process or as a food additive. A particularly useful aspect of such uses is the ability of xcex1-glucuronidase to release from the hemicellulose component glucuronic acid groups to which are bound metal ions such as alkaline or earth alkaline metals. When bound to hemicellulose such ions are not bioavailable, but when the metal-glucuronic acid groups are released the metals are in a form where they can be absorbed from the intestinal mucosa. It has been found that the. addition of xcex1-glucuronidase to food or feed may result in an increase of the content of metal ions in a dialysable form which is at least 5%, such as at least 10%, preferably at least 15% or even at least 20%.
In accordance with the invention there is provided a method of producing a food product comprising adding to a food product mixture an xcex1-glucuronic acid hydrolysing effective amount of the enzyme according to the invention. It will be understood that such a method can be applied to any food product having a content of substrate for the enzyme and in which it is desirable to obtain at least a partial degradation of the hemicellulose to improve the nutritional or sensory quality of the food product or to improve the manufacturing process.
In particular, the above method will be useful in the manufacturing of cereal-based food products having a high content of hemicellulose including as examples breakfast cereals and bakery products such as bread products e.g bakery products based on wholemeal flour. In the manufacturing of a bakery product the enzyme is conveniently added to the dough or to a component of the dough.
As mentioned above, a particularly advantageous effect of adding xcex1-glucuronidase to a dough is that the quality of the finished baked product is enhanced e.g. with respect to bread volume which can be increased by at least 5%, preferably 10%, more preferably by at least 15% such as at least 20% by adding an effective amount of the enzyme. Such an effect can be obtained by the addition of 1-100 units of xcex1-glucuronidase per 1 kg of flour, such as 1-50 units including 10-30 units.
For the above application in food products or in food manufacturing xcex1-glucuronidase may be used as such or in the form of a composition comprising further components which are useful in the particular use. In particular it may be advantageous to use compositions comprising one or more further enzymes which in a particular food product has advantageous effects.
An interesting aspect of the present invention is the use of xcex1-glucuronidase as a means of enhancing the nutritional value of an animal feed such as xe2x80x9cby-productsxe2x80x9d from production of refined products. Thus, large quantities of by-products of plant origin are produced by the agricultural industry e.g. those parts of crops that remain after removal of value giving components. Examples of such by-products include beet pulp, straws, cereal brans or oil seed meals (Dxc3xcsterhxc3x6ft, 1993). These residues still contain considerable amounts of energy, but are often rather indigestible for both animals and humans.
Enzymatic treatment of feedstuffs including treatment with xcex1-glucuronidase may improve their nutritional value by different mechanisms: by degradation of cell walls resulting in improved accessibility or release of intracellular nutrients, by direct utilisation of hydrolysis products of polysaccharides, by elimination of anti-nutritional factors, and by affecting physiological effects exerted by non-starch polysaccharides or by their degradation products (Dxc3xcsterhxc3x6ft, 1993). xcex1-Glucuronidase can improve the in vitro digestibility of hemicellulose-containing vegetable material e.g. wheat and corn cob flour as it is demonstrated in the following examples. A specific effect obtained by adding xcex1-glucuronidase to animal feed is a reduction in residual dry matter after digestion of the feed with digestive enzymes in the presence of xcex1-glucuronidase. Preferably, such a reduction is at least 10%, more preferably at least 15% or even at least 20%.
The application of xcex1-glucuronidase to improve the nutritional value of feed components can be in the form of a preparation containing only this enzyme, but it may also be applied in the form of a composition according to the invention. Thus, it may be advantageous to combine xcex1-glucuronidase with one or more enzymes which degrade, hemicellulose such as a xylanase and/or xcex2-xylosidase. As it demonstrated in the examples, synergistic effects with respect to degrading hemicellulose can be achieved by such enzyme combinations.
It will be understood that use of xcex1-glucuronidase to enhance the digestibility or nutritional value of feed can be in the feed manufacturing process e.g. by pre-treating one or more components of the feed with the enzyme or with the composition containing the enzyme or, alternatively, the enzyme or the enzyme-containing composition can be incorporated in the feed as an additive which will enhance the utilization of the feed in the digestive tract of the animal including the effect that metal ions bound to glucuronic acid is brought into a dialysable and absorbable form. This latter effect implies that the manure from the animals will have a lower content of metals.
A particular application of xcex1-glucuronidase which is contemplated is the addition of the enzyme to feed crops such as grass or corn which are subjected to an ensiling process. For such an application it may be advantageous to combine the enzyme according to the invention with other silage additives such as other enzymes, or microbial inoculants.
The xcex1-glucuronidase may also be used for degradation of plant material used for other purposes than as a feed component. Thus, it may in certain industries be desirable to use the enzyme as an auxiliary agent in the processing of plant materials e.g. as a means of facilitating purification or extraction of specific components or in order to alter the water binding capacity of a plant material. The use of xcex1-glucuronidase to enhance the degradability in waste water plants is also contemplated.
The invention also relates to a method of treating a cellulosic pulp as it is mentioned above. The objectives of such a treatment may be several such as delignification during the pulping, enzyme aided bleaching, improved drainage of pulp from recycled paper and production of dissolved pulps (for rayon, viscose etc.). The term xe2x80x9cdissolving pulpxe2x80x9d refers to a product used for manufacturing of rayon, cellophane, carboxymethyl cellulose, plastics, lacquers, and other cellulose derivatives (Christov and Prior, 1993). E.g. in the production of dissolving pulps for viscose rayon manufacturing it is necessary to remove the xylan selectively (Roberts et al. 1990). In this manufacturing a xe2x80x9ccleanxe2x80x9d cellulose fiber is required and to provide such a raw material a hemicellulose-containing starting materials is advantageously treated with the enzyme or the composition according to the present invention.
Use of recycled paper as raw material in paper manufacturing necessitates de-inking of the recycled paper. Accordingly, the xcex1-glucuronidase according to the invention may be used as a de-inking agent
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