MXPA97009544A - Hexosa recombinant oxidase, a method to produce the same and the use of tal enz - Google Patents

Abstract

A method for producing hexose oxidase by recombinant DNA technology, recombinant hexose oxidase and the use of such an enzyme, in particular in the manufacture of food products such as dough and dairy products, animal husbandry, pharmaceuticals, cosmetics, dental care products and in the manufacture of lactones. Suitable sources of DNA encoding the enzyme are seaweed species including Chondrus crispus, Iridophycus flaccidum and Euthora cristata. In the useful modalities, the recombinant hexose oxidase is produced by Pichia pastoris, Saccharomyces cerevisiae or E. coli.

Description

HEXOSA RECOMBINANT OXIDASE, A METHOD TO PRODUCE THE SAME AND THE USE OF SUCH ENZYME FIELD OF THE INVENTION The invention provides a method for producing hexose oxidase by recombinant DNA technology and the enzyme produced by the method and its use in the food industry and other fields.

PREVIOUS TECHNICAL AND TECHNICAL BACKGROUND The hexose oxidase (D-hexose: 02-oxidoreductase, EC 1.1.3.5) is an enzyme that in the presence of oxygen is able to oxidize the D-glucose and several other reducing sugars including maltose, lactose and cellobiose to their corresponding lactones with hydrolysis subsequent to the respective aldobionic acids. Accordingly, hexose oxidase differs from another oxidoreductase, glucose oxidase which can only convert D-glucose, in that this enzyme can use a wider range of sugar substrates. Oxidation catalyzed by hexose oxidase can be illustrated, e.g. , as follows: D-glucose + 02"^ d-D-gluconolactone + H02, D-galactose + 02 - > d-D-galactogalactone + H202 REF: 26330 Hitherto hexose oxidase (hereinafter referred to as HOX) has been provided by isolating the enzyme from several species of red algae such as Iridophycus flaccidum (Bean and Hassid, 1956) and Chondrus crispus (Sullivan et al., 1973). Additionally, the Euthora cristata algae species has been shown to produce hexose oxidase. It has been reported that hexose oxidase isolated from these natural sources can potentially be used in the preparation of certain food products. Thus, hexose oxidase isolated from Iridophycus flaccidum has been shown to be capable of converting lactose into milk with the production of the corresponding aldobionic acid and has proven to be of potential interest as an acidifying agent in milk, e.g. , to replace the acidifying microbial cultures for such purpose (Rand, 1972). In this regard, hexose oxidase has been mentioned as a more interesting enzyme than glucose oxidase, since the latter enzyme can only be used in milk or in food products that do not contain glucose with the concomitant addition of glucose or, in the case of a dairy product, the lactase enzyme that degrades lactose, whereby lactose is degraded to glucose and galactose. Even if glucose can be obtained in this manner as a substrate for glucose oxidase, it is evident that only 50% of the final lactase products can be used as a substrate by glucose oxidase, and therefore glucose oxidase is not an acidifying agent. efficient in natural milk or dairy products. The ability of oxygen oxidoreductases, including that of hexose oxidase to generate hydrogen peroxide, which has an antimicrobial effect, has been used to improve the storage stability of certain food products including cheese, butter and fruit juice as disclosed in JP-B-73/016612. It has also been suggested that oxidoreductases may be potentially useful as oxygen scavengers or antioxidants in food products. The use of oxidizing agents, such as, for example, in the bakery and mill industries, is known. , iodates, peroxides, ascorbic acid, K-bromate or azodicarbonamide to improve the baking performance of the flour to achieve a dough with improved drawability and which therefore has a suitable strength and stability. The mechanism behind this effect of the oxidizing agents is that the flour proteins, such as eg. , the gluten in wheat flour, contain thiol groups, which, when oxidized, form disulfide bonds so the protein forms a more stable matrix resulting in better dough quality and improvements in volume and structure. the crumb of baked goods.

However, such use of several of the oxidizing agents that can be obtained ordinarily is objected by consumers or is not allowed by the competent bodies and consequently, attempts have been made to find alternatives to these conventional additives for flour and dough, and the prior art has suggested the use of glucose oxidase for the purpose indicated above. Thus, US 2,783,150 discloses the addition of glucose oxidase to the flour to improve the rheological characteristics of the dough. CA 2,012,723 discloses the use of bread improving agents comprising cellulolytic enzymes and glucose oxidase and JP-A-. 084848 suggests the use of a bread improver composition comprising glucose oxidase and lipase. However, the use of glucose oxidase as a dough and bread improver additive has the limitation that this enzyme requires the presence of glucose as a substrate to be effective in a dough system and generally, the glucose content in the flours of Cereal is low. Thus, in the wheat flour the glucose is present in an amount that is in the range of 0-0.4% w / w, that is, the flours may not contain glucose at all. Therefore, the absence or low content of glucose in masses will be a limiting factor for the use of glucose oxidase as a mass-improving agent. In contrast, the maltose content is significantly higher in the freshly prepared dough and additional maltose is formed in the dough due to the activity of the β-amylase which is either inherently present in the flour or is added. The current source of hexose oxidase are partially purified or crude enzyme preparations isolated by extraction of the naturally occurring seaweed species indicated above. However, since the amount of hexose oxidase in the algae is low, it is evident that an enzyme production in this manner is too tedious and expensive to ensure effective commercial production with respect to the cost of the enzyme from these natural sources. In addition, the provision of a sufficiently pure enzyme product at an effective level with respect to cost can not easily be achieved in this way. There is therefore a considerable industrial need to provide an alternative and more effective source with respect to the costs of this industrially valuable enzyme without relying on a natural source and also to provide the enzyme in a pure form, ie without contaminating activities of the enzyme or any other unwanted contaminants including unwanted algae pigments and environmental contaminants that may be present in the marine areas where the algae species that produce the hexose oxidase grow.

In addition, the industrial availability of a food-grade grade of hexose oxidase in sufficient quantities and at cost effective prices will undoubtedly lead to new applications of this enzyme not only in the food industry, but also in other industrial areas as will be discussed then. An example of a new application of hexose oxidase in the food industry is the use thereof as a dough enhancing agent, another example being the use of a polypeptide with hexose oxidase activity or a recombinant organism that produces the polypeptide in the manufacture of lactones. The present invention made it possible for the first time, using recombinant DNA technology, to provide polypeptides with hexose oxidase activity in industrially appropriate amounts and at a level of quality and purity that make the polypeptide with hexose oxidase activity according to the invention highly suitable for any purpose relevant industry including the preparation of food and pharmaceutical products. Accordingly, the invention relates in a first aspect to a method for producing a polypeptide having hexose oxidase activity, which comprises isolating or synthesizing a DNA fragment encoding the polypeptide, introducing said DNA fragment into an appropriate host organism in the which DNA fragment is combined with an appropriate expression signal for the DNA fragment, culturing the host organism under conditions that lead to the expression of the polypeptide with hexose oxidase activity and recovering the polypeptide from the culture medium of the host organism. In a further aspect, the invention relates to a polypeptide in isolated form having hexose oxidase activity, comprising at least one amino acid sequence selected from the group consisting of: (i) Tyr-Glu-Pro-Tyr-Gly-Gly-Val-Pro (SEQ ID NO: 1), (ii) Ala-Ile-Ile-Asn-Val-Thr-Gly-Leu-Val-Glu-Ser-Gly-Tyr-Asp-XXX-Gly-Tyr-X-Val-Ser-Ser, (SEQ ID NO: 2), (iii) Asp-Leu-Pro-Met-Ser-Pro-Arg-Gly-Val-Ile-Ala-Ser-Asn-Leu-X-Phe, (SEQ ID NO: 3), (iv) Asp-Ser-Glu-Gly-Asn-Asp-Gly-Glu-Leu-Phe-X-Ala-His-Thr, (SEQ ID N0: 4), (v) Tyr-Tyr-Phe-Lys, (SEQ ID NO: 5), (vi) Asp-Pro-Gly-Tyr-Ile-Val-Ile-Asp-Val-Asn-Ala-Gly-Thr-X Asp, (SEQ ID NO: 6), (vii) Leu-Gln-Tyr-Gln-Thr-Tyr-Trp-Gln-Glu-Glu ~ Asp, (SEQ ID NO: 7), (viii) X-Ile-Arg-Asp-Phe-Tyr-Glu- Glu-Met, (SEQ ID NO: 8) where X represents an amino acid selected from the group consisting of Ala, Arg, Asn, Asp, Asx, Cys, Gln, Glu, Glx, Gly, His, Lie, Leu, Lys , Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val, and muteins and variants thereof. In still further aspects, the invention relates to a recombinant DNA molecule comprising a DNA fragment encoding a polypeptide having hexose oxidase activity and a microbial cell comprising a recombinant DNA molecule like this. In other aspects, the invention relates to the use of the polypeptide with more indicated hexose oxidase activity. above or to a microbial cell expressing such a polypeptide in the preparation of a food product or an animal feed and the preparation of a pharmaceutical product, a cosmetic or dental care product. In other useful aspects a method is provided for reducing the sugar content of a food product, which comprises adding to the food product an amount of the polypeptide or microbial cell as described herein, which is sufficient to remove at least part of the sugar initially present in said food product, a method for preparing a baked product from a dough, comprising adding the polypeptide with hexose oxidase activity or a microbial cell expressing a polypeptide like this to the dough, and a dough improving composition which comprises the polypeptide or the microbial cell according to the invention and at least one conventional dough component. In another aspect, the invention relates to the use of the polypeptide or a microbial cell according to the invention as an analytical reagent for measuring the content of sugars. In an interesting aspect, the invention also provides the use of a polypeptide or a microbial cell according to the invention in the preparation of a lactone, wherein the polypeptide and / or the microbial cell are applied to, a reactor containing a carbohydrate. which can be oxidized by the polypeptide and operate the reactor under conditions in which the carbohydrate is oxidized.

DETAILED DESCRIPTION OF THE INVENTION The hexose oxidases are produced naturally by several species of marine algae. Such species are found, among others, in the family Gigartinaceae belonging to the order Gigartinales. Examples of algae species that produce hexose oxidase, which belong to the Gigartinaceae are Chondrus crispus and Iridophycus flaccidum. Also species of algae of the order Cryptomeniales, including the species Euthora cristata are potential sources of the polypeptide with hexose oxidase activity according to the invention. Accordingly, such algae species are potentially useful sources of hexose oxidase and of DNA encoding such polypeptides with hexose oxidase activity. As used herein, the term "polypeptide with hexose oxidase activity" denotes an enzyme that oxidizes at least D-glucose, D-galactose, D-mannose, maltose, lactose and cellobiose. When such natural sources are used for the isolation of native hexose oxidase, as was done in the prior art and in the present invention for the purpose, to identify algae material that could be used as a source of mRNA to be used in the construction of CDNA and as the starting point for constructing synthetic DNA oligonucleotide primers, the enzyme is typically isolated from the algae starting material by extraction using an aqueous extraction medium. As starting material for such extraction the algae can be used in their fresh state as they were harvested from the marine area of cultivation or they can be used after drying the fronds, for ex. , by air drying at room temperature or by any suitable industrial drying method such as drying in circulating heated air or by freeze drying. To facilitate the subsequent extraction step, the dried or fresh starting material can be ground, e.g. , by grinding or mixing. Aqueous extraction media are suitable buffer solutions having a pH in the range of 6-8, such as 0.1 M sodium phosphate buffer, 20 mM triethanolamine buffer or 20 mM Tris-HCl buffer. The hexose oxidase is typically extracted from the algae material by suspending the starting material in the buffer and maintaining the suspension at a temperature in the range of 0-20 ° C such as at about 5 ° C for 1 to 10 days, preferably under stirring . The suspended algal material is then separated from the aqueous medium by means of a suitable separation method such as filtration, sieving or centrifugation and the hexose oxidase is subsequently recovered from the filtrate or supernatant. Optionally, the separated algae material is subjected to one more additional extraction steps. As various marine algae contain color pigments such as phycocyanins, it may be required to subject the filtrate or supernatant to an additional purification step whereby these pigments are removed. As an example, the pigments can be removed by treating the filtrate or supernatant with an organic solvent wherein the pigments are soluble and subsequently separating the solvent containing the pigments dissolved from the aqueous medium. The recovery of hexose oxidase from the aqueous extraction medium is carried out by any suitable conventional method that allows the isolation of proteins from the aqueous medium. Such methods, examples of which will be described in detail below, include methods such as ion exchange chromatography, optionally followed by a concentration step such as ultrafiltration. It is also possible to recover the enzyme by adding substances such as eg. , (NH4) 2S04 which causes the protein to precipitate, followed by separation of the precipitate and optionally subjecting it to conditions that allow the protein to dissolve. For the purpose of the invention it is convenient to provide the enzyme in a substantially pure form, e.g. , as a preparation essentially without other proteins or contaminants that are not proteins and therefore, the relatively crude enzyme preparation resulting from the extraction and isolation steps indicated above is preferably subjected to further purification steps such as additional chromatography steps , gel filtration or chromatofocusing as will also be described by way of example below.

As mentioned above, the polypeptide with hexose oxidase activity according to the invention is provided by means of recombinant DNA technology methods that allow it to be produced by culturing in a culture medium a cell of an appropriate host organism comprising a gene coding. for the hexose oxidase and recovering the enzyme from the cells and / or the culture medium. The method for producing hexose oxidase that is provided herein comprises as a first step the isolation or construction of a DNA fragment encoding hexose oxidase. Several strategies are available to provide such a DNA fragment. Thus, the DNA fragment can be isolated as such from an organism that inherently produces hexose oxidase. To identify the location of the coding DNA fragment, it is required to have RNA or DNA probe sequences that under appropriate conditions hybridize to the desired DNA fragment and subsequently isolate a DNA fragment comprising the coding sequence and clone it into a vector of DNA. adequate cloning. Another suitable strategy, which is described in detail in the examples below, is to isolate mRNA from an organism that produces hexose oxidase and use such mRNA as the starting point for the construction of a cDNA library that can then be used for Polymerase chain reaction (PCR) synthesis of DNA based on oligonucleotide primers that are synthesized based on amino acid sequences of hexose oxidase. It was found that such a strategy is adequate to provide a DNA fragment encoding hexose oxidase. By way of example, such a strategy as described in detail below is described in summary form. Synthetic oligonucleotides are prepared based on the peptide sequences, H0X-2 and H0X-3 prepared as described hereinbelow by digestion of endoLys-C of a 40 kD polypeptide of hexose oxidase extracted from Chondrus-crispus. PCR using a first strand cDNA as a template and with a sense H0X-2 primer and an HOX-3 antisense primer produced a 407 bp DNA fragment. This fragment was inserted into an E. coli vector, pT7 Blue and subsequently sequenced. It was found that in addition to the sequences for the HOX-2 and HOX-3 peptides this 407 bp fragment also contained an open reading frame containing the HOX-4 and HOX-5 peptides of the hexose oxidase fragment derived from Chondrus cri spus 40 kD, whose isolation is also described below. Sense and antisense oligonucleotides were synthesized based on the 407 bp fragment, and two fragments of 800 and 1400 bp, respectively, could be subsequently isolated by PCR using cDNA as a template.

These two fragments were cloned into the vector pT7 Blue and subsequently sequenced. The DNA sequence of the 5 'fragment showed an open reading frame containing the HOX-6 peptide which was also isolated from the 40 kD Chondrus crispus hexose oxidase fragment derived from Chondrus crispus. Similarly, the 3 'fragment showed a reading frame containing HOX-1, whose isolation is revealed below, and HOX-7 and HOX-8, both isolated from a hexose oxidase polypeptide derived from Chondrus crispus of 29 kD obtained by digestion of endoLys-C as will also be described below. Based on the combined DNA sequences as mentioned above, an oligonucleotide corresponding to the 5 'end of the putative hox gene and an oligonucleotide corresponding to the 3' end of that gene were synthesized. These two oligonucleotides were used in the PCR using first strand cDNA as a template resulting in a DNA fragment of approximately 1.8 kb. This fragment was cloned into the E. coli vector indicated above and sequenced. The DNA sequence was identical to the combined sequence of the 5 ', 407 bp and 3' end sequences indicated above and it was concluded that this approximately 1.8 kb DNA sequence encodes both hexose oxidase fragments derived from Chondrus crispus , 40 kD and 29 kD.

As will be apparent to the skilled person, the above strategy for isolating a DNA fragment encoding a polypeptide with hexose oxidase activity, including the isolation and characterization of hexose oxidase, can be used for constructions of such fragments encoding hexose oxidases derived from any other natural source other than Chondrus crispus, including the species of marine algae mentioned above, such as from other plants or from microorganisms. Alternatively, the DNA sequence of the DNA fragment encoding the polypeptide with hexose oxidase activity can be synthetically constructed by established standard methods, e.g. , the phosphoamidite method described by Beaucage and Caruthers (1981) or the method described by Matthes et al. (1984). According to the phosphoamidite method the oligonucleotides are synthesized, eg. , in an automatic DNA synthesizer, purified, recognized, ligated and cloned into an appropriate vector. In addition, the DNA fragment can be of mixed and synthetic genomic origin, mixed synthetic and cDNA or mixed genomic and cDNA, prepared by sub-fragment sub-fragments of synthetic, genomic or cDNA origin as appropriate, the subfragments corresponding to several parts of the whole DNA fragment , according to standard techniques.

In a subsequent step of the method according to the invention, the DNA fragment encoding the polypeptide with isolated or synthesized hexose oxidase activity is introduced into an appropriate host organism wherein the DNA fragment is combined in operable form with an expression signal for the DNA fragment. Such an introduction can be carried out by methods that are well known to the skilled person including the construction of a vector having the fragment inserted and transforming the host organism with the vector. Suitable vectors include plasmids that are capable of duplication in the selected host organism. It is also contemplated that the DNA fragment can be integrated into the chromosome of the host organism, e.g. , inserting the fragment into a transportable element such as a transposon, and subjecting a mixture of the selected host organism and the transposon to conditions in which the transposon will be integrated into the chromosome of the host organism and combined with an appropriate expression signal. According to the invention, a DNA fragment encoding the polypeptide with hexose oxidase activity including the gene for the polypeptide, which is produced by methods such as those described above or any alternative method known in the art, can be expressed in enzymatically active form using an expression vector. An expression vector generally includes the components of a typical cloning vector, i.e., an element that allows for autonomous duplication of the vector in the selected host organism and one or more phenotypic markers for selection purposes. An expression vector includes control sequences that encode a promoter, operator, ribosome ligation site, translation initiation signal and optionally a repressor gene or one or more activating genes. To allow secretion of the expressed polypeptide, a sequence of signals upstream of the coding sequence of the gene can be inserted. In the present context, the term "expression signal". it includes any of the control sequences indicated above, repressor or activating sequences and signal sequences. For expression under the control of the control sequences, the gene encoding hexose oxidase is operably linked to the control sequences in an appropriate manner with respect to expression. Promoter sequences that can be incorporated into plasmid vectors, and that can support transcription of the hexose oxidase gene include, but are not limited to, the tac promoter, promoters derived from lambda phage, including the PL and PR promoters. An expression vector carrying the DNA fragment of the invention can be any vector that is capable of expressing the hexose oxidase gene in the selected host organism, and the choice of vector will depend on the host cell into which it has to be introduced. Thus, the vector can be a vector that is autonomously duplicated, ie a vector that exists as an extrachromosomal entity, whose duplication is independent of chromosomal duplication, e.g. a plasmid, a bacteriophage or an extrachromosomal element, a minichromosome or an artificial chromosome. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the genome of the host cell and duplicated together with the chromosome. In the vector, the DNA fragment encoding the. polypeptide with hexose oxidase activity should be combined in operable form with a suitable promoter sequence. The promoter can be any DNA sequence that confers transcriptional activity to the host organism of choice and can be derived from genes encoding proteins that are either homologous or heterologous with respect to the host organism. Examples of suitable promoters for directing the transcription of the DNA fragment of the invention in a bacterial host are the promoter of the lac operon of E. coli, the promoters of the agarase gene dagA of Streptomyces coeli color, the promoters of the a-amylase gene (amyL) of Bacill us licheniformis, the promoters of the maltogenic amylase gene (amyM) of Bacill us stearothemophil us, the promoters of the a-amylase gene (amyQ) of Bacillus amyloliquefaciens, the promoters of the xylA and xylB genes of Bacillus subtilis . For transcription in a fungal species, examples of useful promoters are those derived from the genes encoding the alcohol oxidase of Pichia pastoris, the TAKA amylase from Aspergill us oryzae, the aspartic proteinase from Rhizomucor mi ehei, the neutral a-amylase from Aspergill us niger, the stable a-amylase of A. niger, the glucoamylase of A. niger, the lipase of Rhizomucor miehei, the alkaline protease of Aspergill us oryzae, the triose phosphate isomerase of Aspergill us oryzae or the acetamidase of Aspergillus nidulans . As examples of promoters suitable for expression in a yeast species may be mentioned the Gal 1 and Gal 10 promoters of Saccharomyces cerevisiae. When expressed in a bacterial species such as E. coli, a suitable promoter from a bacteriophage promoter including a T7 promoter or a bacteriophage lambda promoter can be selected. The vector comprising the DNA fragment encoding the polypeptide with hexose oxidase activity may also comprise a selectable marker, e.g. , a gene whose product complements a defect in the host organism such as a mutation that confers an auxotropic phenotype, or the marker can be one that confers resistance to antibiotics or resistance to heavy metal ions. The host organism of the invention comprising either a DNA construct or an expression vector, as described above, is advantageously used as a host cell in the recombinant production of a polypeptide according to the invention. The cell can be transformed with a DNA construct comprising the gene encoding the polypeptide of the invention or, conveniently, by integrating the DNA construct into the host chromosome. An integration like this is considered * generally advantageous since the DNA fragment is more likely to be stably maintained in the cell. The integration of the DNA constructs in the host chromosome can be carried out according to conventional methods such as, for example. , homologous or heterologous recombination or by means of a translocable element. Alternatively, the host organism can be transformed with an expression vector as described above. According to the invention, the host organism can be a cell of a higher organism such as an animal cell, including a mammalian cell, a bird or an insect, or a plant cell. However, in the preferred embodiments, the host organism is a microbial cell, e.g. , a bacterial or fungal cell, including a yeast cell. Examples of suitable bacterial host organisms are gram-positive bacterial species such as Bacillaceae including Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus. megaterium and Bacillus thuringiensis, the Streptomyces species such as Streptomyces murinus, the bacterial species of lactic acid including Lactococcus spp. such as Lactococcus lactis, Lactobacillus spp. including Lactobacillus reuteri, Leuconostoc spp. and Streptococcus spp. Alternatively, strains of a gram-negative bacterial species such as a species belonging to the .Enterojacteriaceae including E. coli or the Pseudomonadaceae may be selected as the host organism. A yeast host organism can be advantageously selected from a species of Saccharomyces including Saccharomyces cerevisiae or a species belonging to the Schizosaccharomyces. Suitable host organisms among filamentous fungi include Aspergillus species, e.g. Aspergillus oryzae, Aspergillus nidulans or Aspergillus niger. Alternatively, strains of a Fusari um species, eg. , Fusarium oxysprum or a Rhizomucor species such as Rhi zomucor miehei can be used as the host organism. In a preferred embodiment a strain of the species Pi chia pastoris is used as the host organism. Some of the useful host organisms indicated above such as fungal species or gram-positive bacterial species can be transformed by means of a process that includes the formation of protoplasts and the transformation of protoplasts followed by regeneration of the cell wall of a way known per se. For the production of the polypeptide with hexose oxidase activity, the cells of the recombinant host organism as described above are cultured under conditions that lead to the expression of the polypeptide in a recoverable form. The medium used to culture the cells can be any conventional means suitable for culturing the host cells in question and obtaining the expression of the polypeptide. Suitable media can be obtained from commercial suppliers or can be prepared according to published recipes. The resulting polypeptide is typically recovered from the culture medium by means of conventional methods including separation of the cells from the medium by centrifugation or filtration, if necessary, after cell disruption, followed by precipitation of the proteinaceous components of the supernatant or filtered, eg , by addition of a salt such as ammonium sulfate, followed by a purification step. An industrially convenient aspect of the invention is that microbial cultures such as e.g. , Bacterial cultures that are used in the preparation of food products or forages can be used as the host organism expressing the gene encoding the polypeptide with hexose oxidase activity. Thus, the cultures, bacterial initiators of lactic acid that are used in the preparation of dairy products or other food products such as meat or wine products and which comprise, e.g. , one or more strains of a lactic acid bacterium selected from any of the bacterial lactic acid species indicated above, can be used as host organisms, whereby hexose oxidase will be produced directly in the food product to which they are added. starter cultures. Similarly, the gene encoding the hexose oxidase according to the invention can be introduced into bacterial starter cultures of lactic acid which are used as added inoculants to forage crops such as grass or corn or to proteinaceous waste products of animal origin such as fish. and waste materials from slaughterhouses for the production of silage to feed the animals. For this purpose, the expression of hexose oxidase by inoculants in silage will imply that the oxygen initially present in the crops or the waste materials to be ensiled will be exhausted, so that anaerobic conditions will be established, which will inhibit the growth of aerobic putrefaction organisms such as gram-negative bacteria and yeasts. It is also considered that yeast cultures such as baker's yeast or yeast cultures that are used in the preparation of alcoholic beverages including wine and beer can be used as host organisms for the gene encoding the polypeptide with hexose oxidase activity. the invention. For example, in the case of such strains of recombinant baking yeasts, the hexose oxidase that is produced will have a mass-improving effect as described below. From the above it is evident that the direct addition of recombinant microbial cultures expressing the hexose oxidase according to the invention to a food product or any other product where hexose oxidase activity is desired, can be used as an alternative to the addition of the isolated enzyme.

In additional industrially important embodiments, recombinant microbial cultures expressing a polypeptide with hexose oxidase activity are used in a bioreactor for the production of the enzyme or for the production of lactones of any of the above-mentioned carbohydrates which can be oxidized by the enzyme with hexose oxidase activity. For this latter application, the cells of the microbial cultures are advantageously immobilized on a solid support such as a polymer material, which is preferably in the form of small particles to provide a large surface to bind the cells. Alternatively, the isolated enzyme may be used for the purpose indicated above, also preferably attached to a solid support material. In this connection, the ligation of the cells or the enzyme can be provided by any conventional method for that purpose. In other useful embodiments of the invention, the polypeptide having hexose oxidase activity can be a fusion product, i.e., a polypeptide that in addition to the amino acid sequences with hexose oxidase activity comprises additional amino acid sequences that have other useful activities. Thus, fusion polypeptides having one or more enzymatic activities in addition to hexose oxidase activity are considered. Such additional enzymatic activities may be chosen from enzymes capable of degrading carbohydrates, such as lactase, amylases including glucoamylases, glucanases, cellulases, hemicellulases, xylanases, lactases or any other oxidoreductase such as glucose oxidase, galactose oxidase or pyranose oxidase, and also between proteases and peptidases, lipases or nucleases. The additional enzymatic sequence (s) to be chosen for integration into a polypeptide with hexose oxidase activity according to the invention depends on the product for which the product is intended. of enzymatically active fusion. Thus, as examples, it is considered that a fusion polypeptide with hexose oxidase activity for use in the preparation of a dairy product advantageously comprises a lactase, a protease or a peptidase, and that a fusion polypeptide provided for improving a mass can comprise any of the carbohydrate degrading enzymes as an associated element of fusion. It is also evident that the microbial cells according to the invention as described above and expressing a fusion polypeptide with hexose oxidase activity having additional enzymatic activities, can be used for inoculation of other food products and animal feed in the manner which was also described above.

It is also considered that a suitable fusion partner may be a sequence which confers on hexose oxidase altered characteristics such as solubility or a sequence which can serve as an "aggregate" group which confers hexose oxidase the ability to bind more strongly or more selectively to a particular solid material for purposes of purification or immobilization of the polypeptide with hexose oxidase activity. Furthermore, it is within the scope of the invention to provide the polypeptide as a chimeric product comprising partial sequences of polypeptides with activity, hexose oxidase derived from different sources and which are encoded by a DNA fragment that is constructed by combining DNA sequences encoding polypeptides with hexose oxidase activity of these different sources in a DNA fragment encoding the entire chimeric polypeptide. In a useful embodiment, the method according to the invention is one in which the DNA fragment encoding the polypeptide with hexose oxidase activity comprises at least one DNA sequence encoding an amino acid sequence selected from the group consisting of: (i) Tyr-Glu-Pro-Tyr-Gly-Gly-Val-Pro (SEQ ID NO: 1), (ii) Ala-Ile-Ile-Asn-Val-Thr-Gly-Leu-Val-Glu-Ser-Gly-Tyr-Asp XXX-Gly-Tyr-X-Val-Ser-Ser, (SEQ ID NO: 2 ), (iii) sp-Leu-Pro-Met-Ser-Pro-Arg-Gly-Val-Ile-Ala-Ser-Asn-Leu-X-Phe, (SEQ ID NO: 3), (iv) Asp- Ser-Glu-Gly-Asn-Asp-Gly-Glu-Leu-Phe-X-Ala-His-Thr, (SEQ ID NO: 4), (v) Tyr-Tyr-Phe-Lys, (SEQ ID NO: 5), (vi) Asp-Pro-Gly-Tyr-Ile-Val-Ile-Asp-Val-Asn-Ala-Gly-Thr-X-Asp, (SEQ ID NO: 6), (vii) Leu-Gln-Tyr-Gln-Thr-Tyr-Trp-Gln-Glu-Glu-Asp, (SEQ ID NO: 7), (viii) X-Ile-Arg-Asp-Phe-Tyr-Glu- Glu-Met, (SEQ ID NO: 8) wherein X represents an amino acid selected from the group consisting of Ala, Arg, Asn, Asp, Asx, Cys, Gln, Glu, Glx, Gly, His, Lie, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val, and muteins and variants thereof. In the present context, the term "variant" is used to designate any modification of a polypeptide sequence with hexose oxidase activity that does not result in a complete loss of hexose oxidase activity. The modifications may include deletion, substitution of amino acid residues present in the polypeptide, whether it is derived from a natural source or in a modified polypeptide sequence or the modification may involve insertion into a polypeptide like this of additional amino acid residues. Substitution of one or more amino acid residues can be done by modifying or substituting the codon or codons that encode the amino acid or amino acids that one wishes to replace, e.g. , by mutagenesis, in particular site-directed mutagenesis, using methods that are known per se. Similarly, the deletion of one or more amino acid residues can be performed by deletion of the corresponding codon or codons in the DNA fragment encoding the polypeptide according to the invention. As mentioned above, the method of agreement < with the invention may include, as a further step, a purification of the preparation of polypeptides initially recovered from the medium and / or culture microorganisms. The purpose of this additional step is to obtain an enzyme preparation in which the polypeptide with hexose oxidase activity is in substantially pure form. The term "substantially pure form" implies that the preparation does not have unwanted contaminants originating in the culture medium, the cells of the host organism of production or substances produced by these cells during cultivation. Thus, for many applications, it is important that the polypeptide preparation resulting from the purification step have substantially no enzymatic hexose oxidase activity.

Purification methods will depend on the degree of purity desired, but will typically be selected from conventional protein purification methods, such as desalting, ion exchange or affinity chromatography procedures, including hydrophobic interaction chromatography and methods. of gel filtration, such as the method described in the following examples. As mentioned above, the invention relates in a further aspect to a polypeptide in isolated form having hexose oxidase activity, comprising at least one of the amino acid sequences indicated above, or muteins and variants thereof as is described above. Preferably, the polypeptide is produced according to the methods described above. Depending on the production method, in particular the host organism in question, the polypeptide according to the invention can be glycosylated to a varying degree or can be expressed for certain purposes advantageously in a substantially non-glycosylated form. In a preferred embodiment of the invention, the polypeptide is one that has functional characteristics identical or partially identical to those of hexose oxidase that occurs naturally in the algae species Chondrus crispus as described in the prior art. It was found that such hexose oxidase extracted from an algae source, when subjected to an SDS-PAGE as described here, it can show protein bands separated by 29, 40 and / or 60 kD. In order to obtain a generally effective use of the polypeptide with respect to cost, it is preferred that the enzyme has an enzymatic activity over a wide pH range. Thus, it is preferred that the hexose oxidase according to the invention show at least one enzymatic activity at a pH in the range of 1-9, such as in the range of 2-9, including the range of 5-9. In this regard it is considered that the pH range of activity or the optimum pH of a naturally derived hexose oxidase can be modified in <; a desired direction and to a desired degree by modifying the enzyme, as described above or by random mutagenesis of a replicon or a host organism comprising the DNA coding for hexose oxidase, followed by the selection of mutants having the characteristics of Altered pH desired. Alternatively, such modifications of the enzyme may be directed to modify the thermotolerance and the optimum temperature for the activity of the polypeptide with hexose oxidase activity, or to change the isoelectric point of the enzyme. In addition, the polypeptide according to the invention is preferably enzymatically active within a wide temperature range such as a range of 10-90 ° C, e.g. , within a range of 15-80 ° C including the range of 20-60 ° C. In particular, it may be preferred for certain specific purposes that the polypeptide with hexose oxidase activity maintain significant residual enzymatic activity at temperatures of 70 ° C or greater, e.g. , when the enzyme is intended to be used in batches where it may be useful to have hexose oxidase activity during at least part of the subsequent baking step. The extent of the application of hexose oxidase depends on the range of carbohydrates that can be used as a substrate. Although hexose oxidase appears to have the highest substrate specificity with respect to hexoses, such as glucose, galactose and mannose, it has been found that the range of carbohydrate substances that can be used as substrates for the polypeptide according to the invention is not is limited to hexoses. Thus, a preferred polypeptide is one that in addition to a high specificity with respect to hexoses also has a high specificity with respect to other carbohydrate substances including disaccharides such as lactose, maltose and / or cellobiose and even substantial specificity with respect to pentoses including as an example xylose, or deoxypentose or deoxyhexose such as rhamnose or fucose. It is of significant practical importance that hexose oxidase, in addition to a high specificity with respect to hexoses and other monosaccharides, also has substantial specificity with respect to disaccharides, in particular lactose present in milk and maltose which, among others, occurs in the flours of cereals and masses. Accordingly, in another preferred embodiment the polypeptide according to the invention is one which in addition to D-glucose oxidizes at least one sugar selected from the group consisting of D-galactose, maltose, cellobiose, lactose, D-mannose, D-fucose and D-xylose. In another preferred embodiment the polypeptide with hexose oxidase activity has an isoelectric point in the range of 4-5. Specifically, the polypeptide may preferably have an isoelectric point of 4.3 ± 0.1 or one of 4. 5 ± 0.1. Generally, the polypeptide according to the invention typically has a molecular weight as determined by gel filtration using Sephacryl 5-200 Superfine (Pharmacia) which is in the range of 100-150 kD. A molecular weight determined by this or equivalent methods is also called apparent molecular weight. Specifically, the polypeptide can have an apparent molecular weight of 110 kD ± 10 kD. In still another aspect, the invention provides a recombinant DNA molecule comprising a DNA fragment encoding a polypeptide having hexose oxidase activity. As described above, such DNA fragment can be isolated from a natural source or can be constructed, e.g. , as described in detail in the examples below. In addition, the coding fragment can also be synthesized based on amino acid sequences of a naturally occurring hexose oxidase. The recombinant molecule can be selected from any of the types of expression vectors as mentioned above. In preferred embodiments, the recombinant DNA molecule comprises a DNA fragment encoding a hexose oxidase polypeptide comprising at least one of the amino acid sequences indicated above (i) to (viii) ,. or a mutein or derivative of such a polypeptide. In a specific embodiment, the recombinant DNA molecule comprises the following DNA sequence: TGAATTCGTG GGTCGAAGAG CCCTTTGCCT CGTCTCTCTG CTACCGTGTA TGTCAAAGGT 60 TCGCTTGCAC ACTGAACTTC ACGATGGCTA CTCTTCCTCA GAAAGACCCC GGTTATATTG 120 TAATTGATGT CAACGCGGGC ACCGCGGACA AGCCGGACCC ACGTCTCCCC TCCATGAAGC 180 AGGGCTTCñA CCGCCGCTGG ATTGGAACTA ATATCGATTT CGTTTATGTC GTGTACACTC 240 CTCAAGGTGC TTGTACTGCA CTTGACCGTG CTATGGAAñA GTGTTCTCCC GGTACAGTCA 300 GGATCGTCTC TGGCGGCCAT TGCTACGAGG ACTTCGTATT TGACGAATGC GTCAAGGCCA 360 TCATCAA.CGT CACTGGTCTC GTTGAGAGTG GTTATGACGA CGATAGGGGT TACTTCGTCA 420 GCAGTGGAGA TACAAATTGG GGCTCCTTCA AGACCTTGTT CAGAGACCAC GGAAGAGTTC 480 TTCCCGGGGG TTCCTGCTAC TCCGTCGGCC TCGGTGGCCA CATTGTCGGC GGAGGTGACG 540 GCATTTTGGC CCGCTTCGAT GGCCTCCCCG TCGATTGGCT CAGCGGCGTG GAGGTCGTCG 600 TTAAGCCAGT CCTCACCGA GACTCGGTAC TCAAGTATGT GCACAAAGAT TCCGAAGGCT 660 ACGACGGGGA GCTCTTTTGG GCACACACAG GTGGCGGTGG CGGAAACTTT GGAATCATCA 720 CCAAATACTA CTTCAAGGAT TTGCCCATGT CTCCACGGGG CGTCATCGCA TCAAATTTAC 780 ACTTCAGCTG GGACGGTTTC ACGAGAGATG CCTTGCAGGA TTTGTTGACA AAGTACTTCA 840 AACTTGCCAG ATGTGATT GG AñGAATACGG TTGGCAAGTT TCAAATCTTC CATCAGGCAG 900 CGGAAGAGTT TGTCATGTAC TTGTATACAT CCTACTCGAA CGACGCCGAG CGCGAAGTTG 960 CCCAAGACCG TCACTATCAT TTGGAGGCTG ACATAGAACA GATCTACAAA. ACATGCGAGC 1020 CCACCAAAGC GCTTGGCGGG CATGCTGGGT GGGCGCCGTT CCCCGTGCGG CCGCGCAAGA 1080 GGCACACATC GCTTGGCGGG CATGCTGGGT GGGCGCCGTT CCCCGTGCGG CCGCGCAAGA 1140 TCACTGAGAC GATCAACGGC TCCGGGCCGA ATCAGCGCGG CAAGTACAAG TCTGCGTACA 1200 TGATCAAGGA TTTCCCGGAT TTCCAGATCG ACGTGATCTG CAAATACCTT ACGGAGGTCC 1260. CGGACGGCTT GACTAGTGCC GAAATGAAGG ATGCCTTACT CCAGGTGGAC ATGTTTGGTT 1320 GTGAGATTCA CAAGGTGGTC TGGGATGCGA CGGCAGTCGC GCAGCGCGAG TACATCATCA 1380 AACTGCAGTA CCAGACATAC TGGGAGGAAG AAGACAAGGA TGCAGTGAAC CTCAAGTGGA 1440 TTAGAGACTT TTACGAGGAG ATGTATGAGC CGTATGGCGG GGTTCCAGAC CCCAACACGC 1500 AGGTGGAAGG TGGTAAAGGT GTGTTTGAGG GATGCTACTT CAACTACCCG GATGTGGACT 1560 TGAACAACTG GAAGA.CGGC AAGTATGGTG CCCTCGAACT TTACTTTTTG GGTAACCTGA 1620 ACCGCCTCAT CAAGGCCAA TGGTTGTGGG ATCCCAACGA GATCTTCACA AACAAACAGA 1680 GCATCCCTAC TAAACCTCTT AAGGAGCCCA AGCAGACGAA ATAGTAGGTC ACAATTAGTC 1740 ATCGACTGAA GTGCAGCTAC TGTCGGATAC GGCGTGATGG TTGCTTTTTA TAAACTTGGT 1800 A 1801 In addition, the invention provides in another aspect a microbial cell comprising the recombinant DNA molecule indicated above. The general description made above of the host organism comprising a DNA fragment encoding the polypeptide according to the invention, comprises a microbial cell like that and therefore, such cells can be selected from any of the groups, families, microbial genera and species mentioned above, ie, the microbial cell can be selected from a bacterial cell, a fungal cell and a yeast cell, including as examples an E. coli cell, a bacterial lactic acid cell, a Saccharomyces cerevisiae cell and a Pichia pastoris cell. The microbial cell according to the invention can be provided, if it is intended for direct addition to a product in which it is desired to have hexose oxidase activity, e.g. , during a manufacturing process, in the form of a microbial culture, preferably in a concentrated form. Thus, a culture like this can advantageously contain the microbial cell according to the invention in a concentration which is preferably in the range of 105 to 1012 per g of culture. The culture can be a fresh culture, i.e., an unfrozen suspension of the cells in a liquid medium or it can be in the form of a frozen or dried culture, e.g. , a culture dried by freezing. The microbial cell can also be immobilized for specific purposes in a solid substrate. As mentioned above, the invention relates in a further aspect to the use of the polypeptide with hexose oxidase activity according to the invention or of a microbial cell expressing such a polypeptide in the preparation of food products. In this context, the term "preparation" should be understood in its broadest sense, so as to comprise the addition of the hexose oxidase or the microbial cell to the ingredients for the food product in question before, during or after any subsequent step of the process, during the packaging and during the storage of the finished product until it is consumed. The food products in which such use is advantageous can be any product in which the final products of the hexose oxidase confer advantageous effects on the food product. Naturally, the desired hexose oxidase activity will only be obtained if the substrate for the enzyme is present in sufficient amounts. The substrate carbohydrates may be inherently present in the food product or the ingredients therefor, or may be added or generated during the preparation process. An example of substrate that is generated during the preparation is the enzymatic degradation of di-, oligo- or polysaccharides to lower sugar substances that are degradable by hexose oxidase, which can occur as a result of the enzymatic activity of the enzymes inherently present in the food product or added during the preparation. In addition, the substrate for the polypeptide with hexose oxidase activity can be generated as a result of the enzymatic activity of an associated fusion element as described above. The desirable effects of hexose oxidase activity on a product containing substrates for the enzyme include the generation of lactones from the sugar substrate, which can subsequently be converted to the corresponding acids, generation of hydrogen peroxide and consumption of oxygen. Typical examples of food products where the hexose oxidase activity may be advantageous include as examples the dairy products, the food products containing starch and non-dairy beverages. Therefore, in the preparation of a range of dairy products it is desired to lower the pH. This is conventionally obtained by inoculating the milk with starter cultures that produce lactic acid. As mentioned above, it is contemplated that hexose oxidase or organisms expressing this enzyme may be used as an alternative means to acidify milk. The same effect may be desirable in other food products that are acidified during the preparation, such as certain meat products or plant products that are commonly acidified by the addition of bacterial starter cultures of lactic acid. The oxygen consumption resulting from hexose oxidase activity has several advantageous implications in relation to the preparation of food and pharmaceutical products. By causing the depletion or removal of oxygen in food or in pharmaceutical products that contain lipids that tend to oxidative decomposition processes, the hexose oxidase can act as a, antioxidant and additionally, the reduction of the oxygen content can inhibit the decomposition organisms, whose growth depends on the presence of oxygen and consequently, the polypeptide with hexose oxidase activity can also act as an agent antimicrobial This latter effect can be used to extend the shelf life of packaged foods where decomposition can be prevented by the incorporation of the polypeptide with hexose oxidase activity according to the invention either in the food product itself or by providing a mixture of the same. hexose oxidase and an appropriate substrate for the same in the package, but separated from the contents of the food product. In a typical example, a mixture like this is added to the inside of a food container such as, for example. , a can or a boat. Accordingly, hexose oxidase according to the invention can be used as an oxygen scavenging agent in a food package. It is evident that the effects indicated above of the polypeptide according to the invention, in the preparation of food products will also be applicable in the preparation of animal forage products. In particular, these effects are suitable for the preparation of silage made from forage crops such as grass or maize, or from animal waste products1 from slaughterhouses or from fish processing plants. Such food products are commonly ensiled with the addition of acids or bacteria that produce acids such as bacterial lactic acid inoculants. To promote the growth of acidifying bacteria and to prevent the growth of aerobic decomposition organisms such as grag-negative bacteria and yeasts, it is essential to have a low oxygen content in the silage material. It is therefore contemplated that the hexose oxidase according to the invention is useful as an agent for the removal of oxygen and acidification in animal forage silage, optionally in the form of compositions additionally comprising one or more conventional silage feed additives. such as lactic acid bacterial inoculants or enzymes that generate low molecular weight sugar substances. A further useful application of the hexose oxidase polypeptide according to the invention is the use of the enzyme to reduce the sugar content of a food product, which comprises adding to the product an amount of the polypeptide or of a microbial cell that produces the polypeptide which is sufficient to eliminate at least part of the sugar initially present in the food product. An application like this can be useful, eg. , in the preparation of diets for diabetic patients in. where a low sugar content is desired, and in the production of wines with a reduced alcohol content. In this last application, the hexose oxidase is preferably added to the must before the inoculation of the yeast. In a further useful aspect, the invention relates to the use of the polypeptide with hexose oxidase activity or of a microbial cell which produces the enzyme according to the invention in the preparation of pharmaceuticals, cosmetics or tooth care products such as toothpastes or toothpastes. The desired effects of hexose oxidase in such products are essentially those described above with respect to foodstuffs and animal fodder.

A particularly interesting use of hexose oxidase according to the invention is its use as a dough-improving agent. It has been found that the addition of the hexose oxidase to the dough results in an increased resistance of the same to the break when the dough is stretched, that is, the enzyme gives the dough an increased resistance, so that it becomes less prone to mechanical deformation. It is considered that, based on the known effects in this respect for glucose oxidase, that this effect of addition of the hexose oxidase according to the invention to a mass is the result of the formation of cross-links between them. thiol groups in sulfur-containing amino acids in flour proteins that occur when the hydrogen peroxide generated by the enzyme in the dough reacts with the thiol groups that are oxidized in this way. Accordingly, the invention also provides a method for preparing a baked product. from a mass, comprising adding to the dough an effective amount of the polypeptide or a microorganism according to the invention that is capable of expressing such a polypeptide, and a dough improving the composition comprising the polypeptide or a micro-organism capable of expressing such a polypeptide in a dough, and at least one component of the conventional dough. In useful embodiments, a composition such as this may further comprise at least one enzyme that improves dough or baking product, e.g. , selected from a cellulase, a hemicellulase, a pentosanase, a lipase, a xylanase, an amylase, a glucose oxidase and a protease. In other aspects of the invention, hexose oxidase is used as an analytical reagent in methods to determine in biological samples and in other samples the concentration of any sugar that can be converted by the enzyme. Typically, the sugar content is measured by determining the amount of final products resulting from the enzymatic conversion of the substrate sugar present in the. shows to be measured. In this regard, it is considered that hexose oxidase can be used directly as a reagent in an in vitro analytical assay or that it can be incorporated into a detector. The invention will now be described by way of illustration in the following examples and the accompanying drawings, in which: Figure 1 depicts a schematic overview of the hexose oxidase purification (HOX) and the two adopted strategies for obtaining sequence information of amino acids, Figure 2 shows native, non-dissociating polyacrylamide gel electrophoresis (native PAGE) of hexose oxidase preparations at different steps of purification. The samples represent the enzyme preparation obtained after the anion exchange chromatography and the concentration (lane 1), after gel filtration (lane 2), and after cation exchange chromatography (lane 3) or chromatofocusing (lane 4). ). The Phast gel (Pharmacia, gel 8-25% gradient) was stained with silver. The molecular weights of the standard proteins (x 10 ~ 3) are indicated on the left. The band corresponding to hexose oxidase, indicated by an arrow, was identified by enzyme staining from another gel in parallel (not shown). The four lanes were run in separate gels. Figure 3 shows the UV profile obtained during the purification of hexose oxidase by gel filtration on Sephacryl S-200 HR as described in the text. Fractions containing hexose oxidase (HOX) activity are indicated by the filled area, Figure 4 shows SDS-PAGE of hexose oxidase purified from Chondrus crispus by anion exchange chromatography on DEAE-Fast Flow Sepharose, gel filtration on Sephacryl followed by cation exchange chromatography on Fast Flow Sepharose (lane 1) or chromatofocusing on a Mono P column (lane 2). The molecular weights of the standard proteins (x 10"3) are indicated on the left: the polypeptides at 60 kD, 40 kD and 29 kD are marked with arrows. Reduced samples were run on a 12% polyacrylamide gel that was stained with Coomassie Brilliant Blue R-250. The two lanes were run on separate gels, Figure 5 shows the isoelectric focus (IEF) of the hexose oxidase. The gel was stained with Coomassie Brilliant Blue R-250 (lane 1) or stained by enzymatic activity as described in the text (lane 2). The positions of the isoelectric point markers run in parallel are shown on the left. The two lanes were run on separate gels, Figure 6 shows the reverse phase CLAP separation of the peptides generated by digestion of the 40 kD HOX polypeptide with endoproteinase Lys-C. The peaks labeled 1, 2, 3, 4 and 5 were subjected to amino acid sequencing, Figure 7 shows the reverse phase CLAP separation of the peptides generated by digestion of the 29 kD HOX polypeptide with endoproteinase Lys-C. The peaks labeled 1 and 2 were subjected to amino acid sequencing, Figure 8 shows a Northern blot analysis of RNA extracted from Chondrus cri spus. The denatured agarose gel was loaded with 30 μg (lane 1) and 3 μg (lane 2), respectively, of total RNA. The arrow on the left indicates the specific hexose oxidase transcript. The positions of the molecular weight markers in kb are shown on the right, Figure 9 shows the construction of plasmid pUP0153 which mediates the expression of the recombinant hexose oxidase in Pichia pastori s. Small arrows indicate the PCR primers. The gray box indicates the hexose oxidase gene, Figure 10 shows the purification of the recombinant hexose oxidase from Pichia pastoris by changing anions in a HiTrap-Q column (step one). The activity of * the alcohol oxidase (AOX) (•) and the activity of the hexose oxidase (HOX) (o) in the collected fractions were tested as described in the text, Figure 11 shows purification of recombinant hexose oxidase from Pichia pastoris by gel filtration on Sephacryl S-200 HR (step two). The alcohol oxidase (AOX) activity (o) and the hexose oxidase (o) activity in the collected fractions were tested as described in the text, Figure 12 shows the construction of plasmid pUPOldl which acts as an intermediate in the expression of hexose oxidase recombinant in E. coli. The small arrows indicate PCR primers (the gray box indicates the hexose oxidase gene), Figure 13 shows SDS-PAGE of recombinant hexose oxidase produced in E. coli. Crude extracts of cells used in a 14% denaturing gel were analyzed. The molecular weights of the standard proteins (x 10"3) are indicated on the left The gel was stained with Coomassie Brilliant Blue R-250. Lane 1 shows extract of E. coli cells with pUPOldl, lane 2 shows Plasmid-less control The arrow shows the hexose oxidase band and Figure 14 shows the construction of plasmid pUP0155 which acts as an intermediate in the expression of recombinant hexose oxidase in Saccharomyces cerevisiae The 'small arrows indicate PCR primers. gray indicates the hexose oxidase gene.

EXAMPLE 1 Purification of hexose oxidase from Chrondrus crispus Fig. 1 shows a schematic overview of the purification and two strategies adopted to obtain information on the amino acid sequence for the enzyme. 1. 1 Dried collection and crushing of Chrondrus crispus The red alga Chrondrus cri spus was collected during April to September on the coast near Greña, Jutland, Denmark, at a depth of 2-5 meters. Freshly harvested algae fronds were rinsed with cold water and stored on ice during transport to the laboratory (<24 hours). The algae was then either dried immediately or stored in a frozen state until further processing. For the purification of the enzyme the material was stored at -18 ° C, while the material intended for mRNA isolation was stored in liquid nitrogen. The fronds of Chondrus crispus were thawed at 4 ° C and air-dried at room temperature (20-25 ° C) for 2-3 days. The dried material was crushed to a fine powder in a Commercial Mixer (model 34BL97, Waring, New Harford, Connecticut, USA). 1. 2. Extraction of the enzyme Approximately 500 g of Chondrus crispus powder were mixed with 2.5 1 of 20 mM Tris-Cl, pH 7.0. The water used in all extraction and purification procedures was obtained from a Milli-Q UF Plus Laboratory water purification system (Millipore). The buffer was previously cooled to 4 ° C. The mixture was kept at 4 ° C for 6-8 days. The extract was collected by filtration through several layers of gauze. The alga material was subjected to repeated extractions that were carried out as the first described above. The material was generally discarded after 5-8 extractions when the residual activity had declined to an almost negligible level. The filtrate was clarified by centrifugation at 10,000 x g in a Sorvall GSA rotor (Sorvall Instruments). The supernatant was filtered through chromatography paper-Whatman (chr 1) and diluted with water to a conductivity of 7-8 mS / cm. The pH was adjusted to 7.5. The extract was then ready for anion exchange chromatography as described below. 1. 3. Hexaose oxidase assay The procedure used was essentially as described by Sullivan and Ikawa, 1973. This test is based on the principle that the hydrogen peroxide formed in the oxidation of sugar in the presence of peroxidase reacts with the orthodontic substance, o-dianisidine for form a dye with absorbance at 402 nm. The test mixture was composed of 1-40 μl of enzyme sample and 850 μl of a test solution containing 370 μl of 0.1 M sodium phosphate buffer, pH 7.0; 462 μl of 0.1 M D-glucose in 0.1 M sodium phosphate buffer, pH 7.0; 9 μl of horseradish peroxidase, 0.1 mg / ml in water (Sigma Chemicals, cat No. P 6782 or Boehringer Mannheim, cat No. 814,393); and 9 μl of o-dianisidine: 2 HCl, 3.0 mg / ml in water (3,3'-dimethoxybenzidine, Sigma Chemicals). After incubation at room temperature for 15 or 30 minutes, the assay was stopped by the addition of one drop of 37% HCl (Merck, p .. a.). 100 μl samples were transferred from the test tubes to the cavities of a microtiter plate (NUNC, Denmark) and the absorbance at 410 nm was read on a Titertek Multiskan II PLUS plate reader (Labsystems / Flow Laboratories, Finland). To ensure that the observed activity was due to hexose oxidase - and not to glucose oxidase - the assay was occasionally performed with D-galactose as the substrate instead of D-glucose. 1. 4. Anion exchange chromatography This step was performed in a BioPilot chromatography system (Pharmacia Biotech, Sweden) connected to a SuperRac fraction collector (LKB-Produkter AB, Sweden). This and the next steps in the purification were performed at room temperature (20-25 ° C), but the fraction collector was placed in a refrigerator so that the collected fractions were stored at 4 ° C until the enzyme assay. Absorbance at 280 nm and conductivity were recorded. The extract was applied on an XK50 / 30 column (Pharmacia, 5.0 x 25 cm) with a bed volume of 500 ml which had been packed with DEAE-Fast Flow Sepharose (Pharmacia) and equilibrated with. buffer A: Tris-Cl 20 mM, pH 7.5. The flow rate was 5 ml / mm during the application of the sample and 10 ml / mm during the subsequent steps of the chromatography. After the application of the sample, the column was washed with 1200 ml of buffer A. The adsorbed proteins were eluted with 2800 ml of a gradient from 0% to 100% buffer B: 20 mM Tris-Cl, 500 mM NaCl, pH 7.5. The fractions of 15 ml were collected during gradient elution. After each chromatographic run the column was regenerated with 500 ml of 0.5 M NaOH, neutralized with 500 ml of 1.0 M Tris-Cl, pH 7.5 and finally equilibrated with 1200 ml of buffer A. The collected fractions were tested with respect to hexose oxidase activity as described above (40 μl sample, 30 min incubation time). The fractions of hexose oxidase activity were combined and stored at 4 ° C. 1. 5. Concentration of fractions containing hexose oxidase activity Several stored fractions of chromatography on DEAE Sepharose were combined and concentrated by ultrafiltration in a system of ultrafiltration cassettes from Millipore Lab (cat # XX420LCSO). The system was equipped with a nominal molecular weight limit (NMWL) membrane cell of 30,000 (cat No. PTTKOLCP2) and was powered by a peristaltic pump. After concentrating at room temperature to about 50 ml, the enzyme preparation was further concentrated to 10-20 ml by centrifugal ultrafiltration at 4 ° C in Centriprep concentrators.

(Amicon, USA, 30,000 nominal molecular weight cut) according to the manufacturer's instructions. The concentrated enzyme solution was stored at 4 ° C. 1. 6. Native polyacrylamide gel electrophoresis (PAGE) The composition of the hexose oxidase preparation obtained by ion exchange chromatography and ultrafiltration was analyzed by native PAGE in a Pharmacia Phast system, see Fig. 2. The gradient gels were run from 8-25% and stained with silver to determine proteins according to the manufacturer's instructions. A kit containing the following molecular weight markers was also obtained from Pharmacia: tireglobulin (669,000); ferritin (440,000); catalase (232,000); lactate dehydrogenase (140,000) and albumin (67,000). Staining to determine hexose oxidase activity was performed as described for glucose oxidase by Sock & Rohringer (1988). In principle, the redox reaction catalyzed by glucose oxidase or hexose oxidase is coupled, with reduction of tetrazolium salt to insoluble, colored formazan. Immediately after electrophoresis / the Phast gel was immersed in 10 ml of freshly prepared staining solution containing: 0.1 M D-glucose (or D-galactose); citric acid / 85 mM sodium phosphate, pH 6.5; 0.2 mg / ml of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl-tetrazolium bromide ("thiazolyl blue", MTT, Sigma Chemicals, cat No. M 2128); and 0.1 mg / ml salt N-methyl-dibenzopyrazine methyl sulfate ("phenazine methosulfate"), PMS, Sigma Chemicals cat. do not. P 9625). The gel was incubated at room temperature in the dark until a blue-violet, colored band was visible (usually 5-90 minutes) and then rinsed in 10% acetic acid, 5% glycerol and air dried. The silver-stained gel is observed in Fig. 2, lane 1. As seen in the figure, numerous proteins were present in this purification step. By staining enzymes, however, only the band marked with an arrow was stained in Fig. 2 (the results are not shown). 1. 7. Gel filtration This step in the purification was carried out in an FPLC system (Pharmacia) equipped with a XK26 / 70 column (2.6 x 66 cm, Pharmacia) with a bed volume of 350 ml. The column was packed with Sephacryl S-200 HR (Pharmacia) according to the manufacturer's instructions. The buffer was 20 mM Tris-Cl, 500 mM NaCl, pH 7.5 and the flow rate was 0.5 ml / min. The UV absorbance 280 nM was recorded. The 2.5 ml fractions were collected with a FRAC-100 fraction collector (Pharmacia) that was placed in a refrigerator (4 ° C) near the FPLC. The concentrated preparation of hexose oxidase was clarified by centrifugation at 30,000 rpm in a 5W60 oscillating cuvette rotor (Beckman) in an ultracentrifuge L7 (Beckman) for 60 min at 4 ° C. An aliquot of 3.0-4.0 ml of the supernatant was mixed with 5% glycerol (Sigma Chemicals, cat # 7757), filtered through a 0.22 μm pore size disposable filter unit (Millipore, cat. SLGV 025 BS) and applied to the column using a sample applicator SA-5 (Pharmacia) connected from the entrance to the column. Fractions showing hexose oxidase activity were identified using the assay method described above (10 μl sample, 15 min incubation time) and stored separately at -18 ° C until further processing. The UV profile and elution position of hexose oxidase is shown in Figure 3. As seen from this figure, a substantial amount of UV absorbing material was removed in this step. Electrophoretic analysis by native PAGE and silver staining (Fig. 2, lane 2) showed that only a few contaminants remained after this step. 1. 8. Determination of the molecular weight of native hexose oxidase by analytical gel filtration The molecular weight of the native hexose oxidase was determined by gel filtration on Sephacryl S-200 Superfine (Pharmacia). The dimensions of the column, buffer, flow and fraction collection were as described above. The blue dextran for determination of the empty volume (v0) of the column and the following standard proteins for column calibration were obtained from Pharmacia: Ovalbumin (43,000), albumin (67,000), catalase (158,000) and aldolase (252,000). A sample containing hexose oxidase was obtained by chromatography on DEAE-Sepharose as described above. Based on the determination of the elution volumes (ve) of the standard proteins and of the hexose oxidase, the corresponding Kav values (ve-v0 / vt-v0) were calculated. Finally, the Kav values of the standard proteins were represented according to the corresponding record values (molecular weight). The Kav of hexose oxidase corresponded to a native molecular weight of approximately 110,000. This is in agreement with Sullivan & Ikawa (1973), who found a molecular weight of approximately 130,000. Kerschensteiner & Klippenstein (1978) reported a molecular weight of 140,000, also determined by gel filtration. 1. 9. Cation exchange chromatography This step was performed in a SMART microarray chromatography system (Pharmacia) equipped with a HRS / 5 column (Pharmacia, 0.5 x 5 cm, 1.0 ml bed volume) packed with Fast Flow S-Sepharose (Pharmacia). The column was equilibrated in buffer A: 50 mM sodium acetate, pH 4.5 (prepared by adjusting 50 mM acetic acid to pH 4.5 with NaOH). Buffer B used for gradient elution contained 50 mM sodium acetate, 500 mM NaCl, pH 4.5. The gel filtration fractions were desalted on prepackaged, prepackaged Sephadex G25 (PD-10, Pharmacia) columns, which were equilibrated and eluted with 25 mM sodium acetate, pH 4.5. Twenty ml of desalted sample derived from 6 fractions of gel filtration with high hexose oxidase activity were applied to the column of a 50 ml Superloop (Pharmacia). at a flow rate of 250 μl / min. The column was then washed with 4 volumes of cushion bed A at the same flow rate. The ligated proteins were eluted with a gradient of buffer A to buffer over 5 ml. The fractions of 250 μl were collected during gradient elution and tested for hexose oxidase activity as described above (1 μl sample, 15 min incubation time) and stored at -18 ° C until further use. The resulting preparation of hexose oxidase was analyzed by native PAGE and silver staining (Fig. 2, lane 3). The hexose oxidase band was now the only significant band, although small amounts of contaminating proteins were also observed. 1. 10. PAGE with analytical sodium dodecylsulfate (SDS-PAGE) S-Sepharose chromatography fractions showing hexose oxidase activity were also analyzed by SDS-PAGE according to Lae mli (1970). Minigeles of 12. 5% acrylamide / bisacrylamide (mixture 37.5: 1) with a thickness of 0.75 mm were run on a Mini-Protean II device (BioRad). The gels were stained with 0.1% Blue Coomassie Brillante R-250, 10% acetic acid, 40% ethanol and decolorized in 10% acetic acid, 30% ethanol. The result of the electrophoresis is shown in Fig. 4, lane 1. The purified preparation of hexose oxidase showed strong bands at relative molecular weights of 40 kD and 29 kD, respectively and weak bands at 60 kD and 25 kD, respectively. In addition, two strong doublet bands at 55 kD and 57 kD were observed. 1. 11. SDS-PAGE followed by transfer and staining to determine carbohydrates The presence of carbohydrate in the hexose oxidase isolated with the Glucan Detection Device DIG (Boehringer Mannheim) was examined, which is designed for the detection of amounts of sugars in micrograms in glucoconjugates in transfers. In principle, the adjacent hydroxyl groups in carbohydrates are oxidized to aldehydes. The digoxigenin is then covalently bound to the aldehyde groups and subsequently detected with an anti-dig-oxygenin-alkaline phosphatase antibody conjugate. The purified hexose oxidase from the cation exchange chromatography was run on a 12% PAGE-SED gel as described above, transferred to nitrocellulose according to standard procedures and stained to determine carbohydrates with the Glycan detection equipment in accordance with the manufacturer's instructions. None of the bands of hexose oxidase at 60 kD, 40 kD, 29 kD and 25 kD was colored. Only the strong doublet band at 57 kD-55 kD was intensely colored (results not shown). The doublet band 57 kD-55 kD was later identified as a residual contaminant as described below. Thus, it could be concluded that none of the hexose oxidase components observed in PAGE-SED were glycosylated. 1. 12. Isoelectric focus The hexose oxidase fractions from S-Sepharose chromatography were combined and concentrated by centrifugal ultrafiltration in Centricon concentrators (Amicon) and analyzed by isoelectric focusing (IEF) on Isogel agarose plates, pH 3-10, according to the instructions of manufacturer (FMC Bioproducts, Rockland, ME, USA). A mixture of pl (FMC Bioproducts) markers was run in parallel with the hexose oxidase samples. The mixture consisted of cytochrome C (pl = 10.2), major / minor band of myoglobin (7.4 / 7.0), carbonic anhydrase (6.1), β-lactoglobulin A / B (5.4 / 5.5), ovalbumin (4.8), glucose oxidase ( 4.2) and amyloglucosidase (3.6). The gels were stained with Coomassie Brilliant Blue R-250. As shown in Figure 5, lane 1, the purified preparation of hexose oxidase was composed of two variants with pl of 4.3 and 4.5, respectively. The purified hexose oxidase was also analyzed by isoelectric focusing on pre-molded polyacrylamide gels, pH 3.5-9.5 (Pharmacia, Ampholine PAG plates) according to the manufacturer's instructions. These gels were stained for enzyme activity by incubation in a dyeing mixture as described above for native polyacrylamide gels. As shown in Figure 5, lane 2, both variants of pl were enzymatically active. 1. 13. Chromatic focusing The observation of several SDS-PAGE bands of hexose oxidase purified in S-Sepharose as the final step aroused the suspicion that one or more bands may represent residual contaminants. In addition, chromatography on S-Sepharose consistently gave low recoveries. Therefore, chromatofocusing was introduced as a final purification step instead of cation exchange chromatography on S-Sepharose. The chromatofocusing was carried out in the "SMART chromatography system equipped with a Mono P HR 5/5 column (0.5 x 5 cm, Pharmacia) with a bed volume of 1 ml and a Superloop of 50 ml for the application of samples. Starting buffer for the separation in the range between pH 5.0 and 3.5 was 25 mM piperazine adjusted to pH 5.5 with HCl The eluent was Polybuffer 74 (Pharmacia) diluted 10 times with water and adjusted to pH 3.5 with HCl. previously and balanced with starting buffer as recommended by the manufacturer, the sample preparation was carried out in the following way: in a typical experiment the best fractions of two runs of gel filtration (fractions 2 x 4, 20 ml) were combined and passed through a 1 ml column of Phenyl Sepharose 6 Fast Flow (high sub, Pharmacia) which had been packaged in a disposable Poly-prep column (Bio-Rad) and equilibrated in the buffer used for gel filtration. (20 mM Tris-Cl, 500 mM NaCl, pH 7.5). This treatment almost completely eliminated the remaining amounts of the red protein phycoerythrin and other colored substances that were adsorbed to the gel matrix at this ionic concentration, and thereby eliminated contaminants that were only partially removed during the other steps of the purification process. The Phenyl Sepharose column was discarded after use. The hexose oxidase activity was recovered quantitatively in the. effluent that was then desalted in disposable, prepackaged Sephadex G-25 columns (PD10, Pharmacia) equilibrated and eluted with starting buffer. Before applying the sample, 1 ml of eluent was pumped onto the column. The flow rate was 0.5 ml / mm. After the application of the sample, the pH gradient was formed by pumping 11 ml of eluent through the column. During the elution of the pH gradient, 44 fractions of 250 μl were collected. Fractions containing hexose oxidase were identified by the assay method described above (1 μl sample, 15 min incubation time) and stored at -18 ° C until further use. The hexose oxidase purified by chromatofocusing was analyzed by native PAGE and silver staining (Fig. 2)., lane 4) by SDS-PAGE and staining with Coomassie Brilliant Blue (Fig. 4, lane 2). In native PAGE the hexose oxidase band was the only significant band, and only very low amounts of contaminants were observed. By SDS-PAGE it was clearly demonstrated that this purification method was able to eliminate the strong double band at 57 kD and 55 kD. The 25 kD band observed after chromatography on S-Sepharose was very weak after chromatofocusing. In conclusion, the hexose oxidase obtained by DEAE chromatography, gel filtration and chromatofocusing showed a band on native PAGE. In SDS-PAGE bands were observed. strong at 40 kD and 29 kD and a weak band at 60 kD. As the intensity of the 60 kD component, with respect to the 40 kD and 29 kD components, varied between the different preparations of the enzyme, it was hypothesized that the 29 kD and 40 kD polypeptides may originate in the proteolytic processing of a precursor of approximately 60 kD. This would agree with the idea of a homodimeric structure of the enzyme with a native molecular weight of 110,000-120,000 as actually found by gel filtration, as described above. In addition, this hypothesis would be consistent with the results obtained by Kerschensteiner and Klippenstein that found a native molecular weight of 140,000 in gel filtration and a subunit molecular weight of 70,800 in SDS-PAGE (Kerschensteiner and Klippenstein, 1978).

EXAMPLE 2 Generation and analysis of amino acid sequence of peptide fragments of hexose oxidase 2. 1. Digestion of purified hexose oxidase with cyanogen bromide This procedure was performed while still using cation exchange chromatography on S-Sepharose as the last purification step. The hexose oxidase obtained by purification in DEAE Sepharose, Sephacryl 3-200 and S-Sepharose were transferred to a volatile buffer by buffer exchange in a pre-packed PC3.2 / 10 fast desalting column containing Superfine Sephadex G-25 (Pharmacia, 0.32 x 10 cm, bed volume 0.8 mi) that was mounted in the SMART system indicated above. The column was equilibrated and eluted with 200 mM ammonium bicarbonate (BDH, AnalaR). To obtain a satisfactory recovery it was necessary to add 500 mM sodium chloride to the sample of hexose oxidase before injection.

The eluted hexose oxidase, with buffer exchange, was distributed in 1.5 ml microcentrifuge tubes and lyophilized in a Speedvac concentrator (Savant Instruments). Cyanogen bromide (CNBr, Pierce), 200 μl of a 10 mg / ml solution in 70% formic acid v / v was added (Promega) (Promega reagents were components of a peptide separation system "Probé DesignMR" cat.

V6030). The tubes were incubated overnight at dark and at room temperature. The solutions were then dried in the speed-vac concentrator, suspended again in 50 μl of water and dried again. 2. 2. Separation of cyanogen bromide fragments by high-resolution SDS-PAGE and membrane electrotransfer of polyvinylidene difluoride (PVDF) The peptides generated by digestion of cyanogen bromide were separated by high resolution PAGE-SED according to Schgüger and von Jagow (1987). This system provides excellent separation of low molecular weight peptides (20-250 amino acid residues). The gel system was composed of a 16.5% separation gel, a 10% spacer gel and a 4% stacking gel, all made using a 29: 1 acrylamide / bisacrylamide mixture from Promega.

The minigels with a thickness of 0.75 mm were run on a Mini-Protean II device (Bio-Rad). Ammonium persulfate and N, N, N ', N' -tetramethyl-ethylenediamine (TEMED) were from Bio-Rad. SDS was from United States Biochemical (ultrapure). Tris was from Fluka (cat # 93350). Tricine and sodium thioglycate were from Promega. Glycine (p.a.), 2-mercaptoethanol (p.a.) and bromophenol blue was from Merck and glycerol from GIBCO BRL (ultrapure). Sodium thioglycolate, 0.1 mM was added to the cathode buffer just before use to avoid chemical blockage of the amino termini of the peptides during separation. The gel was previously run for 60 min at 30 V to allow the thioglycolate to clear any aminorreactive substance. Sample preparation: Dried cyanogen bromide peptide fragments were resuspended in 30 μl of gel loaded buffer containing 63 M Tris-Cl, pH 6.8, SDS 1%, 2.5% 2-mercaptoethanol, 10% glycerol and 0.0012% bromophenol blue. Samples that turned yellow after mixing due to the residual formic acid content were neutralized by the addition of 1-3 μl of 1.0 M Tris base until the blue color was restored. The samples were denatured by heating at 95 ° C for 5 min before application on the gel. A mixture of molecular weight standards of low-range proteins (Promega) with molecular weights between 31,000 and 2,500 were run in parallel with samples of peptides with hexose oxidase activity. The electrophoresis was run at 150 V constant voltage. The electrophoretic transfer to the PVDF membrane was performed in a Mini Trans-Blot electrophoretic transfer cell (Bio-Rad) according to the manufacturer's instructions. Three layers of Problott membrane (Applied Biosystems) cut to size from the gel were briefly wetted in methanol (Merck, p. a.) and then embedded in a transfer buffer (25 mM Tris, 192 mM glycine, pH 8.5, previously cooled to 4 ° C) until the transfer sandwich was set up. After electrophoresis the gel was incubated in transfer buffer for 5 min at 4 ° C and then set up in a transfer sandwich having the following layers: a layer of Whatman paper (3MM chr), two layers of Plobott membrane, the gel of separation of peptides SDS-PAGE, the third layer of Problott, and a final layer of Whatman paper. The sandwich was oriented with the two layers of Problott membrane towards the positive electrode in the set of electrodes. The cooling unit was mounted in the damper chamber before it was filled with a previously cooled transfer buffer and the transfer was then carried out at room temperature for 60 min at 100 V constant voltage. During the transfer the current increased from approximately 270 mA to approximately 400 mA. After transfer the membrane was washed in water for 1 min and then stained for 30-45 sec in 100 ml of freshly prepared staining solution containing 0.1% Coomassie Brilliant Blue R-250 (Bio-Rad), 5% acetic acid (Merck, pa) and 45% methanol (Merck, pa). The membrane was decolorized with 3 changes of approximately 80 ml of freshly prepared 5% acetic acid, 45% methanol for 30-60 sec. each. The membrane was finally washed in 3 water changes to remove the glycine, residual and then dried in air. Well-resolved and relatively abundant bands of molecular weights of approximately 2.5 kD, 9 kD and 16 kD, respectively were removed and subjected to amino acid analysis and sequence analysis. 2. 3. Amino acid analysis and sequencing of a fragment of 9 kD cyanogen bromide of hexose oxidase The amino acid analysis was performed by ion exchange chromatography and post-column derivation with o-phthaldialdehyde. The samples were hydrolyzed at 110 ° C for 20 h in 6 M HCl, 0.05% phenol and 0.05% dithiopropionic acid (Barkholt and Jensen, 1989). The peptides were sequenced in an automated protein / peptide sequencer from Applied Biosystems, model 477A, equipped with on-line PTH analyzer, model 120A and data analysis system. Protein sequencing reagents were obtained from Applied Biosystems. The analysis of the amino acids and the analysis of the sequence of peptides was carried out by Arne L. Jensen, Department of Protein Chemistry, University of Copenhagen, Denmark. The sequence of peptides identified by analysis of the 9 kD fragment is shown in Table 2.1. The initial yield of phenylthiohydantoin-. tyrosine (PTH-Tyr) in step one was 22 pmol. The amino acid composition of the 9 kD fragment is shown in Table 2.2.

Table 2.1 Peptide sequence obtained by sequence analysis of a fragment of 9 kD cyanogen bromide of hexose oxidase Areviatures: Y = Tyr; E = Glu; P = Pro; G = Gly; V = Val Table 2.2. Amino acid composition of a 9 kD cyanogen bromide fragment Not determined 2.4. Preparative SDS-PAGE and membrane electrotransfer PVDF The following procedure was performed to obtain amino acid sequences that were specifically known to come from either the 40 kD or the 29 kD polypeptide of the hexose oxidase preparation. The preparative SDS-PAGE gels were run according to Laemmli (Laemmli, GB, 1970). The minigels containing 12.5% acrylamide / bisacrylamide (mixture 37.5: 1) with a thickness of 0.75 mm were run on a Mini- * Protean II (Bio-Rad) apparatus. The solution of acrylamide (BDH, cat # 44313) and N, N '-methylene-bis-acrylamide (BDH, cat # 44300) was stored on a mixed-bed ion exchange resin (Bio-Rad, cat No. 142-6425). The sources of all the other reagents were as described above. Sample preparation: chromatofocusing fractions were concentrated by centrifugal ultrafiltration at 4 ° C in Ultrafree-MC filter units with NMWL 10,000 and a sample capacity of 400 μl (Millipore, cat # UFC3 LGC25). The retained substance was mixed with a volume of gel that loaded 2X buffer containing 125 mM tris-Cl, pH 6.8, 2% SDS, 5% 2-mercaptoethanol, 20% glycerol and 0.0025% bromophenol blue. Samples that turned yellow after mixing, due to the content of acidic Polybuffer components, were neutralized by the addition of 1-3 μl of 1.0 M Tris base, until the blue color was restored. The samples were denatured by heating at 95 ° C for 5 min and applied in the gel in aliquots of approximately 30 μl per lane. A mixture of molecular weight marker proteins (Bio-Rad) with molecular weights ranging from 97,400 and 14,400 were run in parallel with the hexose oxidase samples. The electrophoresis was run at low current, 10 mA per gel, to minimize the risk of chemical modification, thermally induced, of the sample proteins. The electrophoretic transfer to the PVDF membrane was performed as described above, except that an Immobilon P membrane layer (Millipore, cat No. IPVH 15150) was used instead of three layers of Problott. The sandwich was oriented with the transfer membrane towards the positive electrode in the set of electrodes. After transfer the Immobilon P membrane was rinsed in water for 10 sec and then stained for 45-60 sec in 100 ml of freshly prepared staining solution containing 0.025% Coomassie Brilliant Blue R-250, 5% acetic acid and 40% methanol. The membrane was bleached for 2-3 min in 250 ml of freshly prepared 5% acetic acid, 30% ethanol (96% v / v, Danisco, Denmark). The membrane was finally air dried and stored at 4 ° C. The band pattern in the blot was identical to the model observed in the analytical SDS-PAGE after the final purification by chromatoforming. It showed strong bands 40 kD and 29 kD, in addition to a weak band at 60 kD. The bands at 40 kD and 29 kD were removed from the transfer and used for amino acid analysis and for enzymatic digestion of membrane-bound polypeptides, as described below. The amount of 60 K material was too low to allow any further analysis of this polypeptide. 2. 5. Amino acid analysis of 40 kD and 29 kD polypeptides of hexose oxidase The amino acid compositions of the 40 kD and 29 kD components of hexose oxidase are presented in Table 2.3. 2. 6. Enzymatic digestion of hexose oxidase polypeptides linked to PVDF Digestion of hexose oxidase polypeptides linked to PVDF and extraction of the resulting proteolytic peptides was performed as described by Fernandez et al. (1992) and Fernandez et al. (1994). Digestion of the 40 kD polypeptide of hexose oxidase: eleven bands of 40 K were removed with an estimated total protein content of approximately 5 μg (corresponding to approximately 125 pmol) of the membrane ' PVDF stained with Coomassie blue, decolorized in methanol for 1-2 min and rinsed in water for 2-3 min. The membrane bands were cut into 1 x 1 mm pieces and transferred to microcentrifuge tubes. A control region of the PVDF membrane served as a base control. The cube membrane pieces were embedded in 50 μl of digestion buffer containing 1% (v / v) Hydrogenized Triton X-100 (RTX-100, Sigma Chemicals, Cat. No. X-100R-PC, or Calbiochem, protein grade, cat No. 648464), 10% acetonitrile (Merck, Lichrosolv gradient grade) and 100 mM Tris-Cl, pH 8.0. The proteolytic enzyme selected for digestion was endoproteinase Lys-C (endoLys-C) that cleaves peptide chains on the C-terminal side of the lysine residues. An aliquot of 5 μg of endoLys-C (Boehringer Mannheim, sequencing grade, cat # 1047 825) was reconstituted by the addition of 20 μl of water. Two μl of enzyme solution corresponding to 0.5 μg (enzyme ratio: substrate 1:10) was added. Digestion was performed at 37 ° C for 22-24 h. After digestion the samples were sonicated in an ultrasonic tank (Elma transonic) for 5 min and centrifuged at 1700 rpm in a microcentrifuge for 5 min, and the supernatant was then transferred to a new tube. Consecutive washes with 50 μl of digestion buffer and 100 μl of 0.1% trifluoroacetic acid (TFA, Pierce, cat # 28902) were performed with sonication and centrifugation as described above. All supernatants were combined, resulting in an extract volume of 200 μl. The extracts were maintained at -18 ° C until the peptide was purified. Digestion of the 29 kD hexose oxidase polypeptide was performed as described for the 40 kD component, except that four bands with a total protein content of 2.4 μg (approximately 80 pmoles) were used, according to the amino acid analysis . 2. 7. Purification of peptides generated with endoLvs-C Peptide fragments obtained by digestion of 40 kD and 29 kD polypeptides were separated in the SMART chromatography system. The system was equipped with a μPeak variable wavelength monitor and a fraction collector for 60 ampoules. The reverse phase column used for the separation was a narrow orifice column μRPC C2 / C18 SC2.1 / 120 based on silica (Pharmacia, column dimensions 2.1 x 100 mm, particle size 3 μm, average pore size 125 A ). The buffers were A: 0.1% TFA (Pierce) in Milli-Q water and B: 0.1% TFA in acetonitrile (Merck, Lichrosolv gradient grade). The dampers were filtered and degassed by vacuum filtration on a 0.5 μm fluoroporous filter (Millipore, cat # FHLP04700). The flow rate was 100 μl / min. The UV absorbance in the effluent was monitored at 220 nm, 254 nm and 280 nm. The gradient was 0-30% B (0-65 min), 30-60% B (65-95 min) and 60-80% B (95-105 min). The column was then washed at 80% B for 10 min at 100 μl / min. The 50 μl fractions were collected between t = 15 min and t = 105 min (3 x 60 fractions) and stored at -18 ° C until analysis of the amino acid sequence.

The peptide map obtained after digestion of endoLys-C from the 40 kD polypeptide is shown in Fig. 6. As seen in this figure, digestion and separation in CLAP resulted in several well-resolved peaks with a high signal to noise ratio. A corresponding chromatogram of a control digestion mixture (not shown) indicated that peaks eluting after t = 83 min were non-peptides, peaks derived from reagents, possibly UV absorbing contaminants from hydrogenated Triton X-100 or traces residuals of Coomassie dye. The peaks marked 1-5. in Fig. 6 were selected for amino acid sequencing according to the following criteria: 1) Peak height; 2) Apparent purity; 3) High A28o ratio: A220 and / or high A254: A220 ratio indicating the presence of aromatic amino acid residues, which are very useful for the selection of PCR primer sequences due to their low degeneracy of genetic code; 4) Late elution time, which may indicate a relatively long peptide. The chromatogram of the 29 kD endoLys-C peptides is shown in Fig. 7. Obviously, this hexose oxidase component gave rise to only a few significant peptide fragments compared to the 40 kD component in Fig. 6. When the chromatograms were compared, there was no indication that there was a peptide fragment in both digested substances. This finding suggests that the 40 K and 29 K hexose oxidase components do not have amino acid sequences in common, which would have been the case if the 29 kD chain was generated by proteolytic conversion of the 40 kD polypeptide. (Compared with the digested component of 40 kD, the digested component of 29 kD contained only small amounts of pollutants that eluted later than t = 83 min.) The reason for this may be that the hydrogenated Triton X-100 was used. of Calbiochem for the 29 kD digestion, while the 40 kD digestion was performed with Triton, X-100, from Sigma Chemicals). The fractions corresponding to the peaks marked 1 and 2 in the 49 kD peptide map (Fig. 7) were subjected to amino acid sequencing. 2. 8. Analysis of amino acid sequences of hexose oxidase peptides generated proteolytically The peptide sequences identified by analysis of fractions corresponding to peaks 1-5 in Fig. 6 (peptides HOX-2, HOX-3, HOX-4, HOX-5 and HOX-6) and peaks 1-2 in the Fig. 7 (HOX-7 and HOX-8 peptides) are presented in Table 2.4 below. The initial yields of the PTH amino acids ranged from 46 pmoles of PTH-Tyr in step one in the HOX-5 peptide and 6 pmoles of PTH-Ile in step two in the HOX-8 peptide. As expected from the absorbances at 254 nm and 280 nm, respectively, of the selected peaks all the sequenced peptides contained at least one aromatic amino acid residue.

Table 2.4. Sequences of peptides obtained by peptide sequence analysis of endoproteinase Lys-C derived from hexose oxidase polypeptides of 40 kD and 29 kD Origin of Sequence Identification of amino acid sequence sequence 40K peptides, peak 1 HOX-2 peptide A-I-I-N-V-T-G-L-V-E-S-G-Y-D- -X11-X2) -X3) -G-Y-X-V-S-S 40K, peak 2 HOX-3 peptide D-L-P-M-S-P-R-G-V-I-A-S-N-L-W-F 40K, peak 3 HOX- 4 peptide D-S-E-G-N-D-G-E-L-F-X-A- (H) - -T 40K, peak 4 HOX-5 peptide Y-Y-F-K 40K, peak 5 HOX- 6 peptide D-P-G- Y- I -V- I -D-V-N-A-G-T-P- -D 29K, peak 1 HOX- 7 peptide L-Q-Y-Q-T-Y-W-Q- (E) - (E) - (D) -29K, peak 2 HOX- 8 peptide X-I- (R) -D- F-Y-E-E-M- The tentatively identified residues are shown in parentheses. 1) Residue no. 15 was identified as Asp or Asn. 2) Residue no. 16 was identified as Asp or Ala. 3) Residue no. 17 was identified as Arg or Trp.

HOX-2 peptide = SEQ ID NO: 9 HOX-3 peptide = SEQ ID NO: 10 HOX-4 peptide = SEQ ID NO: 11 HOX-5 peptide = SEQ ID NO: 12 HOX-6 peptide = SEQ ID NO: 13 HOX-7 peptide = SEQ ID NO: 14 HOX-8 peptide = SEQ ID NO: 15 EXAMPLE 3 Isolated hexose oxidase gene from Chondrus crispus 3. 1. Purification of Chondrus crispus RNA Freshly collected fronds of Chondrus críspus were rinsed with cold water and stored immediately in liquid nitrogen until further use. Approximately 15 grams of the stem of Chondrus crispus frozen in liquid nitrogen were homogenized to a fine powder in a mortar. The frozen homogenized material was transferred to a 50 ml tube (Nunc, cat # 339497) containing 15 ml of extraction buffer (8M guanidinium hydrochloride; mM 2- (N-morpholino) ethanesulfonic acid (MES), pH 7.0; 20 mM ethylenediaminetetraacetic acid (EDTA); 50 mM β-mercaptoethanol). The tube was vortexed and kept cool (0 ° C) during the following steps unless other temperatures are indicated. The tube was then centrifuged for 20 minutes at 6,000 x g in a Heraeus Omnifuge 2.0RS and the supernatant containing RNA (approximately 15 ml) was carefully collected and transferred to a pre-cooled 15 ml tube. 1.5 ml of 2 M sodium acetate, pH 4.25, 15 ml of phenol saturated with water and 3 ml of chloroform: isoamyl alcohol (49: 1) were added to the tube containing the RNA extract. The tube was vortexed subsequently vigorously for 1/2 minute and the phases were separated by centrifuging the tube for 20 minutes in an Omnifuge at 6,000 x g. The aqueous phase (ca. 17 ml) was transferred to a 30 ml Corex tube (Sorvall, cat.no. 00156) and an equal volume (i.e., about 17 ml) of cold isopropanol was added. The tube was vortexed again and incubated for at least 1 hour at -20 ° C. The precipitated RNA was pelleted by centrifugation for 20 minutes at 10,000 rpm using a Sorvall RC-5B centrifuge equipped with a previously cooled SS34 rotor. The supernatant was discarded and the pellet RNA was suspended again in 4 ml of 0.3 M sodium acetate, pH 5.5 and 12 ml of 96% ethanol were added. The Corex tube was then vortexed and incubated again for at least 1 hour at -20 ° C followed by a second RNA pellet transformation by centrifugation for 20 minutes as described above. The supernatant was carefully discarded and the RNA pellets suspended again in 2 ml of 0.15 M sodium acetate., pH 5.5. Then 8 ml of 4M sodium acetate, pH 5.5, was added, and the RNA was precipitated on ice for 30 minutes and pelletized again, as described above. The RNA pellet was washed in 70% ethanol and suspended again in 500 μl of water. The RNA suspended again was transferred to a microcentrifuge tube and stored at -20 ° C until further use. The purity and concentration of the RNA were analyzed by agarose gel electrophoresis and by absorption measurements at 260 nm and 280 nm as described in Sambrook et al. (1989). 3. 2. Isolation of polyadenylated RNA from Chondrus crispus Polyadenylated RNA was isolated from total RNA using magnetic beads containing oligo dT (Dynabeads (R) Oligo (dT) 25, in the mRNA Purification Kit, mRNA, Dynal). Approximately 100 μg of total RNA was mixed with 1 mg of Dynabeads (R) Oligo (dT) 25 and polyadenylated RNA was isolated as described in the protocol for the mRNA Purification Kit. The yield of polyadenylated RNA isolated with Dynabeads (R) was between 1 and 3%. Other methods were used in the isolation of polyadenylated RNA from Chondrus cri spus including the use of columns packed with oligo- (dT) -cellulose (Clontech, cat # 8832-2) or pre-packaged columns (Separator Equipment1 of mRNA, Clontech, cat.No.K1040-1) as described in the protocol for Separator Kit ™ mRNA. The yield of polyadenylated RNA isolated from the oligo- (dT) columns was between 0.1 and 1% of the initial total RNA. The polyadenylated RNA isolated on oligo- (dT) columns was used in cDNA synthesis reactions as described above (3.4) but the yield of the first-strand cDNA was very low (less than 1%). The reason for the poor performance and poorer performance of RNA isolated in oligo- (dT) columns compared to RNA purified in Dynabeads (R) could be the presence of carbohydrates or proteoglycans in the total RNA extract. The carbohydrates that contaminated the total RNA preparations have been shown to impede the purification of polyadenylated RNA and to inhibit cDNA synthesis and therefore methods for the purification of carbohydrate-free RNA have been developed (Groppe et al., 1993; et al.). However, polyadenylated RNA purified by these methods was not as effective in cDNA synthesis reactions as the RNA isolated with Dynabeads (R). Accordingly, the polyadenylated RNA purified using Dynabeads (R> was used as a template in the first strand cDNA synthesis reactions (see 3.4 below). 3. 3. Hexose oxidase-specific oligonucleotides Synthetic oligonucleotides- (DNA technology, ApS, Forskerparken, DK-8000 Aarhus C, Denmark) were synthesized based on the amino acid sequences derived from the hexose oxidase peptides HOX-2, HOX-3 and HOX-4 (Table 2.4). Table 3.1 shows the oligonucleotides and their corresponding amino acid sequences. Also shown in Table 3.1 is the DNA sequence of the primers used in DNA sequencing or PCR.

Table 3.1. Synthetic oligonucleotide nucleotide sequences specific for hexose oxidase When Y is C or T, R is A or G; when W is A or T, 5 is C or G; when D is A, G or T, N is A, C, G or T and I = deoxy Inosine.

Hox-2 = SEQ ID NO: 9 Hox2-3 + = SEQ ID NO: 16 Hox-3 = SEQ ID NO: 10 Hox3-2- = SEQ ID NO: 17 Hox-4 = SEQ ID NO: 11 Hox4-l + = SEQ ID NO: 18 Hox-4-2- = SEQ ID NO: 19 HOX5 + = SEQ ID NO: 20 Hox5- = SEQ ID NO: 21 Hox6 + = SEQ ID NO: 22 Hox7- = SEQ ID NO: 23 Hox8- = SEQ ID NO: 24 HoxlO- = SEQ ID NO: 25 Hoxll + = SEQ ID NO: 26 Hoxl2- = SEQ ID NO: 27 Hoxl3- = SEQ ID NO: 28 Hox5'-l = SEQ ID NO: 29 3.4. Synthesis of APNc and polymerase chain reaction (PCR) Polyadenylated RNA was used as a template in the first-strand cDNA synthesis reactions with commercially available equipment. Approximately 1 μg of polyadenylated RNA was reverse transcribed as described in the protocol for the MaratonNR cDNA amplification kit (Clontech, cat # K1802-1) with Hox3-2- or Hox4-2- as primers. In the subsequent PCR amplification the anchor primer or adapter of the kit was also used * of the specific hexose oxidase primers Hox3-2- or Hox4-2-, respectively. The buffers used and the conditions for amplification were essentially as described in the protocol for the MaratonNR cDNA amplification kit. PCR amplification was performed with AmpliTaq (Perkin-Elmer Cetus) using a Perkin-Elmer Thermalcycler 480MR programmed at 30 cycles at 1 min at 94 ° C, 2 min at 55 ° C and 2 min at 72 ° C. Gel electrophoresis of 5 μl of the reaction mixture on a 1% agarose gel (Seaplaque (R) GTG, FMC) showed DNA fragments with approximate sizes of 600 base pairs (bp) with the primer Hox4-2- and 700 bp with the Hox3-2- primer. These DNA fragments were purified from the agarose gel using commercially available equipment (QIAEXNR gel extraction equipment, Cat. No. 20020, QIAGEN) and approximately 100 ng of fragment were ligated to 50 ng of plasmid pT7 Blue as it was described in the protocol for the T-Vector team of pT7 Blue (Novagen, cat.no. 69829-1). Escherichia coli DH5a (Life Technologies, cat # 530-82585A) or E. coil NovaBlue (Novagen) were transformed with the ligation mixture and recombinant, white colonies were further analyzed. Plasmid DNA from such colonies was purified using the Midi kit of QIAGEN plasmids (QIAGEN, cat # 12143) and subjected to DNA sequence analysis using "Sequenase (Sequenase DNA sequencing kit, Version 2.0, USB). of DNA sequencing were subjected to acrylamide gel electrophoresis (Gel-Mix (R) 6 sequencing, Life Technologies.) The DNA sequence analysis of the 700 bp fragment showed an open reading frame with a coding capacity of 234 amino acids Table 3.2 below shows that all peptide sequences of the 40 kD polypeptide, ie HOX-2, HOX-3, HOX-4, HOX-5 and HOX-6, were found in the sequence of 234 amino acids derived from this open reading frame Thus, it was concluded that the 700 bp fragment encoded part of the hexose oxidase gene The DNA sequence of the 600 bp fragment proved to be identical to that of 600 bp proximal of the 700 fragment bp (see Table 3 .2) The Hox2-3 + and Hox3-2- primers were used similarly in cDNA synthesis and PCR amplification experiments. Approximately 50 ng of polyadenylated RNA were reverse transcribed with Hox3-2-as primer as described in the protocol for the RACE 3'-AmplifinderNR kit (Clontech, cat.P.K1801-1). In the subsequent PCR amplification, the primers Hox2-3 + and Hox3-2- were used. The buffers used and the conditions for amplification were essentially as described-for the AmpliTaq polymerase (Perkin-Elmer Cetus) and in the protocol for the RACE 3 '-Amplifinder ™. Gel electrophoresis of 5 μl of the PCR amplification mixture showed a fragment with a size of 407 bp. This fragment was purified, inserted into the pT7 Blue plasmid and sequenced as described above. The DNA sequence of this fragment was shown to be identical to that of the distal 407 bp of the 700 bp fragment. The DNA sequence downstream of the 700 and 407 bp fragments was amplified with the RACE 3 '-Amplifinder ™ (Clontech) equipment using the anchor primer of the kit as the 3' primer and the hexose specific primers HoxS + and Hox4 + as primers 5 'specific genes. The buffers and conditions for amplification were as described above. PCR and analysis of the reaction mixture on agarose gels showed a fragment with the size of approximately 1.3 kb. The fragment was isolated and subjected to DNA sequence analysis as described above. The DNA sequence of this 1.3 kb fragment showed an open reading frame of 357 amino acids. This reading frame of 357 amino acids contained the amino acid sequences of the HOX-1, HOX-3, HOX-4, HOX-5, HOX-7 and HOX-8 peptides. Therefore, it was concluded that the 1.3 kb DNA fragment encoded the 9 kD CNBr fragment, the 29 kD polypeptide and part of the 40 kD polypeptide of hexose oxidase. A specific primer for the 5 'end of hexose oxidase, Hox5'-1, was used together with an oligo- (dT) primer to amplify the open reading frame of putative complete hexose oxidase. The gene was amplified using PCR, inserted in pT7 Blue and sequenced as described above. The DNA sequence of this 1.8 kb fragment was identical to the DNA sequences of the fragments described above with minor differences. As these differences could be caused by misincorporations during PCR amplifications, the entire hexose oxidase gene was amplified and isolated from at least three independent PCR amplifications. Therefore, the DNA sequence presented in Table 3.2 presented below is composed of at least three independently derived DNA sequences to exclude PCR errors in the sequence. The amino acid sequence derived from the open reading frame in the 1.8 kb DNA sequence has been shown to contain all the HOX peptides indicated above, namely HOX-1 to HOX-8. Accordingly, the 1.8 kb DNA sequence encodes the hexose oxidase fragments derived from Chondrus crispus of 9 kD, 29 kD and 40 kD indicated above. The molecular weight of this derived open reading frame polypeptide agrees with the <; presumption that the polypeptide is a subunit (possibly a monomer fragment) of a dimeric hexose oxidase enzyme molecule. 3. 5. Northern blot analysis of Chondrus crispus RNA Total RNA isolated from Chondrus crispus was subjected to Northern blot analysis. The RNA was purified as described above (3.1) and fractionated on a denatured formaldehyde agarose gel and transferred to a HybondC filter (Amersham) as described by Sambrook et al. (1989). Using the primers Hox2-3 + and Hox3-2- a 400 bp DNA fragment was synthesized by PCR as described above (3.4). This fragment was purified from a 1.2% agarose gel (SeaPlaque (R) GTG, FMC) and labeled with 32 P as described by Sambrook et al. (see above). This specific hybridization probe for radioactive hexose oxidase was used for probing in the Northern blot. The conditions for hybridization were: 3.5.1. Prehybridization at 65 ° C for two hours in a buffer containing 10 x Denhardt's solution (0.1% Ficoll, 0.1% polyvinylpyrrolidone, 0.1% bovine serum albumin), 2 x SSC (lx SSC is 0.15 M sodium chloride, sodium citrate 0.015 M, pH 7.0), 0.1% sodium dodecyl sulfate. (SDS), and 50 μg / ml of denatured salmon sperm DNA. 3.5.2. Hybridization at 65 ° C for at least 14 hours in a buffer containing 1 x Denhardt's solution, 2 x SSC, 0.1% dextran sulfate, 50 μg / ml denatured salmon sperm DNA, and 32 P-labeled probe ( approximately 106 dp / ml). The filter was washed twice at 65 ° C for 10 min in 2 x SSC, 0.1% SDS followed by two washes at 65 ° C for 10 min in 1 x SSC, 0.1% SDS. After the final wash for 10 minutes at 65 ° C in 0.2 x SSC, 0.1% SDS, the filter was wrapped in Saran Wrap and exposed to an X-ray film (Kodak XAR2) for two days at -80 ° C using a screen intensifier Siemens-Titan HS. The resulting autoradiogram (Fig. 8) shows that a band with the approximate size of 2 kb was illuminated.

Table 3.2. Nucleotide sequence of the 1.8 kb APN sequence and the open reading frame for a hexose oxidase amino acid sequence of 546 amino acids derived from the APN sequence TGAATTCGTG GGTCGAAGAG CCCTTTGCCT CGTCTCTCTG GTACCGTGTA TGTCAAAGGT 60 TCGCTTGCAC ACTGAACTTC ACG ATG GCT ACT CTT CCT CAG AAA GAC CCC 110 Met Wing Thr Leu Pro Gln Lys Asp Pro 1 5 GGT TAT ATT GTA ATT GAT GTC AAC GCG GGC ACC GCG GAC AAG CCG GAC 158 Gly Tyr He Val He Asp Val Asn Wing Gly Thr Wing Asp Lys Pro Asp 10 15 20 25 CCA CGT CTC CCC TCC ATG AAG CAG GGC TTC AAC CGC CGC TGG ATT GGA 206 Pro Arg Leu Pro Ser Met Lys Gln Gly Phe Asn Arg Arg Trp He Gly 30 35 40 ACT AAT ATC GAT TTC GTT TAT GTC GTG TAC ACT CCT CA GGT GCT TGT 254 Thr Asn He Asp Phe Val Tyr Val Val Tyr Thr Pro Gln Gly Ala Cys 45 50 55 ACT GCA CTT GAC CGT GCT ATG GAA AAG TGT TCT CCC GGT ACA GTC AGG 302 Thr Ala Leu Asp Arg Ala Met Glu Lys Cys Ser Pro Gly Thr Val Arg 60 65 70 ATC GTC TCT GGC GGC CAT TGC TAC GAG GAC TTC GTA TTT GAC GAA TGC 350 He Val Ser Gly Gly His Cys Tyr Glu Asp Phe Val Phe Asp Glu Cys 75 80 85 GTC AAG GCC ATC ATC AAC GTC ACT GGT CTC GTT GAG AGT GGT TAT GAC 398 Val Lys Wing He He Asn Val Thr Gly Leu Val Glu Ser Gly Tyr Asp 90 95 100 105 GAC GAT AGG GGT TAC TTC GTC AGC AGT GGA GAT ACA AAT TGG GGC TCC 446 Asp Asp Arg Gly Tyr Phe Val Ser Ser Gly Asp Thr Asn Trp Gly Ser 110 115 120 TTC AAG ACC TTG TTC AGA GAC CAC GGA AGA GTT CTT CCC GGG GGT TCC 494 Phe Lys Thr Leu Phe Arg Asp His Gly Arg Val Leu Pro Gly Gly Ser 125 130 135 TGC TAC TCC GTC GGC CTC GGT GGC CAC ATT GTC GGC GGA GGT GAC GGC 542 Cys Tyr Ser Val Gly Leu Gly Gly His He Val Gly Gly Gly Asp Gly 140 145 150 ATT TTG GCC CGC TTG CAT GGC CTC CCC GTC GAT TGG CTC AGC GGC GTG 590 He Leu Wing Arg Leu His Gly Leu Pro Val Asp Trp Leu Ser Gly Val 155 160 165 GAG GTC GTC GTT AAG CCA GTC CTC ACC GAA GAC TCG GTA CTC AAG TAT 638 Glu Val Val Val Lys Pro Val Leu Thr Glu Asp Ser Val Leu Lys Tyr 170 175 180 185 GTG CAC AAA GAT TCC GAA GGC AAC GAC GGG GAG CTC TTT TGG GCA CAC 686 Val His Lys Asp Ser Glu Gly Asn Asp Gly Glu Leu Phe Trp Wing His 190 195 200 ACA GGT GGC GGC GGC GCA AAC TTT GGA ATC ATC ACC AAA TAC TAC TTC 734 Thr Gly Gly Gly Gly Asn Phe Gly He He Thr Lys Tyr Tyr Phe 205 210 215 AAG GAT TTG CCC ATG TCT CCA CGG GGC GTC ATC GCA TCA AAT TTA CAC 782 Lys Asp Leu Pro Met Ser Pro Arg Gly Val He Wing Ser Asn Leu His 220 225 230 TTC AGC TGG GAC GGT TTC ACG AGA GAT GCC TTG CAG GAT TTG TTG ACA 830 Phe Ser Trp Asp Gly Phe Thr Arg Asp Ala Leu Gln Asp Leu Leu Thr 235 240 245 AAG TAC TTC AAA CTT GCC AGA TGT GAT TGG AAG AAT ACG GTT GGC AAG 878 Lys Tyr Phe Lys Leu Wing Arg Cys Asp Trp Lys Asn Thr Val Gly Lys 250 255 260 265 TTT CAA ATC TTC CAT CAG GCA GCG GAA GAG TTT GTC ATG TAC TTG TAT 926 Phe Gln He Phe His Gln Wing Wing Glu Glu Phe Val Met Tyr Leu Tyr 270 275 280 ACA TCC TAC TCG AAC GAC GCC GCC CGC GAA GTT GCC CAAC GAC CGT CAC 974 Thr Ser Tyr Ser Asn Asp Wing Glu Arg Glu Val Wing Gln Asp Arg His 285 290 295 TAT CAT TTG GAG GAC GAC ATA GAA CAG ATC TAC AAA ACA TGC GAG CCC 1022 Tyr His Leu Glu Wing Asp He Glu Gln He Tyr Lys Thr Cys Glu Pro 300 305 310 ACC AAA GCG CTT GGC GGG CAT GCT GGG TGG GCG CCG TTC CCC GTG CGG 1070 Thr Lys Ala Leu Gly Gly His Ala Gly Trp Ala Pro Phe Pro Val Arg 315 320 325 CCG CGC AAG AGG CAC ACA TCC AAG ACG TCG TAT ATG CAT GAC GAG ACG 1118 Pro Arg Lys Arg His Thr Ser Lys Thr Ser Tyr Met His Asp Glu Thr 330 335 340 345 ATG GAC TAC CCC TTC TAC GCG CTC ACT GAG ACG ATC AAC GGC TCC GGG 1166 Met Asp Tyr Pro Phe Tyr Wing Leu Thr Glu Thr He Asn Gly Ser Gly 350 355 360 CCG AAT CAG CGC GGC AAG TAC AAG TCT GCG TAC ATG ATC AAG GAT TTC 1214 Pro Asn Gln Arg Gly Lys Tyr Lys Ser Wing Tyr Met He Lys Asp Phe 365 370 375 CCG GAT TTC CAG ATC GAC GTG ATC TGG AAA TAC CTT ACG GAG GTC CCG 1262 Pro Asp Phe Gln He Asp Val He Trp Lys Tyr Leu Thr Glu Val Pro 380 385 390 GAC GGC TTG ACT AGT GCC GAA ATG AAG GAT GCC TTA CTC CAG GTG GAC 1310 Asp Gly Leu Thr Ser Wing Glu Met Lys Asp Ala Leu Leu Gln Val Asp 395 400 405 ATG TTT GGT GGT GAG ATT CAC AAG GTG GTC TGG GAT GCG ACG GTC GTC 1358 Met Phe Gly Gly Glu He His Lys Val Val Trp Asp Ala Thr Ala Val 410 415 420 425 GCG CAG CGC GAG TAC ATC ATC AAA CTG CAG TAC CAG ACA TAC TGG CAG 1406 Wing Gln Arg Glu Tyr He He Lys Leu Gln Tyr Gln Thr Tyr Trp Gln 430 435 440 GAA GAA GAC AAG GAT GCA GTG AAC CTC AAG TGG ATT AGA GAC TTT TAC 1454 Glu Glu Glu Asp Lys Asp Wing Val Asn Leu Lys Trp He Arg Asp Phe Tyr 445 450 455 GAG GAG ATG TAT GAG CCG TAT GGC GGG GTT CCA GAC CCC AAC ACG CAG 1502 Glu Glu Met Tyr Glu Pro Tyr Gly Gly Val Pro Asp Pro Asn Thr Gln 460 465 470 GTG GAG AGT GGT AAA GGT GTG TTT GAG GGA TGC TAC TTC AAC TAC CCG 1550 Val Glu Ser Gly Lys Gly Val Phe Glu Gly Cys Tyr Phe Asn Tyr Pro 475 480 485 GAT GTG GAC TTG AAC AAC TGG AAG AAC GGC AAG TAT GGT GCC CTC GAA 1598 Asp Val Asp Leu Asn Asn Trp Lys Asn Gly Lys Tyr Gly Ala Leu Glu 490 495 500 505 CTT TAC TTT TTG GGT AAC CTG AAC CGC CTC ATC AAG GCC AAA TGG TTG 1646 Leu Tyr Phe Leu Gly Asn Leu Asn Arg Leu He Lys Wing Lys Trp Leu 510 515 520 TGG GAT CCC AAC GAG ATC TTC ACA AAC AAA CAG AGC ATC CCT ACT AAA 1694 Trp Asp Pro Asn Glu He Phe Thr Asn Lys Gln Ser He Pro Thr Lys 525 530 535 CCT CTT AAG GAG CCC AAG CAG ACG AAA TAGTAGGTCA CAATTAGTCA 1741 Pro Leu Lys Glu Pro Lys Gln Thr Lys 540 545 TCGACTGAAG TGCAGCACTT GTCGGATACG GCGTGATGGT TGCTTTTTAT AAACTTGGTA 1801 In the amino acid sequence shown in Table 3 .2 the HOX-1 to HOX-8 peptides are shown with bold or underlined codes. Bold codes indicate amino residues that have been confirmed by amino acid sequencing of peptides. The underlined codes indicate amino acid residues that are derived from the nucleotide sequence, but that have not been confirmed by sequencing of relevant HOX peptides.

HOX-1 are amino acid residues 461-468, HOX-2 residues 92-114, HOX-3 residues 219-234, HOX-4 residues 189-202, HOX-5 residues 215-218, HOX-6 residues 8- 22, HOX-7 residues 434-444 and HOX-8 residues 452-460.

EXAMPLE 4 Production of recombinant hexose oxidase in Pichia pastoris 4. 1. Construction of a vector for the expression of recombinant hexose oxidase in Pichia pastoris The open reading frame that encodes the hexose oxidase of Chondrus crispus as shown in Table 3.2. was inserted into an expression vector of Pichia pastoris, pPIC3 (Research Corporation Technologies, Inc., Tucson, Aricarril 85711-3335). The plasmid contains the alcohol oxidase promoter (aoxl promoter) and the transcriptional termination signal of Pichia pastoris (in Fig. 9)., aoxp and aoxt, respectively). A his4 + gene in the vector enables the selection of recombinant Pi + chia pastoris His + cells. When this cassette of expression is transformed into Pichia pastoris it is integrated into the chromosomal DNA. Pichia pastoris cells harboring an expression cassette with a hexose oxidase gene from Chondrus crispus inserted downstream of the aoxl promoter can be induced to produce hexose oxidase by the addition of the inducer of the aoxyl promoter, methanol. A mutant of Pichia pastoris, KM71, which is defective in the main alcohol oxidase gene, aoxl, can be used as a hexose oxidase gene receptor (Cregg and Madden, 1987; Tschopp et al., 1987). However, Pichia pastoris contains another alcohol oxidase gene, aox2, which can also be induced by methanol. Thus, recombinant Pichia pastoris with a hexose expression cassette will produce two oxidases, hexose oxidase and alcohol oxidase, after the addition of methanol. Prior to the insertion of the hexose oxidase gene into the expression vector pPIC3, the 5 'and 3' sequences of the open reading frame were modified. The first strand cDNA was used as a template in PCR. The synthetic oligonucleotide specific for the 5 'end of the open reading frame, Hox5'-l (Table 3.1) was used as a PCR primer together with a primer (Hox3'-1) specific for the 3' end of the coding sequence. the hexose oxidase of Chondrus crispus. The Hox3'-l primer had the sequence 5'-ACCAAGTTTATAAAAAGCAACCATCAC-3 '. Amplification of the PCR was performed using the GeneAmp PCR reagent kit (R | with AmpliTaq (R) DNA polymerase (Perkin-Elmer Cetus) .The PCR program was 30 cycles at 30 sec at 94 ° C, 30 sec 55 ° C and 2 min at 72 ° C. Gel electrophoresis of the reaction mixture showed a band with the approximate size of 1.7 kb.This 1.7 kb fragment was inserted into the vector pT7 Blue (Novagen) (plasmid pUPO150) and subjected to DNA sequencing. The fragment encoding the hexose oxidase from Chondrus crispus was further subcloned into the Pichia pastoris expression vector pPIC3 (Clare et al., 1991) as shown in Fig. 9. Plasmid pT7 Blue harboring the hexose gene oxidase was restricted with the restriction endonuclease Ndel and the ends were-polished with Klenow DNA polymerase essentially as described by Sambrook et al. (1989). After heat inactivation of the DNA polymerase (Sambrook et al., 1989) the DNA was further restricted with EcoRI and the DNA fragment containing the hexose oxidase gene was purified on an agarose gel as a DNA fragment from EcoRI - blunt end (QIAEXMR, QIAGEN). The expression vector of Pichia pastoris pPIC3 was restricted with the restriction enzymes SnaBI and EcoRI and purified on an agarose gel. The purified vector and the fragment encoding hexose oxidase were ligated and the ligation mixture was transformed into E. coli DH5a (Life Technologies), essentially as described by Sambrook et al. (1989). The resulting expression vector containing the hexose oxidase gene from Chondrus crispus, plasmid pUP0153, was subjected to DNA sequencing to ensure that mutations in the hexose oxidase gene had not occurred during the subcloning procedure. Plasmid pUP0153 was purified from E. coli DH5a and introduced into Pichia pastoris using electroporation (the Pichia Yeast Expression System, Phillips Petroleum Company) or using the Pichia Module (Invitrogen, San Diego, USA cat. K1720-01). The defective mutant using methanol from Pichia pastoris, KM71 (genotype his4, aoxl:: ARG4), (Cregg and Madden, 1987; Tschopp et al., 1987) was used as a receptor. The colonies of. Selected recombinant pichia pastoris on agar plates without histidine were screened for the presence of the hexose oxidase gene using PCR. Specific primers for hexose oxidase (Table 3.1) were used in addition to the primers specific for the alcohol oxidase promoter of Pichia pastoris and the transcription termination signal (Invitrogen, cat Nos. N710-02 and N720-02, respectively) . A sample of KM71 from Pichia pastoris containing pUP0153 was deposited at the Deutsche Sa mlung von Mikroorganismen und Zellkulturen (DSM) (German Collection of Microorganisms and Cell Cultures), Mascheroder Weg lb, D-38124 Braunschweig, Germany, May 23, 1996 under access number DSM 106934. 2. Expression of recombinant hexose oxidase in Pichia pastoris Strain KM71 of Pichia pastoris containing the expression cassette with the hexose oxidase gene inserted between the aoxal promoter and the transcription termination signal was cultivated in shake flasks in MD (1.34 grams per liter of yeast nitrogen base (Difco , cat No. 0919-15-3), 0.4 mg / l of biotin, 0.1% of arginine, and 20 g / 1 of glucose). One liter shake flasks containing 150 ml of culture were incubated on a rotary shaker at 30 ° C, 300 rpm. When the cells reached a density of 0D6Oo = 15-20 the cells were harvested by centrifugation at 6,000 xg for 10 min and resuspended in a similar volume (150 'mi) of induction medium, MM (1.34 g / 1 base of yeast nitrogen, 0.4 mg / l of biotin, 0.1% of arginine and 1% of methanol). After cultivating for 2 days, additional methanol was added (0.5%) to compensate the consumption and evaporation of methanol. Three or four days after induction the cells were harvested by centrifugation (6,000 x g, 10 min) and resuspended in approximately 1/5 of the growth volume of 50 mM Tris-Cl, pH 7.5. The suspended cells were again kept cold until rupture in a FRENCH (R) press (SLM Instruments, Inc., Rochester, N.Y.). The cells were opened in a FRENCH (R) 20K pressure cell at an internal pressure of 20,000 psi. The cell extract was clarified by centrifugation at 10,000 x g for 10 min at 5 ° C. The hexose oxidase containing the supernatant was carefully removed and subjected to purification as described below. 4. 3. Purification of recombinant hexose oxidase from Pichia pastoris 4. 3.1. First step, anion exchange chromatography A clarified homogenization of the homogenization in the FRENCH press (100-150 ml) was subjected to anion exchange chromatography in an FPLC system equipped with two 5 ml HiTrap-Q columns pre-packaged with High Performance Q-Sepharose (Pharmacia). The columns were connected in series and the chromatography was carried out at room temperature. The column was equilibrated in buffer A: 20 mM Tris-Cl, pH 7.5. The flow rate was 1.25 mi during the application of the sample and 2.5 ml during the washing and elution. After the application of the sample the column was washed with 30 ml of buffer A. The adsorbed proteins were then eluted with 200 ml of a gradient from buffer A to buffer B: 20 mM Tris-Cl, 750 mM NaCl, pH 7.5. Fractions of 2 ml were collected during washing and elution of the gradient. The fractions were assayed for hexose oxidase activity as described above in Example 1.3 (10 μl sample, 15 min incubation time). The fractions were also tested for alcohol oxidase (AOX) activity in an assay that was identical to the hexose oxidase assay except that 0.5% methanol was used instead of 0.05 M "glucose as substrate. 10, the activity profiles showed that AOX and HOX coeluted at a salt concentration of approximately 400 mM NaCl The fractions containing hexose oxidase were combined and stored at 4 ° C. 4. 3.2. Second step gel filtration The combined step one in the purification (20-30 ml) was concentrated at approx. 3.5 ml by centrifugal ultracentrifugation at 4 ° C in Centriprep concentrators (Amicon, USA, 30,000 nominal molecular weight cut). The concentrated preparation of hexose oxidase was clarified by centrifugation and the supernatant was mixed with glycerol to a final concentration of 5%. The sample was applied to the column using a sample applicator SA-5 (Pharmacia) connected to the entrance of the column. Gel filtration was performed at 4 ° C on a XK 26/70 column (2.6 x 66 cm, Pharmacia) with a bed volume of 350 ml. The column was packed with Sephacryl S-200 HR (Pharmacia) according to the manufacturer's instructions. The buffer was 20 mM Tris-Cl, 500 mM NaCl, pH 7.5 and the peristaltic pump Pl (Pharmacia) was set at 0.5 ml / min. The UV absorbance at 280 nm was recorded. The 2.5 ml fractions were collected and assayed for hexose oxidase and glucose oxidase activity as described above (10 μl sample, 15 min incubation time). The activity profiles clearly showed that the AOX and HOX activities were separated, see Fig. 11. This-result of the gel filtration was expected since the alcohol oxidase of the methylotrophic yeasts such as Pichia pastoris have a native molecular weight of approximately 600,000 (Sahm and Wagner, 1973), while HOX has a native molecular weight of approximately 110,000-130,000, as described in section 1.8. The elution volume of the recombinant HOX was identical to the volume of elution previously observed in the same column for native HOX isolated directly from Chondrus crispus (sections 1.7 and 1.8). Thus, the recombinant HOX appeared to have the same molecular weight as the native HOX isolated directly from Chondrus crispus. Fractions containing hexose oxides were combined and stored at 4 ° C. 4. 4.3. Third step anion exchange chromatography on a Mono O column The combined second step indicated above was further purified by anion exchange chromatography in an FPLC system equipped with a Mono Q HR 5/5 column (1 ml bed volume). The column 'was equilibrated in buffer A: 20 mM Tris-Cl, pH 7.5. The flow rate was 1 ml / min. The combined step 2 was desalted by gel filtration in buffer A on prepackaged Sephadex G-25 columns (PD-10, Pharmacia). After application of the sample the column was washed with 30 ml of buffer A. The adsorbed proteins were eluted with 20 ml of a gradient from 0% to 100% buffer B: 20 mM Tris-Cl, 500 mM NaCl, pH 7.5. Fractions of 0.5 ml were collected and tested for hexose oxidase activity as described above (10 μl sample, 15 minutes incubation time). Fractions containing hexose oxidase were combined and stored at 4 ° C. 4. 3.4. Fourth step, chromatofocusing The combined third step indicated above was purified by chromatofocusing on a Mono P HR 5/5 column as described above in Example 1.13, except that the adsorption step on Phenyl Sepharose was omitted. When comparing the native hexose oxidase and the recombinant hexose oxidase - both forms obtained by a final purification by chromatofocusing - it was found that the specific activity of the recombinant hexose oxidase of Pichia pastoris was similar to that of the isolated native form of Chondrus crispus. The fractions containing hexose oxidase were analyzed by SDS-PAGE and stained with Coomassie Brilliant Blue R-250 gel as described above The purified preparation of recombinant hexose oxidase was composed of two bands migrating at 40 kD and 29 In conclusion, the recombinant hexose oxidase could be isolated and purified from the host organism Pichia pastoris.In SDS-PAGE the purified, recombinant enzyme presented the same bands at 40 kD and 29 kD as the corresponding native Chondrus crispus enzyme. . 4. 4. Properties of recombinant hexose oxidase from Pichia pastoris Generation and analysis of the amino acid sequence of the peptide fragments of recombinant hexose oxidase (rHOX). The purified rHOX was used for preparative SDS-PAGE and PVDF membrane electrotransfer, as described above in Example 2.4. The resulting 40 kD and 29 kD bands were subjected to enzymatic digestion of hexose oxidase-linked PVDF polypeptides, as described above in Example 2.5. Peptide fragments were separated by reverse phase liquid chromatography as described above in Example 2.7. Well-resolved and abundant peptides were selected from the amino acid sequence analyzes by automated Edman degradation (10 steps), as described above in Example 2.3. The amino acid sequences obtained are shown in Table 4.1.

Table 4.1. Sequences of peptides obtained by peptide sequence analysis of endoproteinase Lys-C derived from 40 kP and 29 kP polypeptides of recombinant hexose oxidase expressed in Pichia pastoris The HOX-9 peptide sequence of the recombinant 40 kD polypeptide showed a sequence identical to Aspx8 to Asbiv in the hexose oxidase sequence of Chondrus crispus as shown in Table 3.2 (SEQ ID NO: 30). Sequence analysis of a peptide sample obtained from the recombinant 29 kD polypeptide showed two residues in each step. The amino acid identifications showed that two peptides present in the sample correspond to Leu434 to Glu4 3 and Tyr388 to Thr397, respectively, in the amino acid sequence of hexose oxidase of Chondrus crispus, see Table 3.2 (SEQ ID NO: 30).

It could therefore be concluded that the peptide sequences obtained from recombinant hexose oxidase were identical to the corresponding amino acid sequence of the hexose oxidase native to Chondrus crispus. Furthermore, it could be concluded that Pichia pastoris transformed with the hexose oxidase gene of Chondrus crispus was able to produce recombinant hexose oxidase. 4. 4.1 Substrate specificity The specificity of the substrate of the recombinant hexose oxidase of Pichia pastoris and hexose oxidase native to Chondrus crispus was compared using a number of sugars at a final concentration of 0.1 M in the test described above.The relative proportions are shown in Table 4.2 .

Table 4.2. Specificity of the substrate of recombinant hexose oxidase expressed in Pichia pastoris and hexose oxidase native to Chondrus crispus As shown in Table 4.2., The substrate specificity of the recombinant hexose oxidase was almost identical to that of the native enzyme. However, although the relative proportion between the disaccharides decreased for both enzymatic forms in the order maltose, cellobiose and lactose, the recombinant enzyme appeared to be less selective in its ability to oxidize these disaccharides. The results for the native enzyme were almost identical to the data previously reported by Sullivan et al. (1973). 4. 4.2. Inhibition by sodium diethyldithiocarbamate Sullivan and Ikawa (1973) reported that the hexose oxidase of Chondrus crispus is strongly inhibited by sodium diethyldithiocarbamate. The recombinant hexose oxidase of Pichia pastoris was compared with the native enzyme of Chondrus crispus with respect to the inhibition by this copper binding compound. The inhibitor was included in the enzymatic assay in two concentrations, 0.1 mM and 0.01 mM, as described by Sullivan and Ikawa (1973). The results are summarized in Table 4.3.

Table 4.3. Comparison of the inhibitory effect of sodium diethyldithiocarbamate on the enzymatic activity of the recombinant hexose oxidase of Pichia pastoris and hexose oxidase native to Chondrus crispus From Table 4.3. it is observed that the recombinant hexose oxidase and the native one were equally sensitive when they were subjected to inhibition by sodium diethyldithiocarbamate.

In addition, the results were similar to the data for the native hexose oxidase reported by Sullivan and Ikawa (1973).

EXAMPLE 5 Production of recombinant hexose oxidase in Escherichia coli . 1. Construction of a vector for the expression of recombinant hexose 'oxidase in Escherichia coli The open reading frame that encodes the hexose oxidase of Chondrus crispus that is presented in Table 3.2 (SEQ ID NO: 30) was inserted into an expression vector of Escherichia coli, pET17b (Novagen, cat.No. 69726-1). The plasmid contains a strongly inducible bacteriophage T7 promoter and a T7 transcription termination signal. The genes inserted between these control elements can be expressed by the addition of isopropyl β-D-thiogalactopyranoside (IPTG) if the plasmid is propagated in special E. coli host cells, e.g. , strain BL21 (DE3) (Novagen, cat No. 69387-1).

The hexose oxidase gene was modified at the 5 'and 3' ends to insert the gene into the expression vector pET17b. The hexose oxidase gene was isolated by PCR with specific primers for the 5 'and 3' ends of the hexose oxidase gene. The 5 'primer (Hox5'-2) had the 5' DNA sequence -ATGAATTCGTGGGTCGAAGAGCCC-3 '(SEQ ID NO: 33) and the specific primer for the 3' end was Hox3'-1. The first strand cDNA of Chondrus crispus was used as a template. Amplification of the PCR was performed with AmpliTaq (R) DNA polymerase (Perkin-Elmer Cetus) as described in Example 4.1. The gel electrophoresis of the reaction mixture showed a band with the approximate size of 1.7 kb. This 1.7 kb fragment was inserted into the vector pT7 Blue (Novagen) giving rise to the plasmid pUP0161. The modification of the 5 'end of the hexose oxidase gene and the subsequent subcloning of the gene into the E. coli expression vector is shown in Figure 12. The 5' end was modified by PCR to insert a NdeJ site just inside the cell. start of the ATG translation. The oligonucleotide, Hox5'-4, with sequence 5'-CAGGAATTCATATGGACTACTCTTCCCCAGAAAG-3 '(SEQ ID? O: 34) was used, together with the oligonucleotide Hoxl3- (SEQ ID? O: 28) (Table 3.1). The amplification of the PCR was as described above in Example 4.1. The reaction mixture was fractionated on a 2% agarose gel and the 180 bp specific fragment of hexose oxidase was purified as described in Example 3.4. The 180 bp fragment was restricted with the restriction endonuclease ClaJ and EcoRI and bound to pUP0161 restricted with the same enzymes giving rise to the plasmid pUP0167. The hexose oxidase gene in plasmid pUP0167 was further subcloned to construct a hexose oxidase expression vector for E. coli. The plasmid pUP0167 was restricted with the enzymes Ndel and BamHl and with the enzymes BamHl and Salí. The first reaction gave rise to a 1.6 kb fragment encoding the 5 'end and the middle part of the hexose oxidase gene while the reaction with the BamH1 and Sali enzymes gave a 200 bp fragment encoding the 3' end of the hexose oxidase gene. The two specific fragments of the hexose oxidase were purified on agarose gels as described in Example 3.4 and ligated to the restricted plasmid pET17b with the restriction endonucleases NdeJ and Xhol. Plasmid pET17b harboring the hexose oxidase gene was denoted pUP0181. The sequencing of AD? showed that no mutation was introduced into the hexose oxidase gene during the isolation and cloning procedure. . 2. Expression of recombinant hexose oxidase in Escherichia coli Plasmid pUPOldl was introduced into strain BL21 (DE3) of E. coli (Novagen) by means of a standard transformation procedure (Sambrook et al., 1989). The cells were cultured in shake flasks in LB medium (Sambrook et al., See above). At a cell density of OD6oo = 0-5 cells were induced to express recombinant hexose oxidase by the addition of 10"3 M IPTG.An hour after the addition of IPTG the cells were harvested" by centrifugation and resuspended in sample buffer and subjected to SDS -PAGE as described above in Example 1.10. The result of the electrophoresis is shown in the Figure 13. The crude extract of E. coli expressing the recombinant hexose oxidase enzyme of plasmid pUPOldl showed a protein band prominent at Mr 62 kD. This 62 kD band had the same molecular weight as the translation product predicted by the open reading frame. The non-transformed E. coli cells showed no such 62 kD protein. A sample of BL21 (DE3) from E. coli containing pUPOldl was deposited at the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM), Mascheroder Weg lb, D-38124 Braunschweig, Germany, on May 23, 1996 under the access number DSM 10692.

EXAMPLE 6 Production of recombinant hexose oxidase in Saccharomyces cerevisiae 6. 1. Construction of a vector for the expression of recombinant hexose oxidase in Saccharomyces cerevisiae The open reading frame encoding the hexose oxidase of Chondrus crispus shown in Table 3.2 (SEQ ID NO: 30) was inserted into a yeast expression vector, pYES2 (Invitrogen, cat No. V825-20). Plasmid pYES2 is a high copy number episomal vector designated for the inducible expression of recombinant proteins in Saccharomyces cerevisiae. The vector contains in the entry line promoter and activating sequences of the S. cerevisiae Gal I gene for tightly regulated, high-level transcription. The transcription termination signal is from the CYC1 gene. The hexose oxidase gene from Chondrus crispus was modified at the 5 'and 3' ends to insert the gene into the expression vector pYES2. The hexose oxidase gene was isolated from plasmid pUPO150 as described in Example 4.1 (Figure 9). The hexose oxidase gene was isolated on a blunt-ended EcoRI DNA fragment as described, and was inserted into the plasmid pYES2 restricted with the enzymes PvuII and EcoRI (Figure 14). The resulting plasmid, pUPI155, was subjected to DNA sequencing to show that no mutation had occurred during the cloning procedure. Plasmid pUP0155 was purified from E. coli DH5a and transformed into S. cerevisiae by electroporation "(Gray and Brendel, 1992) .The strain PAP1500 (genotype a, ura3-52, trpl:: GALI O -CAL4, lys2-801 , leu2? l, his3__200, pep4 :: H153, prbl._ll.6R, canl, GAL) (Pedersen et al., 1996) was used as a receiver. 6. 2. Expression of recombinant hexose oxidase in Saccharamyces cerevisiae The S. cerevisiae strain 1500 containing the plasmid pUP01500 was cultured and induced with 2% galactose as described by Pedersen et al. (nineteen ninety six). Three days after induction the cells were harvested by centrifugation and used as described above in Example 4.2. The crude extract was tested with respect to hexose oxidase actvity using the o-dianisidine assay described above in Example 1.3. Table 6.1 shows that the S. cerevisiae cells harboring the hexose oxidase gene were able to express the active hexose oxidase.

Table 6.1. Production of recombinant hexose oxidase in Saccharomyces cerevisiae 0 = no detectable activity A sample of strain S. cerevisiae 1500 containing the plasmid pUP0155 was deposited in the Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSM), Mascheroder Weg lb, D-38124 Braunschweig, Germany, on May 23, 1996 under the number of DSM access 10694.

REFERENCES 1. Barkholt, V. and A.L. Jensen 1989. Amino Acid Analysis: Determination of Cysteine plus Half-Cystine in Proteins after Hydrochloric Acid Hydrolysis with a Disulfide Compound as Additive. Analytical Biochemistry 177: 318-322. 2. Bean, R. C. and W.Z. Hassid 1956. J. Biol. Chem. 218: 425-436. 3. Clare, J. J, PB Rayment, SP Ballantine, K. "Sreekrishna and MA Romans 1991. High-level expression of tetanus toxin fragment C in Pichia pastoris strains containing multiple tandem integrations of the gene. Bio / Technology 9: 455-460 . 4. Cregg, J. M. and K. N. Madden 1987. Development of transformation systems and construction of methanol-utilization-defective mutants of Pichia pastoris by gene disruption. In: Biological Research on Industrial Yeast, Vol III. Stewart, G. G. et al. (Eds.). pp 1-18. CRC Press, Boca Raton, FL.

. Fernandez, J. et al. 1992. Internal Protein Sequence Analysis: Enzymatic Digestion for Less man 10 μg of Protein Bound to Polyvinylidene Difluoride or Nitrocellulose Membranes. Analytical Biochemistry, 201: 255-264. 6. Fernandez, J. et al. 1994. An Improved Procedure for Enzymatic Digestion of Polyvinylidene Difluoride-Bound Proteins for Internal Sequence Analysis. Analytical Biochemistry, 218: 112-117. 7. Groppe, J. C. and D. E. Morse 1993. Isolation of full-length RNA templates for reverse transcription from tissues rich in RNase and proteoglycans, Anal. Biochem., 210: 337-343. 8. Kerschensteiner, D. A. and G. L. Klippenstein 1978. Purification, Mechanism, and State of Copper in Hexose Oxidase. Federation Proceedings 37: 1816 abstract. 9. Laemmli, U.K. 1970. Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4. Nature (London) 227: 680-685.

. Pedersen P., J. H. Rasmussen, and P. L. J0rgensen. 1996. Expression in high yield of pig alßl Na, K-ATPase and inactive mutants D369N and D807N in Saccharomyces cerevisiae. J. Biol. Chem. 271: 2514-2522. 11. Rand, A.G. 1972. Direct enzyyrnatic conversion of lactose to acid: glucose oxidase and hexose oxidase. Journal of Food Science 37: 698-701. 12. Sahm, H. and Wagner, F. 1973. Microbial assimilation of methanol. Eur. J. Biochem. 36: 250-256. 13. Sambrook, J., E. F. Fritsch and T. Maniatis 1989. Molecular Cloning, A Laboratory Manual 2nd Ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 14. Schágger, H. and G. von Jagow 1987. Tricine-Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis for the Separation of Proteins in the Range from 1 to 100 kDa. Analytical Biochemistry 166: 368-379.

. Sock, J. and R. Rohringer 1988. Activity staining of Blotted Enzymes by Reaction Coupling with Transfer Membrane-Immobilized Auxiliary Enzymes. Analytical Biochemistry 171: 310-319. 16. Sullivan, J. D. and M. Ikawa 1973. Purification and Characterization of Hexose Oxidase from the Red Alga Chondrus crispus. Biochemica et Biophysica Acta 309: 11-22. 17. Tschopp, J. F., G. Sverlow, R. Kosson, W. Craig, and L. Grinna 1987. High-level secretion of glycosylated invertase in the methylotrophic yeast, Pichia pastoris. Bio / Technology 5: 1305-1308. 18. Yeh, K-W, R. H. Juang and J-C. His. A rapid and efficient method for RNA isolation from plants with high carbohydrate content. Focus 13: 102-103 LISTAPO PE SEQUENCES (1. GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Bioteknologisk Institut (B) STREET: Anker Engelunds Vej 1 (C) CITY: Lyngby (D) COUNTRY: Denmark (E) POSTAL CODE (ZIP): 2800 (ii) TITLE OF THE INVENTION: Hexose Recombinant Oxidase, a Method to Produce the Same and the Use of such Enzyme (iii) SEQUENCE NUMBER: 34 (iv) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIA: Flexible Disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Relay # 1.0, Version # 1.25 (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) HEBRA: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1 Tyr Glu Pro Tyr Gly Gly Val Pro 1 5 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 23 amino acids (B) TYPE: amino acid (C) HEBRA: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: peptide (Xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2: Ala He He Asn Val Thr Gly Leu Val Glu Ser Gly Tyr Asp Xaa Xaa 1 5 10 15 Xaa Gly Tyr Xaa Val Ser Ser 20 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) HEBRA: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: Asp Leu Pro Met Pro Pro Arg Gly Val He Wing Ser Asn Leu Xaa Phe 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) HEBRA: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: Asp Ser Glu Gly Asn Asp Gly Glu Leu Phe Xaa Wing His Thr 1 5 10 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) HEBRA: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5; Tyr Tyr Phe Lys 1 (2) INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) HEBRA: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: Asp Pro Gly Tyr He Val He Asp Val Asn Wing Gly Thr Xaa Asp 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 11 amino acids (B) TYPE: amino acid (C) HEBRA: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7 Leu Gln Tyr Gln Thr Tyr Trp Gln Glu Glu Asp 1 5 10 (2) INFORMATION FOR SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) HEBRA: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8; Xaa He Arg Asp Phe Tyr Glu Glu Met 1 5 (2) INFORMATION FOR SEQ ID NO: 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 23 amino acids (B) TYPE: amino acid (C) HEBRA: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9: Ala He He Asn Val Thr Gly Leu Val Glu Ser Gly Tyr Asp Xaa Xaa 1 5 10 15 Xaa Gly Tyr Xaa Val Ser Ser 20 (2) INFORMATION FOR SEQ ID NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 16 amino acids (B) TYPE: amino acid (C) HEBRA: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10: Asp Leu Pro Met Pro Pro Arg Gly Val He Wing Ser Asn Leu Trp Phe 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 14 amino acids (B) TYPE: amino acid (C) HEBRA: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11: Asp Ser Glu Gly Asn Asp Gly Glu Leu Phe Xaa Wing His Thr 1 5 10 (2) INFORMATION FOR SEQ ID NO: 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 4 amino acids (B) TYPE: amino acid (C) HEBRA: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12: Tyr Tyr Phe Lys 1 (2) INFORMATION FOR SEQ ID NO: 13: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 15 amino acids (B) TYPE: amino acid (C) HEBRA: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 13: Asp Pro Gly Tyr He Val As Asp Val Asn Wing Gly Thr Pro Asp 1 5 10 15 (2) INFORMATION FOR SEQ ID NO: 14: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 11 amino acids (B) TYPE: amino acid (C) HEBRA: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 14 Leu Gln Tyr Gln Thr Tyr Trp Gln Glu Glu Asp 1 5 10 (2) INFORMATION FOR SEQ ID NO: 15: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 9 amino acids (B) TYPE: amino acid (C) HEBRA: unknown (D) TOPOLOGY: unknown (ii) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 15: Xaa He Arg Asp Phe Tyr Glu Glu Met 1 5 (2) INFORMATION FOR SEQ ID NO: 16: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (ix) CHARACTERISTICS: (A) NAME / KEY: modified base; N = inosine (B) LOCATION: base pairs 3, 6 and 12 (C) IDENTIFICATION METHOD: commercially available (D) OTHER INFORMATION (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 16: YTNGTNGARW SNGGNTAYGA 20 (2) INFORMATION FOR SEQ ID NO: 17: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (ix) CHARACTERISTICS: (A) NAME / KEY: modified base; N = inosine (B) LOCATION: base pairs 6 and 12 (C) IDENTIFICATION METHOD: commercially available (D) OTHER INFORMATION (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 17: AACCANARRT TNGANGCDAT NAC 23 [2] INFORMATION FOR SEQ ID NO: 18 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (ix) CHARACTERISTICS: (A) NAME / KEY: modified base; N = inosine (B) LOCATION: base pairs 6 and 15 (C) IDENTIFICATION METHOD: commercially available (D) OTHER INFORMATION (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 18: GARGGNAAYG AYGGNGARCT NTT 23 - (2) INFORMATION FOR SEQ ID NO: 19: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (ix) CHARACTERISTICS: (A) NAME / KEY: modified base; N = inosine (B) LOCATION: base pairs 3 and 9 (C) IDENTIFICATION METHOD: commercially available (D) OTHER INFORMATION (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 19: AANAGYTCNC CRTCRTTNCC YTC 23 (2) INFORMATION FOR SEQ ID NO: 20: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 20: ATTGGGGCTC CTTCAAGACC TT 22 (2) INFORMATION FOR SEQ ID NO: 21: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 21: TGATGATTCC AAAGTTTC 18 (2) INFORMATION FOR SEQ ID NO: 22: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 22 TTGGAAGAAT ACGGTTGG 18 (2) INFORMATION FOR SEQ ID NO: 23: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 23: TACTATTTCG TCTGCTTGGG 20 (2) INFORMATION FOR SEQ ID NO: 24 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 24: GAACTCTTCC GTGGTCTCCT 20 (2) INFORMATION FOR SEQ ID NO: 25: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 25: CCACCTGCGT GTTGGGGTCT 20 (2) INFORMATION FOR SEQ ID NO: 26: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 21 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 26: CAGATCTACA AAACATGCGA G 21 (2) INFORMATION FOR SEQ ID NO: 27: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 27: TGTCGCAGAC TGTACTTG 18 (2) INFORMATION FOR SEQ ID NO: 28 (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 28: GAGTGTACAC GACATAAA 18 (2) INFORMATION FOR SEQ ID NO: 29: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 22 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 29: ATGGCTACTC TTCCCCAGAA AG 22 (2) INFORMATION FOR SEQ ID NO: 30: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1801 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 84..1721 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 30: TGAATTCGTG GGTCGAAGAG CCCTTTGCCT CGTCTCTCTG GTACCGTGTA TGTCAAAGGT 60 TCGCTTGCAC ACTGAACTTC ACG ATG GCT ACT CTT CCT CAG AAA GAC CCC 110 Met Wing Thr Leu Pro Gln Lys Asp Pro 1 5 GGT TAT ATT GTA ATT GAT GTC AAC GCG GGC ACC GCG GAC AAG CCG GAC 158 Gly Tyr He Val He Asp Val Asn Wing Gly Thr Wing Asp Lys Pro Asp 10 15 20 25 CCA CGT CTC CCC TCC ATG AAG CAG GGC TTC AAC CGC CGC TGG ATT GGA 206 Pro Arg Leu Pro Ser Met Lys Gln Gly Phe Asn Arg Arg Trp He Gly 30 35 40 ACT AAT ATC GAT TTC GTT TAT GTC GTG TAC ACT CCT CA GGT GCT TGT 254 Thr Asn He Asp Phe Val Tyr Val Val Tyr Thr Pro Gln Gly Ala Cys 45 50 55 ACT GCA CTT GAC CGT GCT ATG GAA AAG TGT TCT CCC GGT ACA GTC AGG 302 Thr Ala Leu Asp Arg Wing Met Glu Lys Cys Ser Pro Gly Thr Val Arg 60 65 70 ATC GTC TCT GGC GGC CAT TGC TAC GAG GAC TTC GTA TTT GAC GAA TGC 350 He Val Ser Gly Gly His Cys Tyr Glu Asp Phe Val Phe Asp Glu Cys 75 80 85 GTC AAG GCC ATC ATC AAC GTC ACT GGT CTC GTT GAG AGT GGT TAT GAC 398 Val Lys Ala He He Asn Val Thr Gly Leu Val Glu Ser Gly Tyr Asp 90 95 100 105 GAC GAT AGG GGT TAC TTC GTC AGC AGT GGA GAT ACA AAT TGG GGC TCC 446 Asp Asp Arg Gly Tyr Phe Val Ser Ser Gly Asp Thr Asn Trp Gly Ser 110 115 120 TTC AAG ACC TTG TTC AGA GAC CAC GGA AGA GTT CTT CCC GGG GGT TCC 494 Phe Lys Thr Leu Phe Arg Asp His Gly Arg Val Leu Pro Gly Gly Ser 125 130 135 TGC TAC TCC GTC GGC CTC GGT GGC CAC ATT GTC GGC GGG GGT GGC 542 Cys Tyr Ser Val Gly Leu Gly Gly His He Val Gly Gly Gly Asp Gly 140 145 150 ATT TTG GCC CGC TTG CAT GGC CTC CCC GTC GAT TGG CTC AGC GGC GTG 590 He Leu Wing Arg Leu His Gly Leu Pro Val Asp Trp Leu Ser Gly Val 155 160 165 GAG GTC GTC GTT AAG CCA GTC CTC ACC GAA GAC TCG GTA CTC AAG TAT 638 Glu Val Val Val Lys Pro Val Leu Thr Glu Asp Ser Val Leu Lys Tyr 170 175 180 185 GTG CAC AAA GAT TCC GAA GGC AAC GAC GGG GAG CTC TTT TGG GCA CAC 686 Val His Lys Asp Ser Glu Gly Asn Asp Gly Glu Leu Phe Trp Wing His 190 195 200 ACA GGT GGC GGC GGC GCA AAC TTT GGA ATC ATC ACC AAA TAC TAC TTC 734 Thr Gly Gly Gly Gly Asn Phe Gly He He Thr Lys Tyr Tyr Phe 205 210 215 AAG GAT TTG CCC ATG TCT CCA CGG GGC GTC ATC GCA TCA AAT TTA CAC 782 Lys Asp Leu Pro Met Ser Pro Arg Gly Val He Wing Ser Asn Leu His 220 225 230 TTC AGC TGG GAC GGT TTC ACG AGA GAT GCC TTG CAG GAT TTG TTG ACA 830 Phe Ser Trp Asp Gly Phe Thr Arg Asp Ala Leu Gln Asp Leu Leu Thr 235 240 245 AAG TAC TTC AAA CTT GCC AGA TGT GAT TGG AAG AAT ACG GTT GGC AAG 878 Lys Tyr Phe Lys Leu Wing Arg Cys Asp Trp Lys Asn Thr Val Gly Lys 250 255 260 265 TTT CAA ATC TTC CAT CAG GCA GCG GAA GAG TTT GTC ATG TAC TTG TAT 926 Phe Gln He Phe His Gln Wing Wing Glu Glu Phe Val Met Tyr Leu Tyr 270 275 280 ACA TCC TAC TCG AAC GAC GCC GCC CGC GAA GTT GCC CAAC GAC CGT CAC 974 Thr Ser Tyr Ser Asn Asp Wing Glu Arg Glu Val Wing Gln Asp Arg His 285 290 295 TAT CAT TTG GAG GAC GAC ATA GAA CAG ATC TAC AAA ACA TGC GAG CCC 1022 Tyr His Leu Glu Wing Asp He Glu Gln He Tyr Lys Thr Cys Glu Pro 300 305 310 ACC AAA GCG CTT GGC GGG CAT GCT GGG TGG GCG CCG TTC CCC GTG CGG 1070 Thr Lys Ala Leu Gly Gly His Ala Gly Trp Ala Pro Phe Pro Val Arg 315 320 325 CCG CGC AAG AGG CAC ACA TCC AAG ACG TCG TAT ATG CAT GAC GAG ACG 1118 Pro Arg Lys Arg His Thr Ser Lys Thr Ser Tyr Met His Asp Glu Thr 330 335 340 345 ATG GAC TAC TTC CCC TTC GCG CTC ACT GAG ACG ATC AAC GGC TCC GGG 1166 Met Asp Tyr Pro Phe Tyr Ala Leu Thr Glu Thr He Asn Gly Ser Gly 350 355 360 CCG AAT CAG CGC GGC AAG TAC AAG TCT GCG TAC ATG ATC AAG GAT TTC 1214 Pro Asn Gln Arg Gly Lys Tyr Lys Ser Wing Tyr Met He Lys Asp Phe 365 370 375 CCG GAT TTC CAG ATC GAC GTG ATC TGG AAA TAC CTT ACG GAG GTC CCG 1262 Pro Asp Phe Gln He Asp Val He Trp Lys Tyr Leu Thr Glu Val Pro 380 385 390 GAC GGC TTG ACT AGT GCC GAA ATG AAG GAT GCC TTA CTC CAG GTG GAC 1310 Asp Gly Leu Thr Ser Ala Glu Met Lys Asp Ala Leu Leu Gln Val Asp 395 400 405 ATG TTT GGT GGT GAG ATT CAC AAG GTG GTC TGG GAT GCG ACG GTC GTC 1358 Met Phe Gly Gly Glu He His Lys Val Val Trp Asp Ala Thr Ala Val 410 415 420 425 GCG CAG CGC GAG TAC ATC ATC AAA CTG CAG TAC CAG ACA TAC TGG CAG 1406 Wing Gln Arg Glu Tyr He He Lys Leu Gln Tyr Gln Thr Tyr Trp Gln 430 435 440 GAA GAA GAC AAG GAT GCA GTG AAC CTC AAG TGG ATT AGA GAC TTT TAC 1454 Glu Glu Glu Asp Lys Asp Wing Val Asn Leu Lys Trp He Arg Asp Phe Tyr 445 450 455 GAG GAG ATG TAT GAG CCG TAT GGC GGG GTT CCA GAC CCC AAC ACG CAG 1502 Glu Glu Met Tyr Glu Pro Tyr Gly Gly Val Pro Asp Pro Asn Thr Gln 460 465 470 GTG GAG AGT GGT AAA GGT GTG TTT GAG GGA TGC TAC TTC AAC TAC CCG 1550 Val Glu Ser Gly Lys Gly Val Phe Glu Gly Cys Tyr Phe Asn Tyr Pro 475 480 485 GAT GTG GAC TTG AAC AAC TGG AAG AAC GGC AAG TAT GGT GCC CTC GAA 1598 Asp Val Asp Leu Asn Asn Trp Lys Asn Gly Lys Tyr Gly Ala Leu Glu 490 495 500 505 CTT TAC TTT TTG GGT AAC CTG AAC CGC CTC ATC AAG GCC AAA TGG TTG 1646 Leu Tyr Phe Leu Gly Asn Leu Asn Arg Leu He Lys Wing Lys Trp Leu 510 515 520 TGG GAT CCC AAC GAG ATC TTC ACA AAC AAA CAG AGC ATC CCT ACT AAA 1694 Trp Asp Pro Asn Glu He Phe Thr Asn Lys Gln Ser He Pro Thr Lys 525 530 535 CCT CTT AAG GAG CCC AAG CAG ACG AAA TAGTAGGTCA CAATTAGTCA 1741 Pro Leu Lys Glu Pro Lys Gln Thr Lys 540 545 TCGACTGAAG TGCAGCACTT GTCGGATACG GCGTGATGGT TGCTTTTTAT AAACTTGGTA 1801 (2) INFORMATION FOR SEQ ID NO: 31: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 546 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 31: Met Wing Thr Leu Pro Gln Lys Asp Pro Gly Tyr He Val He Asp Val 1 5 10 15 Asn Wing Gly Thr Wing Asp Lys Pro Asp Pro Arg Leu Pro Being Met Lys 20 25 30 Gln Gly Phe Asn Arg Arg Trp He Gly Thr Asn He Asp Phe Val Tyr 35 40 45 Val Val Tyr Thr Pro Gln Gly Ala Cys Thr Ala Leu Asp Arg Wing Met 50 55 60 Glu Lys Cys Ser Pro Gly Thr Val Arg He Val Ser Gly Gly His Cys 65 70 75 80 Tyr Glu Asp Phe Val Phe Asp Glu Cys Val Lys Ala He He Asn Val 85 90 95 Thr Gly Leu Val Glu Ser Gly Tyr Asp Asp Asp Arg Gly Tyr Phe Val 100 105 110 Ser Ser Gly Asp Thr Asn Trp Gly Ser Phe Lys Thr Leu Phe Arg Asp 115 120 125 His Gly Arg Val Leu Pro Gly Gly Ser Cys Tyr Ser Val Gly Leu Gly 130 135 140 Gly His He Val Gly Gly Gly Asp Gly He Leu Ala Arg Leu His Gly 145 150 155 160 Leu Pro Val Asp Trp Leu Ser Val Val Glu Val Val Val Val Lys Pro Val 165 170 175 Leu Thr Glu Asp Ser Val Leu Lys Tyr Val His Lys Asp Ser Glu Gly 180 185 190 Asn Asp Gly Glu Leu Phe Trp Wing His Thr Gly Gly Gly Gly Asn 195 200 205 Phe Gly He He Thr Lys Tyr Tyr Phe Lys Asp Leu Pro Met Ser Pro 210 215 220 Arg Gly Val He Wing Ser Asn Leu His Phe Ser Trp Asp Gly Phe Thr 225 230 235 240 Arg Asp Ala Leu Gln Asp Leu Leu Thr Lys Tyr Phe Lys Leu Ala Arg 245 250 255 Cys Asp Trp Lys Asn Thr Val Gly Lys Phe Gln He Phe His Gln Wing 260 265 270 Wing Glu Glu Phe Val Met Tyr Leu Tyr Thr Ser Tyr Ser A = n Asp Ala 275 280 285 Glu Arg Glu Val Wing Gln Asp Arg His Tyr His Leu Glu Wing Asp He 290 295 300 Glu Gln He Tyr Lys Thr Cys Glu Pro Thr Lys Ala Leu Gly Gly His 305 310 315 320 Wing Gly Trp Wing Pro Phe Pro Val Arg Pro Arg Lys Arg His Thr Ser 325 330 335 Lys Thr Ser Tyr Met His Asp Glu Thr Met Asp Tyr Pro Phe Tyr Wing 340 345 350 Leu Thr Glu Thr He Asn Gly Ser Gly Pro Asn Gln Arg Gly Lys Tyr 355 360 365 Lys Ser Wing Tyr Met He Lys Asp Phe Pro Asp Phe Gln He Asp Val 370 375 380 He Trp Lys Tyr Leu Thr Glu Val Pro Asp Gly Leu Thr Ser Ala Glu 385 390 395 400 Met Lys Asp Ala Leu Leu Gln Val Asp Met Phe Gly Gly Glu He His 405 410 415 Lys Val Val Trp Asp Ala Thr Ala Val Ala Gln Arg Glu Tyr He He 420 425 430 Lys Leu Gln Tyr Gln Thr Tyr Trp Gln Glu Glu Asp Lys Asp Ala Val 435 440 445 Asn Leu Lys Trp He Arg Asp Phe Tyr Glu Glu Met Tyr Glu Pro Tyr 450 455 460 Gly Gly Val Pro Asp Pro Asn Thr Gln Val Glu Ser Gly Lys Gly Val 465 470 475 480 Phe Glu Gly Cys Tyr Phe Asn Tyr Pro Asp Val Asp Leu Asn Asn Trp 485 490 495 Lys Asn Gly Lys Tyr Gly Ala Leu Glu Leu Tyr Phe Leu Gly Asn Leu 500 505 510 Asn Arg Leu He Lys Wing Lys Trp Leu Trp Asp Pro Asn Glu He Phe 515 520 525 Thr Asn Lys Gln Ser He Pro Thr Lys Pro Leu Lys Glu Pro Lys Gln 530 535 540 Thr Lys 545 (2) INFORMATION FOR SEQ ID NO: 32: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 32: ACCAAGTTTA TAAAAAGCAA CCATCAC 27 (2) INFORMATION FOR SEQ ID NO: 33: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 33: ATGAATTCGT GGGTCGAAGA GCCC 24 (2) INFORMATION FOR SEQ ID NO: 34: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 33 base pairs (B) TYPE: nucleic acid (C) HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 34: CAGGAATTCA TATGGCTACT CTTCCCCAGA AAG 33 It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, property is claimed as contained in the following:

Claims (44)

REIVINPICATIONS

1. An isolated DNA fragment characterized in that it encodes a polypeptide having hexose oxidase activity.

2. The DNA fragment according to claim 1, characterized in that the fragment encoding a polypeptide comprises at least one amino acid sequence selected from the group consisting of (i) Tyr-Glu-Pro-Tyr-Gly-Gly-Val-Pro (SEQ ID NO: 1), (ii) Ala-Ile-He-Asn-Val-Thr-Gly-Leu-Val-Glu-Ser-Gly-Tyr-Asp-XXX-Gly-Tyr-X-Val-Ser-Ser, (SEQ ID NO: 2), (iii) Asp-Leu-Pro-Met-Ser-Pro-Arg-Gly-Val-Ile-Ala-Ser-Asn-Leu-X-Phe, (SEQ ID NO: 3), (iv) Asp-Ser-Glu-Gly-Asn-Asp-Gly-Glu-Leu-Phe-X-Ala-His-Thr, (SEQ ID NO: 4), (v) Tyr-Tyr-Phe-Lys, (SEQ ID NO: 5), (vi) Asp-Pro-Gly-Tyr-Ile-Val-Ile-Asp-Val-Asn-Ala-Gly-Thr-X Asp, (SEQ ID NO: 6), (vii) Leu-Gln-Tyr-Gln-Thr-Tyr-Trp-Gln-Glu-Glu ~ Asp, (SEQ ID NO: 7), (viii) X-Tle-Arg-Asp-Phe-Tyr-Glu- Glu-Met, (SEQ ID NO: 8) where X represents an amino acid selected from the group consisting of Ala, Arg, Asn, Asp, Asx, Cys, Gln, Glu, Glx, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val, and a mutein or a derivative of such a polypeptide.

3. A DNA fragment according to claim 2, characterized in that it comprises the region coding for the hexose oxidase of the sequence (SEQ ID NO: 30): TGAATTCGTG GGTCGAAGAG CCCTTTGCCT CGTCTCTCTG CTACCGTGTA TGTCAAAGGT 60 TCGCTTGCAC ACTGAACTTC ACGATGGCTA CTCTTCCTCA GAAAGACCCC GGTTATATTG 120. TAATTGATGT CAACGCGGGC ACCGCGGACA AGCCGGACCC ACGTCTCCCC TCCATGAAGC 180 AGGGCTTCAA CCGCCGCTGG ATTGGAACTA ATATCGATTT CGTTTATGTC GTGTACACTC 240 CTCAAGGTGC TTGTACTGCA CTTGACCGTG CTATGGAAAA GTGTTCTCCC GGTACAGTCA 300 GGATCGTCTC TGGCGGCCAT TGCTACGAGG ACTTCGTATT TGACGAATGC GTCAAGGCCA 360 TCATCAACGT CACTGGTCTC GTTGAGAGTG GTTATGACGA CGATAGGGGT TACTTCGTCA 420 GCAGTGGAGA TACAAATTGG GGCTCCTTCA AGACCTTGTT CAGAGACCAC GGAAGAGTTC 480 TTCCCGGGGG TTCCTGCTAC TCCGTCGGCC TCGGTGGCCA CATTGTCGGC GGAGGTGACG 540 GCATTTTGGC CCGCTTCGAT GGCCTCCCCG TCGATTGGCT CAGCGGCGTG GAGGTCGTCG 600 TTAAGCCAGT CCTCACCGAA GACTCGGTAC TCAAGTATGT GCACAAAGAT TCCGAAGGCT 660 ACGACGGGGA GCTCTTTTGG GCACACACAG GTGGCGGTGG CGGAAACTTT GGAATCATCA 720 CCAAATACTA CTTCAAGGAT TTGCCCATGT CTCCACGGGG CGTCATCGCA TCAAATTTAC 780 ACTTCAGCTG GGACGGTTTC ACGAGAGATG CCTTGCAGGA TTTGTTGACA AAGTACTTCA 840 AACTTGCCAG ATGTGATTGG AAGAATACGG TTGGCAAGTT TCAAATCTTC CATCAGGCAG 900 CGGAAGAGTT TGTCATGTAC TTGTATACAT CCTACTCGAA CGACGCCGAG CGCGAAGTTG 960 CCCAAGACCG TCACTATCA T TTGGAGGCTG ACATAGAACA GATCTACAAA ACATGCGAGC 1020 CCACCAAAGC GCTTGGCGGG CATGCTGGGT GGGCGCCGTT CCCCGTGCGG CCGCGCAAGA 1080 GGCACACATC GCTTGGCGGG CATGCTGGGT GGGCGCCGTT CCCCGTGCGG CCGCGCAAGA 1140 TCACTGAGAC GATCAACGGC TCCGGGCCGA ATCAGCGCGG CAAGTACAAG TCTGCGTACA 1200 TGATCAAGGA TTTCCCGGAT TTCCAGATCG ACGTGATCTG CAAATACCTT ACGGAGGTCC 1260 CGGACGGCTT GACTAGTGCC GAAATGAAGG ATGCCTTACT CCAGGTGGAC ATGTTTGGTT 1320 GTGAGATTCA CAAGGTGGTC TGGGATGCGA CGGCAGTCGC GCAGCGCGAG TACATCA1CA 1380 AACTGCAGTA CCAGACATAC TGGGAGGAAG AAGACAAGGA TGCAGTGAAC CTCAAGTGGA 1440 TTAGAGACTT TTACGAGGAG ATGTATGAGC CGTATGGCGG GGTTCCAGAC CCCAACACGC 1500 AGGTGGAAGG TGGTAAAGGT GTGTTTGAGG GATGCTACTT CAACTACCCG GATGTGGACT 1560 TGAACAACTG GAAGAACGGC AAGTATGGTG CCCTCGAACT TTACTTTTTG GGTAACCTGA 1620 ACCGCCTCAT CAAGGCCAAA TGGTTGTGGG ATCCCAACGA GATCTTCACA AACAAACAGA 1680 GCATCCCTAC TAAACCTCTT AAGGAGCCCA AGCAGACGAA ATAGTAGGTC ACAATTAGTC 1740 ATCGACTGAA GTGCAGCTAC TGTCGGATAC GGCGTGATGG TTGCTTTTTA TAAACTTGGT 1800 A 1801

4. A recombinant DNA molecule characterized in that it comprises the DNA fragment according to any of claims 1-3.

5. A recombinant DNA molecule according to claim 4, characterized in that the DNA fragment is operably linked to an expression signal not natively associated with the DNA fragment.

6. A host cell characterized in that it comprises the DNA fragment according to any of claims 1-3, or the DNA molecule according to any of claims 4-5.

7. a cell according to claim 6, characterized in that it is a cell of a microbe selected from the group consisting of a bacterial cell, a fungal cell and a yeast cell.

8. The cell according to claim 7, characterized in that it is selected from the group consisting of an Escherichia coli cell, a bacterial lactic acid cell, a Saccharomyces cerevisiae cell and a Pichia pastoris cell.

9. A method for producing a polypeptide having hexose oxidase activity, the method is characterized in that it comprises culturing a host cell of claim 6 in a culture medium, the host cell comprising the DNA fragment operably linked to an expression signal. , and recover the enzyme from the cells and / or the culture medium.

10. The method in accordance with the claim 9, characterized in that the DNA fragment is derived from marine algae species.

11. The method in accordance with the claim 10, characterized in that the seaweed species are selected from the group consisting of Chondrus crispus, Iridophycus flaccidum and Euthora cristata.

12. The method in accordance with the claim 9, characterized in that the host cell is a microorganism selected from the group consisting of bacterial species, fungal species, and yeast species.

13. The method according to claim 12, characterized in that the host cell is selected from the group consisting of an E. coli cell, a bacterial lactic acid cell, a S. cerevisiae cell and a P. pastoris cell.

14. The method according to claim 9, characterized in that the DNA fragment comprises at least one DNA sequence coding for an amino acid sequence selected from the group consisting of (i) Tyr-Glu-Pro-Tyr-Gly-Gly -Val-Pro (SEQ ID NO: l), (ii) Ala-Ile-Ile-Asn-Val-Thr-Gly-Leu-Val-Glu-Ser-Gly-Tyr-Asp-XXX-Gly-Tyr-X-Val-Ser-Ser, (SEQ ID NO: 2), (iii) Asp-Leu-Pro-Met-Ser-Pro-Arg-Gly-Val-Ile-Ala-Ser-Asn-Leu-X-Phe, (SEQ ID NO: 3), (iv) Asp-Ser-Glu-Gly-Asn-Asp-Gly-Glu-Leu-Phe-X-Ala-His-Thr, (SEQ ID NO: 4), (v) Tyr-Tyr-Phe-Lys, (SEQ ID NO: 5), (vi) Asp-Pro-Gly-Tyr-Ile-Val-Ile-Asp-Val-Asn-Ala-Gly-Thr-X Asp, (SEQ ID NO: 6), (vii) Leu-Gln-Tyr-Gln-Thr-Tyr-Trp-Gln-Glu-Glu-Asp, (SEQ ID NO: 7), (viii) X-Ile-Arg-Asp-Phe-Tyr-Glu- Glu-Met, (SEQ ID NO: 8) wherein X represents an amino acid selected from the group consisting of Ala, Arg, Asn, Asp, Asx, Cys, Gln, Glu, Glx, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp , Tyr and Val, and a mutein or a derivative of such a polypeptide.

15. The method according to claim 9, characterized in that the composition comprises a further step a purification of the polypeptide preparation initially recovered from the culture medium and / or the host cells to obtain a preparation in which the polypeptide is in a substantially pure form .

16. The method according to claim 9, characterized in that the polypeptide having hexose oxidase activity is a fusion product.

17. A preparation characterized in that it comprises a polypeptide having hexose oxidase activity, the polypeptide is a substantially pure form.

18. The preparation according to claim 17, characterized in that the polypeptide is in a substantially non-glycosylated form.

19. The preparation according to claim 17, characterized in that it comprises at least one amino acid sequence selected from the group consisting of (i) Tyr-Glu-Pro-Tyr-Gly-Gly-Val-Pro (SEQ ID NO: 1), (ii) Ala-Ile-Ile-Asn-Val-Thr-Gly-Leu-Val-Glu-Ser-Gly-Tyr-Asp-XXX-Gly-Tyr-X-Val-Ser-Ser (SEQ ID N0: 2) ), (iii) Asp-Leu-Pro-Met-Ser-Pro-Arg-Gly-Val-Ile-Ala-Ser-Asn-Leu-X-Phe (SEQ ID NO: 3), (iv) Asp-Ser-Glu -Gly-Asn-Asp-Gly-Glu-Leu-Phe-X-Ala-His-Thr (SEQ ID NO: 4), (v) Tyr-Tyr-Phe-Lys, (SEQ ID NO: 5), (vi) Asp-Pro-Gly-Tyr-Ile-Val-Ile-Asp-Val-Asn-Ala-Gly-Thr-X Asp (SEQ ID NO: 6), (vii) Leu-Gln-Tyr-Gln-Thr-Tyr-Trp-Gln-Glu-Glu ~ Asp (SEQ ID NO: 7), (viii) X-Ile-Arg-Asp-Phe-Tyr-Glu-Glu -Met (SEQ ID NO: 8) wherein X represents an amino acid selected from the group consisting of Ala, Arg, Asn, Asp, Asx, Cys, Gln, Glu, Glx, • Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr and Val, and a mutein or a variant thereof.

20. The preparation according to claim 17, characterized in that the polypeptide is produced by the method of any of claims 9-17.

21. The preparation according to claim 17, characterized in that the polypeptide has functional characteristics identical or partially identical to those of the hexose oxidase that occurs naturally in Chondrus crispus.

22. The preparation according to claim 17, characterized in that the polypeptide, when subjected to the SDS-PAGE shows separate bands of 29, 40 and / or 60 kD.

23. The preparation according to claim 17, characterized in that the polypeptide shows an enzymatic activity at a pH in the range of 5-9.

24. The preparation in accordance with the. claim 17, characterized in that the polypeptide has an optimum temperature for the enzymatic activity that is in the range of 20-60 ° C.

25. The preparation according to claim 17, characterized in that the polypeptide oxidizes at least one sugar selected from the group consisting of D-glucose, D-galactose, maltose, cellobiose, lactose, D-mannose, D-fucose and D-xylose. .

26. The preparation according to claim 17, characterized in that the polypeptide has an isoelectric point in the range of 4-5.

27. The preparation according to claim 17, characterized in that the polypeptide has an isoelectric point of 4.3 ± 0.1 or 4.5 ± 0.1.

28. The preparation according to claim 17, characterized in that the polypeptide has a molecular weight determined by gel filtration using Sephacryl S-200 (Pharmacia) which is in the range of 100-150 kD.

29. The preparation according to claim 17, characterized in that the polypeptide has an apparent molecule weight of 110 kD ± 10 kD.

30. A polypeptide characterized in that it has hexose oxidase activity and that it is in a substantially pure form.

31. The polypeptide according to claim 30, characterized in that it comprises at least one amino acid sequence selected from the group consisting of (i) Tyr-Glu-Pro-Tyr-Gly-Gly-Val-Pro (SEQ ID NO: l), (ii) Ala-Ile-Ile-Asn-Val-Thr-Gly-Leu-Val-Glu-Ser -Gly-Tyr-Asp- XXX-Gly-Tyr-X-Val-Ser-Ser (SEQ ID N0: 2), (iii) Asp-Leu-Pro-Met-Ser-Pro-Arg-Gly-Val-Ile-Ala-Ser-Asn-Leu-X-Phe (SEQ ID NO: 3) > (iv) Asp-Ser-Glu-Gly-Asn-Asp-Gly-Glu-Leu-Phe-X-Ala-His-Thr (SEQ ID NO: 4), (v) Tyr-Tyr-Phe-Lys, ( SEQ ID NO: 5), (vi) Asp-Pro-Gly-Tyr-Ile-Val-Ile-Asp-Val-Asn-Ala-Gly-Thr-X Asp (SEQ ID NO: 6), (vii) Leu-Gln-Tyr-Gln- Thr-Tyr-Trp-Gln-Glu-Glu ~ Asp (SEQ ID NO: 7), (viii) X-Ile-Arg-Asp-Phe-Tyr-Glu-Glu-Met (SEQ ID NO: 8) wherein X represents an amino acid selected from the group consisting of Ala, Arg, Asn, Asp, Asx, Cys, Gln, Glu, Glx, Gly, His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp , Tyr and Val, and a mutein or a variant thereof.

32. A method for manufacturing a food product, characterized in that the host cell is used according to claim 6, the preparation of claim 17 or the polypeptide of claim 30.

33. The method according to claim 32, characterized in that the food product is selected from the group consisting of a dairy product, a food product containing starch, and a non-dairy beverage.

34. The method in accordance with the claim 32, characterized in that the polypeptide acts as an antimicrobial agent or as an antioxidant agent.

35. The method according to claim 32, characterized in that the polypeptide acts as an agent that removes oxygen in a food container.

36. A method for manufacturing an animal feed, characterized in that it uses the host cell according to claim 6, the preparation of claim 17 or the polypeptide of claim 30.

37. The method according to claim 36, characterized in that the animal feed is silage.

38. A method for reducing the sugar content of a food product, characterized in that it comprises adding to the product an amount of the host cell of claim 6, the preparation of claim 17 or the polypeptide of claim 30, which is sufficient to eliminate at least part of the sugar substantially present in the food product.

39. A method for manufacturing a product selected from the group consisting of a pharmaceutical product, a cosmetic and a dental care product, characterized in that it is used in the host cell according to claim 6, the preparation of claim 17 or the polypeptide of claim 30.

40. A method for preparing a baked product from a dough, characterized in that it comprises adding the host cell according to claim 6, the preparation of claim 17 or the polypeptide of claim 30 to the dough.

41. A dough-improving composition, characterized in that it comprises the host cell according to claim 6, capable of expressing to a polypeptide in the dough, the preparation of claim 17 or the polypeptide of claim 30, and at least one component of conventional dough.

42. The composition according to claim 41, characterized in that it also comprises at least one enzyme selected from the group consisting of a cellulase, a hemicellulase, a xylanase, a pentosanase, an amylase, a lipase and a protease.

43. A method for analyzing the content of a sugar in a sample, characterized in that the host cell according to claim 6, the preparation of claim 17 or the polypeptide of claim 30 is used as an analytical reagent.

44. A method for making a lactone using the. The cell according to claim 6, the preparation of claim 17 or the polypeptide of claim 30, the method is characterized in that it comprises applying the host cell, the preparation and / or the polypeptide to a reactor containing a carbohydrate, the which can be oxidized by the polypeptide and operate the reactor under conditions wherein the carbohydrate is oxidized to a lactone.