Variant polypeptide having beta-hexosyltransferase activity

Abstract

An isolated and / or recombinant polypeptide, wherein the polypeptide has an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 2, comprises at least one substitution of the amino acid residue corresponding to the amino acid in position 50, 295 or 321, wherein said positions are defined with reference to SEQ ID NO: 2.

Description

[0001] DSM IP Assets B.V. 2024P00106WO

[0002] VARIANT POLYPEPTIDE HAVING BETA-HEXOSYLTRANSFERASE ACTIVITY

[0003] Field of the invention

[0004]

[0001] The invention relates to a polypeptide having p-hexosyltransferase activity. The invention further relates to a composition comprising such polypeptide, to use of such polypeptide or polypeptide-containing composition in the preparation of a dairy product, to a process for the production of a dairy product and to the resulting dairy product.

[0005] Background of the invention

[0006]

[0002] Galacto-oligosaccharides (GOS) are known to enhance the growth of Bifidobacteria. This effect is likely achieved through the unique ability of Bifidobacteria to exploit GOS as a carbon source. Dietary supplements with galacto-oligosaccharides are furthermore thought to have a number of long-term disease protecting effects. There is therefore a great interest in developing cheap and efficient methods for producing GOS for improving dietary supplements and dairy products.

[0007]

[0003] GOS can be produced from lactose using the enzyme p-galactosidase which hydrolyses lactose to the monosaccharides D-glucose and D-galactose. However, at high lactose concentrations some p-galactosidases are able to transfer galactose to the hydroxyl groups of D-galactose or D-glucose in a process called transgalactosylation whereby galacto-oligosaccharides (GOS) are produced.

[0008]

[0004] Enzymes capable of transgalactosylation have been isolated from a wide range of microorganisms, including bacteria, fungi and yeasts (Torres et al. (2010) Comprehensive Reviews in Food Science and Food Safety, 9: 438-454). GOS yield from lactose can vary depending on the origin of the enzyme, lactose concentration, pH, enzyme dosage, time and temperature of the incubation, and is therefore difficult to compare. It is known that several basidiomycetous yeasts have potent transgalactosylation activities, and GOS production by Sporobolomyces singularis (formerly called Bullera singularis and recently renamed to Hamamotoa singularis), Cryptococcus laurentii, Sterigmatomyces eleviae, Rhodotorula minuta, and Sirobasidium magnum has been reported. A major difficulty is producing these hexosyltransferases in sufficient amounts, making the commercial production of GOS with these enzymes inhibitory expensive. An obstacle in producing these enzymes is their cellular attachment and poor secretion, necessitating cell-wall extraction and extensive purification of the enzyme with poor yield (e.g. Ishikawa et al. (2005) Journal of Bioscience and Bioengineering, 4:331-339).

[0009]

[0005] Examples of commercial GOS producing enzymes are Nurica (IFF) and Saphera Fiber (Novonesis). However, both enzymes show some disadvantages. W02020 / 117548, WO2015 / 086746 and WO2022189568 describe that stable GOS content can only be obtained if the transgalactosylating enzyme is deactivated. DSM IP Assets B.V. 2024P00106WO

[0010]

[0006] Industry is calling for improved transgalactosylating enzymes. For example, industry is calling for improved transgalactosylating enzymes which can for example be used in the production of fermented milk products without the need for an enzyme inactivation step. Alternatively, industry is looking for transgalactosylating enzymes which result in increased GOS concentration at the end of yogurt fermentation. Yet another desire of industry is to obtain GOS comprising dairy products, such as milk or yoghurt, which have reduced sugar content (DP1 and / or DP2). Yet a further desire of industry is to have a transgalactosylating enzyme which performs well in acid whey, milk protein concentrate or whey protein concentrate, preferably not only for the production of GOS but also for obtaining a product with reduced amounts of mono sugars.

[0011] Summary of the invention

[0012]

[0007] Advantageously new, suitably BHT polypeptides have now been identified which show improved characteristics and / or allow different applications when compared to the parent polypeptide and / or commercial GOS producing enzymes. The BHT polypeptides of the invention are therefore superior compared to the BHT polypeptides and / or other transgalactosylating enzymes of the prior art.

[0013]

[0008] The examples shows that a polypeptide of the invention produces a higher maximum amount of GOS in dairy and / or increased activity and / or can be used in a fermented milk product without a need for inactivation before storage.

[0014]

[0009] Accordingly, in a first aspect, the present invention provides an isolated and / or recombinant polypeptide, wherein the polypeptide has an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 2, comprises at least one substitution of the amino acid residue corresponding to the amino acid in position 295 or 321 , wherein said position is defined with reference to SEQ ID NO: 2.

[0015]

[0010] In a second aspect, the invention provides a composition comprising the polypeptide of the first aspect.

[0016]

[0011] In a third aspect, the invention provides: (i) a nucleic acid sequence encoding the polypeptide of the first aspect; (ii) a nucleic acid construct comprising such a nucleic acid sequence operably linked to one or more control sequences capable of directing the expression of the polypeptide in a host cell; and / or (iii) a recombinant expression vector comprising such a nucleic acid sequence or such a nucleic acid construct.

[0017]

[0012] In a fourth aspect, the invention provides a recombinant host cell comprising the nucleic acid sequence, the nucleic acid construct and / or the recombinant expression vector of the third aspect.

[0018]

[0013] In a fifth aspect, the invention provides a method for producing the polypeptide of the first aspect, comprising expressing the nucleic acid sequence and / or the nucleic acid construct of the third aspect in the recombinant host cell of the fourth aspect. DSM IP Assets B.V. 2024P00106WO

[0019]

[0014] In a sixth aspect, the invention provides a use of a polypeptide of the first aspect or the composition of the second aspect in the preparation of a dairy product.

[0020]

[0015] In a seventh aspect, the invention provides for a process for the production of a dairy product, wherein the process comprises contacting an amount of the polypeptide of the first aspect or a composition of the second aspect with a milk base. Other processes are also described and claimed herein.

[0021]

[0016] In an eighth aspect, the invention provides a dairy product, wherein the dairy product comprises a polypeptide of the first aspect and / or is obtained or obtainable by the process of the seventh aspect or by the use of the sixth aspect.

[0022]

[0017] The above aspects according to the invention advantageously allow for improved (increased) levels of GOS, preferably in situ produced GOS, in a dairy product, improved production processes which for example do not need an enzyme inactivation step or which can be completed in a shorter time and / or storage stable GOS without enzyme inactivation.

[0023] Brief description of the drawings

[0024]

[0018] The invention is illustrated by the following figures:

[0025]

[0019] Figure 1 : GOS-fiber (g / kg) in yoghurt with added BHT (3.4 mg / L) during making and storage.

[0026]

[0020] Figure 2: GOS-fiber (g / L) in milk with added BHT or control enzymes during incubation.

[0027]

[0021] Figure 3: GOS-fiber (g / kg) in yoghurt with added BHT or competitor enzymes during making and storage.

[0028]

[0022] Figure 4: Relative pH profile of the different BHT variants.

[0029] Brief description of the sequence listing

[0030]

[0023] This application contains a Sequence Listing in computer readable form, which is incorporated herein by reference. An overview of the sequences is provided by Table 1 below.

[0031] Table 1 : Overview of sequence listing:

[0032] SEQ ID No: Enzyme / gene Name Origin Type (herein also

[0033] referred to as)

[0034] SEQ ID NO: 1 Full length mature BHT Sporobolomyces singularis Protein (BHT_01)

[0035] SEQ ID NO: 2 Truncated mature BHT Synthetic construct Protein

[0036] (BHT_02) DSM IP Assets B.V. 2024P00106WO

[0037] SEQ ID NO: 3 Variant of truncated mature Synthetic construct Protein (BHT_03) BHT; S295L mutant of SEQ

[0038] ID NO:2

[0039] SEQ ID NO: 4 Variant of truncated mature Synthetic construct Protein (BHT_04) BHT; R321 H mutant of

[0040] SEQ ID NO:2

[0041] SEQ ID NO: 5 Variant of truncated mature Synthetic construct Protein (BHT_05) BHT; S295L + R321 H

[0042] mutant of SEQ ID NO:2

[0043] SEQ ID NO: 6 Signal sequence Alpha-mating factor signal protein sequence

[0044] SEQ ID NO: 7 Coding sequence of SEQ Synthetic construct DNA

[0045] ID NO:2

[0046] SEQ ID NO: 8 Coding sequence of SEQ Synthetic construct DNA

[0047] ID NO:3

[0048] SEQ ID NO: 9 Coding sequence of SEQ Synthetic construct DNA

[0049] ID NO:4

[0050] SEQ ID NO: 10 Coding sequence of SEQ Synthetic construct DNA

[0051] ID NO:5

[0052] Detailed description of the invention

[0053] Definitions

[0054]

[0024] 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.

[0055]

[0025] Throughout the present specification and the accompanying claims, the words "comprise”, "include" and “having” and variations such as "comprises", "comprising", "includes" and "including" are to be interpreted inclusively. That is, these words are intended to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.

[0056]

[0026] The articles "a" and "an" are used herein to refer to one or to more than one (i.e. to one or at least one) of the grammatical object of the article. By way of example, "an element" may mean one element or more than one element.

[0057]

[0027] Unless explicitly indicated otherwise, the various embodiments of the invention described herein can be cross-combined.

[0058]

[0028] The term "milk" is intended to encompass milks from mammals, milks from microbial sources and / or mixtures thereof. Preferably, the milk is from a mammal source. Mammal sources of milk include, but are not limited to cow milk, sheep milk, goat milk, buffalo milk, camel milk, llama milk, horse milk or reindeer milk. In an embodiment, the milk is from a mammal selected from the DSM IP Assets B.V. 2024P00106WO

[0059] group consisting of cow, sheep, goat, buffalo, camel, llama, horse and deer, and combinations thereof. Microbial sources of milk, supplemented with lactose, include milk and milk proteins produced by recombinant micro-organisms in a laboratory (also referred to as “lab-grown milk”) or bioreactor. Preferably the milk is a non-recombinant, naturally occurring and / or naturally produced, milk. Cows are most preferred as a source for milk. Bovine milk is most preferred. In addition, the term "milk" refers to not only whole milk, but also skim milk or any liquid component derived thereof or reconstituted milk.

[0060] The term "milk-base" refers to a base composition, comprising or consisting of milk or milk ingredients, or derived from milk or milk ingredients. The milk-base can be used as a raw material for the dairy product. The milk-base may for example comprise or consist of skimmed or nonskimmed milk, or reconstituted milk. Optionally the milk-base may be concentrated, or in the form of powder, or may be reconstituted from such. By reconstituted milk is herein understood liquid milk obtained by adding liquid, such as water, to a skim milk powder, skim milk concentrate, whole milk powder or whole milk concentrate. Furthermore, the milk-base may or may not have been subjected to a thermal processing operation which is at least as efficient as pasteurization. Preferably the milk-base is from a bovine source. The milk-based substrate may have been subjected to treatments known in the art such as - for example- homogenization, pasteurization, sterilization or an extended shelf life (ESL) treatment or an ultra-heat treatment (UHT).

[0061] The milk-based substrate may be skim milk, full fat milk, semi-skimmed milk, condensed milk, reconstituted (skim) milk, butter milk, milk protein concentrate (MPC), or whey such as sweet whey or acid whey, whey protein concentrate (WPC), whey protein isolate (WPI) or whey permeate. Preferably, the milk-based substrate may be skim milk, full fat milk, semi-skimmed milk, condensed milk, reconstituted (skim) milk, butter milk or milk protein concentrate (MPC).

[0062] The herein used milk-based substrate comprises lactose and can thus also be described as “a lactose comprising milk-based substrate”. In general, cow’s milk comprises approximately 5% lactose. Preferably, the milk-based substrate only comprises the lactose which is naturally present in the milk-based substrate. Preferably, the milk-based substrate does not comprise added lactose. Preferably, the milk-based substrate comprises lactose in the range of 4 to 10 (w / v)% lactose, preferably 4 to 8 (w / v)% and even more preferably 4 to 6 (w / v)% or 4.5 to 5.5 (w / v)% lactose.

[0063]

[0029] Any references to %wAz herein, such as for example references to 12% w / v reconstituted skim milk (RSM), referto weight in grams present per volume of 100 ml solution, for example 12% w / v RSM corresponds to 12 grams of skim milk powder dissolved per 100 ml water.

[0064]

[0030] Beta-galactosidases are enzymes which hydrolyse terminal non-reducing beta-D-galactose residues in beta-D-galactosides, for example lactose is hydrolysed to galactose and glucose. These enzymes belong to the enzyme class EC 3.2.1.23. Besides hydrolysing, this enzyme class is also able to transfer galactose to other sugars and thereby produce galactooligosaccharides (GOS). The different enzymes of class EC 3.2.1 .23 have various preferences for DSM IP Assets B.V. 2024P00106WO

[0065] hydrolytic (beta-galactosidase) activity and transgalactosylase activity and the preference can be expressed for example by the ratio of transgalactosylating activity to hydrolysing activity.

[0066]

[0031] The enzyme having transgalactosylase activity preferably belongs to subclass EC 3.2.1.23. As used herein, the terms “enzyme having transgalactosylase activity”, “enzyme having transgalactosylating activity”, “transgalactosylating enzyme” and “transgalactosylase” are used interchangeably herein and all refer to an enzyme capable of transferring galactose from lactose to a hydroxyl group of, for example, lactose, D-galactose (gal) or D-glucose (glu) whereby galactooligosaccharides are produced.

[0067]

[0032] As used herein, the terms “gene” and “recombinant gene” refer to nucleic acid molecules which include an open reading frame encoding a polypeptide as described herein. A gene may include coding sequences, non-coding sequences, introns and regulatory sequences. That is to say, a “gene”, as used herein, may refer to an isolated nucleic acid molecule as defined herein. Accordingly, the term “gene”, in the context of the present application, does not refer only to naturally-occurring sequences.

[0068]

[0033] As used herein, the terms “polynucleotide” “nucleic acid sequence” or “nucleic acid molecule” are intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA. The nucleic acid may be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids that have altered base-pairing abilities or increased resistance to nucleases.

[0069]

[0034] As used herein, the terms "peptide" and "oligopeptide" are considered synonymous (as is commonly recognized) and each term can be used interchangeably as the context requires to indicate a chain of at least two amino acids coupled by peptidyl linkages. The word "polypeptide" is used herein for chains containing more than seven amino acid residues. All oligopeptide and polypeptide formulas or sequences herein are written from left to right and in the direction from amino terminus to carboxy terminus. The one-letter code of amino acids used herein is commonly known in the art and can be found in Berg, Tymoczko and Stryer, Biochemistry, 6thedition, chapter 2, W.H Freeman and Company, New York, 2007.

[0070]

[0035] The terms “homology” and “percent identity” are used interchangeably herein. For the purpose of this invention, it is defined here that in order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid for optimal alignment with a second amino or nucleic acid sequence). The amino acid or nucleotide residues at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid or nucleotide residue as the corresponding position in the second sequence, then the molecules are identical at that position. DSM IP Assets B.V. 2024P00106WO

[0071] The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions (i.e. overlapping positions) x 100). Preferably, the two sequences are the same length.

[0072]

[0036] A sequence comparison may be carried out over the entire lengths of the two sequences being compared or over fragment of the two sequences. Suitably the comparison can be carried out over the full length of the two sequences being compared. However, sequence identity may be carried out over a region of, for example, twenty, fifty, one hundred or more contiguous amino acid residues.

[0073]

[0037] The skilled person will be aware of the fact that several different computer programs are available to determine the homology between two sequences. For instance, a comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred embodiment, the percent identity between two amino acid or nucleic acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48): 444-453 (1970)) algorithm which has been incorporated into the GAP program in the Accelrys GCG software package (available at http: / / www.accelrys.com / products / gcg / ), using either a Blosum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1 , 2, 3, 4, 5, or 6. The skilled person will appreciate that all these different parameters will yield slightly different results but that the overall percentage identity of two sequences is not significantly altered when using different algorithms.

[0074]

[0038] The protein sequences or nucleic acid sequences of the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the BLASTN and BLASTP programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403 — 10. BLAST protein searches can be performed with the BLASTP program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTP and BLASTN) can be used. See the homepage of the National Center for Biotechnology Information at http: / / www.ncbi.nlm.nih.gov / .

[0075] The polypeptide

[0076]

[0039] In a first aspect, the present invention provides an isolated and / or recombinant polypeptide, wherein the polypeptide has an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 2, comprises at least one substitution of the amino acid residue corresponding to the amino acid in position 295 or 321 , wherein said position is defined with reference to SEQ ID NO: 2. DSM IP Assets B.V. 2024P00106WO

[0077]

[0040] The polypeptide is a variant of an enzyme having transgalactosylase activity originally found in Sporobolomyces singularis and the mature sequence of the wildtype enzyme is shown in SEQ ID NO:1. SEQ ID NO: 2 is a truncated version of SEQ ID NO:1 by deleting a potential cellbinding domain / anchor at the start of the BHT sequence. The herein described and claimed polypeptide is a variant polypeptide of the polypeptide having the amino acid sequence of SEQ ID NO:2. The polypeptide as claimed and described herein is a beta-hexosyltransferase and is also referred to as BHT.

[0078]

[0041] In one of its embodiments, the invention provides an isolated and / or recombinant polypeptide, wherein the polypeptide has an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 2, comprises at least one substitution of the amino acid residue corresponding to the amino acid in position 295, wherein said position is defined with reference to SEQ ID NO: 2.

[0079]

[0042] Alternatively, the invention provides an isolated and / or recombinant polypeptide, wherein the polypeptide has an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 2, comprises at least one substitution of the amino acid residue corresponding to the amino acid in position 321 , wherein said position is defined with reference to SEQ ID NO: 2.

[0080]

[0043] Preferably, the substitution at position 295, is S295L, wherein said positions are defined with reference to SEQ ID NO: 2. Alternatively, the substitution at position 321 is R321H, wherein said positions are defined with reference to SEQ ID NO: 2.

[0081]

[0044] In one of its embodiments, the invention provides an isolated and / or recombinant polypeptide, wherein the polypeptide has an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 2, comprises at least two substitutions of the amino acid residues corresponding to the amino acids in position 295 and 321 , wherein said positions are defined with reference to SEQ ID NO: 2. Preferably, the at least two substitutions are S295L and R321H, wherein said positions are defined with reference to SEQ ID NO: 2

[0082]

[0045] Preferably the polypeptide has an amino acid sequence with equal to or more than 80%, more preferably equal to or more than 85%, more preferably equal to or more than 90%, or most preferably equal to or more than 95%, sequence identity with SEQ ID NO: 2. Hence, provided herein is

[0083] - an isolated and / or recombinant polypeptide, wherein the polypeptide has an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 2, comprises at least one substitution of the amino acid residue corresponding to the amino acid in position 295, wherein said position is defined with reference to SEQ ID NO: 2, wherein the polypeptide has an amino acid sequence with equal to or more than 80%, more preferably equal to or more than 85%, more preferably equal to or more than 90%, or most preferably equal to or more than 95%, sequence identity with SEQ ID NO: 2 DSM IP Assets B.V. 2024P00106WO

[0084] - an isolated and / or recombinant polypeptide, wherein the polypeptide has an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 2, comprises at least one substitution of the amino acid residue corresponding to the amino acid in position 321 , wherein said position is defined with reference to SEQ ID NO: 2, wherein the polypeptide has an amino acid sequence with equal to or more than 80%, more preferably equal to or more than 85%, more preferably equal to or more than 90%, or most preferably equal to or more than 95%, sequence identity with SEQ ID NO: 2

[0085] - an isolated and / or recombinant polypeptide, wherein the polypeptide has an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 2, comprises at least one substitution of the amino acid residue corresponding to the amino acid in position 295, wherein said position is defined with reference to SEQ ID NO: 2 and wherein the substitution is S295L, wherein the polypeptide has an amino acid sequence with equal to or more than 80%, more preferably equal to or more than 85%, more preferably equal to or more than 90%, or most preferably equal to or more than 95%, sequence identity with SEQ ID NO: 2

[0086] - an isolated and / or recombinant polypeptide, wherein the polypeptide has an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 2, comprises at least one substitution of the amino acid residue corresponding to the amino acid in position R321H, wherein said position is defined with reference to SEQ ID NO: 2 and wherein the substitution is R321H, wherein the polypeptide has an amino acid sequence with equal to or more than 80%, more preferably equal to or more than 85%, more preferably equal to or more than 90%, or most preferably equal to or more than 95%, sequence identity with SEQ ID NO: 2

[0087] - an isolated and / or recombinant polypeptide, wherein the polypeptide has an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 2, comprises at least two substitutions of the amino acid residues corresponding to the amino acids in position 295 and 321 , wherein said positions are defined with reference to SEQ ID NO: 2, wherein the polypeptide has an amino acid sequence with equal to or more than 80%, more preferably equal to or more than 85%, more preferably equal to or more than 90%, or most preferably equal to or more than 95%, sequence identity with SEQ ID NO: 2 or

[0088] - an isolated and / or recombinant polypeptide, wherein the polypeptide has an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 2, comprises at least two substitutions of the amino acid residues corresponding to the amino acids in position 295 and 321 , wherein said positions are defined with reference to SEQ ID NO: 2 and wherein the substitutions are S295L and R321H, wherein the polypeptide has an amino acid sequence with equal to or more than 80%, more preferably equal to or more than 85%, more preferably equal to or more than 90%, or most preferably equal to or more than 95%, sequence identity with SEQ ID NO: 2.

[0089]

[0046] Preferably, the claimed isolated and / or recombinant polypeptide has beta-hexosyltransferase activity. Hence, the claimed isolated and / or recombinant polypeptide is capable DSM IP Assets B.V. 2024P00106WO

[0090] of transferring galactose from lactose to a hydroxyl group of, for example, lactose, D-galactose (gal) or D-glucose (glu) whereby galacto-oligosaccharides are produced or in short the claimed isolated and / or recombinant polypeptide uses lactose as a substrate to produce galacto-oligosaccharides (GOS).

[0091]

[0047] Most preferably the polypeptide is:

[0092] - a polypeptide that has an amino acid sequence of SEQ ID NO: 3, SEQ ID NO:4 or SEQ ID NO:5, or

[0093] - has an amino acid sequence with more than 80% sequence identity with SEQ ID NO: 3 and wherein said polypeptide comprises substitution S295L; or

[0094] - has an amino acid sequence with more than 80% sequence identity with SEQ ID NO: 4 and wherein said polypeptide comprises substitution R321H; or

[0095] - has an amino acid sequence with more than 80% sequence identity with SEQ ID NO: 5 and wherein said polypeptide comprises substitutions S295L and R321H.

[0096]

[0048] The polypeptide according to the invention can be an isolated naturally occurring polypeptide or a non-naturally occurring polypeptide. By a non-naturally occurring polypeptide is herein understood a polypeptide that is not naturally produced by any organism. Preferably the polypeptide is a non-naturally occurring polypeptide.

[0097]

[0049] By an "isolated" polypeptide or protein is herein understood a polypeptide or protein removed from its native environment. For example, recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for the purpose of the invention as are recombinant polypeptides which have been substantially purified by any suitable technique. A polypeptide variant according to the invention can be recovered and purified from recombinant cell cultures by methods known in the art.

[0098] The composition comprising the polypeptide

[0099]

[0050] In a second aspect, the invention provides a composition comprising the polypeptide of the first aspect.

[0100]

[0051] More preferably such a composition additionally comprises a polyol, a salt, a sugar and / or an organic acid, preferably said composition has a pH in the range of 3.5 to 8.

[0101]

[0052] The composition comprises the herein described polypeptide and at least one component selected from milk powder, gluten, granulated fat, an additional enzyme, an amino acid, a salt, an oxidant, a pH buffer, a reducing agent, an emulsifier, sodium stearoyl lactylate, calcium stearoyl lactylate, polyglycerol esters of fatty acids and diacetyl tartaric acid esters of mono- and diglycerides, a gum, a flavour, an acid, a starch, a modified starch, a humectant, a polyol and a preservative. DSM IP Assets B.V. 2024P00106WO

[0102]

[0053] A composition as disclosed herein may be a solid (for example a powder or a granulate) or fluid (i.e. liquid) or frozen composition. If liquid, the composition is preferably a solution, suspension or emulsion.

[0103]

[0054] A composition as disclosed herein may comprise one or more compounds selected from the group consisting of: milk powder, gluten, granulated fat, an additional enzyme, an amino acid, a salt (like sodium or potassium chloride), an oxidant, a pH buffer, a reducing agent, an emulsifier, sodium stearoyl lactylate, calcium stearoyl lactylate, polyglycerol esters of fatty acids and diacetyl tartaric acid esters of mono- and diglycerides, a gum, a flavour, an acid, a starch, a modified starch, a humectant, a polyol (like glycerol) and a preservative. The term composition includes a pre-mix.

[0104]

[0055] A composition as disclosed herein may comprise one or more further enzyme(s) such as an amylase such as an alpha-amylase, for example a fungal alpha-amylase, a beta-amylase; a glucanotransferase; a peptidase for example an exopeptidase or endopeptidase; a transglutaminase; a protein glutaminase; a cellulase; a hemicellulase, in particular a pentosanase such as xylanase; protease; a protein disulfide isomerase, e.g., a protein disulfide isomerase as disclosed in WO 95 / 00636; a glycosyltransferase; a peroxidase; a laccase; an oxidase, such as an hexose oxidase, a glucose oxidase, aldose oxidase, pyranose oxidase; a lipoxygenase; L-amino acid oxidase; a glucoamylase, a lactase, a catalase or a phytase and I or an asparaginase. Preferably, a preferred further enzyme is lactase, glucose oxidase or catalase.

[0105]

[0056] A suitable commercial example of an enzyme having hydrolytic activity on lactose (i.e. a lactase) is Maxilact (dsm-firmenich), Lactozyme (Novozymes / Novonesis), Saphera (Novozymes 1 Novonesis), Nolafit (Chr. Hansen I Novonesis), Ha-lactase (Chr. Hansen I Novonesis), Godo YNL- 2 (Dupont) and some other enzymes described as lactases. Other suitable lactases have been described in the scientific literature and / or in patent publications.

[0106] Nucleic acid sequences encoding the polypeptide

[0107]

[0057] In a third aspect, the invention provides: (i) a nucleic acid sequence encoding the polypeptide of the first aspect; (ii) a nucleic acid construct comprising such a nucleic acid sequence operably linked to one or more control sequences capable of directing the expression of the polypeptide in a host cell; and / or (iii) a recombinant expression vector comprising such a nucleic acid sequence or such a nucleic acid construct.

[0108]

[0058] The polypeptides and / or nucleic acid sequences of the present invention can, for example, be generated using the methods and techniques as described in the Examples.

[0109]

[0059] Conveniently, the nucleic acid sequence of the invention can be generated using standard molecular biology techniques well known to those skilled in the art taken in combination with the sequence information provided herein.

[0110]

[0060] For example, using standard synthetic techniques, the required nucleic acid sequence may be synthesized de novo. Such a synthetic process will typically be an automated process. DSM IP Assets B.V. 2024P00106WO

[0111]

[0061] Alternatively, a nucleic acid sequence of the invention may be generated by use of site-directed mutagenesis of an existing nucleic acid sequence, for example a nucleic acid sequence encoding the protein of SEQ ID NO: 2. Site-directed mutagenesis may be carried out using a number of techniques well known to those skilled in the art.

[0112]

[0062] In one such method, mentioned here merely by way of example, PCR is carried out on a plasmid template using oligonucleotide "primers" encoding the desired substitution. As the primers are the ends of newly-synthesized strands, should there be a mis-match during the first cycle in binding the template DNA strand, after that first round, the primer-based strand (containing the mutation) would be at equal concentration to the original template. After successive cycles, it would exponentially grow, and after 25, would outnumber the original, unmutated strand in the region of 8 million: 1 , resulting in a nearly homogeneous solution of mutated amplified fragments. The template DNA may then be eliminated by enzymatic digestion with, for example using a restriction enzyme which cleaves only methylated DNA, such as Dpn1. The template, which is derived from an alkaline lysis plasmid preparation and therefore is methylated, is destroyed in this step, but the mutated plasmid is preserved because it was generated in vitro and is unmethylated as a result. In such a method more than one mutation (encoding a substitution as described herein) may be introduced into a nucleic acid sequence in a single PCR reaction, for example by using one or more oligonucleotides, each comprising one or more mis-matches. Alternatively, more than one mutation may be introduced into a nucleic acid sequence by carrying out more than one PCR reaction, each reaction introducing one or more mutations, so that altered nucleic acids are introduced into the nucleic acid in a sequential, iterative fashion.

[0113]

[0063] A nucleic acid of the invention can be generated using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate mis-matched oligonucleotide primers according to the site-directed mutagenesis technique described above. A nucleic acid sequence derived in this way can be cloned into an appropriate vector and characterized by DNA sequence analysis.

[0114]

[0064] A nucleic acid sequence of the invention may comprise one or more deletions, i.e. gaps, in comparison to nucleic acid sequence encoding a wild type enzyme having transgalactosylating activity, such as the enzyme having amino acid sequence SEQ ID NO: 01. Such deletions / gaps may also be generated using site-directed mutagenesis using appropriate oligonucleotides. Techniques for generating such deletions are well known to those skilled in the art.

[0115]

[0065] Furthermore, oligonucleotides corresponding to or hybridizable to nucleotide sequences according to the invention can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.

[0116]

[0066] Also, complementary nucleic acid sequences are included in the present invention. A nucleic acid sequence which is complementary to another nucleotide sequence is one which is sufficiently complementary to the other nucleotide sequence such that it can hybridize to the other nucleotide sequence thereby forming a stable duplex. DSM IP Assets B.V. 2024P00106WO

[0117]

[0067] The invention also relates to nucleic acid sequences encoding at least one functional domain of a polypeptide variant of the invention. Suitably, such a domain may comprise one or more of the substitutions described herein.

[0118]

[0068] A gene or cDNA coding for the polypeptide of the invention may be cloned and overexpressed in a host organism. Suitable host organisms include Aspergillus, Kluyveromyces, Trichoderma, Escherichia coll, Pichia, Saccharomyces, Yarrowia, Neurospora, Bacillus, Fusarium, Hansenula, Chrysosporium or Candida. Examples of suitable bacterial host organisms are gram positive bacterial species such as Bacillaceae including Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus megaterium and Bacillus thu- ringiensis, Streptomyces species such as Streptomyces murinus, lactic acid bacterial species including Lactococcus spp. such as Lactococcus lactis, Lactobacillus spp. including Lactobacillus reuteri, Leuconostoc spp. and Streptococcus spp. Alternatively, strains of a gram negative bacterial species such as a species belonging to Enterobacteriaceae, including E. coll or to Pseudomonadaceae may be selected as the host organism.

[0119]

[0069] A suitable yeast host organism may advantageously be selected from a species of Saccharomyces including Saccharomyces cerevisiae or a species belonging to Schizosaccharomyces. Further useful yeast host organisms include Pichia spp. such as methylotrophic species hereof, including Pichia pastoris, and Kluyveromyces spp. including Kluyveromyces lactis.

[0120]

[0070] Suitable host organisms among filamentous fungi include species of Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophtora, Neurospora, Penicillium, Thielavia, Tolypocladium or Trichoderma, such as e. g. Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus oryzae, Aspergillus nidulans or Aspergillus niger, including Aspergillus nigervar. awamori, Fusarium bactridioides, Fusa- rium cereals, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium sambucinum, Fusarium sarcochroum, Fusarium sporotrichiodes, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium venenatum, Humicola insolens, Humicola langinosa, Mucor miehei, Myceliophtora thermophila, Neurospora crassa, Penicillium chrysogenum, Penicillium camenbertii, Penicillium purpurogenum, Rhizomucor miehei, Thielavia terestris, Tricho- derma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesii or Trichoderma viride.

[0121]

[0071] In the invention, the polypeptide of the invention may originally be provided in the form of pre-polypeptide or (mature) polypeptide. A corresponding nucleic acid sequence may also be provided, for example a polynucleotide that encodes a pre-polypeptide or (mature) polypeptide is DSM IP Assets B.V. 2024P00106WO

[0122] provided. The nucleic acid sequence encoding for such a pre-polypeptide or (mature) polypeptide-encoding sequence may be optimized for expression in a desired host cell.

[0123]

[0072] The invention therefore also provides a nucleic acid construct comprising such a nucleic acid sequence operably linked to one or more control sequences capable of directing the expression of the polypeptide in a host cell.

[0124]

[0073] Suitable host cells include cells from the above exemplified host organisms. Preferably the polypeptide according to the invention can be produced by or in a host cell, selected from the group consisting of bacteria cells, fungus cells and yeast cells. More preferably the polypeptide according to the invention can be produced by or in a host cell chosen from the group consisting of Aspergillus, Kluyveromyces, Trichoderma, Escherichia coll, Pichia, Saccharomyces, Yarrowia, Neurospora or Bacillus. Most preferably the polypeptide is produced by or in Kluyveromyces, more preferably Kluyveromyces lactis.

[0125]

[0074] The term “operatively linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression of the nucleotide sequence (e.g., in an in vitro transcription / translation system or in a host cell when the vector is introduced into the host cell). The term “regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signal).

[0126]

[0075] Polypeptides of the present invention can for example be produced, with the help of recombinant techniques, in a prokaryotic or eukaryotic host cell, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.

[0127]

[0076] The invention also provides a recombinant expression vector comprising the above nucleic acid sequence and / or the above nucleic acid construct.

[0128]

[0077] As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid”, which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “expression vectors”. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. The terms “plasmid” and “vector” can be used interchangeably herein as the plasmid is the most DSM IP Assets B.V. 2024P00106WO

[0129] commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0130]

[0078] The recombinant expression vectors of the invention preferably comprise a nucleic acid sequence of the invention in a form suitable for, preferably constitutive, expression of the nucleic acid sequence in a host cell, which means that the recombinant expression vector may include one or more regulatory sequences, selected on the basis of the host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed.

[0131]

[0079] The polypeptides and / or nucleic acid sequences of the present invention can, for example, be generated using the methods and techniques as described in the Examples.

[0132]

[0080] In a fourth aspect, the invention provides a recombinant host cell comprising the nucleic acid sequence, the nucleic acid construct and / or the recombinant expression vector of the third aspect. In a fifth aspect, the invention provides a method for producing the polypeptide of the first aspect, comprising expressing the nucleic acid sequence and / or the nucleic acid construct of the third aspect in the recombinant host cell of the fourth aspect.

[0133]

[0081] Preferably the recombinant host cell for these aspects is a host cell as described above. Further preferences are also as described above.

[0134] Use of the polypeptide

[0135]

[0082] In a sixth aspect, the invention provides a use of a polypeptide of the first aspect or the composition of the second aspect in the preparation of a dairy product.

[0136]

[0083] More preferably such use comprises the addition of the polypeptide of the invention or the composition of the invention to a milk base. Preferences for such a milk base are as described above and below.

[0137]

[0084] Use of a polypeptide according to the invention may advantageously lead to production of GOS.

[0138]

[0085] GOS may be produced in situ or ex situ. GOS which is ex situ produced can subsequently be added to a dairy product. Preferably, GOS is in situ produced GOS.

[0139] The process

[0140]

[0086] In a seventh aspect, the invention provides a process for the production of a dairy product, wherein the process comprises contacting the polypeptide of the first aspect or a composition of the second aspect with a milk base. More preferably the polypeptide, respectively the composition, is added or supplemented to the milk-base.

[0141]

[0087] Preferably, the invention provides a process for the production of a dairy product, which process comprises adding or otherwise contacting an effective amount of the polypeptide of the DSM IP Assets B.V. 2024P00106WO

[0142] invention or a composition of the invention to / with a milk-base and allowing the polypeptide to perform its enzymatic function.

[0143]

[0088] In the above and below processes, the milk-base can be derived from a microbial source or a mammal source. Preferably, the milk-base is derived from a mammal source, such as cow, sheep, goat, buffalo, camel, llama, horse or deer milk or any combination thereof. Preferably the milk may be selected from the group of cow's milk, camel's milk, buffalo milk, goat's milk, sheep's milk and a mixture of any such milk types. More preferably the milk-base is from a bovine source. Preferably the milk-base is milk. Preferably the milk is derived from a mammalian source. Most preferably the milk is bovine (“cow”) milk.

[0144]

[0089] The milk-base may suitably comprise or consist of fresh skimmed or non-skimmed milk, or reconstituted milk. Optionally the milk-base may be concentrated or in the form of powder, or may be reconstituted from such. By reconstituted milk is herein understood liquid milk obtained by adding liquid, such as water, to a skim milk powder, skim milk concentrate, whole milk powder or whole milk concentrate. Furthermore, the milk-base may or may not have been subjected to thermal (pre-)processing, such as pasteurization or sterilization. In one preferred embodiment the milk-base has been subjected to thermal (pre-) processing, such as pasteurization or sterilization.

[0145]

[0090] Suitable amounts of a polypeptide as claimed herein can easily be determined by the skilled person. Guidance can be found in the experimental part herein. For yogurt production, preferably, the amount of BHT enzyme or a variant thereof which is added to the milk base is 1.0 to 10 mg BHT protein I L milk base, more preferably 2.0 to 7.0 mg BHT protein I L milk base. For milk production, preferably, the amount of BHT enzyme or a variant thereof which is added to the milk base is 50 to 150 mg BHT protein I L milk base, more preferably 70 to 130 or 80 to 120 or 90 to 110 mg BHT protein I L milk base. In general, the amount of BHT enzyme or a variant thereof which is added to the milk base is 1.0 to 150 mg BHT protein I L milk base. It is clear from the examples as shown herein that the skilled person is able to find a suitable enzyme dosage depending on the starting material and used process conditions.

[0146]

[0091] The polypeptide is allowed sufficient time to produce a certain level of GOS molecules. The exact time will depend on the enzyme used, the amount of enzyme used but also on the used temperature and the lactose concentration in the milk-based substrate. The skilled person is capable to identify an enzyme dosage, incubation temperature and incubation time to allow the production of GOS in the milk-based substrate.

[0147]

[0092] The GOS is preferably produced in situ which means that the GOS is produced in the milkbased substrate from the lactose present in said milk-based substrate. Preferably, a method, use or product according to the invention does not comprises added GOS (i.e. non in situ produced GOS). DSM IP Assets B.V. 2024P00106WO

[0148]

[0093] A GOS can be a disaccharide (Degree of Polymerization (DP) 2, i.e. DP2) (except lactose which is not GOS), a trisaccharide (DP3), tetrasaccharide (DP4), pentasaccharide (DP5) or longer oligosaccharides.

[0149]

[0094] An example of a disaccharide is galactosyl-galactose (gal-gal) or allolactose (galactosyl-beta(1-6)-glucose). An example of a trisaccharide (DP3) is galactosyl-lactose (gal-gal-glc). An example of a tetrasaccharide (DP4) is galactosyl-galactosyl-lactose (gal-gal-gal-glc) An example of a pentasaccharide (DP5) is galactosyl-galactosyl-galactosyl-lactose (gal-gal-gal-gal-glc). An example of an even longer oligosaccharide is (galactose)n-lactose with n>3.

[0150]

[0095] The term “DPx+ GOS” means the sum of GOS molecules having DPx or higher DP. For example the term “DP3+ GOS” means the sum of GOS molecules having DP3, DP4, DP5 etc. DP3+ GOS is also referred here to “GOS-fiber”.

[0151]

[0096] The term GOS as used herein refers to the combination of DP2+ GOS. DP2+ GOS excludes lactose (which is by definition not GOS).

[0152]

[0097] The level of GOS molecules can be determined by using different analysis techniques. For example, one can determine the amount of lactose, glucose and galactose in the lactose comprising milk-based substrate and after incubation with the enzymes, determine the amount of lactose, glucose and galactose using an enzymatic analysis, NMR or HPAEC-PAD Quantification of mono-, disaccharides can also be performed by using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) analysis.

[0153]

[0098] In yet another alternative method, GOS production can be established by reaction of an enzyme on lactose in which the amount of galactose generated is less than the amount of glucose generated at a given time.

[0154]

[0099] The amount of GOS can then be determined by subtracting the sum of the amount of lactose, glucose and galactose (as present in the product) from the sum of the starting amount of lactose, glucose and galactose (as present in the substrate). The difference is then the amount of GOS. In a formula:

[0155] [GOS]product=([lac]+[glu]+[gal])substrate - ([lac]+[glu]+[gla])product

[0156]

[0100] Determining the level of GOS with this formula can be performed in respect of a method of the invention, because the skilled person has the opportunity to determine the level of lactose in the milk-based substrate.

[0157]

[0101] In a further aspect, the invention provides a process for the production of a lactose free dairy product, wherein the process comprises contacting a polypeptide of the first aspect or a composition of the second aspect with a milk base and further comprises contacting the milk base with a lactase. DSM IP Assets B.V. 2024P00106WO

[0158]

[0102] In yet another aspect, the invention provides a process for the production of a dairy product, wherein the process comprises contacting a polypeptide of the first aspect or a composition of the second aspect with a milk base and further comprising contacting the milk base with a glucose oxidase and a catalase.

[0159] The dairy product

[0160]

[0103] In a further aspect, the invention provides a dairy product, wherein the dairy product comprises a polypeptide of the first aspect and / or is obtained or obtainable by any of the processes described above.

[0161]

[0104] Preferably the dairy product is milk, yoghurt, (acid) whey permeate or milk powder.

[0162] Further use of the polypeptide

[0163]

[0105] In a further aspect, the invention provides use of a polypeptide of the first aspect or the composition of the second aspect for improving flexibility in dairy product production conditions at an industrial scale. As shown herein within the experimental part, a polypeptide of the invention reaches its highest GOS fiber concentration in shorter time when compared to commercial GOS producing enzymes. As a result, shorter GOS production times can be used. Hence, the invention provides use of a polypeptide of the first aspect or the composition of the second aspect for reducing GOS production time in GOS comprising dairy product production.

[0164]

[0106] The present invention is further illustrated by the following, non-limiting, Examples.

[0165] Examples

[0166] Methods and materials

[0167] Medium composition

[0168]

[0107] YEP2D medium: 10 g / l yeast extract, 20 g / l bacto-peptone, 40 g / l glucose. pH was set to pH 6.7 with 4N NaOH. Medium was autoclaved for 30 minutes at 110°C.

[0169]

[0108] YEP2D / MES medium: 10 g / l yeast extract, 20 g / l bacto-peptone, 40 g / l glucose, 20 g / l MES. pH was set to pH 6.7 with 4N NaOH. Medium was autoclaved for 30 minutes at 110°C.

[0170]

[0109] YEP2D plates contain YEP2D medium with 1.8-2% agar. Medium was autoclaved for 30 minutes at 110°C and poured in petridishes.

[0171] Strains

[0172]

[0110] K. lactis strain GG799: The Kluyveromyces lactis strain is used as a wild-type strain. The strain is obtained from New England Biolabs, Ipswich, Massachusetts, USA. DSM IP Assets B.V. 2024P00106WO

[0173] Molecular biology techniques

[0174]

[0111] Molecular biology techniques known to the skilled person were used (see: Sambrook & Russell, Molecular Cloning: A Laboratory Manual, 3rd Ed., CSHL Press, Cold Spring Harbor, NY, 2001). Examples of the general design of expression vectors for gene over expression, transformation, use of markers and selective media can be found in W02007060247, WO2010102982 and US4943529 and references herein.

[0175] Activity determination BHT

[0176]

[0112] The activity of BHT was determined using an assay which is essentially similar to the assay described (Workman and Day (1982) Appl. Environm. Microbiol. 44(6) 1289-1295). Assay conditions were slightly adapted and performed at 37 °C in sodium acetate buffer pH 4.40 at a p-nitrophenyl-p-D-glucopyranoside (PNPG) concentration of 4.7mM. After termination of the reaction the pNP released was measured at 405 nm, and the enzyme activity was expressed as nanomole of p-nitrophenol released per second (BHTU).

[0177] Protein quantification for BHT, Nurica and Saphera Fiber

[0178]

[0113] Enzyme protein concentration was determined by qSDS-PAGE relative to a BSA standard. SDS-PAGE gels were stained with SYPRO™ Ruby protein gel stain and scanned with a GelDoc Go imaging system (Bio-Rad).

[0179] GOS analysis

[0180]

[0114] For GOS and monosaccharides (DP1) analysis the following HPLC method was used:

[0181]

[0182] GOS oligosaccharides of different degrees of polymerisation (DP) were quantified relative to a standard such as maltose or lactose. The DP of GOS oligosaccharide peaks was determined using commercial oligosaccharide standards. For GOS-fiber quantification, the GOS species of DP3 and higher were summed up. The values as shown in the Examples were obtained by using lactose as a standard. DSM IP Assets B.V. 2024P00106WO

[0183] Lactose analysis

[0184]

[0114] Lactose was quantified based on lactose standard using the following HPLC method:

[0185]

[0186] Sample preparation was performed by dispersing approximately 1 g sample in 25 mL Milli-Q purified water, vigorously mixing for 30 min on a magnetic stirrer and centrifuging for 10 min at 20,000xg. Approximately 0.5-0.6 mL of the supernatant was transferred into an ultrafiltration tube, avoiding the upper fat layer, and ultrafiltrated through a 10 kDa filter at 14,000 x g, for at least 15 min. The clear filtrate was diluted with Milli-Q water to a lactose concentration of approximately 1 mg / L.

[0187] Example 1 : DNA constructs and transformation

[0188]

[0115] Synthetic DNA constructs were designed to start with a Xhol restriction site, encoding amino acids L and E, followed in frame with DNA encoding a kex-protease cleavage site with amino acids K and R, followed by in frame genes encoding variants of BHT and ending with a Pad restriction site just after the stop codon. As an example, a DNA fragment encoding the truncated BHT sequence is listed as SEQ ID NO: 7 Codon usage was adapted according to the method described in patent application US090286280. All variants were designed in a similar fashion and cloned as Xhol Pad fragments in vector pKLACI (New England Biolabs, Ipswich, Massachusetts, USA).

[0189]

[0116] The resulting open reading frames start with the leader sequence of the K. lactis Mating Factor alpha and progresses over the kex processing site to the BHT variants.

[0190]

[0117] Following the design of synthetic DNA constructs were designed. Amino acid changes were introduced resulting in 189 variants.

[0191]

[0118] Transformation and strain selection were performed by electroporation and selection on acetamide containing plates essentially as described in W02007060247. The plasmids were linearized by digestion with Sacll and transformed to Kluyveromyces lactis strain GG799 by electroporation. Of each construct six transformants were tested for BHT production using shake flask fermentations, and the best producing transformant was selected for further analysis. DSM IP Assets B.V. 2024P00106WO

[0192]

[0119] Starting from 189 variants, we selected 7 variants with superior trans-galactosylation activity with lactose as substrate. These samples were tested in milk and yoghurt application after which some variants were selected for further analysis.

[0193]

[0120] A selection of the new variants and their mutations is depicted in Table 1. Position of the change is indicated in comparison with the truncated mature BHT sequence (SEQ ID NO: 2).

[0194] Example 2: Cultivation, purification and concentration

[0195]

[0121] The Kluyveromyces lactis strains harbouring a variant BHT gene were placed on YEP2D agar plate and grown for 48 hours at 30°C. A pre-culture in 20 ml of YEP2D medium in 100 ml Erlenmeyer flasks was inoculated with the yeast cells taken from the plates. The cultures were grown for 24 hours in an incubator shaker at 30°C and 250 rpm. The amount of pre-culture for inoculation of new 500 ml Erlenmeyer flasks with 100 ml YEP2D / MES medium was calculated to give a OD600=0.01 . These main cultures were grown for 65 hours in an incubator shaker at 30°C and 250 rpm.

[0196]

[0122] Broth from each flask was centrifuged and the supernatant containing the target enzymatic activity was clarified using a dead-end filter. The clear liquid, containing the enzyme, was subsequently concentrated using a 10kDa MWCO PES spiral wound ultra-filtration system. The concentrate was then filtered again and stored frozen until use.

[0197] Example 3: Use of BHT-02, -03 and -04 in FMP

[0198]

[0123] Retail pasteurized semi-skimmed cow milk was fortified with 2.32% skim milk powder, pasteurized for 30 min at 85°C and stored refrigerated over-night. Prior to yoghurt preparation, the milk was equilibrated to 42°C for 30 min. Then yoghurt fermentation was performed in duplicates for each enzyme variant at 42°C. 200 mL milk was inoculated with 2U / WOOL of YS-141 (dsm-firmenich; blend consisting of Streptococcus thermophilus, Lactobacillus delbrueckii bulgaricus) and BHT-02, -03 or -04 enzymes were dosed at 3.4 mg BHT protein / L milk. The inoculated milk was divided in two parts:100 mL was used to monitor milk acidification at 42°C until the end of yoghurt fermentation at pH 4.6 was reached; the remaining 100 mL were aliquoted at ~10mL into Falcon tubes for yoghurt preparation at small scale at 42°C. These aliquots were withdrawn during the yoghurt fermentation after 1 , 2, 3 hr and at the end of fermentation (when pH 4.6 was reached in the control tube - EoF). After reaching pH 4.6 all remaining yoghurt aliquots were stored refrigerated for the period of 1 to 40 days, to determine GOS stability. Upon withdrawal of a sample at given time points (1 hr, 2hr, 3 hr, EoF, 1 day, 7 days, 14 days, 28 days, 40 days) enzymes were immediately inactivated by heating at 90°C for 10 min and stored frozen until analysis for galactose and glucose (DP1), lactose and other disaccharides (DP2) and GOS fiber (DP3 and higher). Control yoghurt preparation without enzymes addition was included in duplicates. DSM IP Assets B.V. 2024P00106WO

[0199]

[0124] Table 2 shows GOS fiber (sum of all oligosaccharides with a degree of polymerization (DP) of 3 and higher) and sugar (DP 1 and 2) concentrations analyzed at the end of yoghurt fermentation (when pH 4.6 reached at 42°C; EoF) and after 40 days shelf-life storage at 4°C forthetop 2 variants (BHT_03 and BHT_04) and wild-type BHT (BHT_02) applied at the concentration 3.4 mg / L milk in comparison with the plain yoghurt using only YS-141 :

[0200] Table 2: Determination of sugar and GOS in yoghurt at the EoF and after 40 days storage

[0201]

[0202] *DP2 is lactose plus other disaccharides

[0203] The GOS fiber concentration reached 11.0-15.3 g / kg at the end of fermentation depending on the BHT variant applied. GOS fiber content was further monitored during yoghurt shelf-life of 40 days stored refrigerated. GOS fiber remained stable, in contrast to the faith of GOS produced with enzymes known in the art, during storage in yoghurt (see Example 5).

[0204] Both variants BHT-03 and BHT-04 showed superior performance in comparison with wild-type BHT-02 with improved GOS fibers concentration at the end of fermentation, which remained stable during shelf-life of 40 days.

[0205] Control yoghurt preparation without enzymes addition showed no GOS formation. The sugar content (all DP1 + DP2) of the yoghurt produced with these enzymes is reduced 30-40% in comparison with a control yoghurt recipe without enzymes, both at the end of fermentation and after 40 days storage (see Table 2).

[0206] GOS-fiber formation during fermentation and stability during storage of the yoghurt is also depicted in Figure 1. Further increasing the BHT concentration at the start of the fermentation led to only a slight increase in GOS-fiber content at EoF for all BHT samples (data not shown), but both BHT-03 and BHT-04 were always improved in GOS-fiber content compared to BHT-02. A similar improvement of GOS fiber production at EoF with variants BHT-03 and BHT-04 compared to BHT-02 was also seen when the enzymes were dosed at 2.0, 4.7 or 6.7 mg protein / L milk.

[0207] Example 4: Use of BHT-02, -03 and -04 in milk DSM IP Assets B.V. 2024P00106WO

[0208]

[0125] GOS production in semi skimmed pasteurized milk (containing 4.8 g lactose, 3.5 g proteins and 1.5 g fat per 100 mL) by BHT variants, Nurica (IFF) and Saphera Fiber (Novozymes) was tested by dosing at 104 mg enzyme protein / L milk (for Nurica this corresponds to 3.2 g / L and for Saphera Fiber with 3.4 g / L of volume dosing). Before the start of the incubation the milk was equilibrated at 8°C. The hydrolysis was performed in 250 ml Schott bottles in an incubator (T= 8°C) at continuous gentle stirring using magnetic stirring plates. A control sample (without addition of enzyme) was included. All BHT, Nurica and Saphera Fiber samples were first diluted to an equal enzyme protein concentration and an equal volume was introduced in each test to prevent volumetric differences between the samples. During the incubation, samples of ~5 ml were taken at different timepoints and heat-treated for 10 minutes at 95°C in order to inactivate the enzyme, cooled on ice and stored at -18°C until analysis.

[0209] Table 3 shows GOS fiber (DP3 and higher) concentrations analyzed after milk incubation for 24 hr for all the samples. At 24 hr incubation both control samples Nurica and Saphera Fiber reach their optimal GOS fiber production at this dosage, so this is a good time point to compare BHT samples with these controls. For the BHT samples, the GOS fiber concentration reached 15-18.3 g / L after 24hrs, wherein both BHT-03 and BHT-04 showed optimal GOS fiber concentration already in the period shorter than 24 hrs (see Figure 2). Also in milk the total sugar reduction (reduction in DP1+DP2) was 30-40%, as was also seen in yoghurt (see Example 3).

[0210] Table 3: Determination of sugar and GOS in milk after 24 hr incubation

[0211]

[0212] < <

[0213] *DP2 is lactose plus other disaccharides

[0214] BHT has higher specificity towards GOS formation than both Nurica and Saphera Fiber, yielding higher amounts of total GOS fiber (DP3-6) within 24 hours but lower amounts of monosaccharides (Table 4 - DP1). Hence, BHT has less hydrolase activity and more transferase activity in milk compared to these commercial references. None of the enzymes reached lactose-free concentration (0.1% or 0.01%). DSM IP Assets B.V. 2024P00106WO

[0215] When the milk incubation was extended to 48 hr, a clear difference between the BHT samples and the controls Nurica and Saphera Fiber was seen. While the GOS fiber content in all BHT samples seems to be stable during this extended incubation period, GOS fiber in milk made with both control enzymes seems to be degraded. Having a higher GOS fiber stability in milk may give flexibility in the production conditions at an industrial scale.

[0216] Example 5: Comparisons of GOS production by BHT with commercial references, in FMP

[0217]

[0126] FMP preparation: Semi-skimmed milk (Plus) was fortified with 2.32 % skimmilk powder and after pasteurization under regular conditions (30 min, 85°C) stored refrigerated over-night. Prior to the yoghurt preparation, milk was equilibrated to 42°C for 30 min. CINAC fermentation (pH profile) was performed in duplicates for each enzyme dosage of Nurica, Saphera Fiber and BHT. Milk was inoculated with 2U / WOOL of YS-141 , a blend consisting of Streptococcus thermophilus and Lactobacillus delbrueckii bulgaricus.

[0218] Table 4 shows the accumulation of GOS fiber at the end of the fermentation (EoF) using different dosages of BHT-02, BHT-03 and control enzymes. When all enzymes were added at the start of the fermentation in similar dosage (e.g. 4.7 mg / L; for Nurica this corresponds to 0.54 g / L and for Saphera Fiber with 0.58 g / L of volume dosing), the BHT enzymes clearly were superior to the control enzymes and accumulated much more GOS fiber. W.r.t. the accumulation of GOS fiber, a dosage of 17.5 mg / L of Nurica is comparable to 6.7 mg / L BHT-02 or 4.7 mg / L BHT-03, and a dosage of 17.5 mg / L of Saphera Fiber is comparable to less than 3.4 mg / L BHT-02 or 2.5 mg / L BHT-03. This experiment and Example 3 show that the BHT enzymes can be used at a much lower dosage than prior art enzymes in the FMP application. Both control enzymes Nurica and Saphera Fiber accumulate much more DP1 , again indicating that these control enzymes are much more hydrolytic then BHT.

[0219] Table 4: Determination of sugar and GOS in yoghurt

[0220]

[0221] DSM IP Assets B.V. 2024P00106WO

[0222] DP2 is lactose plus other disaccharides

[0223] The experiment above indicates that for a fair comparison of the GOS production during fermentation, the control enzymes have to be dosed at much higher amount than the BHT enzymes. Therefore, we repeated this experiment and dosed BHT-02 at 6.7 mg / L, BHT-03 at 4.7 mg / L and Nurica and Saphera Fiber at 17.5 mg / L. Samples were taken during fermentation (1 , 2, 3, 5 hours and EoF) and storage of the yoghurt (1 , 7, 14, 21 , 28 and 40 days) as described in Example 3. Results of this experiment are shown in Figure 3. Due to the difference in enzyme dosage, GOS-fiber accumulation during the yoghurt making was similar in the BHT variants and Nurica, and all yoghurts accumulated ~16 g / kg GOS-fiber. As was already expected from Table 4, Saphera Fiber was clearly less effective in GOS formation.

[0224] During yoghurt storage it became clear that the GOS-fiber made with BHT variants was clearly more stable than that of the controls Nurica and Saphera Fiber. Hence, prior art enzymes need inactivation either before or after yoghurt making to prevent GOS fiber degradation during shelflife, as was also indicated previously (W02020 / 117548, WO2015 / 086746 and WO2022189568). Surprisingly, GOS-fiber in yoghurt produced with BHT is much more stable during shelf-life without enzyme inactivation. Clearly this extra process step is not required when BHT is used for the yoghurt making and storage.

[0225] Example 6: pH activity profile of BHT variants

[0226]

[0127] The activity of the different BHT variants at different pH was measured using the assay with p-nitrophenyl-p-D-glucopyranoside as substrate as described above. For setting the pH buffer A (50 mM citric acid) was mixed with buffer B (100 mM disodium phosphate) to obtain the right pH. Relative activity of each BHT variant compared to the optimum activity at pH5.0 is depicted in Figure 4. The activity profile of BHT-03 was shifted to a slightly less acidic optimum compared to BHT-02.

[0227] Example 7: Use of BHT for GOS production in acid whey

[0228]

[0128] Acid whey is regarded as a waste material for the dairy industry. The production of GOS from acid whey was tested using different enzymes. For this a sample of acid whey powder was obtained from the Greek yoghurt producer Yogulac. Also, different acid whey powder samples from caseinate producers were tested and similar results were found. All acid whey samples contained 70-75% lactose were suspended in water until a concentration of 200 g / L without setting the pH, at which the powders were fully soluble at the incubation temperature of 45 °C. Different lactases (Maxilact (dsm-firmenich), Lactozyme (Novonesis), Biolacta (Amano)) that are known to produce GOS when the start-concentration of lactose is sufficiently high, were found to have no activity on this acid whey solution. Since the pH of the whey solution was found to be 4.2, it was tested if an DSM IP Assets B.V. 2024P00106WO

[0229] acid lactase, like Maxilact A4 (dsm-firmenich), or the BHT variants could be used for production of GOS in acid whey without requiring neutralizing of the solution.

[0230]

[0129] Incubation of enzymes was performed in 100 ml Schott bottles in a water bath at 45°C with continuous magnetic stirring (300 rpm). The enzymes were added based on initial lactose concentration: Maxilact A4 was dosed at 25 ALU / g lactose; BHT was added at 0.17 mg / g lactose.

[0231]

[0130] During the incubation 5 ml samples were taken, inactivated for 15 minutes at 95 °C and stored at 4 °C until analysis. Content of the different sugars was determined as described above and the total amount of GOS was calculated by the sum of all DP2 and higher DP, without lactose.

[0232]

[0131] In both the incubations with Maxilact A4 and with BHT-02 the maximum total GOS was reached after approximately 5 hours incubation and reached 55.7 g / L for Maxilact A4 and 92.7 g / L for BHT-02. Longer incubation times did not yield more GOS product. Variants BHT-03 and BHT-04 showed similar results as BHT-02. These results indicate that the maximum GOS yield on lactose in acid whey with Maxilact A4 is ~25%, while with BHT it is almost 50%.

[0233]

[0132] When the GOS composition of the material was further analysed using the HPLC method described in the Materials and Methods section, it was found that the total GOS in acid whey treated with BHT contained ~45% DP2, 50% DP3 and 5% DP4.

[0234] Example 8: Use of BHT for GOS production in milk or whey protein concentrate

[0235]

[0133] Reduction of lactose by a lactase like Maxilact LGi in whey protein concentrate (WPC) and milk protein concentrate (MPC) leads to a high monosaccharides content such as glucose and galactose (DP1). These monosaccharides can react with proteins in the Maillard reaction leading to changes in colour, organoleptic properties and protein functionality. Lactose-reduced (<0.5% w / w) MPC / WPC produced with a lactase like Maxilact LGi contain DP1 in amount of 4.2-4.4% (w / w) on total solids (TS).

[0236]

[0134] BHT-02 was tested in MPC / WPC to verify if the amount of DP1 can be minimized via in-situ galactooligosacharides (GOS) production. MPC (containing 4.9% w / w lactose) or WPC (containing 4.6 w / w% lactose) was reconstituted in preheated water at 50-60oC to obtain a liquid with 18% TS (total solids) for MPC (pH 6.8 - 7.0) or 22% TS for WPC (pH was adjusted with HCI to 6.3 - 6.4). The homogenous solutions were cooled down to 8oC, and BHT-02 was added at a concentration 17-18 mg / L. Samples were taken after 1 , 3, 6, and 25 hours for DP1 and GOS (DP2+) analysis (as described above). Upon withdrawal of a sample at each time point, the enzyme was immediately inactivated by heating at 95oC for 10 min and stored frozen until analysis. BHT produces 2.1 -2.2 w / w% GOS (DP2+) as main product and a DP1 content of 0.8-0.9% (w / w) on TS. This DP1 content is much lower than the lactose-reduced MPC / WPC produced with a lactase like Maxilact LGi, as described above. MPC / WPC produced with variants BHT-03 and BHT-04 showed similar results as with BHT-02. DSM IP Assets B.V. 2024P00106WO

[0237] Example 9: Use of BHT for GOS production in milk or whey protein concentrate in combination with Maxilact LGi

[0238]

[0135] To stimulate the reduction of the residual lactose in MPC / WPC treated with BHT-02 further, the experiment was performed as in Example 8, but a lactase like Maxilact LGi at concentration 500 NLU / L milk was added either together with BHT (1 step approach), or after 24 hours of BHT incubation (2 steps approach). The 1 step approach resulted in lactose reduction to <0.4 w / w% but it still leads to an accumulation of undesirable DP1 up to 4.1 - 4.2 w / w%. Addition of Maxilact LGi in the 2 steps process results in lactose reduction reaching 0.4-0.5 w / w% after 6 hours coincubation, while the DP1 content remained relatively low at 1.4-1.9 w / w%.

[0239]

[0136] So, BHT combined with Maxilact LGi in the 2 steps process reduces DP1 by 60-70% in comparison to lactose reduced MPC / WPC powders produced with Maxilact LGi only (DP1 content at 4.2-4.4%). GOS, DP1 and lactose content is calculated on TS. The variants BHT-03 and BHT-04 in combination with Maxilact LGi in the 2 steps approach showed similar results as BHT-02.

[0240] Example 10: Use of BHT-03 in FMP with varying milk base and culture blends

[0241]

[0137] GOS - containing yoghurts were prepared as in example 3 with or without starch addition or using fat-free milk base without addition of milk powder. Milk base was inoculated with 2UZ WOOL milk of commercial dsm-firmenich cultures blends FVV-121 , FVV-221 Pioneer-01 , YS-051 , YS-241 consisting of Streptococcus thermophilus and Lactobacillus delbrueckii bulgaricus at 42°C with BHT-03 enzyme at dosage range 2.0 - 4.7 mg / L milk, resulting in similar results as in example 3.

Claims

DSM IP Assets B.V. 2024P00106WOClaims1. An isolated and / or recombinant polypeptide, wherein the polypeptide has an amino acid sequence which, when aligned with the amino acid sequence set out in SEQ ID NO: 2, comprises at least one substitution of the amino acid residue corresponding to the amino acid in position 295 or 321 , wherein said position is defined with reference to SEQ ID NO: 2, wherein the polypeptide has an amino acid sequence with equal to or more than 80%, more preferably equal to or more than 85%, more preferably equal to or more than 90%, or most preferably equal to or more than 95%, sequence identity with SEQ ID NO: 2.

2. The polypeptide according to claim 1 , comprising at least the mutation S295L or R321H, wherein said positions are defined with reference to SEQ ID NO: 2.

3. The polypeptide according to claim 1 , which comprises at least 2 substitutions, preferably S295L and R321H, wherein said positions are defined with reference to SEQ ID NO: 2.

4. The polypeptide according to any of the preceding claims, wherein said polypeptide has p- hexosyltransferase activity.

5. The polypeptide according to any one of the preceding claims, wherein the polypeptide is a polypeptide that has an amino acid sequence of SEQ ID NO: 3, SEQ ID NO:4 or SEQ ID NO:5.

6. A composition comprising the polypeptide according to any one of claims 1 to 5.

7. The composition according to claim 6, wherein such composition additionally comprises a polyol, a salt, a sugar and / or an organic acid, preferably said composition has a pH in the range of 3.5 to 8.

8. A nucleic acid sequence encoding a polypeptide according to any one of claims 1 to 5.

9. A nucleic acid construct comprising the nucleic acid sequence according to claim 8 operably linked to one or more control sequences capable of directing the expression of the polypeptide according to any one of claims 1 to 5 in a host cell.28DSM IP Assets B.V. 2024P00106WO10. A recombinant expression vector comprising the nucleic acid sequence according to claim 9 and / or the nucleic acid construct according to claim 9.

11. A recombinant host cell comprising the nucleic acid sequence according to claim 9, the nucleic acid construct according to claim 9, and / or the recombinant expression vector according to claim 10.

12. A method for producing the polypeptide according to any one of claims 1 to 5 comprising expressing the nucleic acid sequence according to claim 8 and / or the nucleic acid construct according to claim 9 in a recombinant host cell according to claim 11.

13. Use of a polypeptide according to any one of claims 1 to 5 or the composition according to any one of claims 6 to 7 in the preparation of a dairy product.

14. A process for the production of a dairy product, wherein the process comprises contacting a polypeptide according to any one of claims 1 to 5 or a composition according to any one of claims 6 to 7 with a milk base.

15. The process for the production of a lactose free dairy product, wherein the process comprises contacting a polypeptide according to any one of claims 1 to 5 or a composition according to any one of claims 6 to 7 with a milk base and further comprises contacting the milk base with a lactase.

16. A process for the production of a dairy product, wherein the process comprises contacting a polypeptide according to any one of claims 1 to 5 or a composition according to any one of claims 6 to 7 with a milk base and further comprising contacting the milk base with a glucose oxidase and a catalase.

17. A dairy product, wherein the dairy product comprises a polypeptide according to any one of claims 1 to 5 and / or is obtained or obtainable by the process according to any one of claims 14, 15 or 16 and / or by the use according to claim 13.

18. Use of a polypeptide according to any one of claims 1 to 5 or the composition according to any one of claims 6 to 7 for improving flexibility in dairy product production conditions at an industrial scale.

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