NZ720283B2 - Improved enzyme variants of lactase from kluyveromyces lactis - Google Patents

Abstract

The invention relates to a variant polypeptide having lactase activity. The invention also relates to a nucleic acid sequence encoding such a variant polypeptide, to a nucleic acid construct comprising said nucleic acid sequence, to a recombinant expression vector comprising said nucleic acid construct and to a recombinant host cell comprising said expression vector. Further, the invention relates to a method for producing a lactase variant via use of such a host cell. Also, the invention relates to a method of producing a lactase polypeptide variant. The invention further relates to a composition comprising a lactase variant, to use of such a lactase variant or to the use of a lactase variant-containing composition in the preparation of a dairy product, to a process for the production of a dairy product and to the resulting dairy product. uct and to a recombinant host cell comprising said expression vector. Further, the invention relates to a method for producing a lactase variant via use of such a host cell. Also, the invention relates to a method of producing a lactase polypeptide variant. The invention further relates to a composition comprising a lactase variant, to use of such a lactase variant or to the use of a lactase variant-containing composition in the preparation of a dairy product, to a process for the production of a dairy product and to the resulting dairy product.

Description

IMPROVED ENZYME VARIANTS OF LACTASE FROM KLUYVEROMYCES LACTIS Field of the invention The invention relates to a variant polypeptide having lactase activity. The invention also relates to a c acid sequence encoding such a variant polypeptide, to a nucleic acid construct comprising said nucleic acid sequence, to a recombinant expression vector comprising said nucleic acid uct and to a recombinant host cell sing said expression vector. Further, the invention relates to a method for producing a lactase variant via use of such a host cell. Also, the invention relates to a method of producing a lactase ptide variant. The invention further relates to a composition comprising a lactase variant, to use of such a lactase variant or to the use of a lactase variant-containing composition in the preparation of a dairy product, to a process for the production of a dairy product and to the resulting dairy product.

Background of the invention This invention s to lactase. Lactase or beta-galactosidase (E.C: 23) is an enzyme, which catalyzes the hydrolysis of e (a disaccharide) into its component ccharides glucose and galactose. Lactose is present in dairy products and more specifically in milk, skimmed milk, cream, ice cream, fermented milk products such as yogurt, many young cheeses and other dairy products. The breakdown of lactose occurs in the intestinal wall of juvenile mammals (among which are ) by the natural presence of lactase. Only a small part of the adult population has not lost this property and can still digest lactose. The nutritional and functional ms caused by lactose in most adults are caused by a lack of lactase and are well known and described. Members of such populations cannot hydrolyze lactose, which in such cases passes into the large intestine where it results in dehydration, poor calcium absorption, flatulence, belching and cramps, and, in severe cases, even watery explosive diarrhea.

An important industrial application of lactase is in the production of ehydrolyzed milk products for lactose intolerant duals. Such hydrolyzed milk products include pasteurized milk, lk and milk reconstituted from all or part of its original constituents with or without intermediate processing steps such as protein hydrolysis. Treatment with lactase may be done prior to or after the heat-treatment of the milk. The lactase treatment may be done by adding the enzyme to the milk or to one of its lactose-containing constituents.

The solubility properties of lactose are such that it may lead to its crystallization when present at high concentration, leading to a sandy or gritty texture in dairy products such as condensed milk, evaporated milk, dry milk, frozen milk, ice cream, and in confectionary products with a high t of milk. Full or l ysis of lactose by lactase eliminates this problem, providing products with a homogeneous texture and as a result a higher consumer acceptance. r industrial application of lactase is to increase sweet taste in lactose containing products like milk or t. The hydrolysis of lactose in such products results in increased sweet taste as a result of the production of glucose, while the caloric value of the product does not increase. Conversely, the use of lactase may also decrease sugar addition in sweetened dairy products, without compromising the sweet taste.

Another industrial application of lactase is the hydrolysis of lactose products containing dairy components such as bread. Lactose is added in such products to enhance r, retain moisture, provide browning and improve toasting properties.

Hydrolyzed lactose syrups are ing in terms of e.g. enhancing crust-colour development, improving flavour and aroma, modifying texture, extending shelf life and strengthening loaf ure.

Lactose hydrolysis by lactase in fermented milk products such as yoghurt will se sweet taste. Also, when the lactase is added prior to the beginning of the fermentative process, it may increase the rate of acid development and thus reduce processing times. The lactase treatment of milk or milk-derived products such as whey makes such products suitable for application in animal feed and pet food for lactose intolerant animals such as cats. The lactose hydrolysis allows the manufacture of higher concentrated whey and at the same time prevents gut problems, similar to those described r for lactose-deficient patients. Lactose hydrolyzed whey is concentrated to produce a syrup containing 70-75% solids and is used as a food ingredient in ice cream, bakery and confectionary ts.

Lactases have been described and isolated from a large variety or organisms, including micro-organisms. Lactase is often an intracellular component of rganisms like Kluyveromyces and Bacillus . Kluyveromyces and especially K. fragilis and K.lactis , and other yeasts such as those of the genera Candida , Torula and Torulopsis are a common source of yeast lactases, whereas B. coagulans, B. circulans or lactic acid bacteria are well known sources of bacterial lactases. Several cial lactase preparations, derived from these organisms are available such as Maxilact® (from K. lactis, produced by DSM, Delft, the Netherlands). These lactases are so called neutral lactases since they have a pH optimum between pH = 6 and pH = 8.

Although yeast neutral es are often used in industry to produce lactose-free or lactose-reduced dairy products, the cost-in-use for the enzyme treatment is often high.

Main reasons for the relative high cost-in-use of the enzyme are: · In order to maintain hygienic conditions in the production plant the incubation is performed at low temperature. At this temperature the industrially used lactases are not very active and should be added at ve high dosage.

· The currently used lactases are inhibited by its products, especially galactose, at later stages of the incubation with lactase. When products with a low residual lactose tration are required, extra enzyme has to be added to compensate for ion in activity due to galactose accumulation.

· The currently used lactase has a relative low ic activity in milk which es the use of a high enzyme dosage in application.

Consequently, enzyme dosage and costs for ing lactose-reduced and lactose-free products are relative high.

It is t that there is a need for one or multiple lactase variant(s) capable of overcoming at least one of the above-mentioned antages.

Summary of the ion The invention relates to a variant polypeptide having lactase activity, i.e. to a lactase variant. A lactase variant of the invention may have one or more improved properties in comparison with a reference polypeptide, the reference polypeptide typically having lactase activity. A reference polypeptide may be a wild-type lactase, for example the lactase from K. lactis. Variant polypeptides of the invention may be referred to as "lactase variant", an "improved lactase" and the like. Variants of Kluyveromyces neutral e were generated that have properties that lead to a ion of the costin-use of such lactases in the production of lactose-reduced or lactose-free dairy products. A lactase variant with an improved property relevant for dairy production may demonstrate: · a higher specific activity on ONPG; · a higher specific activity on lactose; · a higher activity on lactose in milk at low ature; · a reduction in galactose inhibition; and/or · a higher GOS production in milk.

Each of these improvements may be determined as compared with a reference polypeptide. Moreover, a variant polypeptide of the invention may have at least 2 or 3 or 4 improved properties in comparison with a reference polypeptide. Table 1 provides examples of combinations of improved ties.

According to the invention, there is thus provided a variant polypeptide having lactase activity, wherein the variant has an amino acid sequence which, when aligned with the lactase comprising the ce set out in SEQ ID NO: 2, comprises at least one substitution of an amino acid residue ponding to any of amino acids 233, 257, 258, 263, 274, 284, 297, 415, 440, 483, 619, 621, 622, 633, 862 or 1004 said positions being d with reference to SEQ ID NO: 2 and wherein the variant has one or more altered properties as compared with a reference polypeptide having lactase activity and wherein said variant has at least 60% ce identity with SEQ ID NO:2.

The invention also es: - a nucleic acid sequence encoding a variant of the invention; - a c acid construct comprising such a nuclei c acid sequence operably linked to one or more control sequences capable of directing the expression of a lactase in a suitable expression host; - a recombinant expression vector comprising such a nucleic acid uct; and - a recombinant host cell comprising such an expres sion vector.

The ion also relates to a variant polypeptide having lactase activity, wherein the variant has an amino acid sequence which, when aligned with the lactase comprising the sequence set out in SEQ ID NO: 2, comprises substitution: D233V, D257G/E297G, A258T, N263S/K274E/N284S, T415C, T415A, Y440F, A483S, V619I, I621V, M622L, T633G, L862V and/or A1004P said positions being defined with reference to SEQ ID NO: 2 and wherein the variant has one or more altered properties selected from: increased specific activity on ophenyl-β-D-galactopyranoside (ONPG); increased specific activity on lactose; increased activity on lactose in milk; decreased galactose inhibition; and/or increased galacto-oligosaccharides (GOS) production in milk as compared with a reference polypeptide having lactase activity and wherein said t has at least 80% sequence identity with SEQ ID NO:2, wherein the reference polypeptide is the lactase of SEQ ID NO:2.

The invention also s to a method for producing a lactase comprising cultivating the host cell of the invention under conditions conducive to production of the lactase and recovering the lactase.

Also, the invention relates to a method of ing a lactase polypeptide variant, which method comprises: a) selecting a polypeptide having lactase activity, wherein the polypeptide is the lactase of SEQ ID NO: 2; b) substituting at least one amino acid residue corresponding to any of amino acids 233, 257, 258, 263, 274, 284, 297, 415, 440, 483, 619, 621, 622, 633, 862 or said positions being d with reference to SEQ ID NO: 2; c) optionally tuting one or more further amino acids as defined in b); d) ing the t resulting from steps a)-c); e) determining a property of the variant; and f) selecting a t having an altered property in comparison to the lactase comprising the sequence set out in SEQ ID NO: 2 and selecting a variant having at least 60% sequence ty with SEQ ID NO:2, thereby to produce a lactase polypeptide variant.

The invention also relates to a method of producing a e polypeptide variant, which method comprises: (a) selecting a polypeptide having lactase activity; (b) substituting at least one amino acid residue corresponding to any of D233V, D257G/E297G, A258T, N263S/K274E/N284S, T415C, T415A, Y440F, A483S, V619I, I621V, M622L, T633G, L862V and/or A1004P, said position being defined with reference to SEQ ID NO:2; (c) ally substituting one or more further amino acids as defined in (b); (d) preparing the variant resulting from steps (a)-(c); (e) determining a property of the variant; (f) selecting a variant having an altered property in comparison to the polypeptide of (a), thereby producing a lactase polypeptide t.

Further the invention relates to: - a composition comprising the variant of the inven tion or obtainable by a method of the invention; - use of a variant lactase according to the inventi on or of a composition of the invention in the preparation of a dairy product; - a s for the production of a dairy product, which method comprises comprising adding an effective amount of a variant lactase according to the invention or of a composition of the invention to milk and carrying out appropriate further dairy product manufacturing steps; and - a dairy product obtainable by such process or use .

Brief description of the sequence g SEQ ID NO 1 sets out the nucleic acid sequence of the wild type e gene sequence from K. lactis SEQ ID NO: 2 sets out the amino acid sequence of the lactase sequence from K. lactis . ed description of the invention hout the present specification and the accompanying claims, the words ise", "include" and "having" and variations such as "comprises", "comprising", "includes" and "including" are to be interpreted inclusively. That is, these words are ed to convey the possible inclusion of other elements or integers not specifically recited, where the context allows.

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

Claims (20)

1. A variant polypeptide having lactase activity, wherein the variant has an amino acid sequence which, when aligned with the lactase comprising the sequence set out in 5 SEQ ID NO: 2, comprises substitution D233V, D257G/E297G, A258T, N263S/K274E/N284S, T415C, T415A, Y440F, A483S, V619I, I621V, M622L, T633G, L862V and/or A1004Psaid positions being defined with reference to SEQ ID NO: 2 and wherein the variant has one or more altered properties selected from increased specific activity on o-nitrophenyl-β-D-galactopyranoside (ONPG); 10 increased specific activity on lactose; increased activity on lactose in milk; decreased galactose inhibition; and/or increased galacto-oligosaccharides (GOS) production in milk, as compared with a reference polypeptide having lactase activity and wherein said 15 variant has at least 80% sequence identity with SEQ ID NO:2, wherein the reference polypeptide is the lactase of SEQ ID NO:2.

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

3. A variant polypeptide according to any one of claims 1-2, wherein the variant 20 demonstrates increased specific activity on ONPG as compared with a reference polypeptide having lactase activity and comprises an amino acid sequence which, when aligned with the sequence set out in SEQ ID NO:2, comprises at least one substitution selected from T415C, T415A, A483S, V619I, I621V, M622L or T633G.

4. A variant polypeptide according to any one of claims 1-2, wherein the variant 25 demonstrates increased specific activity on lactose as compared with a reference polypeptide having lactase activity and comprises an amino acid sequence which, when aligned with the sequence set out in SEQ ID NO:2, comprises at least one substitution selected from T415C, T415A, Y440F or A483S.

5. A variant polypeptide according to any one of claims 1-2, wherein the variant 30 demonstrates increased activity on lactose in milk as compared with a reference polypeptide having lactase activity and comprises an amino acid sequence which, when aligned with the sequence set out in SEQ ID NO:2, comprises at least one substitution selected from D233V, D257G, A258T, N263S, K274E, N284S, E297G, Y440F, V619I,

6. T633G, L862V or A1004P. 35 6. A variant polypeptide according to claim 5, which comprises an amino acid sequence which, when aligned with the sequence set out in SEQ ID NO:2, comprises at least two substitutions selected from N263S, K274E or N284S. 50

7. A variant polypeptide according to claim 5, which comprises an amino acid sequence which, when aligned with the sequence set out in SEQ ID NO:2, comprises at least substitutions D257G and E297G.

8. A variant polypeptide according to any one of claims 1-2, wherein the variant 5 demonstrates decreased galactose inhibition as compared with a reference polypeptide having lactase activity and comprises an amino acid sequence which, when aligned with the sequence set out in SEQ ID NO:2, comprises at least one substitution selected from V619I, I621V or M622L.

9. A variant polypeptide according to any one of claims 1-2, wherein the variant 10 demonstrates increased GOS production in milk as compared with a reference polypeptide having lactase activity and comprises an amino acid sequence which, when aligned with the sequence set out in SEQ ID NO:2, comprises at least one substitution selected from V619I or M622L.

10. A variant polypeptide according to any one of the preceding claims which 15 comprises additional substitutions other than those defined in claim 1.

11. A nucleic acid sequence encoding a variant polypeptide according to any one of the preceding claims.

12. A nucleic acid construct comprising the nucleic acid sequence of claim 11 operably linked to one or more control sequences capable of directing the expression of 20 a lactase in a suitable expression host.

13. A recombinant expression vector comprising the nucleic acid construct of claim 12.

14. A recombinant bacterial, fungal or yeast host cell comprising the expression vector of claim 13. 25

15. A method for producing a lactase comprising cultivating the host cell of claim 14 under conditions conducive to production of the lactase and recovering the lactase.

16. A method of producing a lactase polypeptide variant, which method comprises: (a) selecting a polypeptide having lactase activity, wherein the polypeptide is the 30 lactase of SEQ ID NO: 2; (b) substituting at least one amino acid residue corresponding to any of D233V, D257G/E297G, A258T, N263S/K274E/N284S, T415C, T415A, Y440F, A483S, V619I, I621V, M622L, T633G, L862V and/or A1004P, said position being defined with reference to SEQ ID NO:2; 51 (c) optionally substituting one or more further amino acids as defined in (b); (d) preparing the variant resulting from steps (a)-(c); (e) determining a property of the variant; (f) selecting a variant having an altered property in comparison to the polypeptide 5 of (a), thereby producing a lactase polypeptide variant.

17. A composition comprising the variant polypeptide according to any one of claims 1 to 10 or obtained by a method according to claim 16 and at least one component selected from salt, preservative, polyol or metal ions.

18. Use of a variant polypeptide according to any one of claims 1 to 10 or of a 10 composition according to claim 17 in the preparation of a low lactose or lactose free dairy product.

19. A process for the production of a dairy product, which method comprises adding an effective amount of a variant polypeptide according to any one of claims 1 to 10 or of a composition according to claim 17 to a dairy product and allowing the polypeptide to 15 exerts its enzymatic activity.

20. A dairy product comprising the variant polypeptide according to any one of claims 1 to 10.