Improved enzyme variants of lactase from Kluyveromyces lactis

Abstract

The present invention relates to variant polypeptides having lactase activity. The present invention also relates to nucleic acid sequences encoding such variant polypeptides, nucleic acid constructs comprising said nucleic acid sequences, recombinant expression vectors comprising said nucleic acid constructs, and recombinant host cells comprising said expression vectors. Further, the present invention relates to methods for producing lactase variants via the use of such host cells. Moreover, the present invention relates to methods for producing variant lactase polypeptides. The present invention further relates to compositions comprising lactase variants, the use of such lactase variants in the preparation of dairy products or the use of compositions comprising lactase variants, methods for producing dairy products, and the resulting dairy products.

Description

Field of the Invention

[0001] The present invention relates to variant polypeptides having lactase activity. The present invention also relates to nucleic acid sequences encoding such variant polypeptides, nucleic acid constructs comprising said nucleic acid sequences, recombinant expression vectors comprising said nucleic acid constructs and recombinant host cells comprising said expression vectors. Further, the present invention relates to methods for producing lactase variants via the use of such host cells. Moreover, the present invention relates to methods for producing variant lactase polypeptides. The present invention further relates to compositions comprising lactase variants, the use of such lactase variants in the preparation of dairy products or the use of compositions comprising lactase variants, methods for producing dairy products and the resulting dairy products. BACKGROUND OF THE INVENTION

[0003] The present invention relates to lactase. Lactase or β-galactosidase (E.C: 3.2.1.23) is an enzyme that catalyzes the hydrolysis of lactose, a disaccharide, into its component monosaccharides, glucose and galactose. Lactose is present in dairy products and more specifically in milk, skim milk, cheese, ice cream, fermented dairy products such as yogurt, many immature cheeses and other dairy products. In the intestinal wall of young mammals, including humans, the breakdown of lactose occurs by naturally occurring lactase. Only a small fraction of the adult population has not lost this ability and can still digest lactose. In most adults, the nutritional and functional problems caused by lactose are due to lactase deficiency and are well known and described. Members of such populations are unable to hydrolyze lactose, in which case lactose enters the large intestine, where it causes dehydration, poor calcium absorption, flatulence, belching and cramps, and, in severe cases, even watery explosive diarrhea.

[0004] An important industrial application of lactase is in the production of lactose-hydrolyzed dairy products for lactose-intolerant individuals. Such hydrolyzed dairy products include pasteurized milk, UHT milk and milk reconstituted from all or part of its original components with or without intermediate processing steps such as proteolysis. Lactase treatment can be carried out before or after heat treatment of the milk. Lactase treatment can be effected by adding the enzyme to the milk or to one of its lactose-containing components.

[0005] The solubility properties of lactose make it possible for it to crystallize when present at high concentrations, resulting in a sandy or granular texture in dairy products such as condensed milk, evaporated milk, milk powder, frozen milk, ice cream and sweet products with a high milk content. Hydrolysis of lactose, either in whole or in part, by lactase eliminates this problem, thus providing products with a uniform texture and therefore higher consumer acceptance.

[0006] Another industrial application of lactase is to increase the sweetness in lactose-containing products such as milk or yogurt. Hydrolysis of lactose in such products results in increased sweetness due to the formation of glucose, without increasing the calorie value of the product. On the contrary, the use of lactase can also reduce the sugar addition in sweetened dairy products without compromising the sweetness.

[0007] Another industrial application of lactase is the hydrolysis of lactose products containing milk components such as bread. Lactose is added to such products to enhance flavor, retain moisture, provide browning and improve baking properties. The hydrolyzed lactose syrup is expected to, for example, enhance crust color development, improve flavor and aroma, change texture, extend shelf life and strengthen the bread structure.

[0008] Hydrolysis of lactose by lactase in fermented dairy products such as yogurt will increase the sweetness. Similarly, when lactase is added before the start of the fermentation process, the rate of acid development can be increased and thus the processing time can be reduced. Lactase treatment of milk or milk-derived products such as whey makes such products suitable for use in animal feed and pet food for lactose-intolerant animals such as cats. Lactose hydrolysis allows the production of a higher concentration of whey and at the same time prevents intestinal problems similar to those described earlier for lactose-deficient patients. The lactose-hydrolyzed whey is concentrated to produce a syrup containing 70 - 75% solids and used as a food ingredient in ice cream, bread and confectionery products.

[0009] Lactase has been described and isolated from a variety of organisms, including microorganisms. Lactase is often an intracellular component of microorganisms such as the genus Kluyveromyces and Bacillus. Yeasts of the genus Kluyveromyces and especially Kluyveromyces fragilis and Kluyveromyces lactis, and other yeasts such as Candida, Torula and Torulopsis are common sources of yeast lactase, while Bacillus coagulans, Bacillus circulans or lactic acid bacteria are well-known bacterial lactase sources. Several commercial lactase preparations derived from these organisms are available, for example (from Kluyveromyces lactis, produced by DSM in Delft, the Netherlands). These lactases are so-called neutral lactases because they have an optimum pH between pH = 6 and pH = 8.

[0010] Although yeast neutral lactase is often used industrially to produce lactose-free or lactose-reduced dairy products, the cost of using enzyme treatment is often high. The main reasons for the relatively high cost of using the enzyme are:

[0011] ● For maintaining the hygiene conditions in the production plant, cultivation is carried out at low temperature. At this temperature, the lactase used industrially is not very active and should be added in relatively high doses.

[0012] ● The currently used lactase is inhibited by its products, especially galactose, in the later stage of lactase cultivation. When a product with a low residual lactose concentration is required, additional enzyme must be added to compensate for the decrease in activity caused by the accumulation of galactose.

[0013] ● The currently used lactase has a relatively low specific activity in milk, and a high enzyme dose is required in applications.

[0014] Therefore, the enzyme dose and cost for producing lactose-reduced and lactose-free products are relatively high.

[0015] There is clearly a need for one or more lactase variants that can overcome at least one of the above-mentioned drawbacks. Summary of the Invention

[0016] The present invention relates to variant polypeptides having lactase activity, i.e., lactase variants. The lactase variants of the present invention may have one or more improved properties compared to a reference polypeptide, which generally has lactase activity. The reference polypeptide may be a wild-type lactase, such as lactase from Kluyveromyces lactis. The variant polypeptides of the present invention may be referred to as "lactase variants", "improved lactase", etc. Variants of Kluyveromyces neutral lactase have been generated, which have properties that result in a reduced cost of use of such lactase in the production of lactose-reduced or lactose-free dairy products. Lactase variants with improved properties regarding dairy product production may exhibit:

[0017] ● Higher specific activity towards ONPG;

[0018] ● Higher specific activity towards lactose;

[0019] ● Higher activity towards lactose in cold milk;

[0020] ● Reduced galactose inhibition; and / or

[0021] ● Higher GOS production in milk.

[0022] Each of these improvements can be determined compared to the reference polypeptide. Moreover, compared to the reference polypeptide, the variant polypeptides of the present invention may have at least 2 or 3 or 4 improved properties. Table 1 provides examples of combinations of improved properties.

[0023] Accordingly, the present invention provides variant polypeptides having lactase activity, wherein said variant has the following amino acid sequence, which, when aligned with a lactase comprising the sequence set forth in SEQ ID NO:2, comprises corresponding to the

[0024] at least one substitution of an amino acid residue of any one of 233, 257, 258, 263, 274, 284, 297, 415, 440, 483, 619, 621, 622, 633, 862 or 1004 amino acids,

[0025] wherein the positions are defined with reference to SEQ ID NO:2 and wherein the variant has one or more altered properties compared to a reference polypeptide having lactase activity and wherein the variant has at least 60% sequence identity with SEQ ID NO:2.

[0026] The present invention also provides:

[0027] - a nucleic acid sequence encoding a variant of the present invention;

[0028] - a nucleic acid construct comprising such a nucleic acid sequence operably linked to one or more control sequences capable of directing the expression of lactase in a suitable expression host;

[0029] - a recombinant expression vector comprising such a nucleic acid construct; and

[0030] - a recombinant host cell comprising such an expression vector.

[0031] The present invention also relates to a method for producing lactase, which comprises culturing the host cells of the present invention under conditions conducive to the production of lactase and recovering the lactase.

[0032] In addition, the present invention relates to a method for producing a variant of a lactase polypeptide, the method comprising:

[0033] a) selecting a polypeptide having lactase activity;

[0034] b) substituting at least one amino acid residue corresponding to

[0035] any one of 233, 257, 258, 263, 274, 284, 297, 415, 440, 483, 619, 621, 622, 633, 862 or 1004 amino acids,

[0036] wherein the positions are defined with reference to SEQ ID NO:2;

[0037] c) optionally substituting one or more additional amino acids as defined in b);

[0038] d) preparing a variant produced by steps a)-c);

[0039] e) determining the properties of the variant; and

[0040] f) Select variants having altered properties compared to a lactase comprising the sequence listed in SEQ ID NO:2 and select variants having at least 60% sequence identity to SEQ ID NO:2, thereby generating lactase polypeptide variants.

[0041] Furthermore, the present invention relates to:

[0042] - A composition comprising a variant of the present invention or a variant obtainable by the method of the present invention;

[0043] - Use of a variant lactase of the present invention or a composition of the present invention in the preparation of dairy products;

[0044] - A method for producing a dairy product, the method comprising adding an effective amount of a variant lactase of the present invention or a composition of the present invention to milk and performing appropriate further dairy product production steps; and

[0045] - A dairy product obtainable by such a method or use.

[0046] BRIEF DESCRIPTION OF THE SEQUENCE LISTING

[0047] SEQ ID NO:1 lists the nucleic acid sequence of the wild-type lactase gene sequence from Kluyveromyces lactis

[0048] SEQ ID NO:2 lists the amino acid sequence of the lactase sequence from Kluyveromyces lactis DETAILED DESCRIPTION OF THE INVENTION

[0050] Throughout this specification and the appended claims, the words "comprising", "including" and "having" shall be interpreted inclusively. That is, where the context allows, these words are intended to convey that other elements or integers may be included which are not specifically recited.

[0051] As used herein, when not modified by a quantifier, refers to one or more (i.e., one or at least one) objects. For example, "element" can mean one element or more than one element.

[0052] As used herein, "lactase" or β-galactosidase (E.C. 3.2.1.23) is an enzyme that catalyzes the hydrolysis of lactose, a disaccharide, into its component monosaccharides glucose and galactose. Due to the transferase activity of lactase, galacto-oligosaccharides (GOS) can be formed during this reaction.

[0053] Lactase is found in the intestine of young mammals, as well as in plants, fungi, yeast and bacteria.

[0054] The lactase can be a neutral or acidic lactase. In a preferred embodiment, the variant polypeptide of the present invention has neutral lactase activity, i.e., it has an optimal pH between pH = 6 and pH = 8.

[0055] The lactase can be an intracellular or extracellularly produced lactase. In a preferred embodiment, the lactase is an intracellularly produced lactase.

[0056] Genes or cDNAs encoding lactase (such as the variants of the present invention) can be cloned and overexpressed in a host organism. Well-known host organisms that can be used for lactase overexpression include Aspergillus, Kluyveromyces, Trichoderma, Escherichia coli, Pichia, Saccharomyces, Yarrowia, Neurospora, Lactococcus, or Bacillus.

[0057] In the present text, the positions that can be substituted to obtain the variants of the present invention are defined with reference to SEQ ID NO:2 of Kluyveromyces lactis lactase.

[0058] The present invention relates to variant polypeptides having lactase activity as compared to a reference polypeptide having lactase activity. The reference polypeptide can generally be a wild-type polypeptide having lactase activity, such as the lactase of SEQ ID NO:2 or a related sequence. The reference polypeptide can also be referred to as the parental polypeptide or the comparison polypeptide.

[0059] More specifically, the present invention relates to variant polypeptides having lactase activity, wherein the variant has the following amino acid sequence which, when aligned with a lactase comprising the sequence listed in SEQ ID NO:2,

[0060] comprises at least one substitution of an amino acid residue corresponding to any one of the amino acids at positions 233, 257, 258, 263, 274, 284, 297, 415, 440, 483, 619, 621, 622, 633, 862, or 1004, the positions being defined with reference to SEQ ID NO:2 and wherein the variant has one or more altered properties as compared to a reference polypeptide having lactase activity and wherein the variant has at least 60% sequence identity with SEQ ID NO:2.

[0061] The wild-type reference polypeptide can be obtained from any suitable organism. Generally, the wild-type reference polypeptide can be obtained from a microorganism, preferably a microorganism in which lactase is naturally produced.

[0062] Such microorganisms include yeasts such as Kluyveromyces spp. The reference polypeptide may be the wild-type sequence of Kluyveromyces lactis.

[0063] Preferably, the reference polypeptide is the lactase set forth in SEQ ID NO:2.

[0064] Variant polypeptides as described herein are generally non-naturally occurring polypeptides.

[0065] Accordingly, the present invention provides a variant polypeptide having lactase activity, wherein the variant has the following amino acid sequence which, when aligned with a lactase comprising the sequence set forth in SEQ ID NO:2, comprises at least one substitution of an amino acid residue corresponding to any one of the amino acids at positions

[0066] 233, 257, 258, 263, 274, 284, 297, 415, 440, 483, 619, 621, 622, 633, 862 or 1004, as defined with reference to SEQ ID NO:2 and wherein the variant has one or more altered properties as compared to a reference polypeptide having lactase activity (such as the polypeptide of SEQ ID NO:2) and wherein the variant has at least 60% sequence identity with SEQ ID NO:2.

[0067] The variant polypeptide will generally have at least one improved property as compared to the reference polypeptide, particularly with respect to properties regarding the use of the variant polypeptide in a method for preparing dairy products.

[0068] Specifically, the improved property may relate to activity or specific activity or to a reduction in galactose inhibition or to a higher GOS yield in milk.

[0069] Table 1 lists the positions affecting the specific properties of the variant lactases of the present invention.

[0070] Table 1: Preferred substitutions defined relative to SEQ ID NO:2. Indicates different characteristics such as specific activity against ONPG or lactose as substrate, activity in cold milk, reduction of galactose inhibition of lactase activity and higher galacto- oligosaccharide production in milk.

[0071]

[0072] The variant polypeptides of the present invention may exhibit a higher specific activity against ONPG.

[0073] Accordingly, the present invention provides a variant polypeptide having lactase activity, wherein the variant has the following amino acid sequence which, when aligned with a lactase comprising the sequence set forth in SEQ ID NO:2, comprises at least one substitution of an amino acid residue corresponding to any one of the amino acids at positions

[0074] at least one substitution of an amino acid residue of any one of 233, 257, 258, 263, 274, 284, 297, 415, 440, 483, 619, 621, 622, 633, 862 or 1004 amino acids, said positions being defined with reference to SEQ ID NO:2 and wherein the variant has a higher specific activity against ONPG compared to a reference polypeptide having lactase activity (e.g., the polypeptide of SEQ ID NO:2) and wherein the variant has at least 60% sequence identity with SEQ ID NO:2.

[0075] Preferably, the present invention provides a variant polypeptide having lactase activity, wherein the variant has the following amino acid sequence which, when aligned with a lactase comprising the sequence listed in SEQ ID NO:2, comprises at least one substitution of an amino acid residue corresponding to any one of the amino acids at positions 415, 483, 619, 621, 622 or 633,

[0076] said positions being defined with reference to SEQ ID NO:2 and wherein the variant has a higher specific activity against ONPG compared to a reference polypeptide having lactase activity (e.g., the polypeptide of SEQ ID NO:2) and wherein the variant has at least 60% sequence identity with SEQ ID NO:2. Preferably at least one substitution of an amino acid residue corresponding to any one of the amino acids at positions 415 and / or 619, said positions being defined with reference to SEQ ID NO:2.

[0077] More preferably, the present invention provides a variant polypeptide having lactase activity, wherein the variant has the following amino acid sequence which, when aligned with a lactase comprising the sequence listed in SEQ ID NO:2, comprises at least one substitution selected from

[0078] T415C, T415A, A483S, V619I, I621V, M622L or T633G,

[0079] said positions being defined with reference to SEQ ID NO:2 and wherein the variant has a higher specific activity against ONPG compared to a reference polypeptide having lactase activity (e.g., the polypeptide of SEQ ID NO:2) and wherein the variant has at least 60% sequence identity with SEQ ID NO:2. Preferably the substitutions T415C and / or V619I, said positions being defined with reference to SEQ ID NO:2.

[0080] Another variant polypeptide of the present invention may exhibit a higher specific activity against lactose. Since lactose is the natural substrate of lactase in dairy products, the higher specific activity of the variant polypeptide can result in a reduced required dose of the enzyme and thus can lead to lower processing costs. By reducing the enzyme dose in the application, the amount of increased side activities is also reduced, and thus a higher quality final dairy product is expected.

[0081] The present invention thus provides a variant polypeptide having lactase activity, wherein the variant has the following amino acid sequence which, when aligned with a lactase comprising the sequence set forth in SEQ ID NO:2, comprises at least one substitution of an amino acid residue corresponding to any one of the amino acids at positions

[0082] 233, 257, 258, 263, 274, 284, 297, 415, 440, 483, 619, 621, 622, 633, 862 or 1004, as defined with reference to SEQ ID NO:2 and wherein the variant has a higher specific activity against lactose compared to a reference polypeptide having lactase activity (such as the polypeptide of SEQ ID NO:2) and wherein the variant has at least 60% sequence identity with SEQ ID NO:2.

[0083] Preferably, the present invention provides a variant polypeptide having lactase activity, wherein the variant has the following amino acid sequence which, when aligned with a lactase comprising the sequence set forth in SEQ ID NO:2, comprises at least one substitution of an amino acid residue corresponding to any one of the amino acids at positions

[0084] 415, 440 or 483, as defined with reference to SEQ ID NO:2 and wherein the variant has a higher specific activity against lactose compared to a reference polypeptide having lactase activity (such as the polypeptide of SEQ ID NO:2) and wherein the variant has at least 60% sequence identity with SEQ ID NO:2. Preferably at least one substitution of an amino acid residue corresponding to any one of the amino acids at positions 415 and / or 483 (this preference is based on the analysis of lactase variants comprising combinations of substitutions), as defined with reference to SEQ ID NO:2.

[0085] More preferably, the present invention provides a variant polypeptide having lactase activity, wherein the variant has the following amino acid sequence which, when aligned with a lactase comprising the sequence set forth in SEQ ID NO:2, comprises at least one substitution selected from

[0086] T415C, T415A, Y440F or A483S,

[0087] The position is defined with reference to SEQ ID NO:2 and wherein the variant has a higher specific activity against lactose compared to a reference polypeptide having lactase activity (e.g., the polypeptide of SEQ ID NO:2) and wherein the variant has at least 60% sequence identity with SEQ ID NO:2. Preferred substitutions are T415A, T415C and / or A483S (this preference is based on the analysis of lactase variants comprising combinations of substitutions), the positions being defined with reference to SEQ ID NO:2.

[0088] Even more preferably, the present invention provides a variant polypeptide having lactase activity, wherein the variant has the following amino acid sequence which, when aligned with a lactase comprising the sequence listed in SEQ ID NO:2,

[0089] comprises at least two, three, four or five substitutions of amino acid residues corresponding to any one of the amino acids at positions 233, 257, 258, 263, 274, 284, 297, 415, 440, 483, 619, 621, 622, 633, 862 or 1004,

[0090] the positions being defined with reference to SEQ ID NO:2 and wherein the variant has a higher specific activity against lactose compared to a reference polypeptide having lactase activity (e.g., the polypeptide of SEQ ID NO:2) and wherein the variant has at least 60% sequence identity with SEQ ID NO:2.

[0091] Examples of such mutants are mutants 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 described in Table 5.

[0092] Preferably at low temperatures (preferably the low temperature is in the range of 4 - 12°C), a further variant polypeptide of the present invention can exhibit higher activity against lactose in milk. Since lactase is often used in cold milk, the enhanced activity of the variant polypeptide in this particular application can lead to a reduction in the enzyme dosage and thus a reduction in cost. Additionally, the higher activity of the variant polypeptide can lead to a reduction in the milk processing time and thus a reduction in the risk of possible microbial spoilage.

[0093] The present invention thus provides a variant polypeptide having lactase activity, wherein the variant has the following amino acid sequence which, when aligned with a lactase comprising the sequence listed in SEQ ID NO:2,

[0094] at least one substitution of an amino acid residue of any one of 233, 257, 258, 263, 274, 284, 297, 415, 440, 483, 619, 621, 622, 633, 862 or 1004 amino acids, said positions being defined with reference to SEQ ID NO:2 and wherein said variant exhibits enhanced activity against lactose in cold milk as compared to a reference polypeptide having lactase activity (such as the polypeptide of SEQ ID NO:2) and wherein said variant has at least 60% sequence identity with SEQ ID NO:2.

[0095] Preferably, the present invention provides a variant polypeptide having lactase activity, wherein said variant has the following amino acid sequence which, when aligned with a lactase comprising the sequence listed in SEQ ID NO:2, comprises

[0096] at least one substitution of an amino acid residue of any one of 233, 257, 258, 263, 274, 284, 297, 440, 619, 633, 862 or 1004 amino acids,

[0097] said positions being defined with reference to SEQ ID NO:2 and wherein said variant exhibits enhanced activity against lactose in cold milk as compared to a reference polypeptide having lactase activity (such as the polypeptide of SEQ ID NO:2) and wherein said variant has at least 60% sequence identity with SEQ ID NO:2. Preferably (at least) a substitution of the amino acid residue corresponding to amino acid position 440, said position being defined with reference to SEQ ID NO:2 (this preference is based on the analysis of lactase variants comprising combinations of substitutions). Preferred combinations of substitutions are substitutions at positions 440 and 619.

[0098] More preferably, the present invention provides a variant polypeptide having lactase activity, wherein said variant has the following amino acid sequence which, when aligned with a lactase comprising the sequence listed in SEQ ID NO:2, comprises at least one substitution selected from

[0099] D233V, D257G, A258T, N263S, K274E, N284S, E297G, Y440F, V619I, T633G, L862V or A1004P

[0100] The position is defined with reference to SEQ ID NO:2 and wherein the variant exhibits enhanced activity against lactose in cold milk compared to a reference polypeptide having lactase activity (such as the polypeptide of SEQ ID NO:2) and wherein the variant has at least 60% sequence identity with SEQ ID NO:2. Preferably the substitution is (at least) Y440F, the position being defined with reference to SEQ ID NO:2 (this preference is based on the analysis of lactase variants comprising combinations of substitutions). A preferred combination of substitutions is Y440F + V619I.

[0101] Although, the presence of at least one substitution of an amino acid residue corresponding to any one of the amino acids at positions

[0102] 233, 257, 258, 263, 274, 284, 297, 440, 619, 633, 862 or 1004 (the positions being defined with reference to SEQ ID NO:2) is sufficient to obtain a variant polypeptide having lactase activity and further showing enhanced activity against lactose in cold milk, but it is shown herein that double or triple mutant polypeptide variants also exhibit enhanced activity against lactose in cold milk.

[0103] Accordingly, the present invention also provides a variant polypeptide having lactase activity, wherein the variant has the following amino acid sequence which, when aligned with a lactase comprising the sequence set forth in SEQ ID NO:2, comprises at least two substitutions selected from 263, 274 or 284 (more preferably N263S, K274E or N284S), the positions being defined with reference to SEQ ID NO:2 and wherein the variant exhibits enhanced activity against lactose in cold milk compared to a reference polypeptide having lactase activity (such as the polypeptide of SEQ ID NO:2) and wherein the variant has at least 60% sequence identity with SEQ ID NO:2.

[0104] The present invention also provides a variant polypeptide having lactase activity, wherein the variant has the following amino acid sequence which, when aligned with a lactase comprising the sequence set forth in SEQ ID NO:2, comprises substitutions at positions 263, 274 and 284 (more preferably the substitutions are N263S, K274E and N284S)

[0105] The position is defined with reference to SEQ ID NO:2 and wherein the variant exhibits enhanced activity against lactose in cold milk compared to a reference polypeptide having lactase activity (e.g., the polypeptide of SEQ ID NO:2) and wherein the variant has at least 60% sequence identity with SEQ ID NO:2.

[0106] The present invention also provides a variant polypeptide having lactase activity, wherein the variant has the following amino acid sequence, which, when aligned with a lactase comprising the sequence set forth in SEQ ID NO:2, comprises substitutions at positions 257 and 297 (preferably the substitutions are D257G and E297G),

[0107] The position is defined with reference to SEQ ID NO:2 and wherein the variant exhibits enhanced activity against lactose in cold milk compared to a reference polypeptide having lactase activity (e.g., the polypeptide of SEQ ID NO:2) and wherein the variant has at least 60% sequence identity with SEQ ID NO:2.

[0108] The present invention also provides a variant polypeptide having lactase activity, wherein the variant has the following amino acid sequence, which, when aligned with a lactase comprising the sequence set forth in SEQ ID NO:2, the amino acid sequence comprises at least three, four or five substitutions of amino acid residues corresponding to any one of the amino acids at positions

[0109] 233, 257, 258, 263, 274, 284, 297, 415, 440, 483, 619, 621, 622, 633, 862 or 1004

[0110] The position is defined with reference to SEQ ID NO:2 and wherein the variant exhibits enhanced activity against lactose in cold milk compared to a reference polypeptide having lactase activity (e.g., the polypeptide of SEQ ID NO:2) and wherein the variant has at least 60% sequence identity with SEQ ID NO:2.

[0111] Examples of such mutants are mutants 15, 17, 18, 19, 20, 21 or 22 described in Table 5.

[0112] Another mutant polypeptide of the present invention may exhibit reduced galactose inhibition. At low lactose concentration and high galactose concentration, galactose inhibition results in slow lactose hydrolysis at a later time point. Therefore, a lactase with reduced galactose inhibition would be desirable, especially when it is desired to produce dairy products with a lactose concentration below 0.5 g / L.

[0113] The present invention thus provides a variant polypeptide having lactase activity, wherein said variant has the following amino acid sequence which, when aligned with a lactase comprising the sequence set forth in SEQ ID NO:2, comprises at least one substitution of an amino acid residue corresponding to any one of the amino acids at positions

[0114] 233, 257, 258, 263, 274, 284, 297, 415, 440, 483, 619, 621, 622, 633, 862 or 1004, said positions being defined with reference to SEQ ID NO:2 and wherein said variant exhibits reduced galactose inhibition compared to a reference polypeptide having lactase activity (e.g., the polypeptide of SEQ ID NO:2) and wherein said variant has at least 60% sequence identity with SEQ ID NO:2.

[0115] Preferably, the present invention provides a variant polypeptide having lactase activity, wherein said variant has the following amino acid sequence which, when aligned with a lactase comprising the sequence set forth in SEQ ID NO:2, comprises at least one substitution of an amino acid residue corresponding to any one of the amino acids at positions

[0116] 619, 621 or 622, said positions being defined with reference to SEQ ID NO:2 and wherein said variant exhibits reduced galactose inhibition compared to a reference polypeptide having lactase activity (e.g., the polypeptide of SEQ ID NO:2) and wherein said variant has at least 60% sequence identity with SEQ ID NO:2.

[0117] More preferably, the present invention provides a variant polypeptide having lactase activity, wherein said variant has the following amino acid sequence which, when aligned with a lactase comprising the sequence set forth in SEQ ID NO:2, comprises at least one substitution selected from

[0118] V619I, I621V or M622L,

[0119] said positions being defined with reference to SEQ ID NO:2 and wherein said variant exhibits reduced galactose inhibition compared to a reference polypeptide having lactase activity (e.g., the polypeptide of SEQ ID NO:2) and wherein said variant has at least 60% sequence identity with SEQ ID NO:2.

[0120] Another mutant polypeptide of the present invention can exhibit increased GOS production in milk. GOS (galacto-oligosaccharide) is a prebiotic defined as an indigestible food component that beneficially affects the host by stimulating the growth and / or activity of beneficial bacteria in the colon. Not all lactases are equally well-suited for preparing GOS. Another lactase capable of accumulating GOS at the low lactose concentration (<50 g / L) present in milk is desired.

[0121] The present invention thus provides a variant polypeptide having lactase activity, wherein the variant has the following amino acid sequence which, when aligned with a lactase comprising the sequence listed in SEQ ID NO:2, comprises at least one substitution of an amino acid residue corresponding to any one of the amino acids at positions

[0122] 233, 257, 258, 263, 274, 284, 297, 415, 440, 483, 619, 621, 622, 633, 862 or 1004, the positions being defined with reference to SEQ ID NO:2 and wherein the variant exhibits increased GOS production in milk compared to a reference polypeptide having lactase activity (such as the polypeptide of SEQ ID NO:2) and wherein the variant has at least 60% sequence identity with SEQ ID NO:2.

[0123] Preferably, the present invention provides a variant polypeptide having lactase activity, wherein the variant has the following amino acid sequence which, when aligned with a lactase comprising the sequence listed in SEQ ID NO:2, comprises at least one substitution of an amino acid residue corresponding to any one of the amino acids at positions

[0124] 619 or 622,

[0125] the positions being defined with reference to SEQ ID NO:2 and wherein the variant exhibits increased GOS production in milk compared to a reference polypeptide having lactase activity (such as the polypeptide of SEQ ID NO:2) and wherein the variant has at least 60% sequence identity with SEQ ID NO:2.

[0126] More preferably, the present invention provides a variant polypeptide having lactase activity, wherein the variant has the following amino acid sequence which, when aligned with a lactase comprising the sequence listed in SEQ ID NO:2, comprises at least one substitution selected from

[0127] V619I or M622L.

[0128] The position is defined with reference to SEQ ID NO:2 and wherein the variant exhibits increased GOS production in milk as compared to a reference polypeptide having lactase activity (e.g., the polypeptide of SEQ ID NO:2) and wherein the variant has at least 60% sequence identity with SEQ ID NO:2.

[0129] The variant lactase of the present invention may also contain additional modifications at positions other than those specified above compared to the parent, e.g., one or more additional substitutions, additions or deletions. The variants of the present invention may comprise combinations of different types of such modifications. The variant may comprise one, two, three, four, at least 5, at least 10, at least 15, at least 20, at least 25, at least 30 or more such modifications (which may all be of the same type or may be of different types of modifications). Generally, the additional modification may be a substitution. Thus, the present invention also provides a variant polypeptide having lactase activity, wherein the variant has the following amino acid sequence, which, when aligned with a lactase comprising the sequence set forth in SEQ ID NO:2, comprises an amino acid residue corresponding to any one of the amino acids at positions

[0130] 233, 257, 258, 263, 274, 284, 297, 415, 440, 483, 619, 621, 622, 633, 862 or 1004, as defined with reference to SEQ ID NO:2, and wherein the variant has one or more altered properties as compared to a reference polypeptide having lactase activity, and wherein the variant polypeptide comprises additional substitutions other than those defined, and wherein the variant has at least 60% sequence identity with SEQ ID NO:2.

[0131] The variants according to the present invention (e.g., variants having one or more substitutions listed in Table 1 or Table 2) may have at least about 60%, 65%, 70%, 75% or 80% homology / identity with a reference lactase polypeptide (e.g., the lactase of SEQ ID NO:2), e.g., at least about 85% homology with the parent polypeptide, e.g., at least about 90% homology with the parent polypeptide, at least 95% homology with the parent polypeptide, at least about 98% homology with the parent polypeptide or at least about 99% homology with the parent polypeptide. Such variants will generally have one or more substitutions or sets of substitutions listed in Table 1 or Table 2.

[0132] Thus, the present invention also provides a variant polypeptide having lactase activity, wherein the variant has the following amino acid sequence, which, when aligned with a lactase comprising the sequence set forth in SEQ ID NO:2, comprises an amino acid residue corresponding to the

[0133] At least one substitution of an amino acid residue of any one of 233, 257, 258, 263, 274, 284, 297, 415, 440, 483, 619, 621, 622, 633, 862 or 1004 amino acids, said positions being defined with reference to SEQ ID NO:2 and wherein said variant has one or more altered properties compared to a reference polypeptide having lactase activity and wherein said variant has at least 80% sequence identity with SEQ ID NO:2.

[0134] Variants of the invention will generally retain lactase activity. That is, variants of the invention will generally be able to convert lactose to glucose and galactose or variants of the invention will generally be able to convert lactose to glucose and galactose and be able to form GOS. Variants of the invention are variants that are generally able to effect the enzymatic conversion of lactose and can be used in the preparation of dairy products, such as milk or yogurt.

[0135] Preferably, variants of the invention will generally exhibit improved properties compared to the reference lactase polypeptide from which they are derived. Such improved properties will generally be relevant properties if the variant is used as described hereinafter, for example, in a method for preparing a dairy product.

[0136] A polypeptide variant that exhibits improved properties relative to a reference lactase is a polypeptide variant that exhibits a measurable decrease or increase in a relevant property, generally such that the variant is more suitable for use, for example, in a method for producing a dairy product.

[0137] Said property may thus be decreased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or at least 99%. Alternatively, said property may be increased by at least 10%, at least 25%, at least 50%, at least 100%, at least 200%, at least 500% or at least 1000%. In this case the percentage decrease or increase represents the percentage decrease or increase compared to the reference lactase polypeptide. Those skilled in the art are familiar with how to measure such percentage changes - which is a comparison of the activity of the reference lactase and the activity of the variant lactase.

[0138] All variants described herein are included within the terms "polypeptides according to the invention" or "variants according to the invention".

[0139] The terms "peptide" and "oligopeptide" are considered synonymous (as is generally recognized) and each term may be used interchangeably when the context requires reference to a chain of at least two amino acids coupled by peptide bonds. In this document, for chains containing more than about seven amino acid residues, the term "polypeptide" is used. All formulas or sequences of oligopeptides and polypeptides herein are written from left to right and in the direction from the amino terminus to the carboxyl terminus. The single-letter codes for amino acids used herein are well known in the art and can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).

[0140] The polypeptides of the present invention can be in isolated form, such as substantially isolated form. An "isolated" polypeptide or protein means a polypeptide or protein removed from its natural environment. For example, for the purposes of the present invention, recombinantly produced polypeptides and proteins expressed in host cells are considered isolated, and recombinantly produced polypeptides that have been substantially purified using any suitable technique are also considered isolated. The polypeptide variants according to the present invention can be recovered and purified from recombinant cell cultures by methods known in the art.

[0141] The polypeptides of the present invention include the products of chemical synthesis processes, as well as products produced by recombinant techniques from prokaryotic or eukaryotic hosts (including, for example, bacteria, yeast, fungi, higher plants, insects, and mammalian cells). Depending on the host used in the recombinant production process, the polypeptides of the present invention can be glycosylated or can be non-glycosylated. Additionally, the polypeptides of the present invention can also include an initial modified methionine residue, which in some cases results from a host-mediated process.

[0142] The present invention also features bioactive fragments of the polypeptide variants according to the present invention. Such fragments are considered to be encompassed by the term "variants of the present invention".

[0143] Bioactive fragments of the polypeptide variants of the present invention include polypeptides comprising an amino acid sequence that is sufficiently identical to or derived from the amino acid sequence of the variant protein of the present invention, said fragments comprising fewer amino acids than the full-length protein but exhibiting at least one biological activity of the corresponding full-length protein. Generally, the bioactive fragment comprises a domain or motif having at least one activity of the variant protein of the present invention. The bioactive fragments of the proteins of the present invention can be polypeptides that are, for example, 10, 25, 50, 100 or more amino acids in length. Moreover, other bioactive portions lacking other regions of the protein can be prepared by recombinant techniques and evaluated for one or more biological activities against the native form of the polypeptide of the present invention.

[0144] Generally, the protein fragments of the present invention will comprise one or more substitutions as defined herein.

[0145] The present invention also features nucleic acid fragments encoding the above bioactive fragments (said bioactive fragments themselves being variants of the present invention).

[0146] The present invention also provides a nucleic acid sequence encoding a variant polypeptide of the present invention. Thus, the present invention also provides a nucleic acid sequence encoding a variant polypeptide having lactase activity, wherein said variant has the following amino acid sequence, which, when aligned with lactase comprising the sequence set forth in SEQ ID NO:2,

[0147] comprises at least one substitution of an amino acid residue corresponding to any one of the amino acids at positions 233, 257, 258, 263, 274, 284, 297, 415, 440, 483, 619, 621, 622, 633, 862 or 1004, said positions being defined with reference to SEQ ID NO:2 and wherein said variant has one or more altered properties as compared to a reference polypeptide having lactase activity (such as the polypeptide of SEQ ID NO:2) and wherein said variant has at least 60% sequence identity with SEQ ID NO:2.

[0148] The present invention also relates to an isolated polynucleotide encoding at least one functional domain of a polypeptide variant of the present invention. Generally, such domains will comprise one or more substitutions as described herein.

[0149] In one embodiment of the present invention, the nucleic acid sequence according to the present invention encodes a polypeptide comprising the following amino acid sequence, wherein said amino acid sequence has one or more truncations and / or at least one amino acid substitution, deletion and / or insertion as compared to the parental lactase.

[0150] As used herein, the terms "gene" and "recombinant gene" refer to nucleic acid molecules that include an open reading frame encoding a variant as described herein. A gene can include coding sequences, non-coding sequences, introns, and regulatory sequences. That is, as used herein, a "gene" can refer to an isolated nucleic acid molecule as defined herein. Thus, in the context of the present application, the term "gene" refers not only to naturally occurring sequences.

[0151] The nucleic acid molecules of the present invention can be generated using standard molecular biology techniques well known to those of skill in the art in conjunction with the sequence information provided herein.

[0152] For example, using standard synthetic techniques, the desired nucleic acid molecule can be synthesized de novo. Such synthetic processes will typically be automated processes.

[0153] Alternatively, the nucleic acid molecules of the present invention can be generated by using existing nucleic acid molecules, such as site-directed mutagenesis of a wild-type nucleic acid molecule. Many techniques well known to those of skill in the art can be used for site-directed mutagenesis.

[0154] In one such method, mentioned here only by way of example, PCR is performed on a plasmid template using an oligonucleotide "primer" encoding the desired substitution. Since the primer is the end of the newly synthesized strand, there should be a mismatch in the binding to the template DNA strand during the first cycle. After this first round, the primer-based strand (containing the mutation) will be equal in concentration to the original template. After successive cycles, there will be exponential growth, and after 25 cycles in the region of 8 million:1, the mutated amplified fragments will outnumber the original unmutated strands in quantity, resulting in an almost homogeneous solution of the mutated amplified fragments. The template DNA is then eliminated by enzymatic digestion (e.g., using a restriction enzyme that cuts only methylated DNA, such as Dpn1). The template derived from alkaline lysis plasmid preparation and thus methylated is destroyed in this step, but the mutated plasmid is preserved because it is generated in vitro and thus not methylated.

[0155] In such methods, in a single PCR reaction, more than one mutation (encoding a substitution as described herein) can be introduced into a nucleic acid molecule, for example, by using one or more oligonucleotides each containing one or more mismatches. Alternatively, more than one mutation can be introduced into a nucleic acid molecule by performing more than one PCR reaction, with one or more mutations introduced in each reaction, thereby introducing altered nucleic acids into the nucleic acid in a continuous, repetitive manner.

[0156] cDNA, mRNA, or genomic DNA can be used as a template and appropriate mismatched oligonucleotide primers to generate the nucleic acids of the present invention according to the above site-directed mutagenesis techniques. The nucleic acid molecules obtained in this way can be cloned into appropriate vectors and characterized by DNA sequence analysis.

[0157] Compared with the parental lactase, the nucleic acid sequence of the present invention may contain one or more deletions, i.e., gaps. Such deletions / gaps can also be generated using site-directed mutagenesis with appropriate oligonucleotides. Techniques for generating such deletions are well known to those skilled in the art.

[0158] In addition, the nucleotide sequences corresponding to the present invention or oligonucleotides capable of hybridizing with the nucleotide sequences of the present invention can be prepared by standard synthetic techniques, such as using an automated DNA synthesizer.

[0159] In addition, complementary nucleic acid molecules are included in the present invention. A nucleic acid molecule complementary to another nucleotide sequence is a nucleic acid molecule that is sufficiently complementary to another nucleotide sequence to hybridize with another nucleotide sequence and thereby form a stable double-stranded molecule.

[0160] One aspect of the present invention relates to an isolated nucleic acid molecule encoding a variant of the present invention or a bioactive fragment or domain thereof, and a nucleic acid molecule sufficient to be used as a hybridization probe to identify a nucleic acid molecule encoding a polypeptide of the present invention and a fragment of such a nucleic acid molecule suitable for use as a PCR primer for amplification or mutagenesis of nucleic acid molecules (e.g., for the preparation of nucleic acid molecules of the present invention).

[0161] "Isolated polynucleotide" or "isolated nucleic acid" is DNA or RNA that is not immediately adjacent in the genome of the organism from which it is derived to the two coding sequences (one at the 5' end and the other at the 3' end) that flank it in nature. Thus, in one embodiment, an isolated nucleic acid includes some or all of the 5' non-coding (e.g., promoter) sequences flanking the coding sequence. Thus, the term includes, for example, recombinant DNA incorporated into a vector, an autonomously replicating plasmid or virus, or the genomic DNA of a prokaryote or eukaryote, or recombinant DNA existing independently of other sequences, as a separate molecule (e.g., a genomic DNA fragment produced by PCR or restriction endonuclease treatment). It also includes recombinant DNA that is part of a hybrid gene encoding an additional polypeptide that is substantially free of cellular material, viral material, or culture medium (when produced by recombinant DNA techniques) or chemical precursors or other chemicals (when chemically synthesized). Moreover, an "isolated nucleic acid fragment" is a nucleic acid fragment that does not exist as a fragment in nature and would not be found in nature.

[0162] As used herein, the terms "polynucleotide" or "nucleic acid molecule" are intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), and analogs of DNA or RNA generated using nucleotide analogs. The nucleic acid molecule can be single-stranded or double-stranded, but is preferably double-stranded DNA. Nucleic acids can be synthesized using oligonucleotide analogs or derivatives (e.g., inosine or phosphorothioate nucleotides). Such oligonucleotides can be used, for example, to prepare nucleic acids having altered base-pairing properties or increased nuclease resistance.

[0163] Another embodiment of the invention provides an isolated nucleic acid molecule that is antisense to a nucleic acid molecule of the invention.

[0164] The terms "homology" or "percent identity" are used interchangeably herein. For the purposes of the present invention, 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 the first amino acid or nucleic acid to best align it with the second amino acid or nucleic acid sequence). The amino acid or nucleotide residues at the 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. The percent identity between the two sequences is a function of the number of positions having identical residues (i.e., percent identity = number of identical positions / total number of positions (i.e., overlapping positions) x 100). Preferably, the two sequences are of the same length.

[0165] Sequence comparisons can be performed over the entire length of the two sequences being compared or over a segment of the two sequences. Generally, sequence comparisons will be performed over the full length of the two sequences being compared. However, sequence identity can be determined over a region of, for example, twenty, fifty, one hundred or more contiguous amino acid residues.

[0166] Those skilled in the art will appreciate the fact that several different computer programs can be used to determine the homology between two sequences. For example, the comparison of sequences and the determination of the percentage of identity between two sequences can be accomplished using mathematical algorithms. In a preferred embodiment, the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm in the GAP program, which has been incorporated into the Accelrys GCG software package (available at http: / / www.accelrys.com / products / gcg / ), is used to determine the percentage of identity between two amino acid or nucleic acid sequences using the Blosum 62 matrix or the 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. Those skilled in the art will recognize that all these different parameters will produce slightly different results, but the overall percentage of identity between two sequences will not change significantly when using different algorithms.

[0167] The protein or nucleic acid sequences of the present invention can further be used as a "query sequence" to search public databases to (e.g.) 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. The BLAST protein search can be performed using the BLASTP program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to the protein molecules of the present invention. For comparison purposes, to obtain a gapped alignment, gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25(17):3389-3402. When using the BLAST and gapped BLAST programs, the default parameters of the individual programs (e.g., BLASTP and BLASTN) can be used. See http: / / www.ncbi.nlm.nih.gov / the homepage of the National Center for Biotechnology Information above.

[0168] The present invention also provides a nucleic acid construct comprising a nucleic acid sequence encoding a variant polypeptide having lactase activity, wherein the variant has the following amino acid sequence, which, when aligned with lactase comprising the sequence set forth in SEQ ID NO:2, comprises the corresponding to the

[0169] at least one substitution of an amino acid residue of any one of 233, 257, 258, 263, 274, 284, 297, 415, 440, 483, 619, 621, 622, 633, 862 or 1004 amino acids, said positions being defined with reference to SEQ ID NO:2 and wherein said variant has one or more altered properties compared to a reference polypeptide having lactase activity (e.g., the polypeptide of SEQ ID NO:2) and wherein said variant has at least 60% sequence identity with SEQ ID NO:2, wherein said nucleic acid sequence is operably linked to one or more control sequences capable of directing the expression of lactase in a suitable expression host cell.

[0170] Another aspect of the invention relates to a vector, preferably an expression vector, comprising a nucleic acid encoding a variant lactase polypeptide of the invention.

[0171] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop into which an additional DNA segment can be ligated. Another type of vector is a viral vector, into which an additional DNA segment can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cells 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) integrate into the genome of the host cell after being introduced into the host cell and are thereby replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes operably linked thereto. Such vectors are referred to herein as "expression vectors". In general, expression vectors used in recombinant DNA techniques are often in the form of plasmids. The terms "plasmid" and "vector" are used interchangeably herein because plasmids are the most 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) that perform equivalent functions.

[0172] The recombinant expression vector of the present invention comprises the nucleic acid of the present invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vector includes one or more regulatory sequences selected based on the host cell to be used for expression and operably linked to the nucleic acid sequence to be expressed. In a recombinant expression vector, "operably linked" is intended to mean that the target nucleotide sequence is linked to the regulatory sequence in such a way as to permit 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 signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include regulatory sequences that direct constitutive expression of a nucleotide sequence in many types of host cells and regulatory sequences that direct expression of a nucleotide sequence only in a particular type of host cell (e.g., tissue-specific regulatory sequences). Those skilled in the art will recognize that the design of the expression vector can depend on factors such as the choice of host cell to be transformed, the level of expression of the desired protein, etc. The expression vector of the present invention can be introduced into a host cell to produce a protein or peptide encoded by the nucleic acid as described herein (e.g., the lactase variant of SEQ ID NO:2, such as a functional equivalent or fragment, or a fusion protein comprising one or more such variants).

[0173] The recombinant expression vector of the present invention can be designed for expression of the variant protein of the present invention in prokaryotic or eukaryotic cells. For example, the variant protein of the present invention can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors), yeast cells or mammalian cells. Suitable host cells are further discussed in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

[0174] Expression vectors useful in the present invention include vectors of chromosomal origin, episomal origin and viral origin, such as vectors derived from bacterial plasmids, bacteriophages, yeast episomes, yeast chromosomal elements, viruses such as baculoviruses, papovaviruses, vaccinia viruses, adenoviruses, fowlpox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as vectors derived from plasmid and bacteriophage genetic elements (e.g., cosmids and phagemids).

[0175] The DNA insert should be operably linked to a suitable promoter, such as the bacteriophage λ pL promoter, the Escherichia coli lac, trp, and tac promoters, the SV40 early and late promoters, and the retroviral LTR promoter, etc. Other suitable promoters will be known to the person skilled in the art. In a particular embodiment, a promoter that is preferably capable of directing high levels of expression of lactase in filamentous fungi is preferred. Such promoters are known in the art. The expression construct may contain sites for transcription initiation, termination, and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcript expressed by the construct will include a translation initiation AUG at the start and a termination codon appropriately located at the end of the polypeptide to be translated.

[0176] The vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing exogenous nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, transduction, infection, lipofection, cationic lipid-mediated transfection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd, ed. Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), Davis et al. Basic Methods in Molecular Biology (1986), and other laboratory manuals.

[0177] For stable transfection of mammalian cells, it is known that depending on the expression vector and transfection technique used, only a small fraction of cells will integrate the exogenous DNA into their genome. To identify and select these components, a gene encoding a selectable marker (e.g., antibiotic resistance) is usually introduced into the host cell together with the gene of interest. Preferred selectable markers include those that confer resistance to drugs such as G418, hygromycin, and methatrexate. The nucleic acid encoding the selectable marker can be introduced into the host cell on the same vector as the nucleic acid encoding the variant protein of the invention or can be introduced on a separate vector. Cells stably transfected with the introduced nucleic acid can be identified by drug selection (e.g., cells that have incorporated the selectable marker gene will survive while other cells will die).

[0178] Protein expression in prokaryotes is often carried out in Escherichia coli with a vector containing a constitutive or inducible promoter that directs the expression of a fusion or non-fusion protein. Fusion vectors add a number of amino acids to the protein encoded therein, such as to the amino terminus of a recombinant protein. Such fusion vectors generally serve three purposes: 1) to increase the expression of the recombinant protein; 2) to increase the solubility of the recombinant protein; and 3) to facilitate the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in a fusion expression vector, a proteolytic cleavage site is introduced at the junction of the fusion portion and the recombinant protein so that the recombinant protein can be separated from the fusion portion after purification of the fusion protein.

[0179] As shown herein, the expression vector will preferably contain a selectable marker. Such markers include: dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance for culture in E. coli and other bacteria. Representative examples of suitable hosts include bacterial cells such as Escherichia coli, Streptomyces, Salmonella typhimurium, and certain Bacillus species such as Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, and Bacillus clausii; fungal cells such as Aspergillus species such as Aspergillus niger, Aspergillus oryzae, and Aspergillus nidulans, and / or Fusarium species such as Fusarium venenatum, and / or Trichoderma species such as Trichoderma reesei; yeast cells such as Kluyveromyces, such as Kluyveromyces lactis and Kluyveromyces marxianus, and / or Pichia, such as Pichia pastoris, and / or Saccharomyces, such as Saccharomyces cerevisiae, and / or Hansenula, such as Hansenula polymorpha; insect cells such as Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS, and Bowes melanoma; and plant cells. Media and conditions suitable for the above host cells are known in the art.

[0180] Preferred vectors for bacteria are, for example, those disclosed in WO-A1-2004 / 074468 (incorporated herein by reference). Other suitable vectors will be apparent to those skilled in the art.

[0181] Known bacterial promoters suitable for the present invention include those disclosed in WO-A1-2004 / 074468 (incorporated herein by reference).

[0182] Transcription of DNA encoding variants of the present invention in higher eukaryotes can be increased by inserting enhancer sequences into the vector. Enhancers are cis-acting elements of DNA, typically about 10 to 300 bp, which serve to increase the transcriptional activity of a promoter in a specified host cell type. Examples of enhancers include the SV40 enhancer located 100 to 270 bp downstream of the origin of replication, the cytomegalovirus early promoter enhancer, the polyomavirus enhancer downstream of the origin of replication, and the adenovirus enhancer.

[0183] To direct the translated protein into the lumen of the endoplasmic reticulum, the periplasmic space, or the extracellular environment, appropriate secretion signals can be incorporated into the expressed polypeptide. The signal can be endogenous to the polypeptide or it can be a heterologous signal.

[0184] Variants of the present invention can be expressed in a form that enables them to include additional heterologous functional regions (such as secretion signals). Variants of the present invention can also contain, for example, regions of additional amino acids (especially charged amino acids) added to the N-terminus of the polypeptide, for example to enhance stability and persistence within the host cell during purification or during subsequent processing and storage. In addition, peptide moieties can be added to variants of the present invention to facilitate purification, for example by adding histidine residues or T7 tags.

[0185] Variants of the present invention, such as the proteins of the present invention or functional equivalents thereof, such as bioactive portions and fragments thereof, can be operably linked to a non-variant polypeptide (e.g., a heterologous amino acid sequence) to form a fusion protein. For the purposes of this, "non-variant polypeptide" refers to a polypeptide having an amino acid sequence corresponding to a protein that is not substantially homologous to the lactase variant of the present invention.

[0186] In the fusion protein, the variant of the present invention can correspond to the full-length sequence or a bioactive fragment of the polypeptide of the present invention. In a preferred embodiment, the fusion protein of the present invention comprises at least two bioactive portions. In the fusion protein, the term "operably linked" is intended to indicate that the variant polypeptide and the non-variant polypeptide are fused to each other in a manner that is in frame. The non-variant polypeptide can be fused to the N-terminus or the C-terminus of the variant polypeptide.

[0187] The expression and secretion of variant lactase can be enhanced by expressing the variant in the form of a fusion protein. In this regard, the nucleic acid sequence can encode a fusion protein comprising lactase. More specifically, the fusion partner can be glucoamylase or a fragment thereof. In one embodiment, lactase or its fusion protein is secreted through the host cell membrane.

[0188] For example, in one embodiment, the fusion protein is a fusion protein in which the variant sequence is fused to the C-terminus of the GST sequence. Such fusion proteins can facilitate the purification of the recombinant variants according to the present invention. In another embodiment, the fusion protein is a variant of the present invention that contains a heterologous signal sequence at its N-terminus. In certain host cells (e.g., mammalian and yeast host cells), the expression and / or secretion of the variants of the present invention can be increased by using a heterologous signal sequence.

[0189] In another example, the gp67 secretion sequence of the baculovirus envelope protein can be used as a heterologous signal sequence (Current Protocols in Molecular Biology, edited by Ausubel et al., John Wiley & Sons, 1992). Other examples of eukaryotic heterologous signal sequences include the secretion sequences of melittin and human placental alkaline phosphatase (Stratagene; La Jolla, California). In yet another example, useful prokaryotic heterologous signal sequences include the phoA secretion signal (Sambrook et al., ibid.) and the protein A secretion signal (Pharmacia Biotech; Piscataway, New Jersey).

[0190] Signal sequences can be used to facilitate the secretion and isolation of variants of the invention. Signal sequences are generally characterized by a core of hydrophobic amino acids, which are typically cleaved from the mature protein during one or more cleavage events during secretion. Such signal peptides contain processing sites that allow the signal sequence to be cleaved from the mature protein as it passes through the secretory pathway. The signal sequence can direct the secretion of the variant, for example, from a eukaryotic host transformed with an expression vector, and the signal sequence can be cleaved subsequently or simultaneously. The variant of the invention can then be easily purified from the extracellular medium by known methods. Alternatively, a sequence that facilitates purification can be used, for example, by linking the signal sequence to the target variant with a GST domain. Thus, for example, the sequence encoding the variant of the invention can be fused to a tag sequence, such as a sequence encoding a peptide that facilitates the purification of a fusion variant of the invention. In certain preferred embodiments of this aspect of the invention, the tag sequence is a hexahistidine peptide, such as the tag provided in the pQE vector (Qiagen, Inc.), many of which are commercially available. As described, for example, by Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), the hexahistidine provides convenient purification of the fusion protein. The HA tag is another peptide used for purification, which corresponds to an epitope derived from the influenza virus hemagglutinin protein that has been described, for example, by Wilson et al., Cell 37:767 (1984).

[0191] Fusion proteins of the invention can be generated by standard recombinant DNA techniques. For example, DNA fragments encoding different polypeptide sequences are ligated together in-frame according to conventional techniques, such as by ligation using blunt or staggered ends, digestion with restriction enzymes to provide appropriate ends, filling in sticky ends as appropriate, treatment with alkaline phosphatase to avoid unwanted ligation, and enzymatic ligation. In another embodiment, the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers. Alternatively, anchored primers can be used to PCR amplify gene fragments, which can generate complementary overhangs between two successive gene fragments, and subsequent annealing and reamplification can be performed to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, edited by Ausubel et al. John Wiley & Sons: 1992). Moreover, many expression vectors that already encode a fusion moiety (e.g., a GST polypeptide) are commercially available. The nucleic acid encoding the variant can be cloned into such an expression vector such that the fusion moiety is ligated to the variant in-frame.

[0192] The terms "functional equivalent" and "functional variant" are used interchangeably herein. A functional equivalent according to the present invention is an isolated DNA fragment encoding a polypeptide that exhibits a specific function as defined herein for the variant. Thus, functional equivalents also encompass bioactive fragments and are themselves encompassed within the term "variant" of the present invention.

[0193] Preferably, the functional equivalents of the present invention contain one or more of the substitutions described herein. However, in addition to the above substitutions, the functional equivalents may contain one or more modifications.

[0194] Functional nucleic acid equivalents typically may contain silent mutations or mutations that do not alter the biological function of the encoded polypeptide. Thus, the present invention provides nucleic acid molecules encoding variant lactase proteins containing changes in amino acid residues that are not essential for a particular biological activity. Such variant proteins differ in amino acid sequence from the parental lactase sequences from which they are derived while retaining at least one of their biological activities, preferably they retain at least lactase activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence that is substantially homologous to a reference amino acid sequence (such as the sequence shown in SEQ ID NO:2) having at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or higher homology.

[0195] As defined herein, the term "substantially homologous" means that a first amino acid or nucleotide sequence contains a sufficient or minimum number of amino acids or nucleotides that are the same as or equivalent to (e.g., having similar side chains) a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have a common domain. For example, amino acid or nucleotide sequences containing a common domain having about 60%, preferably 65%, more preferably 70%, even more preferably 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity or higher are defined herein as being sufficiently identical.

[0196] Those skilled in the art will recognize that changes can be introduced into the nucleotide sequences according to the present invention by mutation, thereby producing changes in the amino acid sequence of the resulting protein while substantially not altering the function of such protein.

[0197] Accordingly, the lactase variants of the present invention are preferably proteins comprising an amino acid sequence that is at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more homologous to a reference amino acid sequence (such as the sequence shown in SEQ ID NO:2), and generally also retain at least one functional activity of the reference polypeptide. The variants of the present invention can also be identified (for example) by screening for lactase activity in a combinatorial library of mutants of the proteins of the present invention, such as truncated mutants, for example, functional equivalents of the proteins of the present invention. In one embodiment, a diverse library of variants is generated by combinatorial mutagenesis at the nucleic acid level. A diverse library of variants is generated by (for example) enzymatically ligating a mixture of synthetic oligonucleotides into a gene sequence such that a set of degenerate potential protein sequences can be expressed as individual polypeptides or as a larger set of fusion proteins (such as for phage display). There are various methods that can be used to generate a library of potential variants of the polypeptides of the present invention from degenerate oligonucleotide sequences. Methods for synthesizing degenerate oligonucleotides are known in the art (see, for example, Narang (1983) Tetrahedron 39:3; Itakura et al. (1984) Annu. Rev. Biochem. 53:323; Itakura et al. (1984) Science 198:1056; Ike et al. (1983) Nucleic Acid Res. 11:477).

[0198] In addition, a library of fragments of the sequences encoding the polypeptides of the present invention can be used to generate a diverse population of polypeptides for screening for subsequent variant selection. For example, a library of coding sequence fragments can be generated as follows: by treating a double-stranded PCR fragment of a target coding sequence with a nuclease under conditions where each molecule is nicked only approximately once, denaturing the double-stranded DNA, allowing the DNA to renature to form double-stranded DNA that can include sense / antisense pairs from different nicked products, removing single-stranded portions from the re-formed duplexes by treatment with S1 nuclease, and ligating the resulting fragment library into an expression vector. By this method, an expression library encoding different sized N-terminal and internal fragments of the target protein can be obtained.

[0199] In the art, several techniques are known for screening gene products of combinatorial libraries generated by truncated point mutations, and for screening gene products in cDNA libraries having a selected property. The most widely used techniques for screening large gene libraries, suitable for high-throughput analysis, generally include: cloning the gene library into a replicable expression vector, transforming appropriate cells with the resulting vector library, and expressing the combinatorial genes under conditions where detection of the desired activity facilitates isolation of the vector encoding the gene whose product is detected. Recursive ensemble mutagenesis (REM), a technique for enhancing the frequency of functional mutants in a library, can be used in combination with screening assays to identify variants of the proteins of the invention (Arkin and Yourvan (1992) Proc. Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331).

[0200] Fragments of the polynucleotides according to the invention may also comprise polynucleotides that do not encode a functional polypeptide. Such polynucleotides may serve as probes or primers for PCR reactions.

[0201] The nucleic acids according to the invention, whether they encode a functional polypeptide or a non-functional polypeptide, can be used as hybridization probes or polymerase chain reaction (PCR) primers. Uses of the nucleic acid molecules of the invention that do not encode a polypeptide having lactase activity include, in particular: (1) in situ hybridization with metaphase chromosome spreads (e.g., FISH) to provide precise chromosomal localization of the lactase-encoding gene, as described by Verma et al., Human Chromosomes: a Manual of Basic Techniques, Pergamon Press, New York (1988); (2) Northern blot analysis for detecting expression of lactase mRNA in specific tissues and / or cells; and (3) primers and probes that can be used as diagnostic tools to analyze the presence of nucleic acids that can hybridize to such probes or primers in a given biological (e.g., tissue) sample.

[0202] Variants of a given reference lactase can be obtained by the following standard procedures:

[0203] - Synthesis or mutagenesis of the variant (error-prone, doped oligo, spiked oligo)

[0204] - Transformation, for example, in Escherichia coli or Kluyveromyces lactis

[0205] - Culturing of the transformants, selection of the transformants

[0206] - Expression

[0207] - Optional purification and concentration

[0208] - Preliminary screening

[0209] - Identification of improved variants (e.g., with respect to specific activity).

[0210] In one embodiment, the present invention relates to a method for producing a lactase polypeptide variant according to the present invention, the method comprising:

[0211] a) Selecting a reference lactase polypeptide;

[0212] b) Substituting at least one amino acid residue corresponding to any one of 233, 257, 258, 263, 274, 284, 297, 415, 440, 483, 619, 621, 622, 633, 862 or 1004,

[0213] wherein the positions are defined with reference to SEQ ID NO:2;

[0214] c) Optionally substituting one or more additional amino acids as defined in b);

[0215] d) Preparing the variant produced by steps a)-c);

[0216] e) Determining the properties of the variant, such as as shown in the examples;

[0217] f) Selecting a variant having altered properties compared to the reference lactase polypeptide, and wherein the variant has at least 60% sequence identity with SEQ ID NO:2.

[0218] In a preferred embodiment of the method for producing a lactase polypeptide variant according to the present invention, the reference lactase polypeptide has the sequence listed in SEQ ID NO:2.

[0219] More preferably, in step b) of the method according to the present invention, substituting at least one amino acid residue corresponding to

[0220] any one of 233, 257, 258, 263, 274, 284, 297, 415, 440, 483, 619, 621, 622, 633, 862 or 1004,

[0221] wherein the positions are defined with reference to SEQ ID NO:2. The reference polypeptide may have at least about 80% homology with SEQ ID NO:2.

[0222] In another embodiment, the invention features a cell containing a nucleic acid encompassed by the invention, such as a transformed host cell or a recombinant host cell. A "transformed cell" or "recombinant cell" is a cell into which (or into an ancestor of which) a nucleic acid according to the invention has been introduced by means of recombinant DNA technology. This includes both prokaryotic and eukaryotic cells, for example, bacteria, fungi, yeast, etc., and cells from yeast, such as Kluyveromyces lactis, are particularly preferred. Host cells also include, but are not limited to, mammalian cell lines such as CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and choroid plexus cell lines.

[0223] Examples of suitable bacterial host organisms are Gram-positive bacterial species such as the Bacillaceae, including Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus circulans, Bacillus lautus, Bacillus megaterium, and Bacillus thuringiensis; 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 Gram-negative bacterial species such as those belonging to the Enterobacteriaceae, including Escherichia coli, or species belonging to the Pseudomonadaceae can be selected as host organisms.

[0224] Suitable yeast host organisms may advantageously be selected from the species of Saccharomyces, including Saccharomyces cerevisiae, or species belonging to Schizosaccharomyces. Other useful yeast host organisms include Pichia, such as its methylotrophic species, including Pichia pastoris, and Kluyveromyces, including Kluyveromyces lactis.

[0225] Suitable host organisms among filamentous fungi include species of the genera Acremonium, Aspergillus, Fusarium, Humicola, Mucor, Myceliophtora, Neurospora, Penicillium, Thielavia, Tolypocladium or Trichoderma, such as Aspergillus aculeatus, Aspergillus awamori, Aspergillus foetidus, Aspergillus japonicus, Aspergillus oryzae, Aspergillus nidulans or Aspergillus niger, including Aspergillus niger var. awamoriawamori), Fusarium bactridioides, Fusarium 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, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesii or Trichoderma viride.

[0226] Host cells can be selected that can regulate the expression of the inserted sequence or that can modify or process the product of the incorporated nucleic acid sequence in a specific and desired manner. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of the protein product can facilitate the optimal function of the encoded protein.

[0227] A variety of host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems familiar to those skilled in the fields of molecular biology and / or microbiology can be selected to ensure that the modification and processing of the expressed foreign protein are desirable and correct. For this purpose, eukaryotic host cells with intracellular processing mechanisms can be used, which are used for the correct processing of primary transcripts, glycosylation, and phosphorylation of gene products. Such host cells are well known in the art.

[0228] If desired, stably transfected cell lines can produce variants according to the present invention. Many vectors suitable for the stable transfection of mammalian cells are publicly available, and methods for constructing such cell lines are also well known, for example, in Ausubel et al. (ibid.).

[0229] The present invention also discloses a composition comprising a lactase variant according to the present invention. Thus, the present invention provides a composition comprising a variant polypeptide as described herein and at least one component selected from salts (such as sodium chloride or potassium chloride), preservatives, polyols (such as glycerol), and metal ions (such as magnesium or manganese ions).

[0230] The composition may optionally contain other ingredients, such as other enzymes. Such compositions may contain the variant polypeptide of the present invention or variant polypeptides obtainable by the method for identifying variant lactase of the present invention.

[0231] In addition to the variant lactase and one or more additional enzymes (if present), the composition according to the present invention may contain additives conventionally used in the preparation of lactase, such as KCl or glycerol.

[0232] The present invention also relates to the use of the variant polypeptide or the composition of the present invention in the preparation of dairy products.

[0233] The present invention also relates to a method for producing dairy products, which comprises adding an effective amount of the variant polypeptide or composition of the present invention to milk and allowing the variant polypeptide to exert its enzymatic activity.

[0234] The present invention relates to dairy products obtainable by such methods.

[0235] As used herein, dairy products encompass any composition produced from milk, such as casein and / or whey protein. Examples are milk, milk-derived products, fermented dairy products (such as yogurt), condensed milk, UHT milk, evaporated milk, milk powder, frozen milk, ice cream, cheese, butter, buttermilk, whey, and / or curd. The product can also be a hydrolyzate or a product obtained by fractionation of milk or whey, such as caseinates, milk protein concentrate, whey protein concentrate (WPC), whey protein isolate (WPI), or (concentrated) whey permeate and products made therefrom.

[0236] Milk is obtained, for example, from cows, buffaloes, goats, sheep, camels, donkeys, horses, reindeer, moose, or yaks.

[0237] The citation of patent documents or other materials as prior art herein should not be taken as an admission that, as of the priority date of any claim, such document or material was known or that the information contained therein was part of the common general knowledge.

[0238] The present invention will now be illustrated with reference to the following examples, which, however, are not intended to limit the present invention. Examples

[0239] General materials and methods

[0240] Molecular and Genetic Techniques

[0241] Standard genetic and molecular biology techniques are known in the art (e.g., Maniatis et al., “Molecular cloning: a laboratory manual” (1982) Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Miller, “Experiments in molecular genetics” (1972) Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Sambrook and Russell, “Molecular cloning: a laboratory manual” (3rd ed.) (2001) Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press; Ausubel, “Current protocols in molecular biology” (1987) Green Publishing and Wiley Interscience, New York).

[0242] Plasmids and Strains

[0243] pBAD / HisA was obtained from Invitrogen TM (Life Technologies Corporation, Carlsbad, CA, USA). The β-galactosidase-deficient strain Escherichia coli BW25113 (Δ(araD-araB)567, ΔlacZ4787(::rrnB-3), λ - , rph-1, Δ(rhaD-rhaB)568, hsdR514) (Datsenko KA, Wanner BL (2000) Proc Natl Acad Sci USA 97:6640–6645) was used for the expression of Kluyveromyces lactis β-galactosidase variants.

[0244] Culture Media

[0245] 2xPY medium (16 g / l BD BBL TM Phytone TM peptone, 10 g / l yeast extract, 5 g / l NaCl) was used for the growth of E. coli. Antibiotics (100 μg / ml ampicillin) were supplemented to maintain the plasmid. L-arabinose at a final concentration of 0.02% was used to induce gene expression.

[0246] Example 1: DNA Constructs and Transformation

[0247] The synthetic DNA construct was designed to start with a BbsI restriction site that generates NcoI-compatible overhangs and end with a BbsI restriction site that generates HindIII-compatible overhangs after the stop codon. Internal BbsI restriction sites were removed in the design of the synthetic DNA construct. As an example, the DNA fragment encoding the wild-type Kluyveromyces lactis β-galactosidase sequence is listed as SEQ ID NO:1. All variants were designed in a similar manner and cloned as BbsI fragments into the NcoI / HindIII sites of the expression vector pBAD / HisA.

[0248] The amino acid changes introduced in 14 variants are depicted in Table 2. The positions of the changes are indicated compared to the wild-type Kluyveromyces lactis β-galactosidase amino acid sequence (SEQ ID NO:2). Some variants have multiple changes introduced into the amino acid sequence of the β-galactosidase protein, such as variants #8 and #7. The wild-type gene encoding the β-galactosidase protein with no changes was also used for gene cloning and transformation and was later used for comparison with the enzymes produced with the variant genes.

[0249] Table 2: Amino acid changes introduced into the protein sequence of Kluyveromyces lactis β-galactosidase. Amino acids are shown according to the single-letter notation.

[0250]

[0251] Transformation of Escherichia coli BW25113 was performed using the Zymo Research Z-Competent TM E. coli Transformation Kit and Buffer Set (T3001). The transformed E. coli strain was plated on 2xPY agar plates containing 100 μg / ml ampicillin, 0.02% L-arabinose, and 40 μg / ml 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal), and incubated overnight at 30 °C.

[0252] X-gal is a lactose analogue and is hydrolyzed by β-galactosidase. When X-gal is cleaved by β-galactosidase, galactose and 5-bromo-4-chloro-3-hydroxyindole are produced. The latter then spontaneously dimerizes and oxidizes to 5,5'-dibromo-4,4'-dichloro-indigo, which is an insoluble dark blue product. The plates were stored at 4 °C for at least 24 h to allow blue formation after X-Gal hydrolysis. Since the wild-type E. coli strain BW25113 lacks β-galactosidase activity due to the lac operon deletion, the formation of blue confirmed the active expression of the selected β-galactosidase variant. In each construct, β-galactosidase production of three blue-forming transformants was detected using small-scale 24-well cultures (Example 2), and the best-producing transformant was selected for further enzyme characterization.

[0253] Example 2: Culturing and Preparation of β-Galactosidase Samples

[0254] E. coli BW25113 transformants expressing the variant β-galactosidase gene were replicated from the agar plates to a 96-well plate with 200 μl of 2*PY and 100 μg / ml ampicillin (NUNC 267334, NUNC A / S, Roskilde, Denmark), and then incubated overnight at 30 °C, 550 rpm, and 80% humidity in an INFORS HT Microtron shaker (Infors AG, Bottmingen, Switzerland). A 24-well plate (AXYGPDW10ML24CLIDS, Axygen) containing 3 ml of 2*PY and 100 μg / ml ampicillin was inoculated with 15 μl from these precultures TM, Corning, NY 14831 USA). The 24-well plates were covered with a breathable sealing material (6786051, greiner bio-one, Frickenhausen, Germany) and incubated in an INFORS HT Microtron shaker at 30 °C, 550 rpm, and 80% humidity until an optical density at 600 nm of 0.4 - 0.6 was reached. Then, L-arabinose was added to a final concentration of 0.02% and the 24-well plates were further incubated in an INFORS HT Microtron shaker at 20 °C, 750 rpm, and 80% humidity for 20 - 24 hours. The 24-well plates were centrifuged at 2750 rpm and 4 °C for 10 minutes and the supernatant was removed by decanting the plates. The obtained cell pellets were stored at -20 °C for at least 24 hours. The frozen cell pellets were resuspended in 1 ml of extraction buffer (50 mM Tris-HCl pH 7.5, 0.2 mM MgSO4, 2 mg / ml lysozyme, 0.1 mg / ml DNAse I, 1x complete protease inhibitor mixture (without EDTA, Roche)) by vortexing, incubated at room temperature for 30 minutes, and then centrifuged at 2750 rpm and 4 °C for 10 minutes. The supernatant containing overexpressed β-galactosidase (cell-free extract, CFE) was formulated by adding 1 volume of glycerol and used in different activity assays.

[0255] Example 3: Determination of the Amount of Lactase Protein

[0256] The amount of lactase protein produced by E. coli was determined using HP-SEC (Thermo Scientific Dionex UltiMate 3000 Rapid Separation). For this, 2 μl of the cell-free extract (CFE) from Example 1 was loaded onto a BEH200, sec 1.7 μm 4.6X150 mm column (Waters). The mobile phase consisted of 100 mM potassium phosphate buffer (pH 7.32) and was maintained at a flow rate of 0.1 mL / min. The column temperature was maintained at 25 °C while the flow rate was set at 0.1 mL / min. After protein elution, the absorbance at 280 nm was measured. Since the lactase protein is larger than most other proteins in the CFE, it was the first protein peak to elute from the column under these conditions. The area under this peak was calculated and quantified by comparison with a bovine serum albumin (BSA) standard. As described in Examples 4 - 7, this quantification result was used to calculate the (specific) activity of the protein.

[0257] Table 3A: Analysis results of the (specific) activity of variants in various assays described in Examples 4-7. Values significantly (p<0.05) higher or lower (inhibition %) than the average value of the wild-type lactase (6 samples) are marked.

[0258]

[0259] Table 3B: Analysis results of the (specific) activity of variants in various assays described in Examples 4-7. The values in Table 3A are shown as relative values compared to the values of the wild-type enzyme in the same assay.

[0260]

[0261] Example 4: Activity Assay Against ONPG as Substrate

[0262] The activity assay using o-nitrophenyl-β-D-galactopyranoside (ONPG) as a substrate was carried out essentially according to the procedure described in the Food Chemical Codex (FCC 8th Edition, pages 1319 - 1320: Lactase (neutral) β-galactosidase activity).

[0263] The sample generated in Example 2 was diluted 200-fold using Buffer A (100 mM potassium phosphate (pH 6.5) containing 0.05 mM EDTA, 0.1 mM MgSO4, and 0.2% (w / v) Triton X100) until approximately 0.1 neutral lactase units (NLU) / mL. The substrate (50 mg of o-nitrophenyl-β-D-galactopyranoside (Sigma-Aldrich) in 20 mL) was prepared using the same buffer without Triton X100. After preheating the substrate, the following materials were mixed together: 125 μL of substrate and 25 μL of sample. The reaction was allowed to proceed at 37 °C for 10 minutes, after which the reaction was terminated by adding 25 μL of sodium carbonate (30 g / L) and 20 μL of ultrapure water. The resulting absorbance at 405 nm was measured and compared to a calibration curve prepared from o-nitrophenol (ONP). Measurements were carried out on a Konelab clinical analyzer (Thermo Scientific Arena 30). Activity was calculated as described in the Food Chemical Codex and corrected for differences in assay temperature. The correction factor was 1.25 and was determined empirically. The specific activity of different lactase variants was determined by dividing these values by the protein dose in the assay (as described in Example 3) and the results are depicted in Table 3.

[0264] Example 5: Activity Assay Against Lactose as Substrate

[0265] Dilute the sample to approximately 0.4 NLU / mL in buffer B (100 mM sodium phosphate (pH 6.5) containing 0.05 mM EDTA and 1 mM MgSO4). The substrate consists of lactose monohydrate dissolved at 4.8% in buffer B. The enzyme mixture consists of 780 units of horseradish peroxidase (Sigma Aldrich), 0.25 units of glucose oxidase (DSM), and 12.5 mg (+ / - 1 mg) of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS, Sigma Aldrich) in a total of 10 mL of buffer B. The assay is measured relative to serial dilutions of neutral lactase (0.1 - 0.8 NLU / mL). To initiate the reaction, transfer the following materials to a well of a standard microtiter plate: 25 μL of buffer B, 25 μL of sample or standard, and 25 μL of enzyme mixture. After pre-incubating for 10 minutes, add 175 μL of substrate and measure the reaction at 420 nm and 30 °C on an MTP reader (Tecan Infinity M1000) for 30 minutes. Measure the absorbance every 30 seconds and calculate the slope for every 5 data points (2.5 minutes). Calculate the activity using the maximum slope during the entire assay. This maximum slope is expressed as the number of μmol of glucose produced per minute by lactase under the conditions described herein (LACU). Calculate the specific activity of different lactase variants by dividing these values by the protein concentration (mg / ml) in the assay (as described in Example 3). The specific activities (LACU / mg) of these lactase variants are depicted in Table 3. A high specific activity against lactose can result in a lower dosage of the enzyme in potential applications.

[0266] Example 6: Activity Assay in the Presence of Galactose

[0267] Dilute the sample to approximately 0.4 NLU / mL in buffer B. The substrate consists of 4.8% lactose monohydrate dissolved in buffer B. The enzyme mixture consists of 780 units of horseradish peroxidase (Sigma Aldrich), 0.25 units of glucose oxidase (DSM), and 12.5 mg (+ / - 1 mg) of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS, Sigma Aldrich) in a total of 10 mL of buffer B. The inhibition buffer C consists of 1500 mM galactose (purity > 99.9%) in buffer B. The assay is measured relative to serial dilutions of neutral lactase (0.1 - 0.8 NLU / mL). To initiate the reaction, transfer the following materials to a well of a standard microtiter plate: 25 μL of buffer C (except for standards), 25 μL of sample or standard, and 25 μL of enzyme mixture. After pre-incubating for 10 minutes, add 175 μL of substrate and measure the reaction at 420 nm and 30 °C on an MTP reader for 30 minutes. Measure the absorbance every 30 seconds and calculate the slope for every 5 data points. Calculate the activity using the maximum slope throughout the assay. This maximum slope is expressed as the number of μmol of glucose produced per minute by lactase under the conditions described herein (LACGU). Calculate the specific activity (LACGU / mg) of different lactase variants by dividing these values by the protein concentration (mg / ml) in the assay (as described in Example 3) and the results are shown in Table 3.

[0268] Calculate the percent galactose inhibition using the following formula

[0269] % Inhibition = 100 * (x – y) / x

[0270] where x represents the specific activity in LACU / mg lactase as described in Example 5, and y represents the specific activity in LACGU / mg lactase as described in Example 6. The calculated results of % Inhibition are shown in Table 3. When low residual lactose concentrations are required, a low % Inhibition under conditions in applications with low lactase concentration and high galactose concentration can result in higher activity.

[0271] Example 7: Activity Assay in Cold Milk

[0272] 1 mL of commercial semi-skimmed UHT milk (Campina) was mixed with 0.2 mL of the enzyme produced in Example 2 (approx. 20 NLU / mL) in a deep-well microtiter plate. The samples were incubated at 6 °C for 4, 24 or 48 h under static conditions. After such incubation, the reaction was terminated by heat treatment at 90 °C for 6 min, after which the samples were placed directly in a -20 °C freezer until analysis. Sample preparation for NMR was carried out as follows: 48 μL of 4.0 M HCl was added, then the plate was sealed and mixed by tilting. Then, the plate was shaken at 600 rpm for 20 min and subsequently centrifuged at 4750 rpm for 10 min. 0.3 mL was transferred from the clear supernatant to a new plate and combined with 0.2 mL of a solution containing 20 g / L maleic acid (internal standard) and 40 g / L EDTA in D2O. The plate was sealed, mixed by tilting and briefly centrifuged. After lyophilization, the residue was dissolved in 0.05 mL of D2O and lyophilized again. The dried residue was dissolved in 0.7 mL of D2O, lyophilized overnight and redissolved in 0.7 mL of D2O. After careful mixing, the sample was centrifuged at 4750 rpm for 10 min and 0.6 mL was transferred to an NMR tube. Samples were measured on a Bruker Avance III spectrometer operating at a proton frequency of 700 MHz with a probe temperature of 290 K equipped with a cryoprobe. Samples were measured singly with 8 scans and a 30 s delay. From the NMR spectra, the following compounds were quantified: lactose (δ = 4.67 (d)), glucose δ = 4.64 (d), galactose δ = 4.58 (d) and galacto-oligosaccharides (GOS, integration of the area from δ = 4.52 to approximately δ = 4.38). In Table 3, the amount of glucose detected per mg of added enzyme after 4 h of incubation is indicated. Also depicted is the amount of GOS detected after 48 h, when most of the lactose was hydrolyzed and little residual lactose (<0.5 g / l) remained.

[0273] The high lactose hydrolysis activity of the enzyme in cold (4 - 12 °C) milk can lead to a reduction in the enzyme dosage and thus cost in such applications related to the dairy industry. The increased GOS production can lead to a prebiotic effect of the produced milk.

[0274] Example 8: Combinations of Lactase Variants

[0275] Different combinations of mutations in the lactase gene were generated as described in Example 1, with the exception that: multiple amino acid change combinations were in the expression products of the gene constructs. Different variants containing these combined amino acid changes are depicted in Table 4. Compared to the wild-type Kluyveromyces lactis β-galactosidase amino acid sequence (SEQ ID NO:2), the positions of the changes are indicated.

[0276] Table 4: Amino acid changes introduced into the protein sequence of Kluyveromyces lactis β-galactosidase. Amino acids are shown according to the single-letter notation.

[0277]

[0278] Similarly, the modified lactase gene was expressed in E. coli and the lactase protein was isolated as described in Example 2. The amount of the expressed lactase protein was determined as described in Example 3. The activity of these enzyme samples towards lactose hydrolysis was determined as described in Example 5 and compared with the activity of the wild-type enzyme expressed and isolated in exactly the same manner. The activity of the enzyme samples towards lactose hydrolysis in cold milk after 4 hours was also determined as described in Example 7.

[0279] The specific activity of the different lactase variants was calculated by dividing the measured value by the lactase protein concentration (mg / ml) in the assay. The specific activity of these lactase variants in the two assays was expressed as the relative activity compared to the activity of the wild-type enzyme obtained in the same assay. For this purpose, the specific activity of the wild-type lactase was set to 100 in the two assays, and the calculated specific activity of the variants was relative to this. The results of this analysis are depicted in Table 5.

[0280] Table 5: Analysis results of the (specific) activity of variants in each assay. The values are depicted relative to the values found with the wild-type lactase Marked are the values significantly (p<0.05) higher than the average value of the wild-type lactase.

[0281]

[0282] It can be concluded from this analysis that several combinatorial variants showed favorable lactose hydrolysis in both assays.

Claims

1. A variant polypeptide having lactase activity, wherein the amino acid sequence of the variant is a substitution selected from SEQ ID NO:2 as follows: Y440F; Y440F, V619I, T633G; Y440F, V619I, A483S; Y440F, V619I, T633G, A258T, A483S; Y440F, V619I, T415A, A258T, A483S; Y440F, V619I, T633G, L862V; Y440F, V619I, T415A, L862V; Y440F, V619I, T415C, L862V; Y440F, V619I, A483S, L862V; Y440F, T415A; Y440F, V619I, T415C; A258T, V619I, Y440F, E264V, A483S; and A258T, V619I, Y440F, L862V, E264V, A483S; The positions are defined with reference to SEQ ID NO:2 and wherein the variant has one or more altered properties compared to a reference polypeptide having lactase activity, the altered properties being selected from Increased specific activity against lactose; and Increased activity against lactose in milk; And wherein the reference polypeptide is the lactase of SEQ ID NO:

2.

2. The variant polypeptide according to claim 1, wherein the variant polypeptide is a non-naturally occurring polypeptide.

3. A nucleic acid sequence encoding the variant polypeptide according to any one of claims 1-2.

4. A nucleic acid construct comprising the nucleic acid sequence according to claim 3 operably linked to one or more control sequences capable of directing the expression of lactase in a suitable expression host.

5. A recombinant expression vector comprising the nucleic acid construct according to claim 4.

6. A recombinant host cell comprising the expression vector according to claim 5.

7. A method for producing lactase, which comprises culturing the host cell according to claim 6 under conditions conducive to the production of the lactase and recovering the lactase.

8. A method for producing a lactase polypeptide variant according to any one of claims 1-2, the method comprising: (a) Select a polypeptide having lactase activity, wherein the polypeptide is the lactase of SEQ ID NO: 2; (b) Perform a Y440F substitution, the position being defined with reference to SEQ ID NO: 2; (c) Optionally further perform one or more substitutions selected from V619I, T633G, A483S, A258T, T415A, L862V, T415C and E264V; (d) Prepare a variant produced by steps (a)-(c); (e) Determine the properties of the variant; (f) Select a variant having altered properties compared to the polypeptide of (a), thereby producing a lactase polypeptide variant.

9. A composition comprising a variant polypeptide according to any one of claims 1-2 or a variant polypeptide obtainable by the method according to claim 8 and at least one component selected from salts, preservatives, polyols or metal ions.

10. Use of the variant polypeptide according to any one of claims 1-2 or the composition according to claim 9 in the preparation of low-lactose or lactose-free dairy products.

11. A method for producing a dairy product, the method comprising adding an effective amount of the variant polypeptide according to any one of claims 1-2 or the composition according to claim 9 to the dairy product and allowing the polypeptide to exert its enzymatic activity.

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