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Title:
USE OF A MUTAROTASE IN THE PRODUCTION OF DRIED POWDERS
Document Type and Number:
WIPO Patent Application WO/2012/007446
Kind Code:
A1
Abstract:
The invention relates to the use of mutarotase in the production of dried powders, such as whey or milk powder. This invention describes the use of mutarotase (aldose-1-epimerase) in a process for dehydration of foodstuffs. Preferably the dried foodstuff is a dried dairy product, more preferably the dried dairy product is produced from whey. Even more preferable the dried dairy product produced from whey is whey powder or whey permeate powder. The mutarotase can be obtained from an organism, preferably a micro-organism, more preferably from a fungus, even more preferably from a fungus of the genus Aspergillus, most preferably from Aspergillus niger. In general, the foodstuff is first concentrated until the carbohydrates within will become supersaturated, after which the temperature is decreased and optional seeding with carbohydrate crystals initiate crystallization of the carbohydrate within. Preferably the carbohydrate within the food stuff will be a reducing sugar, more preferably the reducing sugar will be lactose. Mutarotase is added before or during the crystallization process.

Inventors:
DEKKER, Petrus, Jacobus, Theodorus (Vonkstraat 2, AE Den Haag, NL-2497, NL)
MUIJLWIJK, Cornelis Marinus (Vossersteeg 20, RK Dalfsen, NL-7722, NL)
Application Number:
EP2011/061817
Publication Date:
January 19, 2012
Filing Date:
July 12, 2011
Export Citation:
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Assignee:
DSM IP ASSETS B.V. (Het Overloon 1, TE Heerlen, NL-6411, NL)
DEKKER, Petrus, Jacobus, Theodorus (Vonkstraat 2, AE Den Haag, NL-2497, NL)
MUIJLWIJK, Cornelis Marinus (Vossersteeg 20, RK Dalfsen, NL-7722, NL)
International Classes:
A23C1/00; A23C9/12; A23C21/02
Domestic Patent References:
Foreign References:
US3721585A
US2100433A
US5951777A
US3964974A
EP0184438A2
EP0284603A1
EP0134048A1
EP0253455A1
EP0096430A1
EP0301670A1
Other References:
HYND J: "Drying of whey.", JOURNAL OF THE SOCIETY OF DAIRY TECHNOLOGY, vol. 33, no. 2, 2 April 1980 (1980-04-02), pages 52-54, XP002616719, SCOTTISH MILK MARKETING BOARD, GALLOWAY CREAMERY, STRANRAER, UK DOI: 10.1111/j.1471-0307.1980.tb01471.x
GANZLE M G ET AL: "Lactose: Crystallization, hydrolysis and value-added derivatives", INTERNATIONAL DAIRY JOURNAL, vol. 18, no. 7, 1 July 2008 (2008-07-01), pages 685-694, XP022701022, ELSEVIER APPLIED SCIENCE, BARKING, GB ISSN: 0958-6946, DOI: DOI:10.1016/J.IDAIRYJ.2008.03.003 [retrieved on 2008-03-07]
ANAL. BIOCHEM. vol. 43, 1971, page 312
BENTLEY, BHATE J. BIOL. CHEM. vol. 235, 1960, pages 1219 - 1233
KINOSHITA BIOCHIM. BIOPHYS. ACTA vol. 662, 1981, page 285
SAMUEL, TANNER NAT. PROD. REP. vol. 19, 2002, pages 261 - 277
MIWA ANALYTICAL BIOCHEMISTRY vol. 45, 1972, pages 441 - 447
MIWA, OKUDA J. BIOCHEM., [Online] vol. 75, 1974, pages 1177 - 1179 Retrieved from the Internet:
HUCHO, WALLENFELS EUR. J. BIOCHEM. vol. 23, 1971, pages 489 - 496
ALTSCHUL ET AL. NUCLEIC ACIDS RESEARCH vol. 25, 1997, pages 3389 - 3402
BENDTSEN, NIELSEN, HEIJNE, BRUNAK J. MOL. BIOL. vol. 340, 2004, pages 783 - 795
RAPER, FENNELL: 'The Genus Aspergillus', 1965, THE WILLIAMS & WILKINS COMPANY pages 293 - 344
MICHAELS, VAN KREVELD NETH. MILK DAIRY J. vol. 20, 1966, page 163
KINOSHITA ET AL. BIOCHIMICA ET BIOPHYSICA ACTA vol. 662, 1981, pages 285 - 290
Attorney, Agent or Firm:
GRIEKEN PLOOSTER, VAN, Izabella Johanna et al. (DSM Intellectual Property, P.O. Box 130, AC Echt, NL-6100, NL)
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Claims:
CLAIMS

1. A method for preparing a dehydrated foodstuff from a starting material solution comprising crystallizing at least part of a carbohydrate in a concentrated starting material solution in the presence of a protein having mutarotase activity and drying the at least partly crystallized concentrated product.

2. A method according to claim 1 , wherein said protein having mutarotase activity is a protein having at least 50% identity with the protein of SEQ ID NO: 1.

3. A method according to claim 1 or 2, wherein said protein having mutarotase activity comprises the amino acid sequence as depicted in SEQ ID NO: 1.

4. A method according to any one of claims 1 to 3, further comprising concentrating said starting material before crystallization.

5. A method according to any one of claims 1 to 4, wherein said dehydrated foodstuff is a dehydrated dairy foodstuff.

6. A method according to claim 5, wherein said dehydrated dairy foodstuff is whey powder or whey permeate powder.

7. A method according to any one of claims 1 to 6, wherein said starting material is whey, acid whey or whey permeate.

8. A method according to claim 5, wherein said dehydrated dairy foodstuff is milk powder.

9. A method according to claim 8, wherein said starting material is milk.

10. A method according to any one of claims 1 to 9, wherein said carbohydrate is lactose.

1 1. Use of mutarotase for preparing a dehydrated foodstuff.

12. A product obtainable by a method according to any one of claims 1 to 10.

Description:
USE OF A MUTAROTASE IN THE PRODUCTION OF DRIED POWDERS

Field of the invention

The invention relates to the use of mutarotase in the production of dried powders, such as whey or milk powder.

Background of the invention

Whey is a valuable and nutritious product of the dairy industry. Most whey is produced as side product in the production of cheese using a rennet in combination with a starter culture. Some whey is however produced by direct acid precipitation of casein by addition of acid or starter culture to the milk. After drainage, the curd is separated from the whey. Whey has relatively high water content that limits its valorization. Dry matter of whey is approximately 6.5%, and fluctuates dependent on the milk and the type of cheese that has been produced from it. On average the whey of most cheeses contains approximately 93% water, 4.3-4.9% lactose, -0.9% protein, -0.5% milk salts, -0.2% lactic acid and <0.1 %-0.5% fat. Besides these major components whey may contain a number of vitamins.

Since the high water content of whey increases the chance of spoilage and is logistically less desirable, it limits the usage of liquid whey. Therefore, a major part of the world whey is now being condensed and/or dried.

The raw fresh whey should be cooled to 5-10°C immediately after it is drained off the cheese vat in order to avoid bacterial decomposition with a troublesome drying as a result. It is advisable to clarify the whey in order to remove casein micelles, which will burn and stick to the evaporator tubes and result in discoloured particles in the final powder. The remaining fat in the whey should also be removed by the separation, as fat may tend to give deposits in the drying chamber.

In the evaporator the whey is preheated to 60-70°C (depending on the running conditions) after which it is pasteurized. By means of the temperature and holding time combination it is possible to control the degree of denaturation of the proteins, and thus it is possible to produce powders meeting specific requirements.

Generally speaking the viscosity of the whey concentrate increases and the non- caking properties become better by increasing the pasteurizing temperature/time. The whey is evaporated to a solid content suitable for the subsequent spray drying process and may vary from 40% to 60% dry matter/solids.

Special attention should also be paid to Ca++ content in the whey. Ca-phosphate is a salt, which will precipitate, if the concentration and temperature increase. Especially, it may form deposits on the tubes, if not removed in a decalcification process.

Removing part of the water from the whey in a hyperfiltration (also referred to as reversed osmosis) plant to 10-15% solids may also be done to increase the output from the evaporator.

A pre-evaporation may also be done, if it is impossible to obtain sufficient whey (>400,000 l/day) to make the whey powder production profitable. Small dairies may then pre-evaporate their whey in an evaporator to approximately 30% solids, cool it and transport the concentrate to a factory centrally located. The transport costs are thereby reduced. The pre-condensed whey may then be mixed with raw whey from the factory's own production, after which the final evaporation takes place followed by the spray drying. It is well known that it is cheaper to remove water in an evaporator than in a spray dryer (about 1 :20), and it is thus not surprising that the efforts in the development work have been concentrated on increasing the solids content from the evaporator. The outlet temperature of the spray dryer must be increased (about 1°C/1 % total solids) which means that the powder temperature will get into the sticking point temperature zone resulting in severe problems in the dryer such as wall deposits and blocked cyclones.

The sticking point temperature depends on the composition of the whey powder and mainly on the following components:

• Lactic acid content and salts

• Amorphous lactose content

• Moisture content Pre-crystallization of the concentrate shifts the sticking temperature upwards, as the crystallization yields a concentrate with much less amorphous lactose to be dried. It is thus possible to use considerably higher feed concentrations and inlet temperatures, without sticking during drying.

In whey powder the lactose is present in an amorphous or glassy state. This form of lactose is extremely hygroscopic absorbing water from the air forming a-lactose monohydrate.

The α-lactose monohydrate crystals, which are not hygroscopic, are formed in liquid whey concentrates by crystallization of the lactose from supersaturated solutions. As the a-form is less soluble than the β-form at a given temperature, the a-form reaches the point of supersaturation first and forms crystals of α-lactose monohydrate.

The removal of lactose from the solution due to the crystallization means that the proportion between a- and β-lactose changes, so that the solution contains more β- lactose than corresponding to the equilibrium.

Due to mutarotation the solution of α-lactose becomes again supersaturated, so that the crystallization continues. This process will continue as long as the solution is supersaturated and will not stop until the saturation point is reached.

Lactose can be supersaturated by:

• Increasing the content of lactose in relation to water content. This is done by evaporation.

• Cooling the solution as lactose becomes less soluble in water at lower temperatures.

A rich crystallization is therefore achieved by concentrating the whey to a solid content as high as possible and by cooling the concentrate.

The rate of mutarotation is directly influenced by the temperature of the solution, and it proceeds relatively fast at high temperatures, whereas it goes very slowly at temperatures near the freezing point. It is thus clear that the conditions promoting mutarotation and crystallization are conflicting.

This means that by cooling the concentrate too fast to a too low temperature the proportion of lactose which will crystallize will be low, even though the crystallization proceeds quickly. This is due to the slow mutarotation resulting in only a small amount of β-lactose being transformed into a- lactose. A temperature compromise is therefore necessary to obtain an optimal degree of crystallization.

The actual obtainable solids content depends on the type of whey, degree of denaturation of the whey proteins, pH of the whey, and the ability of the evaporator. Although the crystallization process is not fully understood, it is a well-known fact that the crystallization takes place on the surface of already existing crystals. In order to promote the crystallization, lactose crystals or well crystallized whey powder is seeded to the supersaturated solution. As the surface area of the seeding material is the important factor, a sufficient amount of small crystals should be added to the concentrate. If lactose crystals (a-lactose monohydrate) are used, approximately 0.1 % w/w should be added to the concentrate.

Cooling of the concentrate is done instantaneously to 30°C in a flash cooler connected to the evaporator after which it is cooled slowly (1 -3°C/h) to about 15°C in specially designed crystallization tanks. This may allow the mutarotation to proceed at a reasonable speed resulting in a rich crystallization. During the whole crystallization time it is of significant importance that the content of the crystallization tank be vigorously agitated continuously. This is done in order to transport supersaturated solution to the surface of the crystals, simultaneously replacing the saturated solution. The agitation also prevents the viscosity of the suspension from getting too high, and furthermore sedimentation of the lactose crystals does not take place.

Spray drying whey powder without precrystallization is mainly used as animal fodder. The hygroscopic tendency is due to the lactose in the powder being in the so- called amorphous stage which means water absorbing and sticky. The outlet temperature and content of residual moisture must therefore be kept low. This means also that the solids content must be kept low during spray drying.

The handling of whey powder products is often difficult due to the lactose component in the powder. Lactose solidifies in an amorphous state during rapid drying in a spray dryer. Amorphous lactose is thermodynamically unstable and hygroscopic, absorbing moisture from the surrounding and subsequently plasticizing. This causes the individual whey powder particles to become sticky. The temperature at which this transition from an amorphous solid-state to a viscous rubbery state occurs is known as the glass transition temperature. The glass transition temperature can fall below ambient temperature when the powder absorbs sufficient moisture during storage, leading to caking of the powder. This presents a problem during subsequent handling processes, such as pneumatic conveying.

Spray drying of precrystallized whey concentrate can be performed under much more economical conditions, as the drying temperature and the solids content can be increased. The total drying costs can thus be reduced. As most of the lactose is crystallized, and therefore can absorb very little water, the powder will be of a much better quality, because the caking tendency has been reduced. Additionally, the whey powder yield will be higher when precrystallized whey concentrate is used in spray drying, due to the inclusion of crystal water.

From the discussion above of the problems that can occur during whey powder production, and whey powder storage, it becomes clear that precrystallization of the lactose would be of great advantage in this industry. However, precrystallization of a large part of the lactose requires a large capital investment in the form of large crystallization tanks, energy input for cooling and extended timing of the process that may increase spoilage of the product and decrease throughput of the plant. Consequently, still a large volume of the whey is still dried without pre-crystallization. An improvement in the crystallization process that would increase throughput and/or decrease cooling will therefore be of great advantage for the whey powder production industry. The present invention provides such an improvement.

All problems described herein also occur when whey permeate, instead of whey concentrate, is dried. Consequently, a precrystallization step is also advantageous for drying whey permeate. As permeate powder is used throughout the world, also in countries where the climate is humid, the powder will absorb moisture from the ambient, if the lactose is not totally crystallized. The remaining amorphous lactose will therefore absorb moisture from the air, when the bag is opened, if the ambient air has a high enough moisture content. The powder will cake, and lumping will be the result leaving an unattractive product to the end-user.

Summary of the invention

This invention describes the use of mutarotase (aldose-1-epimerase) in a process for dehydration of foodstuffs. Preferably the dried foodstuff is a dried dairy product, more preferably the dried dairy product is produced from whey or from milk. Even more preferable the dried dairy product produced from whey is whey powder or whey permeate powder. In another preferred embodiment, the dried dairy product is milk powder. The mutarotase can be obtained from an organism, preferably a microorganism, more preferably from a fungus, even more preferably from a fungus of the genus Aspergillus, most preferably from Aspergillus niger. In general, the foodstuff is first concentrated until the carbohydrates within will become supersaturated, after which the temperature is decreased and optional seeding with carbohydrate crystals initiate crystallization of the carbohydrate within. Preferably the carbohydrate within the food stuff will be a reducing sugar, more preferably the reducing sugar will be lactose. Mutarotase is added before or during the crystallization process.

The use of a mutarotase during the preparation of dried foodstuff has advantages over the prior art in that it can speed up crystallization thereby decreasing residence time in the crystallization tank. A shorter crystallization process will also minimize risks for spoilage of the foodstuff. Additional benefits are an increase in quality and/or uniformity of the dried foodstuff, and the reduction of browning and off-flavor formation during drying or storage of the dried food stuff. Additional, the use of mutarotase during crystallization may lead to a yield increase of the foodstuff.

Description of SEQ ID numbers

SEQ ID NO: 1 protein sequence of an Aspergillus niger mutarotase; a possible processing site in the amino acid sequence of SEQ ID NO: 1 is after amino acid 22 or 23; i.e. the mature protein is defined by the sequence of amino acids 23-403 or 24-403 SEQ ID NO: 2 genomic sequence

SEQ ID NO: 3 coding sequence

SEQ ID NO: 4 forward primer

SEQ ID NO: 5 reverse primer

Detailed description of the invention

In a first embodiment, the invention provides a method for preparing a dehydrated foodstuff from a starting material solution comprising crystallizing at least part of a carbohydrate in a concentrated starting material solution in the presence of a protein having mutarotase activity and drying the at least partly crystallized concentrated product (to obtain a dehydrated foodstuff).

A protein having mutarotase activity / mutarotase

Mutarotase (aldose 1-epimerase) accelerates the establishment of equilibrium between the alpha- and beta-anomers of aldoses, for example between alpha- and beta-glucose or alpha- and beta-galactose. The current main application of the enzyme is in analytical biochemistry for the acceleration of enzymatic detection reactions for aldoses by means of enzymes specific for the alpha- or beta-form, where the establishment of an equilibrium between the two anomers can become the rate- determining step, for example in sugar determination methods with glucose- dehydrogenase, glucose-oxidase, galactose-dehydrogenase or galactose-oxidase. Such determinations are conventional and are described in U.S. Pat. No. 3,964,974 and Anal. Biochem. 43, 312 (1971).

Mutarotase is very widespread in nature. It occurs in various microorganisms (bacteria, yeasts, thread fungi), in plants and in animal tissues.

Bacterial galactose mutarotases play a key role in the Leloir pathway by catalyzing the interconversion of β-D-galactose and a-D-galactose. In several bacterial species, especially in Escherichia coli and in Lactococcus lactis, the enzyme and its gene galM, a cistron of the gal operon, have been studied in detail. In contrast to bacteria, in various yeast species such as Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces lactis and Pachysolen tannophilus the mutarotase activity is assumed to be located at the C-terminal domain of the Gall Op protein, while the N-terminal domain of GaM Op has the UDP-glucose 4-epimerase activity needed for the interconversion of UDP-galactose and UDP-glucose in the galactose metabolism.

Significant enzyme contents which allow isolation of mutarotase on an industrial scale have so far only been found in the kidneys of mammals (cattle, pigs) and the known commercial products are prepared from kidneys. It is known from Bailey (Meth. Enzymol. 1975, page 478) that bovine kidneys contain more than 60 times the mutarotase activity per g of fresh weight than, for example, Escherichia coli.

Until recently little was known about possible genes encoding mutarotase in filamentous fungi. A gal10 gene has been identified in the ascomycete fungus Hypocrea jecorina. The gene codes for an enzyme that only shows strict conservation of the amino acid residues and motifs characteristic of the UDP-glucose 4-epimerases at the N-terminal domain of the yeast homologues. It was, therefore, speculated that the H. jecorina Gall Op lacks the mutarotase activity and that the UDP-glucose 4- epimerase and the mutarotase are two independent enzymes in this organism. Additionally, it was reported that a stress inducible protein, encoded by the cDNA from the zygomycete filamentous fungus Rhizopus nigricans (GenBank Accession No. Y10414), exhibits moderate amino acid sequence similarity in a 1 13 amino acid overlap with the galactose mutarotase from Acinetobacter calcoaceticus and the C-terminal domains of Gall Ops from K. lactis and S. cerevisiae. The alignment analysis also revealed the presence of two histidine residues, strictly conserved among different mutarotases. The mutarotase protein could be isolated from mycelial extracts of R. nigricans and the gene coding for the mutarotase was expressed in insect cells and mutarotase activity of a recombinant expressed enzyme was determined.

Additionally, Bentley and Bhate (1960) (J. Biol. Chem. 235, 1219-1233) have described the partial isolation and characterization of a mutarotase enzyme from the mycelial biomass of the fungus Penicillium notatum. P. notatum mutarotase was shown to be active on both glucose and galactose. In Biochim. Biophys. Acta 662, 285 (1981), Kinoshita et al. describe a process for the microbiological preparation of mutarotase from a strain of Aspergillus niger. According to this, a mutarotase activity of 4.4 mU/ml of culture broth was obtained from the best strain, the Michaelis constant was 50 mM, and the pH optimum was in the range 5-7. The Aspergillus mutarotase isolated by Kinoshita et al. has an estimated size of 260,000 Da, which appears to be a homodimer existing of 2 subunits of 130,000 Da. Like with Penicillium notatum, the Aspergillus enzyme was isolated from the mycelium of the fungus, this time using a homogenizer and glass-bead agitation. These results show that all the currently described fungal mutarotases are naturally located intracellular^.

The Aspergillus niger mutarotase described by Kinoshita et al. was also tested in the mutarotation of different sugars. While the enzyme was shown to be highly active on both glucose and galactose, on other sugars (among which was lactose) stimulation of mutarotation compared to the spontaneous reaction was only small. Similar results have been found with the Penicillium notatum mutarotase and mutarotase from hog kidney. The mechanistic aspects of mutarotation have been reviewed by Samuel and Tanner (Nat. Prod. Rep. 2002, 19, 261-277). Theoretically, mutarotation could be brought about by breaking any of the four bonds at C-1 of an aldose. The C(1)-0(ring) bond could be broken by a ring-opening reaction involving general acid/general base catalysis. Alternatively, the C(1)-H bond could be broken in a mechanism involving transient oxidation. However, this would require the presence of a cofactor. A third possibility is a mechanism similar to that of glycosidases, and involves cleavage of the C(1)-0(anomeric) bond with the addition of water. In the mutarotase isolated from Penicillium notatum no significant exchange of the C-1 oxygen or hydrogen has been observed and no primary deuterium kinetic isotope effect has been observed at C-1. This argues against the last two mechanisms. The formation of β-D-galactofuranose as a minor product when a-D-galactopyranose is allowed to equilibrate in water in the presence and absence of galactose mutarotase supports the mechanism involving ring opening. Thus, it seems that both the non-enzymatic and enzymatic reactions occur via an initial ring opening followed by ring closure with addition of the opposite face of the aldehyde. An enzymatic base of pKa 5.5 has been implicated as the catalytic residue. E. coli galactose mutarotase has been cloned, over-expressed and purified to homogeniety. It has been shown to be a part of the gal operon and is involved in the Leloir pathway. The enzyme lacks metal ions and oxidoreduction cofactors. Using the recombinant mutarotase, it has been shown that the enzyme catalyzes both the ring opening as well as closure. Mutagenesis studies on two conserved histidine residues suggest that one of them might be involved in catalysis.

The phrase "a protein having mutarotase activity" refers to a protein which is capable of converted a sugar in alpha conformation to a sugar in beta conformation and vice versa. Whether or not a protein has mutarotase activity is for example determined by assays that are known in the art (Miwa (1972) Analytical Biochemistry 45, 441-447; Miwa and Okuda (1974) J. Biochem. 75, 1 177-1 179; www.bbienzymes.com/userfiles/file/ap46mutarotase01.pdf). An example of such a mutarotase assay is described in Example 2. The protein does not necessarily have to be annotated as a mutaroase or as an aldose 1-epimerase. It might well be that a protein domain having mutarotase activity is fused to another protein sequence or protein domain and that the fusion product is not annotated as being a mutarotase.

In a preferred embodiment, the invention provides a method for preparing a dehydrated foodstuff from a starting material solution comprising crystallizing at least part of a carbohydrate in a concentrated starting material solution in the presence of a protein having mutarotase activity and drying the at least partly crystallized concentrated product (to obtain a dehydrated foodstuff), wherein said protein having mutarotase activity is a protein having at least 50% identity with the protein of SEQ ID NO: 1.

In yet another preferred embodiment, the invention provides a method for preparing a dehydrated foodstuff from a starting material solution comprising crystallizing at least part of a carbohydrate in a concentrated starting material solution in the presence of a protein having mutarotase activity and drying the at least partly crystallized concentrated product (to obtain a dehydrated foodstuff), wherein said protein has at least mutarotase activity for said carbohydrate, i.e. the carbohydrate that needs to be crystallized. For example, the invention provides a method for preparing a dehydrated foodstuff from a starting material solution comprising crystallizing at least part of a carbohydrate in a concentrated starting material solution in the presence of a protein having mutarotase activity and drying the at least partly crystallized concentrated product (to obtain a dehydrated foodstuff), wherein said protein has at least mutarotase activity for lactose.

In another embodiment, the invention provides a method for preparing a dehydrated foodstuff from a starting material solution comprising crystallizing at least part of a carbohydrate in a concentrated starting material solution in the presence of a protein having mutarotase activity and drying the at least partly crystallized concentrated product (to obtain a dehydrated foodstuff) and wherein said protein having mutarotase activity is immobilized on a carrier. Preferably said carrier and immobilized protein having mutarotase activity can easily be separated from the intermediate product.

All currently known mutarotases are intracellular proteins. Consequently, productivity of such enzymes is poor and industrial production of such enzymes for the use in the food, pharma or chemical industry is not (economically) feasible. Mutarotase isolated from porcine kidney is available but is very expensive and only suitable for laboratory scale testing. For instance, the use of mutarotase in the development of glucose sensors has been described. Furthermore, all described mutarotases are poorly active on lactose and generally prefer mono-hexoses or pentoses as substrate. E.g. the mutarotase from Escherichia coli has a Km of 475 mM for lactose, while the Km for glucose and galactose is 15 and 6.5 mM respectively (Hucho and Wallenfels (1971) Eur. J. Biochem. 23, 489-496). Consequently, the efficient mutarotation of lactose is not possible with currently available enzymes.

Here we describe the overexpression of a mutarotase that is active on lactose as substrate. The efficient production of this enzyme makes its use in crystallization of carbohydrates for the food, pharma or chemical industry (economically) feasible.

The nucleotide sequence of the gene expressing the mutarotase protein used in the experimental part has been published as NCBI Reference Sequence NT_166519.1. The protein sequence deduced from this gene sequence is publically available under accession number CAK44320.1 at GenBank and is furthermore presented as SEQ ID NO: 1 in the sequence listing accompanying this patent application. This protein sequence has 25-30% sequence identity when it is compared with galactose mutarotases from different mammalia, such as mouse, rat, human, bovine or porcine, using the pBLAST algorithm on the NCBI server (http://www.ncbi.nlm.nih.gov/). Identity of the Aspergillus niger mutarotase amino acid sequence with putative mutarotase from bacteria run up to 32% when using the same algorithm. Similar identities can be found with putative mutarotases from plants. Identity of the Aspergillus mutarotase with the yeast Gall Op is only 25%.

These relative poor identities with known mutarotases indicate that the Aspergillus niger mutarotase is drastically different from all currently described mutarotases. For the one skilled in the art it will be obvious that the Aspergillus niger mutarotase can be substituted for any other mutarotase active in the application, for the purpose of this invention. Preferably such other mutarotase is a secreted mutarotase and even more preferably such mutarotase is a secreted fungal mutarotase. In another embodiment such mutarotase would be active on carbohydrates, preferably active on sugars, and more preferably on lactose. Advantageously the protein having mutarotase activity of the invention is an extracellular enzyme. Moreover the protein having mutarotase activity is preferably of a fungal origin. An exoenzyme, or extracellular enzyme, is an enzyme that is secreted by a cell and that works outside that cell. It is usually used for breaking up large molecules that would not be able to enter the cell otherwise. It is an enzyme that organisms secrete into the environment which acts outside the microbe. The opposite of an exoenzyme is called an endoenzyme or intracellular enzyme.

The present invention provides a method for applying the protein having mutarotase activity which has been identified in the fungus Aspergillus niger in carbohydrate crystallization.

Advantageously the present invention meets the demand for a protein having mutarotase activity that can be produced in high amounts. Preferably, such a protein having mutarotase activity is secreted from a host cell. Active secretion is of paramount importance for an economical production process because it enables the recovery of the enzyme in an almost pure form without going through cumbersome purification processes. Overexpression of such an actively secreted protein having mutarotase activity by a food grade fungal host such as Aspergillus, yields a food grade enzyme and a cost effective production process, and is therefore preferable. The presently secreted protein having mutarotase activity is for the first time found in filamentous fungi. Processes are disclosed for the production of a protein having mutarotase activity in large amounts by the food-grade production host Aspergillus niger.

From an economic point of view there exists a clear need for an improved means of producing a protein having mutarotase activity in high quantities and in a relatively pure form, compared to the poor productivity of the mammalian and bacterial mutarotases. A preferred way of doing this is via the overproduction of such a mutarotase using recombinant DNA techniques. A particularly preferred way of doing this is via the overproduction of a fungal derived protein having mutarotase activity and a most preferred way of doing this is via the overproduction of an Aspergillus derived protein having mutarotase activity. To enable the latter production route unique sequence information of an Aspergillus derived mutarotase is essential. More preferable the whole nucleotide sequence of the encoding gene has to be available. An improved means of producing the newly identified secreted protein having mutarotase activity in high quantities and a relatively pure form is via the overproduction of the Aspergillus encoded enzyme using recombinant DNA techniques. A preferred way of doing this is via the overproduction of such a secreted protein having mutarotase activity in a food grade host microorganism. Well known food grade microorganisms include Aspergilli, Trichoderma, Fusarium, Streptomyces, Bacilli and yeasts such as Saccharomyces and Kluyveromyces. An even more preferred way of doing this is via overproduction of the secreted Aspergillus derived protein having mutarotase activity in a food grade fungus such as Aspergillus. Most preferred is the over production of the secreted protein having mutarotase activity in a food grade fungus in which the codon-usage of the mutarotase-encoding gene has been optimized for the food grade expression host used. In general, to enable the latter optimization routes, unique sequence information of a secreted protein having mutarotase activity is desirable. More preferable the whole nucleotide sequence of the protein having mutarotase activity encoding gene has to be available. Once the gene encoding a secreted protein having mutarotase activity is transformed in a preferred host, selected strains can be used for fermentation and isolation of the secreted protein having mutarotase activity protein from the fermentation broth.

A polypeptide, used in a method of the invention, which has mutarotase activity (i.e. protein having mutarotase activity) may be in an isolated form. As defined herein, an isolated polypeptide is an endogenously produced or a recombinant polypeptide which is essentially free from other non-mutarotase polypeptides, and is typically at least about 20% pure, preferably at least about 40% pure, more preferably at least about 60% pure, even more preferably at least about 80% pure, still more preferably about 90% pure, and most preferably about 95% pure, as determined by SDS-PAGE. The protein having mutarotase activity may be isolated by centrifugation, filtration and chromatographic methods, or any other technique known in the art for obtaining pure proteins from crude solutions. It will be understood that the protein having mutarotase activity may be mixed with carriers or diluents which do not interfere with the intended purpose of the protein having mutarotase activity, and thus the protein having mutarotase activity in this form will still be regarded as isolated. It will generally comprise the protein having mutarotase activity in a preparation in which more than 20%, for example more than 30%, 40%, 50%, 80%, 90%, 95% or 99%, by weight of the proteins in the preparation is a protein having mutarotase activity.

Preferably, the protein having mutarotase activity is obtainable from a microorganism which possesses a gene encoding an enzyme with mutarotase activity. More preferably the protein having mutarotase activity is secreted from a microorganism. Even more preferably the microorganism is fungal, and optimally is a filamentous fungus. Preferred donor organisms are thus of the genus Aspergillus, such as those of the species Aspergillus niger.

In a preferred embodiment, the invention provides a method for preparing a dehydrated foodstuff from a starting material solution comprising crystallizing at least part of a carbohydrate in a concentrated starting material solution in the presence of a protein having mutarotase activity and drying the at least partly crystallized concentrated product (to obtain a dehydrated foodstuff), wherein said protein having mutarotase activity has an amino acid sequence which has a degree of amino acid sequence identity to amino acids 1 to 403 of SEQ ID NO: 1 of at least 50% or 60%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, still more preferably at least 95%, and most preferably at least 97, 98 or 99%, and which has at least mutarotase activity for the carbohydrate that needs to be crystallized.

For the purposes of the present invention, the degree of identity between two or more amino acid sequences is determined by BLAST P protein database search program (Altschul et al., 1997, Nucleic Acids Research 25: 3389-3402) with matrix Blosum 62 and an expected threshold of 10.

In a more preferred embodiment, the invention provides a method for preparing a dehydrated foodstuff from a starting material solution comprising crystallizing at least part of a carbohydrate in a concentrated starting material solution in the presence of a protein having mutarotase activity and drying the at least partly crystallized concentrated product (to obtain a dehydrated foodstuff), wherein said protein having mutarotase activity comprises the amino acid sequence set forth in SEQ ID NO: 1 or a fragment thereof. In general, the amino acid sequence shown in SEQ ID NO: 1 is preferred.

Also part of the invention is the use of a part of the amino acid sequence shown in SEQ ID NO: 1 in the claimed method. Secreted enzymes, like the amino acid sequence of SEQ ID NO: 1 , are often synthesized including a pre- or signal-sequence and / or a pro-sequence. These sequences are often removed from the protein either during or after the secretion process. The mature secreted protein therefore often does not contain these pre- and pro-sequences anymore. A processed version of SEQ ID NO: 1 lacking possible pre- or pro-sequences can also be used in a method of the invention. A possible processing site in the amino acid sequence of SEQ ID NO: 1 is after amino acid 22 or 23. The mature enzyme will in this case start at amino acid number 23 or 24. Other modifications from the amino acid sequence of SEQ ID NO: 1 due to further processing are allowed as long that they do not disturb the activity of the enzyme.

In yet another preferred embodiment, the invention provides a method for preparing a dehydrated foodstuff from a starting material solution comprising crystallizing at least part of a carbohydrate in a concentrated starting material solution in the presence of a protein having mutarotase activity and drying the at least partly crystallized concentrated product (to obtain a dehydrated foodstuff), wherein said protein having mutarotase activity comprises a naturally occurring variant or species homologue of the polypeptide of SEQ ID NO: 1.

A variant is a polypeptide that occurs naturally in, for example, fungal, bacterial, yeast or plant cells, the variant having mutarotase activity and a sequence substantially similar to the protein of SEQ ID NO: 1. The term "variants" refers to polypeptides which have the same essential character or basic biological functionality as the mutarotase of SEQ ID NO: 1 , and includes allelic variants. Preferably, a variant polypeptide has at least the same level of mutarotase activity as the polypeptide of SEQ ID NO: 1. Variants include allelic variants either from the same strain as the polypeptide of SEQ ID NO: 1 or from a different strain of the same genus or species.

Similarly, a species homologue of the protein having mutarotase activity is an equivalent protein of similar sequence which is a mutarotase and occurs naturally in another species.

Variants and species homologues can be found using homology search of the amino acid sequence of SEQ ID NO: 1 against protein or nucleotide databases, using methods known in the art. Also possible is to use a probe (designed on the corresponding nucleotide sequence of the protein of SEQ ID NO: 1) to probe DNA libraries made from yeast, bacterial, fungal or plant cells in order to obtain clones expressing variants or species homologues of the polypeptide of SEQ ID NO: 1. The methods that can be used to isolate variants and species homologues of a known gene are extensively described in literature, and known to those skilled in the art. These genes can be manipulated by conventional techniques to generate a polypeptide of the invention which thereafter may be produced by recombinant or synthetic techniques known per se.

The sequence of the polypeptide of SEQ ID NO: 1 and of variants and species homologues can also be modified to provide polypeptides for use in a method of the invention. Amino acid substitutions may be made, for example from 1 , 2 or 3 to 10, 20 or 30 substitutions. The same number of deletions and insertions may also be made. These changes may be made outside regions critical to the function of the protein having mutarotase activity, as such a modified protein having mutarotase activity will retain its mutarotase activity.

A method according to the invention can also use a protein having mutarotase activity which protein is a fragment of the above mentioned full length polypeptide and of variants thereof, including fragments of the sequence set out in SEQ ID NO: 1. Such fragments will typically retain activity as a mutarotase (at least for the carbohydrate that needs to be crystallized). Fragments may be at least 50, 100 or 200 amino acids long or may be this number of amino acids short of the full length sequence shown in SEQ ID NO: 1.

A protein having mutarotase activity can, if necessary, be produced by synthetic means although usually they will be made recombinantly as described below. Synthetic and recombinant polypeptides may be modified, for example, by the addition of histidine residues or a T7 tag to assist their identification or purification, or by the addition of a signal sequence to promote their secretion from a cell.

Thus, the variants sequences may comprise those derived from strains of Aspergillus other than the strain from which the polypeptide of SEQ ID NO: 1 was isolated. Variants can be identified from other Aspergillus strains by looking for mutarotase activity and cloning and sequencing as described herein. Variants may include the deletion, modification or addition of single amino acids or groups of amino acids within the protein sequence, as long as the peptide maintains the basic biological functionality of the mutarotase of SEQ ID NO: 1. Amino acid substitutions may be made, for example from 1 , 2 or from 3 to 10, 20 or 30 substitutions. The modified a protein having mutarotase activity will generally retain activity as a mutarotase. Conservative substitutions may be made; such substitutions are well known in the art.

Shorter or longer polypeptide sequences can also be used in a method according to the invention. For example, a peptide of at least 50 amino acids or up 100, 150, 200, 300, 400, 500, 600, 700 or 800 amino acids in length is considered to fall within the scope of the invention as long as it demonstrates the basic biological functionality of the mutarotase of SEQ ID NO: 1. In particular, but not exclusively, this aspect of the invention encompasses the situation in which the protein is a fragment of the complete protein sequence.

The present invention also relates to a polynucleotide which encodes a protein having mutarotase activity, said polynucleotide comprises a polynucleotide sequence which encodes amino acid SEQ ID NO: 1.

For the present invention it is especially relevant that the protein having mutarotase activity is actively secreted into the growth medium. Secreted proteins are normally originally synthesized as pre-proteins and the pre-sequence (signal sequence) is subsequently removed during the secretion process. The secretion process is basically similar in prokaryotes and eukaryotes: the actively secreted pre-protein is threaded through a membrane, the signal sequence is removed by a specific signal peptidase, and the mature protein is (re)-folded. Also for the signal sequence a general structure can be recognized. Signal sequences for secretion are located at the amino- terminus of the pre-protein, and are generally 15-35 amino-acids in length. The amino- terminus preferably contains positively charged amino-acids, and preferably no acidic amino-acids. It is thought that this positively charged region interacts with the negatively charged head groups of the phospholipids of the membrane. This region is followed by a hydrophobic, membrane-spanning core region. This region is generally 10-20 amino- acids in length and consists mainly of hydrophobic amino-acids. Charged amino-acids are normally not present in this region. The membrane spanning region is followed by the recognition site for signal peptidase. The recognition site consists of amino-acids with the preference for small-X-small. Small amino-acids can be alanine, glycine, serine or cysteine. X can be any amino acids. Using such rules an algorithm has been written that is able to recognize such signal sequences from eukaryotes and prokaryotes (Bendtsen, Nielsen, von Heijne and Brunak. (2004) J. Mol. Biol., 340:783-795). The SignalP program to calculate and recognize signal sequences in proteins is generally available (http://www.cbs.dtu.dk/services/SignalP/).

Relevant for the present invention is that signal sequences can be recognized from the deduced protein sequence of a sequenced gene. If a gene encodes a protein where a signal sequence is predicted using the SignalP program, the chance that this protein is secreted is high.

In a further embodiment, the present invention provides a method for preparing a dehydrated foodstuff from a starting material solution comprising crystallizing at least part of a carbohydrate in a concentrated starting material solution in the presence of a protein having mutarotase activity and drying the at least partly crystallized concentrated product (to obtain a dehydrated foodstuff), wherein said protein having mutarotase activity is encoded by polynucleotides which hybridize or are capable of hybrizing under low stringency conditions, more preferably medium stringency conditions, and most preferably high stringency conditions, with (i) the nucleic acid sequence of SEQ ID NO:2 or SEQ ID NO:3 or (ii) a nucleic acid fragment comprising at least a portion of SEQ ID NO:2 or SEQ ID NO:3, or (iii) having bases differing from the bases of SEQ ID NO:2 or SEQ ID NO:3; or (iv) with a nucleic acid strand complementary to SEQ ID NO:2 or SEQ ID NO:3.

The term "capable of hybridizing" means that the target polynucleotide can hybridize to the nucleic acid used as a probe (for example, the nucleotide sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3, or a fragment thereof, or the complement of SEQ ID NO: 2 or SEQ ID NO: 3, or a fragment thereof) at a level significantly above background. The nucleotide sequence may be RNA or DNA, including genomic DNA, synthetic DNA or cDNA. Preferably, the nucleotide sequence is DNA and most preferably, a genomic DNA sequence. Typically, a polynucleotide comprises a contiguous sequence of nucleotides which is capable of hybridizing under selective conditions to the coding sequence or the complement of the coding sequence of SEQ ID NO: 2 or SEQ ID NO:3. Such nucleotides can be synthesized according to methods well known in the art.

Such nucleotides can hybridize to the coding sequence or the complement of the coding sequence of SEQ ID NO: 3 at a level significantly above background. Background hybridization may occur, for example, because of other cDNAs present in a cDNA library. The signal level generated by the interaction between polynucleotide as described herein and the coding sequence or complement of the coding sequence of SEQ ID NO: 3 is typically at least 10 fold, preferably at least 20 fold, more preferably at least 50 fold, and even more preferably at least 100 fold, as intense as interactions between other polynucleotides and the coding sequence of SEQ ID NO: 3. The intensity of interaction may be measured, for example, by radiolabelling the probe, for example with 32 P. Selective hybridization may typically be achieved using conditions of low stringency (0.3M sodium chloride and 0.03M sodium citrate at about 40°C), medium stringency (for example, 0.3M sodium chloride and 0.03M sodium citrate at about 50°C) or high stringency (for example, 0.3M sodium chloride and 0.03M sodium citrate at about 60°C).

A polynucleotide as described herein also includes synthetic genes that can encode for the polypeptide of SEQ ID NO: 1 or variants thereof. It is sometimes preferable to adapt the codon usage of a gene to the preferred bias in a production host. Techniques to design and construct synthetic genes are generally available (i.e. http://www. dnatwopointo.com/).

Modifications

Polynucleotides as described herein may comprise DNA or RNA. They may be single or double stranded. They may also be polynucleotides which include within them synthetic or modified nucleotides including peptide nucleic acids. A number of different types of modifications to polynucleotides are known in the art. These include a methylphosphonate and phosphorothioate backbones, and addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the present invention, it is to be understood that the polynucleotides described herein may be modified by any method available in the art.

It is to be understood that skilled persons may, using routine techniques, make nucleotide substitutions that do not affect the polypeptide sequence encoded by the polynucleotides as described herein to reflect the codon usage of any particular host organism in which the polypeptides as described herein are to be expressed.

The coding sequence of SEQ ID NO: 3 or the genomic sequence of SEQ ID NO: 2 may be modified by nucleotide substitutions, for example from 1 , 2 or 3 to 10, 25, 50, 100, or more substitutions. The polynucleotide of SEQ ID NO: 3 or SEQ ID NO: 2 may alternatively or additionally be modified by one or more insertions and/or deletions and/or by an extension at either or both ends. The modified polynucleotide generally encodes a polypeptide which has mutarotase activity. Degenerate substitutions may be made and/or substitutions may be made which would result in a conservative amino acid substitution when the modified sequence is translated, for example as discussed with reference to polypeptides later.

Homologues

A nucleotide sequence which is capable of selectively hybridizing to the complement of the DNA coding sequence of SEQ ID NO:3 or the genomic sequence of SEQ ID NO: 2 is included for use in a method of the invention and will generally have at least 50% or 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the coding sequence of SEQ ID NO:3 or the genomic sequence of SEQ ID NO: 2 over a region of at least 60, preferably at least 100, more preferably at least 200 contiguous nucleotides or most preferably over the full length of SEQ ID NO:3 or SEQ ID NO: 2. Likewise, a nucleotide which encodes an active mutarotase and which is capable of selectively hybridizing to a fragment of a complement of the DNA coding sequence of SEQ ID NO:3 or the genomic sequence of SEQ ID NO: 2, is also embraced for use in a method of the invention.

Any combination of the above mentioned degrees of identity and minimum sizes may be used to define polynucleotides as described herein, with the more stringent combinations (i.e. higher identity over longer lengths) being preferred. Thus, for example, a polynucleotide which is at least 80% or 90% identical over 60, preferably over 100 nucleotides, forms one aspect of the invention, as does a polynucleotide which is at least 90% identical over 200 nucleotides.

The BLAST N algorithm can be used to calculate sequence identity or to line up sequences (such as identifying equivalent or corresponding sequences, for example on their default settings).

Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: //www .ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits act as seeds for initiating searches to find HSPs containing them. The word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extensions for the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 1 1 , the BLOSUM62 scoring matrix alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similarity between two sequences. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a sequence is considered similar to another sequence if the smallest sum probability in comparison of the first sequence to the second sequence is less than about 1 , preferably less than about 0.1 , more preferably less than about 0.01 , and most preferably less than about 0.001.

Primers and Probes

Polynucleotides as described herein include and may be used as primers, for example as polymerase chain reaction (PCR) primers, as primers for alternative amplification reactions, or as probes for example labeled with a revealing label by conventional means using radioactive or non-radioactive labels, or the polynucleotides may be cloned into vectors. Such primers, probes and other fragments will be at least 15, for example at least 20, 25, 30 or 40 nucleotides in length. They will typically be up to 40, 50, 60, 70, 100, 150, 200 or 300 nucleotides in length, or even up to a few nucleotides (such as 5 or 10 nucleotides) short of the coding sequence of SEQ ID NO: 3 or the genomic sequence of SEQ ID NO: 2.

In general, primers will be produced by synthetic means, involving a step-wise manufacture of the desired nucleic acid sequence one nucleotide at a time. Techniques for accomplishing this and protocols are readily available in the art. Longer polynucleotides will generally be produced using recombinant means, for example using PCR cloning techniques. This will involve making a pair of primers (typically of about 15-30 nucleotides) to amplify the desired region of the mutarotase to be cloned, bringing the primers into contact with mRNA, cDNA or genomic DNA obtained from a yeast, bacterial, plant, prokaryotic or fungal cell, preferably of an Aspergillus strain, performing a polymerase chain reaction under conditions suitable for the amplification of the desired region, isolating the amplified fragment (e.g. by purifying the reaction mixture on an agarose gel) and recovering the amplified DNA. The primers may be designed to contain suitable restriction enzyme recognition sites so that the amplified DNA can be cloned into a suitable cloning vector, such as described in Example 1.

Alternatively, synthetic genes can be constructed that encompass the coding region of the secreted mutarotase or variants thereof. Polynucleotides that are altered in many positions, but still encode the same protein can be conveniently be designed and constructed using these techniques. This has as advantage that the codon usage can be adapted to the preferred expression host, so productivity of the protein in this host can be improved. Also the polynucleotide sequence of a gene can be changed to improve mRNA stability or reduced turnover. This can lead to improved expression of the desired protein or variants thereof. Additionally, the polynucleotide sequence can be changed in a synthetic gene such that mutations are made in the protein sequence that have a positive effect on secretion efficiency, stability, proteolytic vulnerability, temperature optimum, specific activity or other relevant properties for industrial production or application of the protein. Companies that provide services to construct synthetic genes and optimize codon usage are generally available.

Such techniques may be used to obtain all or part of the polynucleotides encoding a protein having mutarotase activity. Introns, promoter and trailer regions can be used and may also be obtained in an analogous manner (e.g. by recombinant means, PCR or cloning techniques), starting with genomic DNA from a fungal, yeast, bacterial plant or prokaryotic cell.

The polynucleotides or primers may carry a revealing label. Suitable labels include radioisotopes such as 32 P or 35 S, fluorescent labels, enzyme labels, or other protein labels such as biotin. Such labels may be added to polynucleotides or primers as described herein and may be detected using techniques known to persons skilled in the art. Polynucleotides or primers (or fragments thereof) labeled or unlabelled may be used in nucleic acid-based tests for detecting or sequencing a mutarotase or a variant thereof in a fungal sample. Such detection tests will generally comprise bringing a fungal sample suspected of containing the DNA of interest into contact with a probe comprising a polynucleotide or primer as described herein under hybridizing conditions, and detecting any duplex formed between the probe and nucleic acid in the sample. Detection may be achieved using techniques such as PCR or by immobilizing the probe on a solid support, removing any nucleic acid in the sample which is not hybridized to the probe, and then detecting any nucleic acid which is hybridized to the probe. Alternatively, the sample nucleic acid may be immobilized on a solid support, the probe hybridized and the amount of probe bound to such a support after the removal of any unbound probe detected.

The probes as described herein may conveniently be packaged in the form of a test kit in a suitable container. In such kits the probe may be bound to a solid support where the assay format for which the kit is designed requires such binding. The kit may also contain suitable reagents for treating the sample to be probed, hybridizing the probe to nucleic acid in the sample, control reagents, instructions, and the like. The probes and polynucleotides of the invention may also be used in microassay.

Preferably, the polynucleotide as described herein is obtainable from the same organism as the polypeptide, such as a fungus, in particular a fungus of the genus Aspergillus.

Production of polynucleotides

Polynucleotides which do not have 100% identity with SEQ ID NO: 3 or SEQ ID NO: 2 can be obtained in a number of ways. Thus, variants of the mutarotase sequence described herein may be obtained for example, by probing genomic DNA libraries made from a range of organisms, such as those discussed as sources of the polypeptides as described herein. In addition, other fungal, plant or prokaryotic homologues of mutarotase may be obtained and such homologues and fragments thereof in general will be capable of hybridising to SEQ ID NO:3 or SEQ ID NO: 2. Such sequences may be obtained by probing cDNA libraries or genomic DNA libraries from other species, and probing such libraries with probes comprising all or part of SEQ ID NO: 2 or 3 under conditions of low, medium to high stringency (as described earlier). Nucleic acid probes comprising all or part of SEQ ID NO: 2 or 3 may be used to probe cDNA or genomic libraries from other species, such as those described as sources for a polypeptide having mutarotase activity.

Species homologues may also be obtained using degenerate PCR, which uses primers designed to target sequences within the variants and homologues which encode conserved amino acid sequences. The primers can contain one or more degenerate positions and will be used at stringency conditions lower than those used for cloning sequences with single sequence primers against known sequences.

Alternatively, such polynucleotides may be obtained by site directed mutagenesis of the mutarotase sequences or variants thereof. This may be useful where, for example, silent codon changes to sequences are required to optimize codon preferences for a particular host cell in which the polynucleotide sequences are being expressed. Other sequence changes may be made in order to introduce restriction enzyme recognition sites, or to alter the property or function of the polypeptides encoded by the polynucleotides.

The invention also describes double stranded polynucleotides comprising a polynucleotide as described above and its complement.

The present invention also describes polynucleotides encoding the polypeptides of the invention described above. Since such polynucleotides will be useful as sequences for recombinant production of a protein having mutarotase activity, it is not necessary for them to be capable of hybridising to the sequence of SEQ ID NO: 2 or SEQ ID NO: 3, although this will generally be desirable. Otherwise, such polynucleotides may be labeled, used, and made as described above if desired.

Recombinant Polynucleotides

The invention also describes vectors comprising a polynucleotide as described above, including cloning and expression vectors, and in another aspect methods of growing, transforming or transfecting such vectors into a suitable host cell, for example under conditions in which expression of a polypeptide as, or encoded by a sequence as, described above. Provided also are host cells comprising a polynucleotide or vector oas herein described wherein the polynucleotide is heterologous to the genome of the host cell. The term "heterologous", usually with respect to the host cell, means that the polynucleotide does not naturally occur in the genome of the host cell or that the polypeptide is not naturally produced by that cell. Preferably, the host cell is a yeast cell, for example a yeast cell of the genus Kluyveromyces, Pichia, Hansenula or Saccharomyces or a filamentous fungal cell, for example of the genus Aspergillus, Penicillium, Trichoderma or Fusarium.

Vectors

The vector into which the expression cassette as described herein is inserted may be any vector that may conveniently be subjected to recombinant DNA procedures, and the choice of the vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously replicating vector, i.e. a vector which exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, such as a plasmid. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicates together with the chromosome(s) into which it has been integrated.

Preferably, when a polynucleotide as described herein is in a vector it is operably linked to a regulatory sequence which is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector. The term "operably linked" refers to a juxtaposition wherein the components described are in a relationship permitting them to function in their intended manner. A regulatory sequence such as a promoter, enhancer or other expression regulation signal "operably linked" to a coding sequence is positioned in such a way that expression of the coding sequence is achieved under production conditions.

The vectors may, for example in the case of plasmid, cosmid, virus or phage vectors, be provided with an origin of replication, optionally a promoter for the expression of the polynucleotide and optionally an enhancer and/or a regulator of the promoter. A terminator sequence may be present, as may be a polyadenylation sequence. The vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a neomycin resistance gene for a mammalian vector. Vectors may be used in vitro, for example for the production of RNA or can be used to transfect or transform a host cell.

The DNA sequence encoding a protein having mutarotase activity is preferably introduced into a suitable host as part of an expression construct in which the DNA sequence is operably linked to expression signals which are capable of directing expression of the DNA sequence in the host cells. For transformation of the suitable host with the expression construct transformation procedures are available which are well known to the skilled person. The expression construct can be used for transformation of the host as part of a vector carrying a selectable marker, or the expression construct is co-transformed as a separate molecule together with the vector carrying a selectable marker. The vectors may contain one or more selectable marker genes.

Preferred selectable markers include but are not limited to those that complement a defect in the host cell or confer resistance to a drug. They include for example versatile marker genes that can be used for transformation of most filamentous fungi and yeasts such as acetamidase genes or cDNAs (the amdS, niaD, facA genes or cDNAs from A.nidulans, A.oryzae, or A.niger), or genes providing resistance to antibiotics like G418, hygromycin, bleomycin, kanamycin, phleomycin or benomyl resistance (benA). Alternatively, specific selection markers can be used such as auxotrophic markers which require corresponding mutant host strains: e.g. URA3 (from S.cerevisiae or analogous genes from other yeasts), pyrG or pyrA (from A.nidulans or A.niger), argB (from A.nidulans or A.niger) or trpC. In a preferred embodiment the selection marker is deleted from the transformed host cell after introduction of the expression construct so as to obtain transformed host cells capable of producing the polypeptide which are free of selection marker genes.

Other markers include ATP synthetase subunit 9 (oliC), orotidine-5'-phosphate- decarboxylase (pvrA), the bacterial G418 resistance gene (useful in yeast, but not in filamentous fungi), the ampicillin resistance gene (E. coli), the neomycin resistance gene (Bacillus) and the E. coli uidA gene, coding for glucuronidase (GUS). Vectors may be used in vitro, for example for the production of RNA or to transfect or transform a host cell.

For most filamentous fungi and yeast, the expression construct is preferably integrated into the genome of the host cell in order to obtain stable transformants. However, for certain yeasts suitable episomal vector systems are also available into which the expression construct can be incorporated for stable and high level expression. Examples thereof include vectors derived from the 2 μηι, CEN and pKD1 plasmids of Saccharomyces and Kluyveromyces, respectively, or vectors containing an AMA sequence (e.g. AMA1 from Aspergillus). When expression constructs are integrated into host cell genomes, the constructs are either integrated at random loci in the genome, or at predetermined target loci using homologous recombination, in which case the target loci preferably comprise a highly expressed gene. A highly expressed gene is a gene whose mRNA can make up at least 0.01 % (w/w) of the total cellular mRNA, for example under induced conditions, or alternatively, a gene whose gene product can make up at least 0.2% (w/w) of the total cellular protein, or, in case of a secreted gene product, can be secreted to a level of at least 0.05 g/l.

An expression construct for a given host cell will usually contain the following elements operably linked to each other in consecutive order from the 5'-end to 3'-end relative to the coding strand of the sequence encoding the polypeptide of the first aspect: (1) a promoter sequence capable of directing transcription of the DNA sequence encoding the polypeptide in the given host cell, (2) preferably, a 5'- untranslated region (leader), (3) optionally, a signal sequence capable of directing secretion of the polypeptide from the given host cell into the culture medium, (4) the DNA sequence encoding a mature and preferably active form of the polypeptide, and preferably also (5) a transcription termination region (terminator) capable of terminating transcription downstream of the DNA sequence encoding the polypeptide.

Downstream of the DNA sequence encoding a polypeptide for use in a method of the invention, the expression construct preferably contains a 3' untranslated region containing one or more transcription termination sites, also referred to as a terminator. The origin of the terminator is less critical. The terminator can for example be native to the DNA sequence encoding the polypeptide. However, preferably a bacterial terminator is used in bacterial host cells, a yeast terminator is used in yeast host cells and a filamentous fungal terminator is used in filamentous fungal host cells. More preferably, the terminator is endogenous to the host cell in which the DNA sequence encoding the polypeptide is expressed.

Enhanced expression of the polynucleotide encoding a protein having mutarotase activity may also be achieved by the selection of heterologous regulatory regions, e.g. promoter, signal sequence and terminator regions, which serve to increase expression and, if desired, secretion levels of the protein of interest from the chosen expression host and/or to provide for the inducible control of the expression of the polypeptide of the invention. Aside from the promoter native to the gene encoding a protein having mutarotase activity, other promoters may be used to direct expression of said protein. The promoter may be selected for its efficiency in directing the expression of the polypeptide of the invention in the desired expression host.

Promoters/enhancers and other expression regulation signals may be selected to be compatible with the host cell for which the expression vector is designed. For example, prokaryotic promoters may be used, in particular those suitable for use in E.coli strains. When expression of a protein having mutarotase activity is carried out in mammalian cells, mammalian promoters may be used. Tissues-specific promoters, for example hepatocyte cell-specific promoters, may also be used. Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR), the rous sarcoma virus (RSV) LTR promoter, the SV40 promoter, the human cytomegalovirus (CMV) IE promoter, herpes simplex virus promoters or adenovirus promoters.

Suitable yeast promoters include the S. cerevisiae GAL4 and ADH promoters and the S. pombe nmt1 and adh promoter. Mammalian promoters include the metallothionein promoter which can be induced in response to heavy metals such as cadmium. Viral promoters such as the SV40 large T antigen promoter or adenovirus promoters may also be used. All these promoters are readily available in the art.

Mammalian promoters, such as β-actin promoters, may be used. Tissue-specific promoters, in particular endothelial or neuronal cell specific promoters (for example the DDAHI and DDAHII promoters), are especially preferred. Viral promoters may also be used, for example the Moloney murine leukaemia virus long terminal repeat (MMLV LTR), the rous sarcoma virus (RSV) LTR promoter, the SV40 promoter, the human cytomegalovirus (CMV) IE promoter, adenovirus, HSV promoters (such as the HSV IE promoters), or HPV promoters, particularly the HPV upstream regulatory region (URR). Viral promoters are readily available in the art.

A variety of promoters can be used that are capable of directing transcription in the host cells as described herein. Preferably the promoter sequence is derived from a highly expressed gene as previously defined. Examples of preferred highly expressed genes from which promoters are preferably derived and/or which are comprised in preferred predetermined target loci for integration of expression constructs, include but are not limited to genes encoding glycolytic enzymes such as triose-phosphate isomerases (TPI), glyceraldehyde-phosphate dehydrogenases (GAPDH), phosphoglycerate kinases (PGK), pyruvate kinases (PYK), alcohol dehydrogenases (ADH), as well as genes encoding amylases, glucoamylases, proteases, xylanases, cellobiohydrolases, β-galactosidases, alcohol (methanol) oxidases, elongation factors and ribosomal proteins. Specific examples of suitable highly expressed genes include e.g. the LAC4 gene from Kluyveromyces sp., the methanol oxidase genes (AOX and MOX) from Hansenula and Pichia, respectively, the glucoamylase (glaA) genes from A.niger and A.awamori, the A.oryzae TAKA-amylase gene, the A.nidulans gpdA gene and the T.reesei cellobiohydrolase genes.

Examples of strong constitutive and/or inducible promoters which are preferred for use in fungal expression hosts are those which are obtainable from the fungal genes for xylanase (xlnA), phytase, ATP-synthetase subunit 9 (oliC), triose phosphate isomerase (tpi), alcohol dehydrogenase (AdhA), amylase (amy), amyloglucosidase (AG - from the glaA gene), acetamidase (amdS) and glyceraldehyde-3-phosphate dehydrogenase (gpd) promoters.

Examples of strong yeast promoters which may be used include those obtainable from the genes for alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, lactase, 3-phosphoglycerate kinase, plasma membrane ATPase (PMA1) and triosephosphate isomerase.

Examples of strong bacterial promoters which may be used include the amylase and SPo2 promoters as well as promoters from extracellular protease genes.

Promoters suitable for plant cells which may be used include napaline synthase (nos), octopine synthase (ocs), mannopine synthase (mas), ribulose small subunit (rubisco ssu), histone, rice actin, phaseolin, cauliflower mosaic virus (CMV) 35S and 19S and circovirus promoters.

The vector may further include sequences flanking the polynucleotide giving rise to RNA which comprise sequences homologous to ones from eukaryotic genomic sequences, preferably fungal genomic sequences, or yeast genomic sequences. This will allow the introduction of the polynucleotides into the genome of fungi or yeasts by homologous recombination. In particular, a plasmid vector comprising the expression cassette flanked by fungal sequences can be used to prepare a vector suitable for delivering the polynucleotides to a fungal cell. Transformation techniques using these fungal vectors are known to those skilled in the art. Host Cells and Expression

In a further aspect the invention describes a process for preparing a protein having mutarotase activity which comprises cultivating a host cell transformed or transfected with an expression vector as described above under conditions suitable for expression by the vector of a coding sequence encoding said protein, and recovering the expressed protein. Polynucleotides as described herein can be incorporated into a recombinant replicable vector, such as an expression vector. The vector may be used to replicate the nucleic acid in a compatible host cell. Thus in a further embodiment, the invention describes a method of making a polynucleotide as described herein by introducing a polynucleotide as described herein into a replicable vector, introducing the vector into a compatible host cell, and growing the host cell under conditions which bring about the replication of the vector. Suitable host cells include bacteria such as E. coli, yeast, mammalian cell lines and other eukaryotic cell lines, for example insect cells such as Sf9 cells and (e.g. filamentous) fungal cells.

Preferably a protein having mutarotase activity is produced as a secreted protein in which case the DNA sequence encoding a mature form of the polypeptide in the expression construct may be operably linked to a DNA sequence encoding a signal sequence. In the case where the gene encoding the secreted protein has in the wild type strain a signal sequence preferably the signal sequence used will be native (homologous) to the DNA sequence encoding the polypeptide. Alternatively the signal sequence is foreign (heterologous) to the DNA sequence encoding the polypeptide, in which case the signal sequence is preferably endogenous to the host cell in which the DNA sequence is expressed. Examples of suitable signal sequences for yeast host cells are the signal sequences derived from yeast MFalpha genes. Similarly, a suitable signal sequence for filamentous fungal host cells is e.g. a signal sequence derived from a filamentous fungal amyloglucosidase (AG) gene, e.g. the A.niger glaA gene. This signal sequence may be used in combination with the amyloglucosidase (also called (gluco) amylase) promoter itself, as well as in combination with other promoters. Hybrid signal sequences may also be used within the context of the present invention.

Preferred heterologous secretion leader sequences are those originating from the fungal amyloglucosidase (AG) gene (glaA - both 18 and 24 amino acid versions e.g. from Aspergillus), the MFalpha gene (yeasts e.g. Saccharomyces, Pichia and Kluyveromyces) or the alpha-amylase gene (Bacillus).

The vectors may be transformed or transfected into a suitable host cell as described above to provide for expression of a protein having mutarotase activity. This process may comprise culturing a host cell transformed with an expression vector as described above under conditions suitable for expression of said protein, and optionally recovering the expressed protein.

The invention thus further describes host cells transformed or transfected with or comprising a polynucleotide or vector as described above. Preferably the polynucleotide is carried in a vector which allows the replication and expression of the polynucleotide. The cells will be chosen to be compatible with the said vector and may for example be prokaryotic (for example bacterial), or eukaryotic fungal, yeast or plant cells.

The invention encompasses processes for the production of a protein having mutarotase activity by means of recombinant expression of a DNA sequence encoding said protein. For this purpose the DNA sequence as described herein can be used for gene amplification and/or exchange of expression signals, such as promoters, secretion signal sequences, in order to allow economic production of the polypeptide in a suitable homologous or heterologous host cell. A homologous host cell is herein defined as a host cell which is of the same species or which is a variant within the same species as the species from which the DNA sequence is derived.

Suitable host cells are preferably prokaryotic microorganisms such as bacteria, or more preferably eukaryotic organisms, for example fungi, such as yeasts or filamentous fungi, or plant cells. In general, yeast cells are preferred over filamentous fungal cells because they are easier to manipulate. However, some proteins are either poorly secreted from yeasts, or in some cases are not processed properly (e.g. hyperglycosylation in yeast). In these instances, a filamentous fungal host organism should be selected.

Bacteria from the genus Bacillus are very suitable as heterologous hosts because of their capability to secrete proteins into the culture medium. Other bacteria suitable as hosts are those from the genera Streptomyces and Pseudomonas. A preferred yeast host cell for the expression of the DNA sequence encoding the polypeptide is one of the genus Saccharomyces, Kluyveromyces, Hansenula, Pichia, Yarrowia, or Schizosaccharomyces. More preferably, a yeast host cell is selected from the group consisting of the species Saccharomyces cerevisiae, Kluyveromyces lactis (also known as Kluyveromyces marxianus var. lactis), Hansenula polymorpha, Pichia pastoris, Yarrowia lipolytica, and Schizosaccharomyces pombe.

Most preferred for the expression of the DNA sequence encoding a protein having mutarotase activity, however, are filamentous fungal host cells. Preferred filamentous fungal host cells are selected from the group consisting of the genera Aspergillus, Trichoderma, Fusarium, Disporotrichum, Penicillium, Acremonium, Neurospora, Thermoascus, Myceliophtora, Sporotrichum, Thielavia, and Talaromyces. More preferably a filamentous fungal host cell is of the species Aspergillus oyzae, Aspergillus sojae or Aspergillus nidulans or is of a species from the Aspergillus niger Group (as defined by Raper and Fennell, The Genus Aspergillus, The Williams & Wilkins Company, Baltimore, pp 293-344, 1965). These include but are not limited to Aspergillus niger, Aspergillus awamori, Aspergillus tubigensis, Aspergillus aculeatus, Aspergillus foetidus, Aspergillus nidulans, Aspergillus japonicus, Aspergillus oryzae and Aspergillus ficuum, and also those of the species Trichoderma reesei, Fusarium graminearum, Penicillium chrysogenum, Acremonium alabamense, Neurospora crassa, Myceliophtora thermophilum, Sporotrichum cellulophilum, Disporotrichum dimorphosporum and Thielavia terrestris.

Examples of preferred expression hosts are fungi such as Aspergillus species (in particular those described in EP-A-184,438 and EP-A-284,603) and Trichoderma species; bacteria such as Bacillus species (in particular those described in EP-A- 134,048 and EP-A-253,455), especially Bacillus subtilis, Bacillus licheniformis, Bacillus amyloliquefaciens, Pseudomonas species; and yeasts such as Kluyveromyces species (in particular those described in EP-A-096,430 such as Kluyveromyces lactis and in EP- A-301 ,670) and Saccharomyces species, such as Saccharomyces cerevisiae.

Host cells include plant cells, and the invention therefore further describes transgenic organisms, such as plants and parts thereof, which contain one or more cells as described herein. The cells may heterologously express a protein having mutarotase activity or may heterologously contain one or more of the polynucleotides as described herein. The transgenic (or genetically modified) plant may therefore have inserted (typically stably) into its genome a sequence encoding a protein having mutarotase activity. The transformation of plant cells can be performed using known techniques, for example using a Ti or a Ri plasmid from Agrobacterium tumefaciens. The plasmid (or vector) may thus contain sequences necessary to infect a plant, and derivatives of the Ti and/or Ri plasmids may be employed.

The host cell may overexpress a protein having mutarotase activity, and techniques for engineering over-expression are well known and can be used in the present invention. The host may thus have more than one copy of the polynucleotide as described herein.

Alternatively, direct infection of a part of a plant, such as a leaf, root or stem can be effected. In this technique the plant to be infected can be wounded, for example by cutting the plant with a razor, puncturing the plant with a needle or rubbing the plant with an abrasive. The wound is then inoculated with the Agrobacterium. The plant or plant part can then be grown on a suitable culture medium and allowed to develop into a mature plant. Regeneration of transformed cells into genetically modified plants can be achieved by using known techniques, for example by selecting transformed shoots using an antibiotic and by sub-culturing the shoots on a medium containing the appropriate nutrients, plant hormones and the like.

Culture of host cells and recombinant production

The invention also describes cells that have been modified to express the mutarotase or a variant thereof. Such cells include transient, or preferably stably modified higher eukaryotic cell lines, such as mammalian cells or insect cells, lower eukaryotic cells, such as yeast and filamentous fungal cells or prokaryotic cells such as bacterial cells.

It is also possible for a protein having mutarotase activity to be transiently expressed in a cell line or on a membrane, such as for example in a baculovirus expression system.

The production of a protein having mutarotase activity can be effected by the culturing of microbial expression hosts, which have been transformed with one or more polynucleotides as described herein, in a conventional nutrient fermentation medium.

The recombinant host cells as described herein may be cultured using procedures known in the art. For each combination of a promoter and a host cell, culture conditions are available which are conducive for expression the DNA sequence encoding a protein having mutarotase activity. After reaching the desired cell density or titre of a protein having mutarotase activity the culturing is ceased and the protein having mutarotase activity is recovered using known procedures.

The fermentation medium can comprise a known culture medium containing a carbon source (e.g. glucose, maltose, molasses, etc.), a nitrogen source (e.g. ammonia, ammonium sulphate, ammonium nitrate, ammonium chloride, etc.), an organic nitrogen source (e.g. yeast extract, malt extract, peptone, etc.) and inorganic nutrient sources (e.g. phosphate, magnesium, potassium, zinc, iron, etc.). Optionally, an inducer (dependent on the expression construct used) may be included or subsequently be added.

The selection of the appropriate medium may be based on the choice of expression host and/or based on the regulatory requirements of the expression construct. Suitable media are well-known to those skilled in the art. The medium may, if desired, contain additional components favoring the transformed expression hosts over other potentially contaminating microorganisms.

The fermentation may be performed over a period of from 0.5-30 days. Fermentation may be a batch, continuous or fed-batch process, at a suitable temperature in the range of between 0°C and 45°C and, for example, at a pH from 2 to 10. Preferred fermentation conditions include a temperature in the range of between 20°C and 37°C and/or a pH between 3 and 9. The appropriate conditions are usually selected based on the choice of the expression host and the protein to be expressed.

After fermentation, if necessary, the cells can be removed from the fermentation broth by means of centrifugation or filtration. After fermentation has stopped or after removal of the cells, a protein having mutarotase activity may then be recovered and, if desired, purified and isolated by conventional means. The protein having mutarotase activity can be purified from fungal mycelium or from the culture broth into which the mutarotase is released by the cultured fungal cells.

Preferably, a protein having mutarotase activity is produced from a fungus, more preferably from an Aspergillus, most preferably from Aspergillus niger.

Modifications

A protein having mutarotase activity may be chemically modified, e.g. post- translationally modified. For example, they may be glycosylated (one or more times) or comprise modified amino acid residues. They may also be modified by the addition of histidine residues to assist their purification or by the addition of a signal sequence to promote secretion from the cell. The protein having mutarotase activity may have amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or a small extension that facilitates purification, such as a poly-histidine tract, an antigenic epitope or a binding domain.

A protein having mutarotase activity may be labelled with a revealing label. The revealing label may be any suitable label which allows the polypeptide to be detected. Suitable labels include radioisotopes, e.g. 125 l, 35 S, enzymes, antibodies, polynucleotides and linkers such as biotin.

A protein having mutarotase activity may be modified to include non-naturally occurring amino acids or to increase the stability of the polypeptide. When the proteins or peptides are produced by synthetic means, such amino acids may be introduced during production. The proteins or peptides may also be modified following either synthetic or recombinant production.

A protein having mutarotase activity may also be produced using D-amino acids. In such cases the amino acids will be linked in reverse sequence in the C to N orientation. This is conventional in the art for producing such proteins or peptides.

A number of side chain modifications are known in the art and may be made to the side chains of a protein having mutarotase activity. Such modifications include, for example, modifications of amino acids by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 , amidination with methylacetimidate or acylation with acetic anhydride.

The sequences as described herein may also be used as starting materials for the construction of "second generation" enzymes. "Second generation" mutarotases are mutarotases, altered by mutagenesis techniques (e.g. site-directed mutagenesis or gene shuffling techniques), which have properties that differ from those of wild-type mutarotase or recombinant mutarotase such as those produced by the present invention. For example, their temperature or pH optimum, specific activity, substrate affinity or thermostability may be altered so as to be better suited for use in a particular process.

Amino acids essential to the activity of the mutarotase of the invention, and therefore preferably subject to substitution, may be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis. In the latter technique mutations are introduced at every residue in the molecule, and the resultant mutant molecules are tested for biological activity (e.g. mutarotase activity) to identify amino acid residues that are critical to the activity of the molecule. Sites of enzyme-substrate interaction can also be determined by analysis of crystal structure as determined by such techniques as nuclear magnetic resonance, crystallography or photo-affinity labeling.

Gene shuffling techniques provide a random way to introduce mutations in a polynucleotide sequence. After expression the isolates with the best properties are re- isolated, combined and shuffled again to increase the genetic diversity. By repeating this procedure a number of times, genes that code for fastly improved proteins can be isolated. Preferably the gene shuffling procedure is started with a family of genes that code for proteins with a similar function. The family of polynucleotide sequences as described herein would be well suited for gene shuffling to improve the properties of secreted mutarotases.

Alternatively classical random mutagenesis techniques and selection, such as mutagenesis with NTG treatment or UV mutagenesis, can be used to improve the properties of a protein having mutarotase activity. Mutagenesis can be performed directly on isolated DNA, or on cells transformed with the DNA of interest. Alternatively, mutations can be introduced in isolated DNA by a number of techniques that are known to the person skilled in the art. Examples of these methods are error-prone PCR, amplification of plasmid DNA in a repair-deficient host cell, etc.

The use of yeast and filamentous fungal host cells is expected to provide for post-translational modifications (e.g. proteolytic processing, myristilation, glycosylation, truncation, and tyrosine, serine or threonine phosphorylation) as may be needed to confer optimal biological activity on recombinant produced protein having mutarotase activity.

Preparations

A protein having mutarotase activity may be in an isolated form. It will be understood that the protein may be mixed with carriers or diluents which will not interfere with the intended purpose of the protein and still be regarded as isolated. A protein having mutarotase activity may also be in a substantially purified form, in which case it will generally comprise the protein in a preparation in which more than 70%, e.g. more than 80%, 90%, 95%, 98% or 99% of the proteins in the preparation is a a protein having mutarotase activity.

A protein having mutarotase activity may be provided in a form such that they are outside their natural cellular environment. Thus, they may be substantially isolated or purified, as discussed above, or in a cell in which they do not occur in nature, for example a cell of other fungal species, animals, plants or bacteria.

In a most preferred embodiment, the invention provides a method for preparing a dehydrated foodstuff from a starting material solution comprising crystallizing at least part of a carbohydrate in a concentrated starting material solution in the presence of a protein having mutarotase activity and drying the at least partly crystallized concentrated product (to obtain a dehydrated foodstuff), wherein said protein having mutarotase activity comprises the amino acid sequence as depicted in SEQ ID NO: 1 and more preferably said protein having mutarotase activity comprises the amino acids 23-403 or 24-403 as depicted in SEQ ID NO: 1 .

The amount of enzyme (i.e. protein having mutarotase activity) added before or during crystallization can easily be determined by the skilled person and is for example within the range of 0.1 to 100 U/ml. Preferably, the protein having mutarotase activity is active during crystallization. The phrase "the protein having mutarotase activity is active during crystallization" is used to refer to the fact that at least 50% of the total amount of added enzyme is active during crystallization. Even more preferably, at least 60, 70 or 80% of the total amount of added enzyme is active during crystallization. Most preferred at least 85, 90 or 95% of the total amount of added enzyme is active during crystallization.

The use of a mutarotase in a method of the invention provides a big advantage for, for example, whey processing companies. A method of the invention allows the use of pre-crystallization in (for example whey) powder production facilities which increases throughput and/or decrease cooling.

The phrase "crystallizing at least part of a carbohydrate in a concentrated starting material solution in the presence of a protein having mutarotase activity" is used to indicate that the protein having mutarotase activity is added during concentration of the carbohydrates into a supersaturated solution or during or after transfer of the obtained concentrated carbohydrate solution to crystallization tanks. Alternatively, the protein having mutarotase activity can be added at the same time the seed crystals are added. The protein having mutarotase activity may also be added when crystallization has already started. For example, the protein having mutarotase activity is added when a certain amount of crystals is already formed.

In general, carbohydrates in the food stuff are first concentrated into a supersaturated solution by evaporation, after which temperature decrease and optional seeding with carbohydrate crystals initiate crystallization. Mutarotase is added before or during the crystallization process.

The use of a mutarotase during the preparation of a dried food stuff has advantages over the prior art in that it can speed up crystallization thereby decreasing residence time in the crystallization tank. A shorter crystallization process will also minimize risks for spoilage of the food stuff. Additional benefits are an increase in quality and/or uniformity of the dried food stuff, and the reduction of browning and off- flavor formation during drying or storage of the dried food stuff. Additional, the use of mutarotase during crystallization may lead to a yield increase of the food stuff.

The process of carbohydrate crystallization before drying of a food stuff generally involves concentration of the food stuff in an aqueous solution by evaporation at high temperature or low pressure. By decreasing the temperature again, more water evaporation or the addition of alcohols to the solution, the solution can become supersaturated for the carbohydrate. In such a supersaturated solution the different anomers of the reducing sugar will be in equilibrium. Crystallization may than be initiated by seeding crystals to the supersaturated solution, preferably with seeding- crystals of one of the anomers of the reducing sugar. Additionally, continuous crystallization methods have been described in the art. By careful adjustment of the crystallization conditions a regular crystal growth can be obtained.

It was realized as part of this invention that mutarotase will have a positive effect on the crystallization of reducing sugars in a food stuff by means of one or more different mechanisms: Since one of the anomers of a reducing sugar will crystallize more readily than the other, the supersaturation of this anomer will decrease. A decrease in supersaturation will slow down the crystallization rate. Maintaining high crystal growth rate will therefore require again an increase in supersaturation which can be achieved by e.g. cooling the solution, further evaporation or extra addition of alcohols. All these treatments are costly. Additionally, a decrease in temperature or evaporation will increase the viscosity of the solution, thereby decreasing crystal growth rate by reduced mass transfer of the anomer in solution to the growing crystal surface. Addition of mutarotase can prevent a drastic decrease in supersaturation of the crystallizing anomer of the reducing sugar, by replenishing the anomer that crystallizes, by mutarotation of the other anomer(s). Consequently, in the presence of mutarotase, crystal growth can occur at sufficient rate without additional measures such as excessive cooling or at an increased rate when the solution is cooled or otherwise treated to increase supersaturation.

Crystal growth of reducing sugars is known to be negatively influenced by molecules that resemble the crystallizing anomer. E.g. it was shown that the presence of β-lactose has a strong negative effect on the growth of crystals of a-lactose. It is thought that β-lactose competes with a-lactose, and is build into growing a-lactose crystals and thereby prevents further growth of the crystals (Michaels and van Kreveld (1966) Neth. Milk Dairy J. 20, 163). Reducing sugars are crystallized from supersaturated solutions where the different anomers are in equilibrium. Crystallization of one of the anomers will lead to change in the equilibrium, increasing the relative concentration of the competing anomer. Due to this, this change in equilibrium of the anomers will further decrease crystal growth rate. Addition of mutarotase during crystallization will restore the equilibrium between the anomers and decrease the concentration of the non-crystallizing anomer, thereby speeding up crystal growth.

As a result, adjustment of the process conditions in the presence of mutarotase allows steering at an increased crystallization rate, increased crystal yield and increased process temperature, and will thereby decrease costs of the crystallization process. Spray drying when a larger part of the carbohydrate is crystallized can be performed under much more economical conditions, as the drying temperature and the solids content can be increased before contamination of the spray dryer occurs. The total drying costs can thus be reduced. Additionally, powders with higher crystal content are less hygroscopic and less prone to caking during storage. The term "dehydrated food stuff" or "dried foodstuff" (the terms are used interchangeably herein) refers to a foodstuff from which most (preferably at least 95%) of the water has been removed. All milk powder has to meet a requirement for residual moisture. For skim milk it is usually 4%, for whole milk usually 2.5% and for whey 5%. There may naturally be deviations from country to country.

The term "starting material" is used to refer to the material which was used to prepare the foodstuff from.

The term "concentrated starting material" is used to refer to starting material which has first been concentrated by any means known to the skilled person, such as evaporation at high temperature or at low pressure, freeze concentration, ultra filtration techniques or centrifugation, dependent on the nature of the starting material and the requirements of the end product. The production of concentrated material in the food industry has been described extensively in literature and equipment to perform this procedure can be bought at a number of suppliers (e.g. GEA-Niro). In a preferred embodiment, the invention provides a method for preparing a dehydrated foodstuff from a starting material solution comprising crystallizing at least part of a carbohydrate in a concentrated starting material solution in the presence of a protein having mutarotase activity and drying the at least partly crystallized concentrated product (to obtain a dehydrated foodstuff), further comprising concentrating said starting material before crystallization.

In a preferred embodiment, the produced dehydrated foodstuff is a dried dairy foodstuff and the invention thus provides a method for preparing a dehydrated foodstuff from a starting material solution comprising crystallizing at least part of a carbohydrate in a concentrated starting material solution in the presence of a protein having mutarotase activity and drying the at least partly crystallized concentrated product (to obtain a dehydrated foodstuff), wherein said dehydrated foodstuff is a dehydrated dairy foodstuff.

In an even more preferred embodiment, said dehydrated dairy foodstuff is whey powder or whey permeate powder and the starting material is whey, acid whey or whey permeate. The invention thus also provides: - a method for preparing a whey powder from a whey solution comprising crystallizing at least part of a carbohydrate in a concentrated whey solution in the presence of a protein having mutarotase activity and drying the at least partly crystallized whey (to obtain a whey powder)

- a method for preparing a whey powder from an acid whey solution comprising crystallizing at least part of a carbohydrate in a concentrated acid whey solution in the presence of a protein having mutarotase activity and drying the at least partly crystallized whey (to obtain a whey powder)

- a method for preparing a whey permeate powder from a whey permeate solution comprising crystallizing at least part of a carbohydrate in a concentrated whey permeate solution in the presence of a protein having mutarotase activity and drying the at least partly crystallized whey permeate (to obtain a whey permeate powder).

In another preferred embodiment, said dehydrated foodstuff is milk powder and the starting material is milk or a derivative thereof (for example skimmed milk, semi- skimmed or full fat milk). The invention thus also provides a method for preparing milk powder from milk or a derivative thereof comprising crystallizing at least part of a carbohydrate in concentrated milk or a derivative thereof in the presence of a protein having mutarotase activity and drying the at least partly crystallized concentrated milk (to obtain milk powder).

Carbohydrates relevant for this invention are preferably reducing sugars where two or more anomeric forms can exist, more preferably reducing sugars where only one anomeric form is crystallized. Even more preferred are reducing sugars such as glucose (dextrose), fructose, galactose, xylose or lactose. In a most preferred embodiment, the invention provides a method for preparing a dehydrated foodstuff from a starting material solution comprising crystallizing at least part of a carbohydrate in a concentrated starting material solution in the presence of a protein having mutarotase activity and drying the at least partly crystallized concentrated product (to obtain a dehydrated foodstuff), wherein said carbohydrate is lactose.

In another embodiment, the invention provides use of mutarotase for preparing a dehydrated foodstuff. In another aspect, the invention provides a product obtainable by any of the methods described above. Preferably, such a product is whey powder, whey permeate powder or milk powder.

Hereafter the invention is illustrated by the following non-limiting examples.

EXAMPLES Example 1

Cloning and expression of the Aspergillus niger mutarotase gene

Aspergillus niger CBS513.88 was grown for 3 days at 30 degrees Celsius in PDB (Potato dextrose broth, Difco) and chromosomal DNA was isolated from the mycelium using the Q-Biogene kit (catalog nr. 6540-600; Omnilabo International BV, Breda, the Netherlands), using the instructions of the supplier. This chromosomal DNA was used for the amplification of the coding sequence of the mutarotase gene using PCR.

To specifically amplify the mutarotase gene MUT from the chromosomal DNA of Aspergillus niger CBS513.88, two PCR oligonucleotide primers were designed (SEQ ID NO: 4 and SEQ ID NO: 5). Primer sequences were partly obtained from a sequence that was found in the genomic DNA of Aspergillus niger CBS513.88 and published under number AM270022.1 in Genbank (SEQ ID NO: 2). The MUT protein sequence can be found back under the number An02g09090 (SEQ ID NO: 1). We describe here for the first time the efficient expression and characterization of a secreted Aspergillus mutarotase, which can be easily over-expressed and secreted in amounts that are relevant for applications in the food industry.

SEQ ID NO: 4: 5'-CCCTTAATTAACTCATAGGCATCATGCATGTCAAAAGCTACCTGTC-3' SEQ ID NO: 5: 5'-TTAGGCGCGCCTTACACAGTACCGAACTGGTAAG-3'

The first, direct PCR primer (SEQ ID NO: 4) contains 23 nucleotides MUT coding sequence starting at the ATG start codon, preceded by a 23 nucleotides sequence including a Pad restriction site. The second, reverse primer (SEQ ID NO: 5) contains 23 nucleotides complementary to the reverse strand of the region at the MUT stop codon, preceded by an Asc\ restriction site. Using these primers we were able to amplify a 1.2 kb sized fragment with chromosomal DNA from Aspergillus niger CBS513.88 as template. The thus obtained 1.2 kb sized fragment was isolated, digested with Pad and Asc\ and purified. The Pad / Asc\ fragment comprising the MUT coding sequences was exchanged with the Pad / Asc\ phyh fragment from pGBFIN-5 (WO 99/32617). Resulting plasmid is the MUT expression vector named pGBFINMUT. The expression vector pGBFINMUT was linearized by digestion with Not\, which removes all E. coli derived sequences from the expression vector. The digested DNA was purified using phenol: chloroform: isoamylalcohol (24:23: 1) extraction and precipitation with ethanol. These vectors were used to transform Aspergillus niger CBS513.88. An Aspergillus niger transformation procedure is extensively described in WO 98/46772. It is also described how to select for transformants on agar plates containing acetamide, and to select targeted multicopy integrants. Preferably, A. niger transformants containing multiple copies of the expression cassette are selected for further generation of sample material. For the pGBFINMUT expression vector, 30 A. niger transformants were purified; first by plating individual transformants on selective medium plates followed by plating a single colony on PDA (potato dextrose agar: PDB + 1.5% agar) plates. Spores of two individual transformants were collected after growth for 1 week at 30 degrees Celsius. Spores were stored refrigerated and were used for the inoculation of liquid media.

The two A. niger transformant strains were used for generation of sample material by cultivation of the strains in shake flask cultures. A useful method for cultivation of A. niger strains and separation of the mycelium from the culture broth is described in WO 98/46772. Cultivation medium was in CSM-MES (150 g maltose, 60 g Soytone (Difco), 15 g (NH 4 ) 2 S0 4 , 1 g NaH 2 P0 4 H 2 0, 1 g MgS0 4 7H 2 0, 1 g L-arginine, 80 mg Tween-80, 20 g MES pH6.2 per liter medium). 5 ml samples were taken from duplicate cultivations on day 4-8 of the fermentation, centrifuged for 10 min at 5000 rpm in a Hereaus labofuge RF and supernatants were stored at -20°C until further analyses.

It became clear that the transformants containing the pGBFINMUT vector had a surprisingly efficient secretion of a protein of apparent molecular weight of approximately 60 kDa when analyzed with SDS-PAGE. This protein is named MUT hereafter. The size of the expressed protein is much larger than the molecular weight that is predicted from the protein sequence, which is approximately 45 kDa. We presume that after removal of the signal sequence, MUT is heavily glycosylated before secretion from Aspergillus niger. The size of the MUT protein is completely different from an aldose-1-epimerase from Aspergillus niger mycelium that has been described before (Kinoshita et al (1981) Biochimica et Biophysica Acta 662, 285-290). The molecular weight of this enzyme, that was isolated from a cell lysate instead of secreted, was determined to be 260 kDa, being a homo-dimer with individual subunits of 130 kDa.

Selected strains can be used for isolation and purification of a larger amount of secreted Aspergillus mutarotase, when fermentation and down-stream processing is scaled up. This enzyme can than be used for further analysis, and for the use in diverse industrial applications.

Example 2

Fermentation, purification, and characterization of fungal mutarotase

Fresh Aspergillus niger MUT transformant #1 spores were used to inoculate shake flasks with 2 liters modified CSM medium (8% maltose, 3% bactosoytone, pH 5.1). After three days cultivation at 30°C, the cells were killed off by adding 3.5 g/l (final concentration) sodium benzoate and keeping at 30 °C for 6 hours. 10 g/l CaCI 2 and 45 g/l filter-aid Dicalite BF were added to the culture broth, and filtration was carried out in one step using filter cloth and filters Z-2000 and Z-200 (Pall). The filter cake remaining on the filter was washed with 1.1 liters of sterile milliQ water. The culture filtrate including the wash was sterile-filtered using 0.22 μηι GP Express PLUS Membrane (Millipore). The sterile filtrate was concentrated by ultrafiltration on a Pellicon-2 mini Cassette Biomax 5k (Millipore). The ultra-filtrate was purified with AIEX chromatography using a Q-sepharose FF XK 16/20 column (gel volume approx 30 ml). The following buffer system was used: Buffer A: 20 mM Na-phosphate, pH 7, Buffer B: 20 mM Na- phosphate, pH 7 + 1 M NaCI. The purification cycle was: equilibration with buffer A (5 column volumes), loading of the sample, washing with buffer A (3 column volumes), gradient elution from buffer A to buffer B in 20 column volumes. Flow rate 5 ml/min. The eluate was retrieved in several fractions. The fractions with minor contaminations were pooled, washed with 20 mM Na-phosphate pH 8.2, and loaded on the same column equilibrated with 20 mM Na-phosphate pH 8.2. MUT was bound to the column and eluted with 20 mM Na-phosphate pH 8.2, 1 M NaCI. The fractions with minor contaminations were pooled and used for subsequent activities determinations. The purity of the MUT sample was checked by SDS-gel electrophoresis and was found to be >90% pure. This enzyme solution was stabilized using the addition of glycerol up to 50% and L-methionine up to 0.1 %, and stored at -20 °C until use.

Enzymatic characterization.

The specific activity of MUT was measured using the standard NAD and glucose dehydrogenase coupled assay. When a-D-glucose is converted to β-D-glucose by mutarotase in the presence of β-D-glucose dehydrogenase and NAD, β-D-glucose is oxidized to D-glucono^-lactone with equimolar reduction of NAD to NADH. Therefore, optical density measurement of NADH at 340 nm (e340 = 6220 mol-1 cm-1) was used for indirect determination of mutarotase activity.

The reaction mixture contained the following components: 0.465 ml of 50 mM Tris- HCI buffer (pH 7.5) with 3 mM NAD and 10 units of glucose dehydrogenase; 0.025 ml MUT solution (0.004 mg/ml). The reaction was initiated by the addition 0.010 ml of freshly prepared 100 mM D-glucose to the reaction mix at the ambient temperature. The increase in absorption at 340 nm was measured for several minutes. A blank was determined by using the same mixture without MUT solution, and subtracted from the value obtained with MUT. The amount of mutarotase in the assay was adjusted to give an absorption change between 0.01 and 0.06 per min under these conditions. One unit of the specific mutarotase activity was defined as μηιοΐβ substrate converted/min/mg protein at 22 °C, pH 7.5, after correction for the nonenzymatic rate.

When the specific activity of the isolated and purified MUT from Aspergillus niger was measured using this assay, it turned out to be approximately 50 U/mg.

In order to test the thermostability of the isolated mutarotase, the enzyme solution was incubated for 2, 4, 6 and 8 hours at 55 °C before measuring the activity at 22 °C using the assay described above. No decrease in activity was noticed after this preincubation, showing that the mutarotase was stable under these conditions.

Example 3

Use of mutarotase in the crystallization of lactose

Monitor crystallization of a-lactose in MTP-plate

A supersaturated aqueous solution of lactose (50% w/w) was prepared from a-lactose monohydrate powder by dissolution in demineralised water, ensured through occasional shaking and heating to -80 degrees Celsius in a microwave for approximately 30 seconds in a closed vessel to prevent evaporation of the solvent. The solution was then placed in a water bath at 40°C with shaking for 30 min to ensure that mutarotation equilibrium was achieved. In the crystallization experiment the lactose solution was buffered at pH 6.9 by the addition of a 0.1 M sodium phosphate buffer solution until 5.3 mM.

60 U or 120 U of MUT was added per ml of the supersaturated lactose solution in a 96 well microtiter plate (MTP), mixed and incubated at 25 degrees Celsius. Five glass beads (diameter 1 mm) were added per well and the solutions were incubated under shaking. The development of lactose crystals was followed in time for 5 hours using a TECAN Genius apparatus, by measuring the optical density (OD) of the solution at 600 nm every 35 seconds. A similar experiment was performed with the addition of demineralised water instead of MUT, to follow spontaneous crystallization under these conditions. All incubations were performed in triplicate to correct for experimental error. Time required to obtain an optical density of 1 and 2 were monitored, and is depicted in Table 1. Especially the time to reach OD2 is significantly shorter in all the samples that used mutarotase. This shows that mutarotase will stimulate the crystallization of lactose under these conditions.

Table 1 : Time required to reach the indicated OD in the presence or absence of mutarotase. Average gives the average time required to reach the indicated OD. SD gives the standard deviation of the obtained time points in triplicate measurements.

The presence of glass beads in the incubation stimulates the nucleation of lactose and enhances the rate of crystallization in this experiment. To monitor the crystallization in the absence of agitation by glass beads, the experiment was repeated with and without 60 U/ml mutarotase in a 48-well MTP plate. Also in this experiment a clear effect of mutarotase on the rate of lactose crystallization could be detected. On average the time to reach an OD of 0.5 was 1.5 hours in the presence of mutarotase, while OD0.5 was only reached after 5 hours in the absence of mutarotase. OD1 was reached after 3.5 hours with, and 5.5 hours without added mutarotase.

Analysis of sugars by HPLC-RI

The amount of lactose in the soluble phase is analyzed using a HPLC system equipped with an Aminex HPX-87P column (BioRad). Separation of the various sugars is based on a combination of size exclusion, ion-exchange and reversed phase mechanisms. Elution is performed with MQ water at a flow of 0.6 ml/min for 22 min, while column temperature is 85°C. A refractive index (Rl) detector (internal oven at 35°C, sensitivity 32) is used for monitoring the sugars.

Before analysis the crystal suspension is centrifuged for 3 min at 10.000 rpm. The supernatant is analyzed after proper dilution. Lactose is quantified using standard solutions. The lactose content of the supernatant is determined after 6 hours incubation of a supersaturated lactose solution in the presence or absence of mutarotase.

Example 4

Use of mutarotase in the dissolution of crystalline a-lactose

Two gram of crystalline alpha-lactose (Sigma) was mixed with 10 ml 5 mM potassium phosphate buffer pH6.8. The slurry was divided over two beakers and to one of the beakers 75 U of MUT was added. The dissolution of α-lactose was followed in time under constant stirring of the slurry in both beakers, at 20 degrees Celsius. The lactose in the beaker that had obtained the mutarotase was dissolved significantly faster than the control beaker where no mutarotase had been added.

Example 5

Use of porcine kidney mutarotase in the crystallization of lactose

A similar experiment as described in Example 3 is performed. The only relevant difference between these experiments is the use of porcine kidney mutarotase instead of Aspergillus niger mutarotase for these experiments. Porcine kidney mutarotase can be purchased from Biozyme Laboratories Ltd (product code MUR1 F). The activity of MUR1 F can be determined using the method described in Example 2. A similar amount of mutarotase activity can be used to stimulate the crystallization of a-lactose, exactly as described in Example 3.