Method for producing GOS syrup

Abstract

The present invention relates to isolated polypeptides having lactase activity which is beta-galactosidase activity (EC 3.2.1.108, 3.2.1.23) which show residual activity after ultra high temperature treatment of milk after which the activity increases during incubation at 23°C due to refolding and also relates to polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides. Further, the invention relates to a method of producing galacto-oligosaccharides GOS applying the lactases of the invention.

Description

[0001] 16154-EP-EPA

[0002] Method for producing GOS syrup

[0003] Reference to a Sequence Listing

[0004] This application contains a Sequence Listing in computer readable form, which is

[0005] 5 incorporated herein by reference.

[0006] Background of the Invention

[0007] Field of the Invention

[0008] The present invention relates to polypeptides having lactase activity, variants of said

[0009] 10 enzymes, and to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods of producing and using the polypeptides. Further, the invention relates to a method of producing galacto-oligosaccharides (GOS) using the lactase enzymes of the invention.

[0010] Description of the Related Art

[0011] 15

[0012] Lactose free UHT milk

[0013] Most lactose-reduced or lactose-free milk-based products are produced in batch processes, i.e. adding lactase to milk and then incubating the milk at cold temperatures (usually less than 10°C) for enough time to reduce the lactose content to less than 0.01% or less than 0.1 % (which in most countries allows for labelling of, e.g., milk as lactose-free), followed by heat treatment such as pasteurization, UHT or ESL (extended shelf-life of up to 35 days) treatments. Some of the draw-backs linked to the batch application of lactases include:

[0014] (1) the incubation time can be up to 24 h and sometimes more, which raises concerns regard-ing capacity and capital investments in new incubation tanks to respond to the growing

[0015] 25 demand for lactose free dairy products.

[0016] (2) Prolonged incubation times give psychotropic microbes a chance to grow and secrete their enzymes (particularly proteases), some of which are heat stable and may deteriorate the quality of the final product during the shelf-life.

[0017] (3) Lactose hydrolysis followed by severe heat treatment, such as in UHT applications,

[0018] 30 will result in Maillard reaction being propagated at an accelerated rate. When lactose is hydrolyzed to glucose and galactose, the concentration of sugars having reducing ends is doubled and espe-cially galactose is much more reactive than lactose.

[0019] Recently and specifically in the case of lactose-free ESL and UHT milk drinks, advanced process engineering technologies in the form of aseptic dosing equipment have gained a lot of

[0020] 35 interest and made it possible to overcome many of the concerns linked to the batch process. Such aseptic dosing equipment makes it possible to add the lactase after the ESL / UHT treatment. Examples of such aseptic dosing equipment are the Tetra Pak® Aldose system, Tetra Pak® Flexdose System, and GEA Varidose system. The advantages of these systems are: 16154-EP-EPA

[0021] (1 ) Dosing smaller quantities of sterile lactases with high precision into the milk stream after the heat treatment step, allowing lactose hydrolysis to take place during the first few days of stor-age.

[0022] (2) Decreasing the extent of Maillard reaction, browning and the formation of

[0023] 5 Advanced Gly-cation End products (AGEs) in lactose-free milk drinks, especially when proper controlled storage conditions are applied.

[0024] (3) Omitting the pre-incubation step compared to the batch process and thus solving the ca-pacity issues and decreasing the risk of activity of psychotropic microbes.

[0025] Despite the above advantages of aseptic dosing systems, there are a number of

[0026] 10 drawbacks asso-ciated with their use, such as:

[0027] (1) Since the lactases are added after the heat treatment step, their formulation should be of the highest possible purity. This is because any detrimental side activity in their formulation may have very negative impact on the end product during the long shelf-life, especially in the case of UHT products.

[0028] 15 (2) In the case of Tetra Pak® Flexdose and GEA Varidose systems, the lactase enzyme should be aseptically filled in sterile buckets and bags.

[0029] (3) The above requirements in points (1) and (2) mean these sterile lactases cost more per unit of activity compared to lactases used in the batch process.

[0030] (4) The average capital cost of these aseptic dosing systems is substantial.

[0031] (5) There are additional running costs linked to the consumables when using Tetra Pak® Flexdose and GEA Varidose systems. An example of this is changing the hose and needle every time an aseptic bucket is changed.

[0032] (6) In the Tetra Pak® Aldose system, the enzyme formulation is not initially sterile. It is dilut-ed with water and then filtered in line (in the dairy) using at least two filters to ensure

[0033] 25 sterility of the enzyme stream before mixing it with the milk stream. This can be problematic, as the filters in-stalled in-line may become blocked by poorly filterable enzyme formulation, which may cause dif-ficulties during operation. Even when the filterability of the enzyme formulation works as expected, there is still a need to change the filters regularly (usually daily).

[0034] A quick, smooth, easy to implement, trouble-free, cost-effective solution for using lactases

[0035] 30 in lac-tose-free UHT and ESL products, which overcomes all the above limitations of both batch and aseptic dosing processes, does not yet exist.

[0036] W02009 / 071539 (Novozymes) relates to a method of producing a dairy product using an enzyme having lactase activity. Disclosed is a method of producing a low-lactose milk product by treating a milk-based substrate with lactase at high temperature, i.e., at least 60°C, at least 62°C,

[0037] 35 at least 63°C, at least 64°C, at least 65°C, at least 67°C, at least 70°C, or at least 75°C.

[0038] WO2018 / 189238 (Chr. Hansen) discloses beta-galactosidases which are said to be stable with relatively high activity at a broad range of temperatures and pH values. Disclosed is a method for producing a dairy product by treating a milk-based substrate with a beta- 16154-EP-EPA galactosidase, wherein the treatment or part of the treatment may take place at high temperature. A lactose concentration of less than 0.2% lactose may be obtained in 3-30 minutes after adding the beta-galactosidase.

[0039] W02020 / 176734 (DuPont) relates to a method for reducing the amount of lactose in a

[0040] 5 milk-based substrate by contacting the substrate with a lactase, such as a thermostable lactase, at high tem-perature. Disclosed is a method for production of a lactose free dairy product from a milk-based substrate with an enzyme having neutral lactase activity wherein more than 20% lactase activity remains in the milk-based substrate after pasteurization at 72°C for 15 seconds. Such pasteuriza-tion is also sometimes referred to as high-temperature, short-time (HTST)

[0041] 10 pasteurization.

[0042] Deeth (2017) “Optimum Thermal Processing for Extended Shelf-Life (ESL) Milk”, Foods 6(11): 102 has reviewed the optimum thermal processing for Extended Shelf-Life (ESL) milk. Deeth ex-plains that ESL or ultra-pasteurized milk is produced by thermal processing using conditions be-tween those used for traditional high-temperature, short-time (HTST)

[0043] 15 pasteurization and those used for ultra-high-temperature (UHT) sterilization. ESL milk should have a refrigerated shelf-life of more than 30 days. To achieve this, the thermal processing must be quite intense. Unlike the temperature-time conditions for pasteurization, which are specified in most countries to be at least 72°C for at least 15 s, there are generally no such specified conditions for ESL processing. According to Deeth (2017), reported commercial processing conditions for ESL milk are mostly in the range of 123-127°C for 1-5 seconds. U.S. regulations define the process of “ultra-pasteurization” as heating milk at a temperature of at least 138°C for at least 2 seconds.

[0044] European patent application No. 21216998.1 discloses a method of producing lactose- reduced, heat-treated, milk-based product, e.g. milk, using an enzyme having lactase activity

[0045] 25 without the need to perform an extensive pre-incubation of the milk-based substrate with the enzyme and without the need to use aseptic dosing systems to add the enzyme after the heat treatment. In this method, after the heat-treatment, such as a UHT treatment, the enzyme has some residual activi-ty which ensures lactose degradation to the desired low lactose level during storage in the cold or at ambient temperature.

[0046] 30 UHT treatment may be, e.g., heat treatment for 30 seconds at 130°C, for 3-4 seconds at 140°C or for 1 second at 145°C.

[0047] There is still a need for improved methods for producing lactose-reduced, heat-treated, milk-based products such as UHT or ESL milk, as well as for lactase enzymes that are suitable for such methods.

[0048] 35

[0049] Galacto-oligosaccharide (GOS) production

[0050] Beta-galactosidase, also known as lactase, is an enzyme known to hydrolyse the terminal nonreducing beta-D-galactose residues in beta-D-galactosidases. More particularly, under normal 16154-EP-EPA reaction conditions, the enzyme hydrolyses its lactose substrate to the component monosaccharides D-glucose and D-galactose. Under certain conditions, certain betagalactosidases have the ability to transfer galactose to the hydroxyl group of either glucose or galactose to form galacto-oligosaccharides (GOS) in a process called transgalactosylation.

[0051] 5 Galactooligosaccharides (GOS) can be produced from lactose through the enzymatic transgalactosylation reaction of beta-galactosidase. It has been shown that GOS can promote the growth of bifidobacteria, healthy microbes, in the large intestine of humans. Therefore, it is beneficial and desirable to produce milk products comprising high amounts of GOS.

[0052] It has been reported that the production of GOS from lactose increases with the concentration of

[0053] 10 lactose.

[0054] GOS can be produced ex-situ from solutions of lactose and added to dairy products as an ingredient. This will add calories to the dairy product, and further the addition of GOS is to be labelled on the final product. This goes against the general “clean label” trend, i.e. the preference of many consumers of “natural” products comprising only ingredients which are perceived as

[0055] 15 natural with no or few additives.

[0056] It’s therefore preferred to produce the GOS in-situ, i.e. to make the GOS in the milk product from the lactose already present in the milk. Producing GOS in situ at high levels has been difficult though.

[0057] In EP 0458358 A1 , skim milk is concentrated to a total solid content of 20-50% by weight. By incubating the concentrated milk with beta-galactosidase at a temperature of 20-50°C followed by spray-drying, it’s speculated that skim milk powder containing a total amount of about 5-15% by weight of GOS (presumably DP2+) can be obtained. Assuming a total content of free carbohydrates in the concentrated skim milk (lactose and derivatives) of about 50% of the dry matter, this corresponds to a relative amount of 10-30% DP2+ GOS w / w of the total free

[0058] 25 carbohydrates. Production of skim milk powder comprising a total amount of 10.5% DP2+ GOS w / w is exemplified.

[0059] Chen et al. (2002), “Optimization of the enzymic process for manufacturing low-lactose milk containing oligosaccharides”, Process Biochemistry 38, 801-808, have compared use of betagalactosidase to transform the lactose in milk into GOS directly with the application of

[0060] 30 ultrafiltration techniques to separate lactose from milk proteins and then transform the lactose in the permeate into GOS. When using concentrated milk containing 16.7% lactose, a milk product could be obtained by incubating at 47°C for 1.4 h wherein 22.8% w / w of the lactose was converted to DP3+ GOS. Whereas by removing milk proteins by ultrafiltration and thereby increasing the lactose concentration in the permeate to 25.3% by evaporation, a permeate could be obtained

[0061] 35 by incubating at 50°C for 3.5 h wherein as much as 31.1 % w / w of the lactose was converted to 16154-EP-EPA

[0062] DP3+ GOS.

[0063] WO 2015 / 086746 discloses efficient conversion of low-concentration lactose (at most 15% w / v) to GOS by treatment of milk-based media comprising, e.g., skim milk, cream and lactose with a truncated beta-galactosidase from Bifidobacterium bifidum consisting of 887 amino acids.

[0064] 5 Disclosed is also a milk-based substrate which comprises GOS after the enzymatic treatment in a total amount of 0.1 to 10% (w / v). The examples show production of GOS from milk-based media to which lactose has been added. Incubation was at 45°C or lower and a total amount of up to 3.7% (w / v) DP3+ GOS was produced.

[0065] 10 The object of the present invention is to provide lactases that have sufficient thermostability and are good at refolding after denaturation, thus making them well suited for production of lactose free milkproducts and for GOS production resulting in a desirable distribution of GOS di-saccharides comprising preferably beta 1-4 bonds.

[0066] Summary of the Invention

[0067] The present invention provides polypeptides having beta-galactosidase activity, selected from the group consisting of:

[0068] (a) a polypeptide having at least 90 % sequence identity to mature polypeptide of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 10;

[0069] (b) a polypeptide having at least 90 % sequence identity to amino acids 1-944 of SEQ ID NO: 6, amino acids 1-837 of SEQ ID NO: 7, amino acids 1-1225 of SEQ ID NO: 10;

[0070] (c) a polypeptide encoded by a polynucleotide having at least 90 % sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15;

[0071] 25 (d) a polypeptide derived from SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 10, a mature polypeptide of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 10 by having 1-30 alterations (e.g., substitutions, deletions and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in

[0072] 30 particular substitutions;

[0073] (e) a polypeptide derived from the polypeptide of (a), (b), (c), or (d) wherein the N- and / or C-terminal end has been extended by addition of one or more amino acids; and

[0074] (f) a fragment of the polypeptide of (a), (b), (c), or (d); wherein the polypeptide has beta-galactosidase activity.

[0075] 35 16154-EP-EPA

[0076] The invention further provides a method for producing galacto-oligosaccharides (GOS) comprising contacting a polypeptide having beta-galactosidase activity with lactose and wherein the polypeptide is selected from the group consisting of:

[0077] (a) a polypeptide having at least 75% sequence identity to SEQ ID NO: 6;

[0078] 5 (b) a polypeptide having at least 75% sequence identity to a mature polypeptide of SEQ ID NO: 6;

[0079] (c) a polypeptide derived from SEQ ID NO: 6, a mature polypeptide of SEQ ID NO: 6, or 1-944 of SEQ ID NO: 6 by having 1-30 alterations (e.g., substitutions, deletions and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or

[0080] 10 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions;

[0081] (d) a polypeptide derived from the polypeptide of (a), (b), or (c), wherein the N- and / or C-terminal end has been extended by addition of one or more amino acids; and

[0082] (e) a fragment of the polypeptide of (a), (b), (c), or (d); and wherein the polypeptide has beta-galactosidase activity;

[0083] (a) a polypeptide having at least 75% sequence identity to SEQ ID NO: 7;

[0084] (b) a polypeptide having at least 75% sequence identity to a mature polypeptide of SEQ ID NO: 7;

[0085] (c) a polypeptide derived from SEQ ID NO: 7, a mature polypeptide of SEQ ID NO: 7, or 1-837 of SEQ ID NO: 7 by having 1-30 alterations (e.g., substitutions, deletions and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions;

[0086] 25 (d) a polypeptide derived from the polypeptide of (a), (b), or (c), wherein the N- and / or C-terminal end has been extended by addition of one or more amino acids; and

[0087] (e) a fragment of the polypeptide of (a), (b), (c), or (d); and wherein the polypeptide has beta-galactosidase activity;

[0088] 30 (a) a polypeptide having at least 75% sequence identity to SEQ ID NO: 10;

[0089] (b) a polypeptide having at least 75% sequence identity to a mature polypeptide of SEQ ID NO: 10;

[0090] (c) a polypeptide derived from SEQ ID NO: 10, a mature polypeptide of SEQ ID NO: 10, or 1-1225 of SEQ ID NO: 10 by having 1-30 alterations (e.g., substitutions, deletions

[0091] 35 and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions; 16154-EP-EPA

[0092] (d) a polypeptide derived from the polypeptide of (a), (b), or (c), wherein the N- and / or C-terminal end has been extended by addition of one or more amino acids; and

[0093] (e) a fragment of the polypeptide of (a), (b), (c), or (d); and wherein the polypeptide has beta-galactosidase activity;

[0094] 5

[0095] (a) a polypeptide having at least 75% sequence identity to SEQ ID NO: 1 ;

[0096] (b) a polypeptide having at least 75% sequence identity to a mature polypeptide of SEQ ID NO: 1 ;

[0097] (c) a polypeptide derived from SEQ ID NO: 1 , a mature polypeptide of SEQ ID NO: 1 ,

[0098] 10 or 1-820 of SEQ ID NO: 1 by having 1-30 alterations (e.g., substitutions, deletions and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions;

[0099] (d) a polypeptide derived from the polypeptide of (a), (b), or (c), wherein the N- and / or

[0100] 15 C-terminal end has been extended by addition of one or more amino acids; and

[0101] (e) a fragment of the polypeptide of (a), (b), (c), or (d); and wherein the polypeptide has beta-galactosidase activity; and

[0102] (a) a polypeptide having at least 75% sequence identity to SEQ ID NO: 3;

[0103] (b) a polypeptide having at least 75% sequence identity to a mature polypeptide of SEQ ID NO: 3;

[0104] (c) a polypeptide derived from SEQ ID NO: 3, a mature polypeptide of SEQ ID NO: 3, or 1-826 of SEQ ID NO: 3 by having 1-30 alterations (e.g., substitutions, deletions and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or

[0105] 25 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions;

[0106] (d) a polypeptide derived from the polypeptide of (a), (b), or (c), wherein the N- and / or C-terminal end has been extended by addition of one or more amino acids; and

[0107] (e) a fragment of the polypeptide of (a), (b), (c), or (d); and wherein the polypeptide

[0108] 30 has beta-galactosidase activity.

[0109] The invention further provides a composition comprising the polypeptides of the invention, polynucleotides encoding the polypeptides of the invention, a nucleic acid construct or expression vector comprising the polynucleotides, and a recombinant host cell comprising the nucleic acid

[0110] 35 construct or expression vector. 16154-EP-EPA

[0111] In another aspect the invention relates to a method of producing a polypeptide having betagalactosidase activity, comprising cultivating the recombinant host cell of the invention under conditions conducive for production of the polypeptide.

[0112] 5 A further aspect of the invention relates to a milk-based product comprising GOS produced in- situ from lactose present in the milk, by the method of the invention.

[0113] Definitions

[0114] In accordance with this detailed description, the following definitions apply. Note that the

[0115] 10 singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0116] Unless defined otherwise or clearly indicated by context, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0117] 15

[0118] Lactase: The term “lactase” means a glycoside hydrolase having the ability to hydrolyze the disaccharide lactose into constituent galactose and glucose monomers. The group of lactases comprises but is not limited to enzymes assigned to subclass EC 3.2.1.108. Enzymes assigned to other subclasses, such as, e.g., EC 3.2.1.23 or EC 3.2.1.21 , may also be lactases in the context of the present invention. A lactase in the context of the invention may have other activities than the lactose hydrolyzing activity, such as for example a transgalactosylating activity. In the context of the invention, the lactose hydrolyzing activity of the lactase may be referred to as its lactase activity, its beta-galactosidase activity or its hydrolyzing activity. In the context of the present invention, the polypeptide having lactase / beta-galactosidase activity preferably belongs

[0119] 25 to the enzyme class EC 3.2.1.23 or EC 3.2.1.108, preferably 3.2.1.23.

[0120] Lactase Activity: Lactase activity may be determined using, e.g., a LAU(B) assay. The activity in LAU(B) of a specific lactase may be determined by direct measurement of o-nitrophenyl (ONP) released from o-nitrophenyl p-D-galactopyranoside (ONPG) in a buffer containing 1.46 mg / ml

[0121] 30 substrate in 0.05 M MES, 1 mM MgSO4 7H2O, 450 mg / L Brij 35 at pH6.5 and 30°C. Af-ter 600 seconds incubation, the reaction is stopped by adding 0.2 M Na2CO3 and the released ONP is measured at 405 nm after 126 seconds incubation. The activity is obtained by comparing to a standard curve run with a lactase of known activity, and the activity of the unknown sample calculated from this. The lactase of known activity may, e.g., be Saphera®, available from No-

[0122] 35 vozymes A / S, Denmark. Lactase activity may also be determined by measuring the amount of lactose hydrolysis in milk, e.g. by the method described in Example 1 in the paragraph “Analysis of residual lactose content” using HPAEC-PAD where the lactose peak is related to a lactose standard with known concentration. The lactose hydrolysis can then be related to the amount of 16154-EP-EPA lactase added, e.g. per mg enzyme protein or per mole enzyme. Other methods for measuring lactase activity are known and used routinely in the art.

[0123] Catalytic domain: The term “catalytic domain” means the region of an enzyme containing

[0124] 5 the catalytic machinery of the enzyme. cDNA: The term "cDNA" means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is processed through a series of steps,

[0125] 10 including splicing, before appearing as mature spliced mRNA.

[0126] Coding sequence: The term “coding sequence” means a polynucleotide, which directly specifies the amino acid sequence of a polypeptide. The boundaries of the coding sequence are generally determined by an open reading frame, which begins with a start codon, such as ATG, GTG, or TTG, and ends with a stop codon, such as TAA, TAG, or TGA. The coding sequence

[0127] 15 may be a genomic DNA, cDNA, synthetic DNA, or a combination thereof.

[0128] Control sequences: The term “control sequences” means nucleic acid sequences involved in regulation of expression of a polynucleotide in a specific organism or in vitro. Each control sequence may be native ( / .e., from the same gene) or heterologous ( / .e., from a different gene) to the polynucleotide encoding the polypeptide, and native or heterologous to each other. Such control sequences include, but are not limited to leader, polyadenylation, prepropeptide, propeptide, signal peptide, promoter, terminator, enhancer, and transcription or translation initiator and terminator sequences. At a minimum, the control sequences include a promoter, and transcriptional and translational stop signals. The control sequences may be provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences

[0129] 25 with the coding region of the polynucleotide encoding a polypeptide.

[0130] Disaccharide: The term “disaccharide” as used herein means two monosaccharide units bound together by a covalent bond known as a glycosidic linkage formed via a dehydration reaction, resulting in the loss of a hydrogen atom from one monosaccharide and a hydroxyl group from the other. In one aspect, the disaccharide is cellobiose, fucose, lactose, lactulose, maltose,

[0131] 30 rhamnose, or sucrose, most preferably lactose.

[0132] Expression: The term “expression” means any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification, and secretion.

[0133] Expression vector: An "expression vector" refers to a linear or circular DNA construct

[0134] 35 comprising a DNA sequence encoding a polypeptide, which coding sequence is operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host. Such control sequences may include a promoter to effect transcription, an optional operator sequence 16154-EP-EPA to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, enhancers and sequences which control termination of transcription and translation.

[0135] Extension: The term “extension” means an addition of one or more amino acids to the amino and / or carboxyl terminus of a polypeptide, wherein the “extended” polypeptide has beta¬

[0136] 5 galactosidase activity.

[0137] Fragment: The term “fragment” means a polypeptide, a catalytic domain, having one or more amino acids absent from the amino and / or carboxyl terminus of the mature polypeptide, catalytic domain, wherein the fragment has beta-galactosidase (lactase) activity.

[0138] Fusion polypeptide: The term “fusion polypeptide” is a polypeptide in which one

[0139] 10 polypeptide is fused at the N-terminus and / or the C-terminus of a polypeptide of the present invention. A fusion polypeptide is produced by fusing a polynucleotide encoding another polypeptide to a polynucleotide of the present invention, or by fusing two or more polynucleotides of the present invention together. Techniques for producing fusion polypeptides are known in the art, and include ligating the coding sequences encoding the polypeptides so that they are in frame

[0140] 15 and that expression of the fusion polypeptide is under control of the same promoter(s) and terminator. Fusion polypeptides may also be constructed using intein technology in which fusion polypeptides are created post-translationally (Cooper et al., 1993, EMBO J. 12: 2575-2583; Dawson et al., 1994, Science 266: 776-779). A fusion polypeptide can further comprise a cleavage site between the two polypeptides. Upon secretion of the fusion protein, the site is cleaved releasing the two polypeptides. Examples of cleavage sites include, but are not limited to, the sites disclosed in Martin et al., 2003, J. Ind. Microbiol. Biotechnol. 3: 568-576; Svetina et al., 2000, J. Biotechnol. 7Q: 245-251 ; Rasmussen-Wilson et al., 1997, Appl. Environ. Microbiol. 63: 3488-3493; Ward et al., 1995, Biotechnology 13: 498-503; and Contreras et al., 1991 , Biotechnology 9: 378-381 ; Eaton et al., 1986, Biochemistry 25: 505-512; Collins-Racie et al.,

[0141] 25 1995, Biotechnology 13: 982-987; Carter eta / ., 1989, Proteins: Structure, Function, and Genetics 6: 240-248; and Stevens, 2003, Drug Discovery World 4: 35-48.

[0142] Heterologous: The term "heterologous" means, with respect to a host cell, that a polypeptide or nucleic acid does not naturally occur in the host cell. The term "heterologous" means, with respect to a polypeptide or nucleic acid, that a control sequence, e.g., promoter, of

[0143] 30 a polypeptide or nucleic acid is not naturally associated with the polypeptide or nucleic acid, i.e., the control sequence is from a gene other than the gene encoding the mature polypeptide.

[0144] Host Strain or Host Cell: A "host strain" or "host cell" is an organism into which an expression vector, phage, virus, or other DNA construct, including a polynucleotide encoding a polypeptide of the present invention has been introduced. Exemplary host strains are

[0145] 35 microorganism cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing the polypeptide of interest and / or fermenting saccharides. The term "host cell" includes protoplasts created from cells. 16154-EP-EPA

[0146] Hybrid polypeptide: The term hybrid polypeptide means a polypeptide comprising domains from two or more polypeptides, e.g., a binding module from one polypeptide and a catalytic domain from another polypeptide. The domains may be fused at the N-terminus or the C-terminus.

[0147] 5 Introduced: The term "introduced" in the context of inserting a nucleic acid sequence into a cell, means "transfection", "transformation" or "transduction," as known in the art.

[0148] Isolated: The term “isolated” means a polypeptide, nucleic acid, cell, or other specified material or component that has been separated from at least one other material or component, including but not limited to, other proteins, nucleic acids, cells, etc. An isolated polypeptide,

[0149] 10 nucleic acid, cell or other material is thus in a form that does not occur in nature. An isolated polypeptide includes, but is not limited to, a culture broth containing the secreted polypeptide expressed in a host cell.

[0150] Mature polypeptide: The term “mature polypeptide” means a polypeptide in its mature form following N-terminal and / or C-terminal processing (e.g., removal of signal peptide). In one

[0151] 15 embodiment the mature polypeptide is amino acids 1-944 of SEQ ID NO: 6. In one embodiment the mature polypeptide is amino acids 1-837 of SEQ ID NO: 7. In one embodiment the mature polypeptide is amino acids 1-1225 of SEQ ID NO: 10. In one embodiment the mature polypeptide is amino acids 1-820 of SEQ ID NO: 1. In one embodiment the mature polypeptide is amino acids 1-826 of SEQ ID NO: 3.

[0152] Mature polypeptide coding sequence: The term “mature polypeptide coding sequence” means a polynucleotide that encodes a mature polypeptide having lactase activity. In one aspect, the mature polypeptide coding sequence is nucleotides 1 to 2832 of SEQ ID NO: 13. In one aspect, the mature polypeptide coding sequence is nucleotides 1 to 2511 of SEQ ID NO: 14. In one aspect, the mature polypeptide coding sequence is nucleotides 1 to 3675 of SEQ ID NO: 15.

[0153] 25 Native: The term "native" means a nucleic acid or polypeptide naturally occurring in a host cell.

[0154] Nucleic acid: The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids may be single stranded or double stranded and may be chemical modifications. The terms "nucleic acid" and

[0155] 30 "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences that encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in 5'-to-3' orientation.

[0156] Nucleic acid construct: The term "nucleic acid construct" means a nucleic acid molecule,

[0157] 35 either single- or double-stranded, which is isolated from a naturally occurring gene or is modified to contain segments of nucleic acids in a manner that would not otherwise exist in nature or which is synthetic, and which comprises one or more control sequences operably linked to the nucleic acid sequence. 16154-EP-EPA

[0158] Operably linked: The term "operably linked" means that specified components are in a relationship (including but not limited to juxtaposition) permitting them to function in an intended manner. For example, a regulatory sequence is operably linked to a coding sequence such that expression of the coding sequence is under control of the regulatory sequence.

[0159] 5 Purified: The term “purified” means a nucleic acid, polypeptide or cell that is substantially free from other components as determined by analytical techniques well known in the art (e.g., a purified polypeptide or nucleic acid may form a discrete band in an electrophoretic gel, chromatographic eluate, and / or a media subjected to density gradient centrifugation). A purified nucleic acid or polypeptide is at least about 50% pure, usually at least about 60%, about 65%,

[0160] 10 about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, about 99.6%, about 99.7%, about 99.8% or more pure (e.g., percent by weight or on a molar basis). In a related sense, a composition is enriched for a molecule when there is a substantial increase in the concentration of the molecule after application of a purification or enrichment technique. The

[0161] 15 term "enriched" refers to a compound, polypeptide, cell, nucleic acid, amino acid, or other specified material or component that is present in a composition at a relative or absolute concentration that is higher than a starting composition.

[0162] In one aspect, the term "purified" as used herein refers to the polypeptide or cell being essentially free from components (especially insoluble components) from the production organism. In other aspects, the term "purified" refers to the polypeptide being essentially free of insoluble components (especially insoluble components) from the native organism from which it is obtained. In one aspect, the polypeptide is separated from some of the soluble components of the organism and culture medium from which it is recovered. The polypeptide may be purified ( / .e., separated) by one or more of the unit operations filtration, precipitation, or chromatography.

[0163] 25 Accordingly, the polypeptide may be purified such that only minor amounts of other proteins, in particular, other polypeptides, are present. The term "purified" as used herein may refer to removal of other components, particularly other proteins and most particularly other enzymes present in the cell of origin of the polypeptide. The polypeptide may be "substantially pure", i.e., free from other components from the organism in which it is produced, e.g., a host

[0164] 30 organism for recombinantly produced polypeptide. In one aspect, the polypeptide is at least 40% pure by weight of the total polypeptide material present in the preparation. In one aspect, the polypeptide is at least 50%, 60%, 70%, 80% or 90% pure by weight of the total polypeptide material present in the preparation. As used herein, a "substantially pure polypeptide" may denote a polypeptide preparation that contains at most 10%, preferably at most 8%, more preferably at

[0165] 35 most 6%, more preferably at most 5%, more preferably at most 4%, more preferably at most 3%, even more preferably at most 2%, most preferably at most 1%, and even most preferably at most 0.5% by weight of other polypeptide material with which the polypeptide is natively or recombinantly associated. 16154-EP-EPA

[0166] It is, therefore, preferred that the substantially pure polypeptide is at least 92% pure, preferably at least 94% pure, more preferably at least 95% pure, more preferably at least 96% pure, more preferably at least 97% pure, more preferably at least 98% pure, even more preferably at least 99% pure, most preferably at least 99.5% pure by weight of the total polypeptide material

[0167] 5 present in the preparation. The polypeptide of the present invention is preferably in a substantially pure form ( / .e., the preparation is essentially free of other polypeptide material with which it is natively or recombinantly associated). This can be accomplished, for example by preparing the polypeptide by well-known recombinant methods or by classical purification methods.

[0168] Recombinant: The term "recombinant" is used in its conventional meaning to refer to the

[0169] 10 manipulation, e.g., cutting and rejoining, of nucleic acid sequences to form constellations different from those found in nature. The term recombinant refers to a cell, nucleic acid, polypeptide or vector that has been modified from its native state. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell, or express native genes at different levels or under different conditions than found in nature. The term “recombinant”

[0170] 15 is synonymous with “genetically modified” and “transgenic”.

[0171] Recover: The terms "recover" or “recovery” means the removal of a polypeptide from at least one fermentation broth component selected from the list of a cell, a nucleic acid, or other specified material, e.g., recovery of the polypeptide from the whole fermentation broth, or from the cell-free fermentation broth, by polypeptide crystal harvest, by filtration, e.g., depth filtration (by use of filter aids or packed filter medias, cloth filtration in chamber filters, rotary-drum filtration, drum filtration, rotary vacuum-drum filters, candle filters, horizontal leaf filters or similar, using sheed or pad filtration in framed or modular setups) or membrane filtration (using sheet filtration, module filtration, candle filtration, microfiltration, ultrafiltration in either cross flow, dynamic cross flow or dead end operation), or by centrifugation (using decanter centrifuges, disc stack

[0172] 25 centrifuges, hyrdo cyclones or similar), or by precipitating the polypeptide and using relevant solid-liquid separation methods to harvest the polypeptide from the broth media by use of classification separation by particle sizes. Recovery encompasses isolation and / or purification of the polypeptide.

[0173] Sequence identity: The relatedness between two amino acid sequences or between two

[0174] 30 nucleotide sequences is described by the parameter “sequence identity”.

[0175] Method 1 :

[0176] For purposes of the present invention, the sequence identity between two amino acid sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the

[0177] 35 Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. In order for the Needle 16154-EP-EPA program to report the longest identity, the -nobnef option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows:

[0178] (Identical Residues x 100) / (Length of Alignment - Total Number of Gaps in Alignment)

[0179] For purposes of the present invention, the sequence identity between two polynucleotide

[0180] 5 sequences is determined as the output of “longest identity” using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NLIC4.4)

[0181] 10 substitution matrix. In order for the Needle program to report the longest identity, the nobrief option must be specified in the command line. The output of Needle labeled “longest identity” is calculated as follows:

[0182] (Identical Deoxyribonucleotides x 100) / (Length of Alignment - Total Number of Gaps in Alignment)

[0183] 15 Method 2:

[0184] For purposes of the present invention, the sequence identity between two amino acid sequences is determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The percent sequence identity is calculated as follows:

[0185] (Identical Residues x 100) / (Length of the Shortest Sequence in the Alignment)

[0186] The sequence identity between two polynucleotide sequences can be determined using

[0187] 25 the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix. The percent sequence identity is calculated as

[0188] 30 follows:

[0189] (Identical Deoxyribonucleotides x 100) / (Length of the Shortest Sequence in the Alignment Method 3:

[0190] For purposes of the present invention, the sequence identity between two amino acid sequences is determined using Needleman-Wunsch algorithm (Needleman and Wunsch, 1970,

[0191] 35 J. Mol. Biol. 48: 443-453) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably version 6.6.0 or later. The parameters used are a gap open 16154-EP-EPA penalty of 10, a gap extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The percent identity is calculated as follows:

[0192] (Identical Residues x 100) / (Length of the Alignment)

[0193] The sequence identity between two polynucleotide sequences can be determined using

[0194] 5 the same Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, supra), preferably version 6.6.0 or later. The parameters used are a gap open penalty of 10, a gap extension penalty of 0.5, and the EDNAFULL (EMBOSS version of NCBI NLIC4.4) substitution matrix. The percent sequence identity is calculated as follows:

[0195] (Identical Deoxyribonucleotides x 100) / (Length of the Alignment)

[0196] Signal Peptide: A "signal peptide" is a sequence of amino acids attached to the N- terminal portion of a protein, which facilitates the secretion of the protein outside the cell. The mature form of an extracellular protein lacks the signal peptide, which is cleaved off during the

[0197] 15 secretion process.

[0198] Subsequence: The term “subsequence” means a polynucleotide having one or more nucleotides absent from the 5' and / or 3' end of a mature polypeptide coding sequence, wherein the subsequence encodes a fragment having lactase activity.

[0199] Variant: The term “variant” means a polypeptide havinglactase activity comprising a man¬

[0200] 20 made mutation, i.e., a substitution, insertion (including extension), and / or deletion (e.g., truncation), at one or more positions. A substitution means replacement of the amino acid occupying a position with a different amino acid; a deletion means removal of the amino acid occupying a position; and an insertion means adding 1-5 amino acids (e.g., 1-3 amino acids, in particular, 1 amino acid) adjacent to and immediately following the amino acid occupying a position.

[0201] Wild-type: The term "wild-type" in reference to an amino acid sequence or nucleic acid sequence means that the amino acid sequence or nucleic acid sequence is a native or naturally- occurring sequence. As used herein, the term "naturally-occurring" refers to anything (e.g., proteins, amino acids, or nucleic acid sequences) that is found in nature. Conversely, the term

[0202] 30 "non-naturally occurring" refers to anything that is not found in nature (e.g., recombinant nucleic acids and protein sequences produced in the laboratory or modification of the wild-type sequence). 16154-EP-EPA

[0203] Detailed Description of the Invention

[0204] The present invention relates to beta-galactosidase polypeptides useful in the production of e.g., Lactose free UHT milk or Galacto-oligosaccharide (GOS) production.

[0205] 5

[0206] Particularly the present invention provides lactases that have sufficient thermo-stability and are good at refolding after denaturation, thus making them well suited for production of lactose free milkproducts and for GOS production resulting in a desirable distribution of GOS di-saccharides comprising preferably beta 1-4 bonds.

[0207] 10

[0208] In certain applications, combinations of polypeptides having predominantly transgalactosylating activity and predominantly hydrolysing activity may be contemplated. This may be especially useful when there is a desire to reduce residual lactose after treatment with the polypeptide having beta-galactosidase activity, for example at low lactose levels.

[0209] 15 When considering the reaction of the polypeptide in e.g. milk, carbohydrates are initially present in the form of lactose, a disaccharide composed of galactose and glucose that is found in milk. In the formation of GOS, successive galactose molecules are added to lactose, and then after prolonged incubation a mixture of the various carbohydrates is present (glucose, galactose and ~30 different di- and polysaccharides).

[0210] The term “disaccharide” as used herein means two monosaccharide units bound together by a covalent bond known as a glycosidic linkage formed via a dehydration reaction, resulting in the loss of a hydrogen atom from one monosaccharide and a hydroxyl group from the other. In one aspect, the disaccharide is cellobiose, fucose, lactose, lactulose, maltose, rhamnose, or sucrose, most preferably lactose.

[0211] 25 As used herein, the term “transgalactosylase” means an enzyme that is able to transfer galactose to the hydroxyl groups of D-galactose (Gal) or D-glucose (Glc) whereby galactooligosaccharides are produced. In one embodiment, transgalactosylase activity is identified by reaction of the enzyme on lactose in which the amount of galactose generated is less than the amount of glucose generated at a given time.

[0212] 30 More particularly, the transgalactosylase activity or preference for an enzyme to hydrolyze lactose or to produce GOS can be evaluated as the amount of glucose minus galactose generated at any given time during reaction or by direct quantification of GOS generated during the reaction.

[0213] When evaluating the transgalactosylating activity versus beta-galactosidase activity of an

[0214] 35 enzyme, the beta-galactosidase activity is measured as concentration of galactose generated at any time point during the reaction. 16154-EP-EPA

[0215] In the present context, the GOS production of a polypeptide is measured as

[0216] (Glucose - Galactose)

[0217] Galactose i.e., the ratio of transgalactosylating activity to beta-galactosidase activity.

[0218] 5 Preferably, the ratio of transgalactosylating activity to beta-galactosidase activity is at least 1 , at least 2.5, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , or at least 12 as measured in high lactose conditions.

[0219] Polypeptides having beta-galactosidase activity useful according to the present invention may be of animal, of plant or of microbial origin. Preferred polypeptides are obtained from microbial sources, in particular from a filamentous fungus or yeast, or from a bacterium.

[0220] In the context of the present invention the main aspect is identifying beta-galactosidases that works particularly for production of GOS syrups resulting in a desirable distribution of GOS disaccharides comprising preferably beta 1-4 bonds.

[0221] 15 Therefore, in an aspect the present invention relates to a method for producing galactooligosaccharides (GOS) comprising contacting a polypeptide having beta-galactosidase activity with lactose wherein the polypeptide is selected from the group consisting of:

[0222] (a) a polypeptide having at least 75% sequence identity to SEQ ID NO: 6;

[0223] (b) a polypeptide having at least 75% sequence identity to a mature polypeptide of

[0224] 20 SEQ ID NO: 6;

[0225] (c) a polypeptide derived from a mature polypeptide of SEQ ID NO: 6, or amino acids 1-944 of SEQ ID NO: 6 by having 1-30 alterations (e.g., substitutions, deletions and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions;

[0226] (d) a polypeptide derived from the polypeptide of (a), (b), or (c), wherein the N- and / or C-terminal end has been extended by addition of one or more amino acids; and

[0227] (e) a fragment of the polypeptide of (a), (b), (c), or (d); and wherein the polypeptide has beta-galactosidase activityactivity.

[0228] 30

[0229] In an aspect, the polypeptide has a sequence identity of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at 16154-EP-EPA least 96%, at least 97%, at least 98%, at least 99%, or 100% to SEQ ID NO: 6 or a mature polypeptide of SEQ ID NO: 6.

[0230] In another aspect, the polypeptide has a sequence identity of at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%,

[0231] 5 at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to amino acids 1-944 of SEQ ID NO: 6.

[0232] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 6 or a mature polypeptide thereof.

[0233] 10 The polypeptide preferably comprises, consists essentially of, or consists of the amino acid 1-944 of SEQ ID NO: 6.

[0234] In another aspect the invention relates to a method for producing galacto-oligosaccharides (GOS) comprising contacting a polypeptide having beta-galactosidase activity with lactose wherein the

[0235] 15 polypeptide is selected from the group consisting of:

[0236] (a) a polypeptide having at least 75% sequence identity to SEQ ID NO: 7;

[0237] (b) a polypeptide having at least 75% sequence identity to a mature polypeptide of SEQ ID NO: 7;

[0238] (c) a polypeptide derived from a mature polypeptide of SEQ ID NO: 7, or 1-837 of SEQ ID NO: 7 by having 1-30 alterations (e.g., substitutions, deletions and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions;

[0239] (d) a polypeptide derived from the polypeptide of (a), (b), or (c), wherein the N- and / or

[0240] 25 C-terminal end has been extended by addition of one or more amino acids; and

[0241] (e) a fragment of the polypeptide of (a), (b), (c), or (d); and wherein the polypeptide has beta-galactosidase activityactivity.

[0242] 30 In an aspect, the polypeptide has a sequence identity of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to SEQ ID NO: 7 or a mature polypeptide of SEQ ID NO: 7.

[0243] 35 In another aspect, the polypeptide has a sequence identity of at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, 16154-EP-EPA at least 96%, at least 97%, at least 98%, at least 99%, or 100% to amino acids 1-837 of SEQ ID NO: 7.

[0244] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 7 or a mature polypeptide thereof.

[0245] 5 The polypeptide preferably comprises, consists essentially of, or consists of the amino acid 1-837 of SEQ ID NO: 7.

[0246] In another aspect the invention relates to a method for producing galactooligosaccharides (GOS) comprising contacting a polypeptide having beta-galactosidase activity

[0247] 10 with lactose wherein the polypeptide is selected from the group consisting of:

[0248] (a) a polypeptide having at least 75% sequence identity to SEQ ID NO: 10;

[0249] (b) a polypeptide having at least 75% sequence identity to a mature polypeptide of SEQ ID NO: 10;

[0250] (c) a polypeptide derived from SEQ ID NO: 10, a mature polypeptide of SEQ ID NO:

[0251] 15 10, or 1-1225 of SEQ ID NO: 10 by having 1-30 alterations (e.g., substitutions, deletions and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions;

[0252] (d) a polypeptide derived from the polypeptide of (a), (b), or (c), wherein the N- and / or C-terminal end has been extended by addition of one or more amino acids; and

[0253] (e) a fragment of the polypeptide of (a), (b), (c), or (d); and wherein the polypeptide has beta-galactosidase activityactivity.

[0254] 25 In an aspect, the polypeptide has a sequence identity of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to SEQ ID NO: 10 or a mature polypeptide of SEQ ID NO: 10.

[0255] 30 In another aspect, the polypeptide has a sequence identity of at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to amino acids 1-1225 of SEQ ID NO: 10.

[0256] 35 The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 10 or a mature polypeptide thereof.

[0257] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid 1-1225 of SEQ ID NO: 10. 16154-EP-EPA

[0258] In another aspect the invention relates to a method for producing galactooligosaccharides (GOS) comprising contacting a polypeptide having beta-galactosidase activity with lactose wherein the polypeptide is selected from the group consisting of:

[0259] (a) a polypeptide having at least 75% sequence identity to SEQ ID NO: 1 ;

[0260] 5 (b) a polypeptide having at least 75% sequence identity to a mature polypeptide of SEQ ID NO: 1 ;

[0261] (c) a polypeptide derived from SEQ ID NO: 1 , a mature polypeptide of SEQ ID NO: 1 , or 1-820 of SEQ ID NO: 1 by having 1-30 alterations (e.g., substitutions, deletions and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or

[0262] 10 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions;

[0263] (d) a polypeptide derived from the polypeptide of (a), (b), or (c), wherein the N- and / or C-terminal end has been extended by addition of one or more amino acids; and

[0264] (e) a fragment of the polypeptide of (a), (b), (c), or (d); and wherein the polypeptide

[0265] 15 has beta-galactosidase activityactivity.

[0266] In an aspect, the polypeptide has a sequence identity of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to SEQ ID NO: 1 or a mature polypeptide of SEQ ID NO: 1.

[0267] In another aspect, the polypeptide has a sequence identity of at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%,

[0268] 25 at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to amino acids 1-820 of SEQ ID NO: 1.

[0269] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 1 or a mature polypeptide thereof.

[0270] 30 The polypeptide preferably comprises, consists essentially of, or consists of the amino acid 1-820 of SEQ ID NO: 1.

[0271] In another aspect the invention relates to a method for producing galactooligosaccharides (GOS) comprising contacting a polypeptide having beta-galactosidase activity

[0272] 35 with lactose wherein the polypeptide is selected from the group consisting of:

[0273] (a) a polypeptide having at least 75% sequence identity to SEQ ID NO: 3;

[0274] (b) a polypeptide having at least 75% sequence identity to a mature polypeptide of SEQ ID NO: 3; 16154-EP-EPA

[0275] (c) a polypeptide derived from SEQ ID NO: 3, a mature polypeptide of SEQ ID NO: 3, or 1-826 of SEQ ID NO: 3 by having 1-30 alterations (e.g., substitutions, deletions and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26

[0276] 5 or 27 or 28 or 29 or 30 alterations, in particular substitutions;

[0277] (d) a polypeptide derived from the polypeptide of (a), (b), or (c), wherein the N- and / or C-terminal end has been extended by addition of one or more amino acids; and

[0278] (e) a fragment of the polypeptide of (a), (b), (c), or (d); and wherein the polypeptide has beta-galactosidase activityactivity.

[0279] In an aspect, the polypeptide has a sequence identity of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at

[0280] 15 least 96%, at least 97%, at least 98%, at least 99%, or 100% to SEQ ID NO: 3 or a mature polypeptide of SEQ ID NO: 3.

[0281] In another aspect, the polypeptide has a sequence identity of at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%,

[0282] 20 at least 96%, at least 97%, at least 98%, at least 99%, or 100% to amino acids 1-826 of SEQ ID NO: 3.

[0283] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 3 or a mature polypeptide thereof.

[0284] The polypeptide preferably comprises, consists essentially of, or consists of the amino

[0285] 25 acid 1-826 of SEQ ID NO: 3.

[0286] In another embodiment the present invention relates to a method for producing galactooligosaccharides (GOS) comprising contacting a polypeptide having beta-galactosidase activity with lactose, wherein the GOS is produced in-situ, in the milk product from the lactose already

[0287] 30 present in the milk.

[0288] Polypeptides Having Lactase Activity

[0289] The present invention relates to polypeptides having lactase activity.

[0290] 35 In an aspect, the invention relates to polypeptides having lactase activity, wherein the polypeptide is selected from the group consisting of:

[0291] (a) a polypeptide having at least 90% sequence identity to SEQ ID NO: 6; 16154-EP-EPA

[0292] (b) a polypeptide having at least 90% sequence identity to a mature polypeptide of SEQ ID NO: 6;

[0293] (c) a polypeptide derived from SEQ ID NO: 6, a mature polypeptide of SEQ ID NO: 6, or 1-944 of SEQ ID NO: 6 by having 1-30 alterations (e.g., substitutions, deletions and / or

[0294] 5 insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions;

[0295] (d) a polypeptide derived from the polypeptide of (a), (b), or (c), wherein the N- and / or C-terminal end has been extended by addition of one or more amino acids; and

[0296] 10 (e) a fragment of the polypeptide of (a), (b), (c), or (d); and wherein the polypeptide has beta-galactosidase activityactivity.

[0297] In an aspect, the polypeptide has a sequence identity of at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or

[0298] 15 100% to SEQ ID NO: 6 or a mature polypeptide of SEQ ID NO: 6.

[0299] In another aspect, the polypeptide has a sequence identity of at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to amino acids 1-944 of SEQ ID NO: 6.

[0300] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 6 or a mature polypeptide thereof.

[0301] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid 1-944 of SEQ ID NO: 6.

[0302] In an aspect, the invention relates to polypeptides having lactase activity, wherein the polypeptide

[0303] 25 is selected from the group consisting of:

[0304] (a) a polypeptide having at least 90% sequence identity to SEQ ID NO: 7;

[0305] (b) a polypeptide having at least 90% sequence identity to a mature polypeptide of SEQ ID NO: 7;

[0306] (c) a polypeptide derived from SEQ ID NO: 7, a mature polypeptide of SEQ ID NO: 7,

[0307] 30 or 1-837 of SEQ ID NO: 7 by having 1-30 alterations (e.g., substitutions, deletions and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions;

[0308] (d) a polypeptide derived from the polypeptide of (a), (b), or (c), wherein the N- and / or

[0309] 35 C-terminal end has been extended by addition of one or more amino acids; and 16154-EP-EPA

[0310] (e) a fragment of the polypeptide of (a), (b), (c), or (d); and wherein the polypeptide has beta-galactosidase activityactivity.

[0311] 5 In an aspect, the polypeptide has a sequence identity of at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to SEQ ID NO: 7 or a mature polypeptide of SEQ ID NO: 7.

[0312] In another aspect, the polypeptide has a sequence identity of at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to amino acids 1-837 of SEQ ID NO: 7.

[0313] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 7 or a mature polypeptide thereof.

[0314] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid 1-837 of SEQ ID NO: 7.

[0315] 15

[0316] In an aspect, the invention relates to polypeptides having lactase activity, wherein the polypeptide is selected from the group consisting of:

[0317] (a) a polypeptide having at least 90% sequence identity to SEQ ID NO: 10;

[0318] (b) a polypeptide having at least 90% sequence identity to a mature polypeptide of

[0319] 20 SEQ ID NO: 10;

[0320] (c) a polypeptide derived from SEQ ID NO: 10, a mature polypeptide of SEQ ID NO: 10, or 1-1225 of SEQ ID NO: 10 by having 1-30 alterations (e.g., substitutions, deletions and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25

[0321] 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions;

[0322] (d) a polypeptide derived from the polypeptide of (a), (b), or (c), wherein the N- and / or C-terminal end has been extended by addition of one or more amino acids; and

[0323] (e) a fragment of the polypeptide of (a), (b), (c), or (d); and wherein the polypeptide has beta-galactosidase activityactivity.

[0324] 30

[0325] In an aspect, the polypeptide has a sequence identity of at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to SEQ ID NO: 10 or a mature polypeptide of SEQ ID NO: 10.

[0326] 35 In another aspect, the polypeptide has a sequence identity of at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to amino acids 1-1225 of SEQ ID NO: 10. 16154-EP-EPA

[0327] The polypeptide preferably comprises, consists essentially of, or consists of the ammo acid sequence of SEQ ID NO: 10 or a mature polypeptide thereof.

[0328] The polypeptide preferably comprises, consists essentially of, or consists of the amino acid 1-1225 of SEQ ID NO: 10.

[0329] 5

[0330] Essential amino acids in a polypeptide can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, 1989, Science 244: 1081-1085). In the latter technique, single alanine mutations are

[0331] 10 introduced at every residue in the molecule, and the resultant molecules are tested for lactase activity to identify amino acid residues that are critical to the activity of the molecule. See also, Hilton et al., 1996, J. Biol. Chem. 271 : 4699-4708. The active site of the enzyme or other biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction, or

[0332] 15 photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., 1992, Science 255: 306-312; Smith et al., 1992, J. Mol. Biol. 224: 899- 904; Wlodaver et al., 1992, FEBS Lett. 309: 59-64. The identity of essential amino acids can also be inferred from an alignment with a related polypeptide, and / or be inferred from sequence homology and conserved catalytic machinery with a related polypeptide or within a polypeptide or protein family with polypeptides / proteins descending from a common ancestor, typically having similar three-dimensional structures, functions, and significant sequence similarity. Additionally or alternatively, protein structure prediction tools can be used for protein structure modelling to identify essential amino acids and / or active sites of polypeptides. See, for example, Jumper et al., 2021 , “Highly accurate protein structure prediction with AlphaFold”, Nature 596: 583-589.

[0333] 25 Single or multiple amino acid substitutions, deletions, and / or insertions can be made and tested using known methods of mutagenesis, recombination, and / or shuffling, followed by a relevant screening procedure, such as those disclosed by Reidhaar-Olson and Sauer, 1988, Science 241 : 53-57; Bowie and Sauer, 1989, Proc. Natl. Acad. Sci. USA 86: 2152-2156; WO 95 / 17413; or WO 95 / 22625. Other methods that can be used include error-prone PCR, phage

[0334] 30 display (e.g., Lowman eta!., 1991 , Biochemistry 30: 10832-10837; US 5,223,409; WO 92 / 06204), and region-directed mutagenesis (Derbyshire et al., 1986, Gene 46: 145; Ner et al., 1988, DNA 7: 127).

[0335] Mutagenesis / shuffling methods can be combined with high-throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides expressed by host cells

[0336] 35 (Ness et al., 1999, Nature Biotechnology 17: 893-896). Mutagenized DNA molecules that encode active polypeptides can be recovered from the host cells and rapidly sequenced using standard methods in the art. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide. 16154-EP-EPA

[0337] Sources of Polypeptides Having Lactase Activity

[0338] A polypeptide having lactase activity of the present invention may be obtained from

[0339] 5 microorganisms of a bacterial genus. For purposes of the present invention, the term “obtained from” as used herein in connection with a given source shall mean that the polypeptide encoded by a polynucleotide is produced by the source or by a strain in which the polynucleotide of the invention has been inserted. In one aspect, the polypeptide obtained from a given source is secreted extracellularly.

[0340] 10 In one aspect the lactase polypeptide of the invention is derived form a Streptococcus sp. In another aspect the the lactase polypeptide of the invention is derived from a Streptomycers sp.

[0341] In another aspect, the polypeptide is a polypeptide obtained from a Streptococcus sp. , e.g., a polypeptide obtained from Streptococcus infantis.

[0342] In another aspect, the polypeptide is a polypeptide obtained from a Streptomyces sp.,

[0343] 15 e.g., a polypeptide obtained from Streptomyces cirratus.

[0344] The polypeptides may be identified and obtained from other sources including microorganisms isolated from nature (e.g., soil, composts, water, etc.) or DNA samples obtained directly from natural materials (e.g., soil, composts, water, etc.) using the above-mentioned probes. Techniques for isolating microorganisms and DNA directly from natural habitats are well known in the art. A polynucleotide encoding the polypeptide may then be obtained by similarly screening a genomic DNA or cDNA library of another microorganism or mixed DNA sample. Once a polynucleotide encoding a polypeptide has been detected with the probe(s), the polynucleotide can be isolated or cloned by utilizing techniques that are known to those of ordinary skill in the

[0345] 25 art (see, e.g., Davis et al., 2012, Basic Methods in Molecular Biology, Elsevier).

[0346] Polynucleotides

[0347] The present invention also relates to polynucleotides encoding a polypeptide of the

[0348] 30 present invention, as described herein.

[0349] The polynucleotide may be a genomic DNA, a cDNA, a synthetic DNA, a synthetic RNA, a mRNA, or a combination thereof. The polynucleotide may be cloned from a strain of Streptococcus or Streptomyces, or a related organism and thus, for example, may be a polynucleotide sequence encoding a variant of the polypeptide of the invention.

[0350] 35 In one embodiment the polynucleotide encoding the polypeptide of the present invention is isolated from a Streptococcus cell.

[0351] In one embodiment the polynucleotide encoding the polypeptide of the present invention is isolated from a Streptomyces cell. 16154-EP-EPA

[0352] In a particularlembodiment the mature polypeptide coding sequence is selected from the group consisting of: a polypeptide encoded by a polynucleotide having at least 90 % sequence identity, at least 91 %,

[0353] 5 at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the mature polypeptide coding sequence of SEQ ID NO: 13; a polypeptide encoded by a polynucleotide having at least 90 % sequence identity, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the mature polypeptide coding sequence of SEQ ID NO: 14; and a

[0354] 10 polypeptide encoded by a polynucleotide having at least 90 % sequence identity, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the mature polypeptide coding sequence of SEQ ID NO: 15.

[0355] The polynucleotide may also be mutated by introduction of nucleotide substitutions that

[0356] 15 do not result in a change in the amino acid sequence of the polypeptide, but which correspond to the codon usage of the host organism intended for production of the enzyme, or by introduction of nucleotide substitutions that may give rise to a different amino acid sequence. For a general description of nucleotide substitution, see, e.g., Ford et al., 1991 , Protein Expression and Purification 2: 95-107.

[0357] In an aspect, the polynucleotide is isolated.

[0358] In another aspect, the polynucleotide is purified.

[0359] Nucleic Acid Constructs

[0360] The present invention also relates to nucleic acid constructs comprising a polynucleotide

[0361] 25 of the present invention, wherein the polynucleotide is operably linked to one or more control sequences that direct the expression of the coding sequence in a suitable host cell under conditions compatible with the control sequences.

[0362] The polynucleotide may be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the polynucleotide prior to its insertion into a vector may be

[0363] 30 desirable or necessary depending on the expression vector. Techniques for modifying polynucleotides utilizing recombinant DNA methods are well known in the art.

[0364] Expression Vectors

[0365] The present invention also relates to recombinant expression vectors comprising a

[0366] 35 polynucleotide of the present invention, a promoter, and transcriptional and translational stop signals. The various nucleotide and control sequences may be joined together to produce a recombinant expression vector that may include one or more convenient restriction sites to allow for insertion or substitution of the polynucleotide encoding the polypeptide at such sites. 16154-EP-EPA

[0367] Alternatively, the polynucleotide may be expressed by inserting the polynucleotide or a nucleic acid construct comprising the polynucleotide into an appropriate vector for expression. In creating the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

[0368] 5 The recombinant expression vector may be any vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures and can bring about expression of the polynucleotide. The choice of the vector will typically depend on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

[0369] 10 The vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector may contain any means for assuring self-replication. Alternatively, the vector may be one that, when introduced into the host cell, is integrated into the genome and replicated together

[0370] 15 with the chromosome(s) into which it has been integrated. Furthermore, a single vector or plasmid or two or more vectors or plasmids that together contain the total DNA to be introduced into the genome of the host cell, or a transposon, may be used.

[0371] The vector preferably contains one or more selectable markers that permit easy selection of transformed, transfected, transduced, or the like cells. A selectable marker is a gene the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.

[0372] The vector preferably contains at least one element that permits integration of the vector into the host cell's genome or autonomous replication of the vector in the cell independent of the genome.

[0373] 25 For integration into the host cell genome, the vector may rely on the polynucleotide’s sequence encoding the polypeptide or any other element of the vector for integration into the genome by homologous recombination, such as homology-directed repair (HDR), or non- homologous recombination, such as non-homologous end-joining (NHEJ).

[0374] For autonomous replication, the vector may further comprise an origin of replication

[0375] 30 enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replicator mediating autonomous replication that functions in a cell. The term “origin of replication” or “plasmid replicator” means a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0376] More than one copy of a polynucleotide of the present invention may be inserted into a

[0377] 35 host cell to increase production of a polypeptide. For example, 2 or 3 or 4 or 5 or more copies are inserted into a host cell. An increase in the copy number of the polynucleotide can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the polynucleotide where cells containing 16154-EP-EPA amplified copies of the selectable marker gene, and thereby additional copies of the polynucleotide, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

[0378] 5

[0379] Methods of Production

[0380] The present invention also relates to methods of producing a polypeptide of the present invention, comprising (a) cultivating a recombinant host cell of the present invention under conditions conducive for production of the polypeptide; and optionally, (b) recovering the

[0381] 10 polypeptide.

[0382] The host cell is cultivated in a nutrient medium suitable for production of the polypeptide using methods known in the art. For example, the cell may be cultivated by shake flask cultivation, or small-scale or large-scale fermentation (including continuous, batch, fed-batch, or solid-state, and / or microcarrier-based fermentations) in laboratory or industrial fermentors in a suitable

[0383] 15 medium and under conditions allowing the polypeptide to be expressed and / or isolated. Suitable media are available from commercial suppliers or may be prepared according to published compositions (e.g., in catalogues of the American Type Culture Collection). If the polypeptide is secreted into the nutrient medium, the polypeptide can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from cell lysates.

[0384] The polypeptide may be detected using methods known in the art that are specific for the polypeptide, including, but not limited to, the use of specific antibodies, formation of an enzyme product, disappearance of an enzyme substrate, or an assay determining the relative or specific activity of the polypeptide.

[0385] The polypeptide may be recovered from the medium using methods known in the art,

[0386] 25 including, but not limited to, collection, centrifugation, filtration, extraction, spray-drying, evaporation, or precipitation. In one aspect, a whole fermentation broth comprising the polypeptide is recovered. In another aspect, a cell-free fermentation broth comprising the polypeptide is recovered.

[0387] The polypeptide may be purified by a variety of procedures known in the art to obtain

[0388] 30 substantially pure polypeptides and / or polypeptide fragments (see, e.g., Wingfield, 2015, Current Protocols in Protein Science’, 80(1): 6.1.1-6.1.35; Labrou, 2014, Protein Downstream Processing, 1129: 3-10).

[0389] In an alternative aspect, the polypeptide is not recovered.

[0390] 35

[0391] The present invention is further described by the following examples that should not be construed as limiting the scope of the invention. 16154-EP-EPA

[0392] Examples

[0393] Polypeptides having bata-galactosidase activity tested in the below examples:

[0394] SEQ ID NO: 1 lactase from Neobacillus bataviensis

[0395] 5 SEQ ID NO: 2 lactase from Neobacillus niacini

[0396] SEQ ID NO: 3 lactase from Paenibacillus antarcticus

[0397] SEQ ID NO: 4 lactase from Neobacillus mesonae

[0398] SEQ ID NO: 5 lactase from Bacillus sp S3

[0399] SEQ ID NO: 6 lactase from Streptococcus sp HMSC056C01

[0400] 10 SEQ ID NO: 7 lactase from Streptococcus infantis

[0401] SEQ ID NO: 8 lactase from Neobacillus kokaensis (truncated version of SEQ ID NO: 9).

[0402] SEQ ID NO: 9 lactase from Neobacillus kokaensis (disclosed in TrEMBL:E5RWQ2).

[0403] SEQ ID NO: 10 lactase from Streptomyces cirratus

[0404] SEQ ID NO: 11 lactase from Neobacillus kokaensis (Lactase from Genofocus Co Ltd disclosed

[0405] 15 in W02016 / 190613 and as TrEMBL:A0A6M5K904 - Lactazym B).

[0406] SEQ ID NO: 12 Lactase know as BIF-917 from Bifidobacterium bifidum disclsoed as SEQ ID NO: 1 in WO2015 / 086746 (DuPont).

[0407] Lactase Biolacta N5 (commercial product from Amano Enzymes Inc.). Lactase Nurica™ (comercial producto from IFF).

[0408] The examples below show lactose conversion measured as residual activity in milk.

[0409] Example 1 :

[0410] UHT treatment

[0411] 25 The enzymes of Table 1 were added to skimmed milk and a UHT treatment was performed followed by incubation at 23°C for 0.5 hrs and for 72 hrs with measurement of lactase activity and lactose being performed after each of these two intervals as described below.

[0412] 12.7 mg enzyme protein (ep) per litre skimmed milk was used of the enzymes shown in Table 1. Sodium azide was added to a final concentration of 0.025% (w / v) in all milk tested to

[0413] 30 avoid microbial growth since due to handling etc. the tubes are not fully sterile despite the UHT step. The milk samples were applied to a lab. scale UHT setup as described below. A syringe with a 10 ml milk sample was connected to a long Teflon tube that was immersed sequentially in four baths. The first bath was in silicone at 90°C with 3 m of the Teflon tube (0.8 mm inner diameter), the second connected bath was in silicone at 140°C with 50 cm of the Teflon tube, the

[0414] 35 third silicone bath was at 70°C with 3 m of Teflon tube, and the final bath was an ice / water bath (0°C) with 1 m of Teflon tube to cool the milk. A flow of 3 ml / min was applied to the syringe ensuring that the milk samples were incubated at 90°C for 30 sec, 140°C for 5 sec and 70°C for 30 sec before cooling for 10 sec in the ice / water bath, thereby cooling the milk to a temperature 16154-EP-EPA in the range of 0-10°C. Finally, the milk was collected in tubes after the ice / water bath, and the samples were incubated at 23°C for 0.5 h and 72 h, respectively, and then assayed for residual activity.

[0415] 5 Residual lactase activity assay

[0416] Samples were centrifuged at 20,600 g in a precooled centrifuge for 45 min at 5°C and the supernatant was diluted with 20 mM sodium succinic acid and 0.01 % Triton X-100, pH 6.5, so an absorbance reading below 1 .5 at 405 nm could be measured. Twenty-five pl of each sample was mixed with 175pl of ONPG substrate (1.67 mg / ml ONPG (o-NitroPhenyl p-D-

[0417] 10 galatopyranoside, ~5.5 mM), 0.05 M MES, 1 mM MgSO4, 150 mM KCI, 0.01 % Triton X-100, pH 6.5) and incubated for 2.5 hr at 40°C and stopped by adding 50pl Na2CO3 + 5 mM Na4EDTA and measured at 405 nm. Residual activity in % was calculated using the formulae = ((Abs405heat-treated sample > Abs405blank) * dilution factor) I ((Abs405untreated sample- Abs405blank) * dilution factor) * 100%.

[0418] 15

[0419] The heat-treated sample is enzyme mixed with skimmed milk and sodium azide and subjected to UHT treatment and subsequent cooling as described above followed by incubation at 23°C for 0.5 h and 72 h. The untreated sample is the same enzyme mixed with skimmed milk and sodium azide but without UHT treatment and incubation. Both of these samples were diluted to obtain an absorption at 405 nm in the range of 0.5-1.5. The blank is sample without enzyme and the same dilution factor has been used as for the sample with enzyme. The results are shown in Table 1 below.Analysis of lactose content

[0420] The analysis of residual lactose was performed by high-performance anion exchange chromatography coupled with pulsed amperometry detector (HPAEC-PAD).

[0421] 25

[0422] Sample preparation for HPAEC-PAD:

[0423] The enzyme was inactivated by adding 5 ul glacial acetic acid to 1 ml milk sample and heated to 90°C for 5 min and centrifugated at 14200 rpm for 10 min. 50 ul sample was transferred to 5 ml Eppendorf tube containing 500 ul MQ water. 10 ul Carrez I solution was added and mixed,

[0424] 30 and then 10 ul Carrez II solution was added and mixed. Then, 4.43 ml MQ water (total volume of 5 ml) was added and mixed. Centrifugation was carried out at 14200 rpm for 5 min. The supernatant was diluted x5 with Milli Q water. These samples were analyzed on HPAEC-PAD.

[0425] Lactose determination using HPAEC-PAD

[0426] 35 The analysis performed is essential as described in Leeuwen S, Kuipers B, Dijkhuizen L, Kamerling J. Comparative structural characterization of 7 commercial galacto-oligosaccharide (GOS) products, Carbohydrate Research, 425 (2016) 48-58 with minor modifications, e.g., with a shorter gradient. Dionex ICS-6000 workstation (Dionex, Amsterdam, The Netherlands) was 16154-EP-EPA used, equipped with a CarboPac PA1 4 x 50 mm Guard Column (Dionex, product no. 043096) followed by CarboPac PA1 4 x 250 mm (Dionex, product no. 035391) and an ICS-6000 DC ECD detector (Dionex), using a complex gradient of A: Milli-Q water, B: 600 mM NaOAc in 100 mM NaOH, C: 100 mM NaOH, and D: 50 mM NaOAc. The fractionations were performed at 1.0 5 mL / min with 85% A, 0% B, 10% C, and 5% D in 25 min linear gradient to 10% A, 0% B, 40%C, and 50% D, followed by a 2-min linear gradient to 0% A, 25% B, 75% C and 0% D, directly followed by 5 min washing with 100% B and reconditioning for 15 min with 85% A, 0% B, 10% C, and 5% D. A lactose standard was used to determine the amount of lactose in the enzyme treated samples.

[0427] .0

[0428] Table 1 : Residual lactase activities in milk after UHT treatment followed by 0.5 h and 72 hours incubation at 23°C.

[0429] Table 2: Lactose content (%, i.e. gram / 100 gram) in milk after UHT treatment followed by 0.5 h and 72 hours incubation at 23°C 16154-EP-EPA

[0430] Table 1 , columns 2 and 3, show residual activity of the enzymes at 0.5 hrs and 72 hrs, respectively, after UHT treatment. There are various degrees of residual activity, and all (expect

[0431] 5 SEQ ID NO: 11 and SEQ ID NO: 9) surprisingly have an increased residual activity after 72 hrs compared to 0.5 hrs which indicate that the enzyme refolds after UHT treatment. Several enzymes have > 50% residual activity after 72 h incubation whereas SEQ ID NO: 11 and SEQ ID NO: 9 have low residual activity and a slight decrease in residual activity from 0.5 h to 72 h. Table 2 shows the % (g / 100g) lactose amount after UHT treatment. As expected only minor amount of hydrolysis have occurred after 0.5 hr incubation due to this short incubation time (initial lactose amount is 4.7%). After 72 hr storage at room temperature the amount of lactose has decreased to various lactose levels which is due to the amount of lactase survival after UHT and the inherent efficiency of lactose hydrolysis (i.e. , specific activity for lactose in milk matrix) for each lactase. Thus, the combination of high survival after UHT and high specific activity at room

[0432] 15 temperature results in the most efficient lactose hydrolysis after 72 hr (column 3, Table 2). The most efficient enzymes are SEQ ID NO: 5, SEQ ID NO: 4 and SEQ ID NO: 1 , in an application without any needed preincubation before UHT and the lactase can convert the lactose to GOS, galactose and glucose in the final container in an efficient way.

[0433] 20

[0434] Example 2: Production of GOS from lactose

[0435] GOS syrup application at 50°C for 24 h.

[0436] To evaluate GOS produced at 50°C, 50 pl 1 g / l enzyme protein is mixed with 950 pl preheated 55.4 % lactose*H20 (w / w), 20 mM sodium succinate, HEPES buffer pH 7.0 in an eppendorftube which gives a final concentration of 50 % lactose (w / w). This mixture is then incubated at 50°C with 1000 rpm for 24 h and applied on ice. Inactivation of the enzyme is performed by diluting the 1 ml GOS syrup product with 9 ml 5 mM H2SO4 and incubated for 5 min at 80°C. After cooling to room temperature, an aliquot (1 ml) was applied to Aminex-HPX-87H for quantitative

[0437] 30 determination of carbohydrate (see below). For samples applied to HPAEC-PAD a further 400x 16154-EP-EPA dilution with milli Q water (i.e. 4000x dilution in total) is made and applied on a PA1 column (see below).

[0438] Quantitative determination of Glc, Gal, DP2, DP3, DP4 and DP4+

[0439] 5 Dionex HPLC system ICS-5000 Rl (Aminex-HPX-87H Ion Exclusion Column with 5 mM H2SO4 as isocratic mobile phase) is used for quantitative determination of Gal, Glc, DP2 and DP3+

[0440] Quantitative determination of Lac and GOS di-saccharides (DP2-GOS)

[0441] High-Performance Anion-Exchange Chromatography with Pulsed Amperometric Detection 10 (HPAEC-PAD) using a PA1 column for quantitative determination of Gal, Glc, Lac and DP2- GOS's + qualitative detection of various DP2+ is performed as follows.

[0442] Quantitative determination of Lac, b-1-6-Gal-Gal, 1-6-Gal-Glc, 1-3-Gal-Gal, 1-4-Gal-Gal and 1- 3-Gal-Glc (+1-2-Gal-Glc) was made so a ratio of GOS di-saccharides (DP2G) and lactose could be determined. This DP2G / Lac ratio is then used to calculate the amount of DP2G in the DP2 15 peak determined by the Dionex HPLC system ICS-5000 Rl method. Thus, Lac + DP2G = DP2 in the column below.

[0443] Table 3 16154-EP-EPA

[0444] The most efficient enzymes to produce DP3+ (GOS fiber) is SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 10 having appr. 41% DP3+ using the described assay condition (see Table 3).

[0445] Thus, these three enzymes are most promising to make GOS sirup efficiently as these enzymes can produce > 40% GOS fiber with only 50 mg enzyme protein per liter solution (50%

[0446] 5 w / w lactose) incubated for 24 h at 50°C.

[0447] The invention described and claimed herein is not to be limited in scope by the specific aspects herein disclosed, since these aspects are intended as illustrations of several aspects of 10 the invention. Any equivalent aspects are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. In the case of conflict, the present disclosure including definitions will control.

Claims

16154-EP-EPAClaimsWhat is claimed is:5 1. A method for producing galacto-oligosaccharides (GOS) comprising contacting a polypeptide having beta-galactosidase activity with lactose and wherein the polypeptide is selected from the group consisting of:(a) a polypeptide having at least 75% sequence identity to SEQ ID NO: 6;(b) a polypeptide having at least 75% sequence identity to a mature polypeptide of10 SEQ ID NO: 6;(c) a polypeptide derived from SEQ ID NO: 6, a mature polypeptide of SEQ ID NO: 6, or 1-944 of SEQ ID NO: 6 by having 1-30 alterations (e.g., substitutions, deletions and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions;(d) a polypeptide derived from the polypeptide of (a), (b), or (c), wherein the N- and / or C-terminal end has been extended by addition of one or more amino acids; and(e) a fragment of the polypeptide of (a), (b), (c), or (d); and wherein the polypeptide has beta-galactosidase activity;(a) a polypeptide having at least 75% sequence identity to SEQ ID NO: 7;(b) a polypeptide having at least 75% sequence identity to a mature polypeptide of SEQ ID NO: 7;(c) a polypeptide derived from SEQ ID NO: 7, a mature polypeptide of SEQ ID NO: 7,25 or 1-837 of SEQ ID NO: 7 by having 1-30 alterations (e.g., substitutions, deletions and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions;(d) a polypeptide derived from the polypeptide of (a), (b), or (c), wherein the N- and / or30 C-terminal end has been extended by addition of one or more amino acids; and(e) a fragment of the polypeptide of (a), (b), (c), or (d); and wherein the polypeptide has beta-galactosidase activity;(a) a polypeptide having at least 75% sequence identity to SEQ ID NO: 10;35 (b) a polypeptide having at least 75% sequence identity to a mature polypeptide of SEQ ID NO: 10;(c) a polypeptide derived from SEQ ID NO: 10, a mature polypeptide of SEQ ID NO: 10, or 1-1225 of SEQ ID NO: 10 by having 1-30 alterations (e.g., substitutions, deletions16154-EP-EPA and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions;(d) a polypeptide derived from the polypeptide of (a), (b), or (c), wherein the N- and / or5 C-terminal end has been extended by addition of one or more amino acids; and(e) a fragment of the polypeptide of (a), (b), (c), or (d); and wherein the polypeptide has beta-galactosidase activity;(a) a polypeptide having at least 75% sequence identity to SEQ ID NO: 1 ;10 (b) a polypeptide having at least 75% sequence identity to a mature polypeptide of SEQ ID NO: 1 ;(c) a polypeptide derived from SEQ ID NO: 1 , a mature polypeptide of SEQ ID NO: 1 , or 1-820 of SEQ ID NO: 1 by having 1-30 alterations (e.g., substitutions, deletions and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or15 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions;(d) a polypeptide derived from the polypeptide of (a), (b), or (c), wherein the N- and / or C-terminal end has been extended by addition of one or more amino acids; and(e) a fragment of the polypeptide of (a), (b), (c), or (d); and wherein the polypeptide has beta-galactosidase activity; and(a) a polypeptide having at least 75% sequence identity to SEQ ID NO: 3;(b) a polypeptide having at least 75% sequence identity to a mature polypeptide of SEQ ID NO: 3;25 (c) a polypeptide derived from SEQ ID NO: 3, a mature polypeptide of SEQ ID NO: 3, or 1-826 of SEQ ID NO: 3 by having 1-30 alterations (e.g., substitutions, deletions and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions;30 (d) a polypeptide derived from the polypeptide of (a), (b), or (c), wherein the N- and / or C-terminal end has been extended by addition of one or more amino acids; and(e) a fragment of the polypeptide of (a), (b), (c), or (d); and wherein the polypeptide has beta-galactosidase activity.35 2. The method of claim 1 , wherein the polypeptide having beta-galactosidase activity is selected from the group consisting of:(a) a polypeptide having a sequence identity of at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%,16154-EP-EPA at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to SEQ ID NO: 6, a mature polypeptide of SEQ ID NO: 6, or amimo acids 1-944 of SEQ ID NO: 6;(b) a polypeptide having a sequence identity of at least 80%, at least 81 %, at least 82%,5 at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to SEQ ID NO: 7, a mature polypeptide of SEQ ID NO: 7, or amimo acids 1-837 of SEQ ID NO: 7;(c) a polypeptide having a sequence identity of at least 80%, at least 81 %, at least 82%,10 at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to SEQ ID NO: 10, a mature polypeptide of SEQ ID NO: 10, or amimo acids 1-1225 of SEQ ID NO: 10;(d) a polypeptide having a sequence identity of at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to SEQ ID NO: 1 , a mature polypeptide of SEQ ID NO: 1 , or amimo acids 1-820 of SEQ ID NO: 1 ; and(e) a polypeptide having a sequence identity of at least 80%, at least 81 %, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to SEQ ID NO: 3, a mature polypeptide of SEQ ID NO: 3, or amimo acids 1-826 of SEQ ID NO: 3.25 3. The method of any of claims 1-2, wherein the polypeptide comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 10, SEQ ID NO: 1 , or SEQ ID NO: 3, or mature polypeptides thereof.

4. The method of any of claims 1-3, wherein at least 40% GOS fiber is produced with 50 mg30 enzyme protein per liter solution (50% w / w lactose) incubated for 24 h at 50°C.

5. The method of claim 1 , wherein the GOS is produced in-situ, in a milk product from the lactose already present in the milk.35 6. A polypeptide having beta-galactosidase activity, selected from the group consisting of:(a) a polypeptide having at least 90 % sequence identity to mature polypeptide of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 10;16154-EP-EPA(b) a polypeptide having at least 90 % sequence identity to amino acids 1-944 of SEQ ID NO: 6, amino acids 1-837 of SEQ ID NO: 7, amino acids 1-1225 of SEQ ID NO: 10;(c) a polypeptide encoded by a polynucleotide having at least 90 % sequence identity to the mature polypeptide coding sequence of SEQ ID NO: 13, SEQ ID NO: 14, or SEQ5 ID NO: 15;(d) a polypeptide derived from SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 10, a mature polypeptide of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 10 by having 1-30 alterations (e.g., substitutions, deletions and / or insertions at one or more positions, e.g., 1 or 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or10 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 alterations, in particular substitutions;(e) a polypeptide derived from the polypeptide of (a), (b), (c), or (d) wherein the N- and / or C-terminal end has been extended by addition of one or more amino acids; and(f) a fragment of the polypeptide of (a), (b), (c), or (d);15 wherein the polypeptide has beta-galactosidase activity.

7. The polypeptide of claim 6, wherein the polypeptide having beta-galactosidase activity is selected from the group consisting of:(a) a polypeptide having a sequence identity of at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to SEQ ID NO: 6, a mature polypeptide of SEQ ID NO: 6, or amimo acids 1-944 of SEQ ID NO: 6;(b) a polypeptide having a sequence identity of at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to SEQ ID NO: 7, a mature polypeptide of SEQ ID NO: 7, or amimo acids 1-837 of SEQ ID NO: 7;25 (c) a polypeptide having a sequence identity of at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to SEQ ID NO: 10, a mature polypeptide of SEQ ID NO: 10, or amimo acids 1-1225 of SEQ ID NO: 10.30 8. The polypeptide of any of claims 6-7, comprising, consisting essentially of, or consisting of SEQ ID NO: 6, SEQ ID NO: 7, or SEQ ID NO: 10, or the mature polypeptides thereof.

9. The polypeptide of any of claims 6-8, wherein the mature polypeptide coding sequence is selected from the group consisting of a polypeptide encoded by a polynucleotide having at least35 90 % sequence identity, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% to the mature polypeptide coding sequence of SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NO: 15.16154-EP-EPA10. The polypeptide of any one of claims 6-9, wherein sequence identity is determined by Sequence Identity Determination Method 1.

11. A composition comprising the polypeptide of any one of claims 6-10.

512. A polynucleotide encoding the polypeptide of any one of claims 6-10.

13. A nucleic acid construct or expression vector comprising the polynucleotide of claim 12, operably linked to one or more control sequences that direct the production of the polypeptide in an expression host.

14. A recombinant host cell comprising the nucleic acid construct or expression vector of claim 13.15 15. A method of producing a polypeptide having beta-galactosidase activity, comprising cultivating the recombinant host cell of claim 14 under conditions conducive for production of the polypeptide.

16. A milk product comprising GOS produced in-situ by lactases present in the milk by contacting a polypeptide having beta-galactosidase activity with lactose according to claim 1.25

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