Galactolipase, as determined by the galactolipase / phospholipase ratio and / or galactolipase / lipase activity ratio, and its uses in bakeries.

Abstract

The present invention provides a polypeptide having galactolipase activity, characterized in that the ratio of galactolipase activity to phospholipase activity is 100.0 or more, and / or the ratio of galactolipase activity to lipase activity is 150.0 or more. The specification also provides isolated nucleic acids encoding such a polypeptide having galactolipase activity, expression vectors containing such nucleic acids, and host cells containing such nucleic acids or expression vectors. Furthermore, the specification provides the use of the polypeptide having galactolipase activity as a food additive or in the preparation of food products, and methods for preparing food products using the polypeptide having galactolipase activity. Finally, the specification provides dough products, batter products, or bakery products obtained by the use or methods described herein.

Description

Technical Field

[0005] ,

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[0006]

[0001] The present invention relates to novel polypeptides having galactolipase activity, particularly polypeptides having galactolipase activity with improved properties for use in dough, which can be used as an alternative to emulsifiers, but are not limited thereto.

Background Art

[0002] In the past few decades, due to the demand for extending the shelf life and consistent quality of baked goods, various additives have been used in the bakery industry. These additives, also called bread improvers, include, for example, emulsifiers, enzymes, soy flour, oxidants, and reducing agents, and are important for improving the machinability of dough, shortening bench time, and improving the shelf life, volume, crust color, crumb whiteness, aroma, and flavor of baked goods. <000001A><000001B><000001C>One of the important aspects in the baking process is fermentation control and dough handling. In fact, if the dough ferments too much or is subjected to shock during handling, the quality of the baked product may be significantly impaired. Therefore, those skilled in the art are constantly seeking ways to improve the "tolerance" of the dough. <000001D><000001E><000001F>Therefore, the tolerance of dough can be defined as the ability to withstand various processing stresses such as excessive fermentation time during fermentation, mechanical shock, and freezing. If the tolerance is improved, bakers can obtain bakery products of better and more uniform quality. Tolerance is usually related to the volume of the baked product. The volume is related, among other things, to the presence of a strongly developed gluten network. Emulsifiers are known to help give strength to this network. <000001G><000001H><000001I>Diacetyl tartaric acid esters of monoglycerides (DATEM) are emulsifiers widely used as bread improvers. DATEM improves the volume and texture of bread, as well as the stability of the dough. <000001J><000001K><000001L>In situ formation of emulsifying molecules can also be achieved by other means. For example, over the past 30 years, lipases have been used to improve several properties of baked goods, such as dough stability. Lipases hydrolyze triglyceride esters to produce mono- or di-glycerides, glycerol, and free fatty acids. Lipases enhance dough stability, improving the volume, texture, and shelf life of bread. In the 1990s, first-generation lipases (e.g., Thermomyces lanuginosus lipase) typically hydrolyze the ester bonds between glycerides and fatty acids at the 1st and 3rd positions of triglycerides, producing free fatty acids and mono-glycerides, increasing polar lipids in the dough. While strengthening the gluten network, excessive application can harden the dough and reduce the volume of the bread. Second-generation lipases (e.g., Fusarium oxysporum lipase) act on both polar and nonpolar lipids in wheat flour, producing even more polar components such as lysolecitin and digalactosylmonocylglycerol (DGMG). Third-generation lipases (e.g., engineered Thermomyces lanuginosus lipase) have a low affinity for short-chain fatty acids, expanding the gluten network and increasing the wall thickness, while decreasing the density of the gluten, thereby improving the properties of the baked product.

[0007] While it has been documented that lipases and / or phospholipases improve dough tolerance, their use remains highly unpredictable due to differences in their specificity, hydrolysis products, potential synergies, and process conditions and substrates. This is particularly evident in baked goods made from whole-wheat-based doughs, where a satisfactory enzymatic solution has yet to be reported. Today, consumers prefer “healthier” baked goods with higher nutritional value. Compared to refined wheat flour, using whole-wheat flour in baked goods makes more fiber, protein, and vitamins available, allowing for an improved nutritional profile. [Overview of the Initiative] [Problems that the invention aims to solve]

[0008] Therefore, there remains a demand for new compositions and methods that further improve properties such as volume of baked products (made with whole wheat flour) by improving the resilience of the dough or batter. [Means for solving the problem]

[0009] The inventors have surprisingly discovered polypeptides that possess galactolipase activity, low or nearly complete lipase activity, and / or low or nearly complete phospholipase activity. These polypeptides have a positive effect on the properties of dough or batter (e.g., dough resilience) and / or the properties of baked products obtained from such dough or batter (e.g., volume after mechanical impact), particularly when the dough or batter contains whole wheat flour.

[0010] Therefore, the first aspect provides a polypeptide having galactolipase activity, characterized in that the ratio of galactolipase activity to phospholipase activity is 100.0 or more, and / or the ratio of galactolipase activity to lipase activity is 150.0 or more.

[0011] In certain embodiments, the ratio of galactolipase activity to phospholipase activity is 1000.0 or greater. In certain embodiments, the ratio of galactolipase activity to lipase activity is 300.0 or higher, preferably 500.0 or higher, and more preferably 1000.0 or higher.

[0012] In a particular embodiment, the polypeptide having galactolipase activity is selected from the following: (a) A polypeptide comprising a sequence having at least 80.0% sequence identity with the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, preferably SEQ ID NO: 4; (b) A polypeptide comprising a sequence encoded by a polynucleotide having at least 80.0% sequence identity with the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 3; (c) A fragment of polypeptide (a) or (b) having galactolipase activity, or; (d) A polypeptide having at least 90.0% structural similarity to a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, preferably SEQ ID NO: 4.

[0013] In certain embodiments, the three-dimensional protein coordinates of the atoms in the amino acid sequence are defined by the set of atomic structure coordinates shown in Figure 1, or have a root mean square deviation of 1.8 Å or less obtained from the set of atomic structure coordinates shown in Figure 1.

[0014] In certain embodiments, the polypeptide is Clostridium galactlipase. A further aspect is the use of polypeptides having galactolipase activity as taught herein as food additives or in the preparation of food products.

[0015] In certain embodiments, the food product is (a) dough or dough product; (b) batter or batter product; or (c) bakery or patisserie product. In certain embodiments, the food additive is a bread improver.

[0016] In certain embodiments, the food product further comprises whole grain flour. In certain embodiments, the uses taught herein are for increasing (enhancing) the resistance of a fabric or batter.

[0017] A further aspect is the provision of a method for preparing a food product, comprising the step of mixing a polypeptide having the galactolipase activity taught herein with a food material. In certain embodiments, the food product is a bakery or pastry product, and the polypeptide having galactolipase activity taught herein is added to the dough or batter.

[0018] In certain embodiments, the food product further comprises whole grains. A further aspect provides a food product, a dough product, a batter product or a bakery product obtained by the uses or methods taught herein.

[0019] A further aspect provides an isolated nucleic acid encoding a polypeptide having galactolipase activity taught herein, preferably comprising a sequence having at least 80.0% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 3.

[0020] A further aspect provides an expression vector comprising a nucleic acid taught herein. A further aspect provides a host cell comprising a nucleic acid taught herein or an expression vector taught herein, preferably a bacterial, fungal or yeast cell.

[0021] A further aspect provides a method for identifying a polypeptide having galactolipase activity for improving dough tolerance, the method comprising determining whether the polypeptide has at least 90.0% structural similarity to the structure of a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, preferably SEQ ID NO: 4.

[0022] A further aspect provides a method for identifying a polypeptide having galactolipase activity for improving dough tolerance, the method comprising determining whether the three-dimensional protein coordinates of the atoms of the amino acid sequence of the polypeptide have a root mean square deviation obtained from the set of atomic structure coordinates shown in FIG. 1 of 1.8 Å or less. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] [Figure 1-1] Provide a table containing the atomic structure coordinates of amino acid residues 1 to 349 of sequence number 4 predicted by AlphaFold. Column 1: Record type (ATOM), Column 2: Atomic serial number, Column 3: Atom name, Column 4: Residue name, Column 5: Chain identifier, Column 6: Residue sequence number, Column 7: Atomic coordinate X (in Å), Column 8: Atomic coordinate Y (in Å), Column 9: Atomic coordinate Z (in Å), Column 10: Occupancy, Column 11: Temperature factor, Column 12: Element symbol [Figure 1-2] Provide a table containing the atomic structure coordinates of amino acid residues 1 to 349 of sequence number 4 predicted by AlphaFold. Column 1: Record type (ATOM), Column 2: Atomic serial number, Column 3: Atom name, Column 4: Residue name, Column 5: Chain identifier, Column 6: Residue sequence number, Column 7: Atomic coordinate X (in Å), Column 8: Atomic coordinate Y (in Å), Column 9: Atomic coordinate Z (in Å), Column 10: Occupancy, Column 11: Temperature factor, Column 12: Element symbol [Figure 1-3] Provide a table containing the atomic structure coordinates of amino acid residues 1 to 349 of sequence number 4 predicted by AlphaFold. Column 1: Record type (ATOM), Column 2: Atomic serial number, Column 3: Atom name, Column 4: Residue name, Column 5: Chain identifier, Column 6: Residue sequence number, Column 7: Atomic coordinate X (in Å), Column 8: Atomic coordinate Y (in Å), Column 9: Atomic coordinate Z (in Å), Column 10: Occupancy, Column 11: Temperature factor, Column 12: Element symbol [Figure 1-4] Provide a table containing the atomic structure coordinates of amino acid residues 1 to 349 of sequence number 4 predicted by AlphaFold. Column 1: Record type (ATOM), Column 2: Atomic serial number, Column 3: Atom name, Column 4: Residue name, Column 5: Chain identifier, Column 6: Residue sequence number, Column 7: Atomic coordinate X (in Å), Column 8: Atomic coordinate Y (in Å), Column 9: Atomic coordinate Z (in Å), Column 10: Occupancy, Column 11: Temperature factor, Column 12: Element symbol [Figure 1-5]This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-6] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-7] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-8] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-9] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-10] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-11] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-12] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-13] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-14]This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-15] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-16] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-17] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-18] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-19] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-20] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-21] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-22] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-23]This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-24] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-25] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-26] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-27] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-28] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-29] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-30] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-31] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-32]This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-33] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-34] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-35] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-36] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-37] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-38] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-39] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-40] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-41]This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-42] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-43] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-44] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-45] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-46] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-47] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-48] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-49] This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 1-50]This table provides the atomic structural coordinates of amino acid residues 1-349 of Sequence ID No. 4 as predicted by AlphaFold. Column 1: Record Type (ATOM), Column 2: Atomic Serial Number, Column 3: Atomic Name, Column 4: Residue Name, Column 5: Chain Identifier, Column 6: Residue Sequence Number, Column 7: Atomic Coordinate X (in Å units), Column 8: Atomic Coordinate Y (in Å units), Column 9: Atomic Coordinate Z (in Å units), Column 10: Occupancy, Column 11: Temperature Coefficient, Column 12: Element Symbol [Figure 2-1] The sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 is provided. [Figure 2-2] The sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 is provided. [Figure 2-3] The sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 is provided. [Figure 2-4] The sequence shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7 is provided. [Modes for carrying out the invention]

[0024] Before describing the methods and apparatus used in the present invention, it should be understood that the present invention is not limited to the specific methods, components, or apparatus described, and these methods, components, and apparatus are naturally subject to change. Furthermore, it should be understood that the scope of the present invention is limited only by the appended claims, and therefore the terms used herein are not intended to be limiting.

[0025] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which the present invention pertains. Any methods and materials similar or equivalent to those described herein may be used in carrying out or testing the present invention, but preferred methods and materials are described below.

[0026] In this specification and the appended claims, the singular forms "a," "an," and "the" include both singular and plural forms unless the context clearly indicates otherwise. As used herein, the terms “comprising,” “comprises,” and “comprised of” are synonymous with “including,” “includes,” or “containing,” and are comprehensive or open-ended, and do not exclude any additional undescribed components, elements, or methods or processes. Where this specification refers to a product or process that “includes” a particular feature, component, or process, this indicates the possibility that other features, components, or processes may also exist, but may also refer to embodiments that include only the described features, components, or processes. The terms “comprising,” “comprises,” and “comprised of” also include the term “consisting of.”

[0027] The enumeration of numbers within a range includes all values ​​and fractions within that range, as well as the cited endpoints. The terms “about” and “approximately,” used when referring to measurable values ​​such as parameters, quantities, or periods, are intended to include variations of ±10%, preferably ±5%, more preferably ±1%, and even more preferably ±0.1% from a specified value, as applicable to the inventions disclosed herein. It should be understood that the values ​​themselves that the terms “about” or “approximately” refer to are also disclosed.

[0028] Surprisingly, the inventors have found that polypeptides possessing galactolipase activity and having low or nearly complete lipase and / or phospholipase activity have a positive effect on the properties of the dough or the baked products obtained therefrom. Even more surprisingly, it has been found that using polypeptides with such galactolipase activity improves the dough's resilience, especially when the dough contains whole wheat flour.

[0029] Therefore, the first aspect of the present invention is to provide a polypeptide (or galactolipase) having galactolipase activity, characterized in that the ratio of galactolipase activity to phospholipase activity of the polypeptide (or galactolipase) (i.e., galactolipase activity to phospholipase activity) is 100.0 or more, and / or the ratio of galactolipase activity to lipase activity of the polypeptide (or galactolipase) is 150.0 or more.

[0030] In other words, this specification provides a polypeptide (or galactlipase) having galactlipase activity, characterized in that the galactlipase activity / phospholipase activity (ratio of galactlipase activity to phospholipase activity) is 100.0 or higher, and / or the galactlipase activity / lipase activity is 150.0 or higher.

[0031] In some embodiments, the ratio of galactolipase activity to phospholipase activity is 300.0 or more, preferably 500.0 or more, and more preferably 1000.0 or more. In some embodiments, the ratio of galactolipase activity to lipase activity is 300.0 or more, preferably 500.0 or more, and more preferably 1000.0 or more. In some embodiments, the ratio of galactolipase activity to phospholipase activity is 100.0 or more, preferably 300.0 or more, more preferably 500.0 or more, and even more preferably 1000.0 or more, and the ratio of galactolipase activity to lipase activity is 150.0 or more, preferably 300.0 or more, more preferably 500.0 or more, and even more preferably 1000.0 or more.

[0032] Preferably, these ratios are obtained when determining (measuring) enzyme activity as described herein. Furthermore, in certain embodiments, galactolipase activity is determined at 30°C and / or phospholipase activity is determined at 30°C.

[0033] In the context of the present invention, the terms "polypeptide having galactolipase activity" or "galactolipase" (EC 3.1.1.26) refer to an enzyme whose primary enzymatic activity is to catalyze the cleavage of acyl ester bonds in galactolipids, such as monogalactosyldiacylglycerol (MGDG) or digalactosyldiacylglycerol (DGDG), to form free fatty acids (FFA), digalactosylglycerol, and / or lysogalactolipids (LysoGL). Galactolipase activity can be evaluated using various methods, such as pHstat titration using dioctanoyl galactolipide-bile salt mixed micelles, barostat using a dilauroyl galactolipide monolayer deployed on an air-water interface, and UV absorption using a novel monogalactosyldiacylglycerol (MGDG) substrate coated on a microtiter plate containing α-eleostearic acid as a chromophore. Preferably, galactolipase activity can be measured using digalactosyldiacylglycerol (DGDG) as the substrate. The release of free fatty acids by hydrolysis of digalactosyldiacylglycerol by galactolipase is measured by spectrophotometric analysis at 550 nm. 1-galactolipase activity (SDGU) is defined as the amount of enzyme required to release 1 nmol of free fatty acids per minute at 30°C and pH 7.5. For example, galactolipase activity can be determined as shown in Example 1.

[0034] This term further includes polypeptide variants or mutants having amino acid sequence changes, e.g., amino acid deletions, additions, and / or substitutions, as described elsewhere in this specification, relative to the corresponding natural polypeptide. This term refers to both full-length polypeptides and parts or fragments of polypeptides, e.g., naturally occurring polypeptides resulting from the processing of such full-length polypeptides, as described elsewhere in this specification.

[0035] In the context of this invention, the term "lipase" (EC 3.1.1.3) refers to an enzyme having triacylglycerol lipase activity as its primary enzymatic activity. Lipase activity can be evaluated by different methods such as fluorescence assay, colorimetric assay, or titration. Preferably, lipase activity can be measured using p-nitrophenyl palmitate (pNPP) as a substrate. The release of yellow p-nitrophenol by hydrolysis of p-nitrophenyl palmitate by lipase is measured by spectrophotometric assay at 414 nm. One milliunit of lipase (LmU) is defined as the amount of enzyme required to release 1 nanomolar (nmole) of p-nitrophenol from p-nitrophenyl palmitate per minute at 45°C and pH 7.5. For example, lipase activity can be determined as shown in Example 1.

[0036] In the context of this invention, the term "phospholipase" refers to an enzyme having hydrolytic activity against one or both carboxylic acid ester bonds in phospholipids as its primary enzymatic activity, such as phospholipase A1 or phospholipase A2 (EC 3.1.1.32 or EC 3.1.1.4, respectively). Phospholipase activity can be measured by various methods, such as the Phospholipase Activity Assay Kit (colorimetric method, Abcam), the Secretory Phospholipase Activity Assay Kit (fluorescence method, Abcam), and the EnzChek® Phospholipase A1 / A2 Assay Kit (Life Technologies). Preferably, phospholipase activity can be measured using 1,2-dioleoyl-sn-glycero-3-phospho-rac-1-glycerol (DOPG) as a substrate. The release of free fatty acids by hydrolysis of 1,2-dioleoyl-sn-glycero-3-phospho-rac-1-glycerol by phospholipase is measured by spectrophotography at 550 nm. One phospholipase activity unit (NefU) is defined as the amount of enzyme required to release 1 nmol of free fatty acids per minute at 30°C and pH 7.5. For example, phospholipase activity can be determined as shown in Example 1.

[0037] In a particular embodiment, the polypeptides having galactolipase activity taught herein are selected from the following: (a) A polypeptide comprising, or comprising, a variant of a polypeptide having galactolipase activity as taught herein, for example, an amino acid sequence defined in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, preferably SEQ ID NO: 2 or SEQ ID NO: 4, more preferably SEQ ID NO: 4 (i.e., the amino acid sequence) and a sequence having at least 80.0%, preferably at least 95.0%, sequence identity; (b) A polypeptide that essentially comprises or comprises a sequence encoded by a polynucleotide comprising a nucleotide sequence defined in SEQ ID NO: 1, SEQ ID NO: 3, or SEQ ID NO: 5, preferably SEQ ID NO: 1 or SEQ ID NO: 3, more preferably SEQ ID NO: 3, and a sequence having at least 80.0%, preferably at least 95.0%, sequence identity; (c) A fragment of polypeptide (a) or (b) having galactolipase activity, and / or (d) A polypeptide having at least 90.0%, preferably at least 95.0%, structural similarity to a polypeptide that essentially comprises or comprises an amino acid sequence defined in SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, preferably SEQ ID NO: 2 or SEQ ID NO: 4, more preferably SEQ ID NO: 4.

[0038] The term “variant” of a protein, polypeptide, peptide, or nucleic acid generally refers to a protein, polypeptide, or peptide, or nucleic acid whose amino acid sequence or nucleotide sequence is substantially identical (i.e., largely identical but not completely identical) to the sequence of the protein, polypeptide, peptide, or nucleic acid, for example, at least about 80.0% identical, or at least about 85.0% identical, for example preferably at least about 90.0% identical, for example at least 91.0% identical, 92.0% identical, more preferably at least about 93.0% identical, for example at least 94.0% identical, even more preferably at least about 95.0% identical, for example at least 96.0% identical, even more preferably at least about 97.0% identical, for example at least 98.0% identical, and most preferably at least 99.0% identical. Preferably, the variant can demonstrate such a degree of identity to the described protein, polypeptide, peptide, or nucleic acid (i.e., overall sequence identity) when the entire sequence of the described protein, polypeptide, peptide, or nucleic acid is searched by sequence alignment.

[0039] In the context of this invention, the term "sequence identity" refers to the relationship between two amino acid or nucleotide sequences. For the purposes of this invention, sequence identity can be determined by performing a sequence alignment that is known in itself, and by using an appropriate algorithm for determining sequence identity. Examples of exemplary but non-exclusive algorithms include those based on the Basic Local Alignment Search Tool (BLAST) first described by Altschul et al. 1990 (J Mol Biol 215:403-10), such as the "Blast 2 sequences" algorithm described by Tatusova and Madden 1999 (FEMS Microbiol Lett 174:247-250), using the published default settings or other appropriate settings (e.g., for the BLASTN algorithm: gap start cost = 5, gap extension cost = 2, mismatch penalty = -2, match reward = 1, gap x_dropoff = 50, expected value = 10.0, word size = 28; or for the BLASTP algorithm: matrix = Blosum62 (Henikoff et al., 1992, Proc Natl Acad Sci 89:10915-10919), gap start cost = 11, gap extension cost = 1, expected value = 10.0, word size = 3 or the "Blast 2 sequences" algorithm described by Tatusova and Madden 1999 (FEMS Microbiol Lett 174:247-250).

[0040] One example of a procedure for determining the percentage of identity between a specific amino acid sequence and the amino acid sequence of a query polypeptide involves aligning the two amino acid sequences using the BLASTP algorithm (with the BLAST-2-Sequences option), which is available as a web application on the NCBI website (https: / / ftp.ncbi.nlm.nih.gov / blast / executables / blast+ / LATEST / ) or as a standalone program (BLAST version 2.13.0) with appropriate algorithm parameters. If homology is found between the two sequences being compared, the output will show the homologous region as an aligned sequence. If homology is not found between the two sequences being compared, the output will not show an aligned sequence. After alignment, the number of matches is determined by counting the number of positions where identical amino acid residues exist in both sequences. The percentage of identity (homology) is calculated by dividing the number of matches by the length of the query polypeptide and multiplying the result by 100. Percentile identity values ​​may be rounded to one decimal place, but this is not always necessary. For example, 78.11, 78.12, 78.13, and 78.14 may be rounded down to 78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 may be rounded up to 78.2. Furthermore, note that the detailed display of each segment of the alignment output by Bl2seq already conveniently displays the percentage of identity.

[0041] Sequence identity can also be determined using the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J Mol Biol 48:443-453), as implemented in the Needle program of the EMBOSS package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. 16:276-277), preferably version 5.0.0 or later. The parameters used may be a gap start penalty of 10, a gap extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version BLOSUM62) substitution matrix for amino acid sequence comparison. The Needle "longest identity" output (obtained using the -nobrief option) can be used as the percentage identity and calculated as follows: (Number of identical residues × 100) / (Alignment length - Total number of gaps in the alignment).

[0042] Sequence identity can also be determined using FASTA36 sequence comparison software, such as that described in Pearson WR, Finding protein and nucleotide similarities with FASTA, Curr Protoc Bioinformatics, 2016 Mar 24;53:3.9.1-3.925.

[0043] A variant of a protein, polypeptide, or peptide may contain the addition, deletion, or substitution of one or more (e.g., several) amino acids compared to (i.e., with respect to) the corresponding protein or polypeptide. Substitution means replacing an amino acid occupying a position with a different amino acid; deletion means removing an amino acid occupying a position; and insertion means adding an amino acid adjacent to and immediately following an amino acid occupying a position.

[0044] In some embodiments, the galactolipases (i.e., polypeptides having galactolipase activity) taught herein essentially consist of, or consist of, a sequence defined by SEQ ID NO: 2 (including the signal peptide), SEQ ID NO: 4 (without the signal peptide or a mature form of galactolipase), or SEQ ID NO: 6 (without the signal peptide or a mature form of galactolipase), preferably SEQ ID NO: 2 or SEQ ID NO: 4, more preferably SEQ ID NO: 4, and a sequence having at least 80.0%, at least 85.0%, preferably at least 90.0%, more preferably at least 91.0%, at least 92.0%, at least 93.0%, at least 94.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 99.0%, or 100.0% sequence identity.

[0045] Sequence ID 6 has 17 amino acid substitutions compared to Sequence ID 4 (and may be encoded by the nucleotide sequence defined in Sequence ID 5). More specifically, the T residue at position 33 of Sequence ID 4 is replaced with an I residue, the K residue at position 52 of Sequence ID 4 is replaced with an R residue, the Y residue at position 120 of Sequence ID 4 is replaced with a D residue, the D residue at position 132 of Sequence ID 4 is replaced with an N residue, the S residue at position 181 of Sequence ID 4 is replaced with an N residue, the A residue at position 184 of Sequence ID 4 is replaced with an S residue, the G residue at position 194 of Sequence ID 4 is replaced with an A residue, and the K residue at position 239 of Sequence ID 4 is replaced with a Q residue. As a result, the P residue at position 278 of SEQ ID NO: 4 is replaced with an L residue, the G residue at position 298 of SEQ ID NO: 4 is replaced with an A residue, the S residue at position 306 of SEQ ID NO: 4 is replaced with a T residue, the R residue at position 318 of SEQ ID NO: 4 is replaced with an I residue, the K residue at position 330 of SEQ ID NO: 4 is replaced with an A residue, the M residue at position 337 of SEQ ID NO: 4 is replaced with an I residue, the T residue at position 341 of SEQ ID NO: 4 is replaced with an S residue, and the N residue at position 342 of SEQ ID NO: 4 is replaced with an M residue.

[0046] In certain embodiments, polypeptides having galactolipase activity as taught herein include, or are essentially derived from, or are derived from, a sequence that differs by up to 38, up to 37, up to 36, up to 35, up to 34, up to 33, up to 32, up to 31, up to 30, up to 25, up to 20, up to 15, up to 10, up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid from the sequence defined by SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, preferably SEQ ID NO: 2 or SEQ ID NO: 4. The term "different from" may also refer to amino acid substitutions, additions and / or deletions, preferably substitutions.

[0047] In certain embodiments, the polypeptide variant taught herein is a functionally active variant of the polypeptide taught herein. Throughout this specification, the term “fragment” as used with respect to peptides, polypeptides, or proteins generally means a portion of a peptide, polypeptide, or protein, for example, typically a cleaved form of the N-terminus and / or C-terminus of a peptide, polypeptide, or protein. Preferably, a fragment may comprise at least about 30.0%, e.g., at least about 50.0% or at least about 70.0%, preferably at least about 80.0%, e.g., at least about 85.0%, more preferably at least about 90.0%, even more preferably at least about 95.0%, or about 99.0% of the amino acid sequence length of the peptide, polypeptide, or protein. For example, within a range not exceeding the full length of the peptide, polypeptide, or protein, a fragment may comprise a sequence of five or more consecutive amino acids, or ten or more consecutive amino acids, or twenty or more consecutive amino acids, or thirty or more consecutive amino acids, e.g., forty or more consecutive amino acids, e.g., fifty or more consecutive amino acids, of the corresponding full-length peptide, polypeptide, or protein.

[0048] These terms include, but are not limited to, any mechanism in vivo and / or in vitro, such as alternative transcription or translation, exo- and / or endo-protease degradation, exo- and / or endo-nuclease degradation, or degradation of peptides, polypeptides, proteins, or nucleic acids, such as fragments resulting from physical, chemical, and / or enzymatic protease or nuclease degradation.

[0049] In certain embodiments, the fragment is a functionally active fragment of a galactolipase (or a polypeptide having galactolipase activity). Exemplary and not limited to, the functionally active fragments of galactolipases taught herein retain, at least in part, one or more aspects of the functionality of the corresponding natural or wild-type galactolipase taught herein. For example, references to the functionality of galactolipases taught herein may refer, in particular, to their galactolipase activity, their extremely low or absent phospholipase activity and / or lipase activity, and / or their ability to enhance dough tolerance, especially when the dough contains whole wheat flour.

[0050] As used herein, the term “functionally active” means that a fragment and / or variant retains at least partially the intended functionality or biological activity of the corresponding peptide, polypeptide, or protein. References to “functionality” or “activity” of a peptide, polypeptide, or protein may generally encompass, but are not limited to, one or more aspects of the functionality or biological activity of that peptide, polypeptide, or protein, such as one or more aspects of biochemical activity, enzymatic activity, interaction activity, ligand activity, and / or structural activity in cells, tissues, organs, or organisms.

[0051] Preferably, a functionally active fragment or variant may retain at least about 20.0%, for example at least about 25.0%, or at least 30.0%, or at least about 40.0%, or at least about 50.0%, for example at least 60.0%, more preferably at least about 70.0%, for example at least 80.0%, even more preferably at least about 85.0%, even more preferably at least about 90.0%, most preferably at least about 95.0%, or about 100.0%, of the intended biological activity or functionality compared to the corresponding peptide, polypeptide, or protein. In certain embodiments, a functionally active fragment or variant may exhibit higher biological activity or functionality compared to the corresponding peptide, polypeptide, or protein, for example, at least about 100.0%, at least about 150.0%, at least about 200.0%, at least about 300.0%, at least about 400.0%, or at least about 500.0% of the intended biological activity or functionality compared to the corresponding peptide, polypeptide, or protein. For example, if the activity of a particular peptide, polypeptide, or protein can be readily measured in an assay with a quantitative output, such as an enzyme assay, a functionally active fragment or variant of that peptide, polypeptide, or protein may produce a signal. This signal is at least about 20.0%, or at least about 25.0%, or at least about 30.0%, or at least about 40.0%, or at least about 50.0%, or at least 60.0%, more preferably at least about 70.0%, or at least 80.0%, or at least about 85.0%, or at least about 90.0%, or at least about 95.0%, or at least about 100.0%, or at least about 150.0%, or at least about 200.0%, or at least about 300.0%, or at least about 400.0%, or at least about 500.0% of the signal produced by the corresponding peptide, polypeptide, or protein.

[0052] In certain embodiments, the three-dimensional protein coordinates of the atoms in the amino acid sequence of a galactolipase (i.e., a polypeptide having galactolipase activity) taught herein are defined by the set of atomic structure coordinates shown in Figure 1, or have a root mean square deviation from the set of atomic structure coordinates shown in Figure 1 (i.e., defining atoms 1 to 2812) of 1.8 Å or less, 1.7 Å or less, 1.6 Å or less, 1.5 Å or less, 1.0 Å or less, or 0.5 Å or less. In other words, in certain embodiments, the three-dimensional protein coordinates of the atoms in that amino acid sequence are defined by the set of atomic structure coordinates shown in Figure 1, or fall within a deviation of 1.8 Å from the coordinates of the corresponding atoms in Figure 1 (i.e., defining atoms 1 to 2812). Furthermore, in certain embodiments, the galactolipases taught herein (i.e., polypeptides having galactolipase activity) have a structure having the atomic structural coordinates shown in Figure 1 (i.e., defining atoms 1 to 2812), or have root mean square deviations of the coordinates relative to the conserved main chain atoms of the described amino acid sequence of 1.8 Å or less, 1.7 Å or less, 1.6 Å or less, 1.5 Å or less, 1.0 Å or less, or 0.5 Å or less.

[0053] In this specification, “root mean square deviation” is the square root of the arithmetic mean of the squares of the deviations from the mean, and is a method of representing deviations or variations from the structural coordinates described herein. This disclosure includes all embodiments that involve conservative substitutions of the described amino acid residues that result in the same structural coordinates within the described root mean square deviations.

[0054] The three-dimensional protein coordinates of atoms in the amino acid sequence of a polypeptide, protein, or peptide may be determined, for example, by X-ray diffraction of the polypeptide, protein, or peptide crystal, or predicted using AlphaFold software (v2.3.1 Monomere, available at https: / / github.com / deepmind / alphafold). AlphaFold software takes a protein sequence, such as SEQ ID NO: 4, as input. The system extracts various features from the protein sequence, including information about amino acid identity, their positions within the sequence, and their surrounding environment. AlphaFold uses a neural network to predict the distances between amino acid pairs within the protein sequence. Based on these predicted distances, AlphaFold predicts the angles between amino acids. AlphaFold then calculates torsion angles, which describe the rotation of each amino acid around its main chain axis. Using the predicted torsion angles and other parameters, AlphaFold determines the precise coordinates of each atom within the protein. Finally, AlphaFold "folds" the protein into a three-dimensional structure using the predicted atomic coordinates. The system then uses a second neural network to refine the structure and improve the accuracy of the predicted atomic coordinates.

[0055] "Structural coordinates" are Cartesian coordinates that correspond to the spatial relationships between atoms within a molecule or molecular complex. Structural coordinates can be obtained experimentally, such as through X-ray crystallography or NMR techniques, or derived through modeling (homology modeling or de novo modeling) using protein sequences as input. Various software programs can graphically represent sets of structural coordinates, allowing for a three-dimensional representation of a molecule or molecular complex. The structural coordinates of the structures described herein may be modified from the original set shown in Figure 1 through mathematical operations such as inversion or integer addition and subtraction. Therefore, it should be recognized that the structural coordinates of this invention are relative and not specifically limited by the actual x, y, and z coordinates in Figure 1. In some embodiments, the galactolipases (i.e., polypeptides having galactolipase activity) taught herein have at least 90.0%, at least 91.0%, or at least 92.0% structural similarity, preferably at least 93.0%, at least 94.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 99.0%, or 100.0% structural similarity, to the structure of a polypeptide essentially comprising or derived from the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, or SEQ ID NO: 6, preferably SEQ ID NO: 2 or SEQ ID NO: 4.

[0056] The polypeptide, protein, or peptide structures referred to herein are the (predicted) three-dimensional (3D) structures of the polypeptide, protein, or peptide. This may include the secondary and / or tertiary structure levels of the protein structure.

[0057] In the context of this invention, the term "structural similarity" corresponds to the relationship between two protein structures, defined as their topological similarity. For the purposes of this invention, the structural similarity between two protein structures can be determined using a TM score calculated using the TM-align algorithm (Zhang and Skolnick, 2004, PROTEINS:Structure, Function, and Bioinformatics 57: 702-710), preferably version 20190822 or later, as implemented in the TM-align Debian-based package (tm-align, described in Zhang and Skolnick, 2005, Nucleic Acid Res 33:2302-2309). The TM score is normalized by the length of the reference protein. In particular, the reference protein is SEQ ID NO: 2 or SEQ ID NO: 4.

[0058] In some embodiments, the galactolipases (i.e., polypeptides having galactolipase activity) taught herein have a TM score of at least 0.90, more preferably at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or 1.0 when compared to the structure of SEQ ID NO: 2 or the structure of SEQ ID NO: 4 (coordinates shown in Figure 1). In other words, a TM score of 0.9 corresponds to a structural similarity of 90.0%, and a TM score of 1.0 corresponds to a structural similarity of 100.0%.

[0059] Polypeptides having galactolipase activity taught herein can be obtained from any microorganism of any genus. In certain embodiments, the polypeptide having galactolipase activity taught herein is Clostridium galactolipase (i.e., obtained from a strain of Clostridium), for example, but not limited to, Clostridium saccharobutylicum galactolipase.

[0060] Polypeptides possessing galactolipase activity as taught herein can be obtained and / or identified from other microbial sources or by other means, for example, by searching sequence databases for polypeptides predicted to belong to the enzyme class of EC number EC 3.1.1.26, or polypeptides with unknown functions that have topological / structural / sequence similarity / identity with SEQ ID NO: 2 or SEQ ID NO: 4.

[0061] A further aspect is to provide an isolated nucleic acid capable of encoding or encoding a polypeptide having galactolipase activity as taught herein, which preferably comprises a sequence having at least 80.0%, at least 85.0%, preferably at least 90.0%, more preferably at least 91.0%, at least 92.0%, at least 93.0%, at least 94.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 99.0%, or 100.0% sequence identity with SEQ ID NO: 1 (including signal peptide) or SEQ ID NO: 3 (not including signal peptide).

[0062] A further aspect is the provision of expression vectors containing nucleic acids taught herein. Those skilled in the art will understand that, assuming the expression and secretion of a polypeptide having galactolipase activity as taught herein by a host cell, the nucleic acid encoding the polypeptide having galactolipase activity as taught herein preferably encodes a precursor form of the polypeptide having galactolipase activity that includes an N-terminal signal peptide sequence. Therefore, the nucleic acid may encode a fragment (i.e., including the signal peptide) of the precursor polypeptide of the polypeptide having galactolipase activity as taught herein. In certain embodiments, the nucleic acid encodes a polypeptide having galactolipase activity that essentially comprises, or comprises, an amino acid sequence having at least 25.0%, at least 30.0%, at least 35.0%, at least 40.0%, at least 45.0%, at least 50.0%, at least 55.0%, at least 60.0%, at least 65.0%, at least 70.0%, at least 75.0%, at least 80.0%, at least 85.0%, at least 90.0%, at least 95.0%, or at least 99.0% sequence identity with the amino acid sequence described herein. Herein, the amino acid sequence is preceded at the N-terminus by a signal peptide having the amino acid sequence MKTKVCKICVFFIACFSIFLINGIQAKA (Sequence ID 7). Alternatively, the nucleic acid encoding the polypeptide having galactolipase activity as taught herein may be contained within a vector providing the signal peptide. The signal peptide may be homogeneous or heterogeneous, depending on the host cell used to produce the polypeptide having galactolipase activity as taught herein. Furthermore, for the expression of the polypeptide having galactolipase activity as taught herein in prokaryotes, protease cleavage site motifs may be present on the C-terminal side of the signal peptide and on the N-terminal side of the polypeptide having galactolipase activity as taught herein.

[0063] A further aspect provides a host cell comprising a nucleic acid or an expression vector as taught herein, preferably a bacterial, fungal, or yeast cell.

[0064] Furthermore, this specification provides for the use of polypeptides having galactolipase activity as taught herein in the preparation of food products. In certain embodiments, the food product is (a) dough or dough product, (b) batter or batter product, or (c) bakery or patisserie product. Furthermore, the use of polypeptides having galactlipase activity as taught herein as food additives is provided herein. In certain embodiments, the food additive is a bread improver. In other words, polypeptides having galactlipase activity as taught herein can be conveniently used as part of a composition such as a bread improver. Bread improvers (also called dough modifiers, dough improvers, improvers, or flour treatments) are typically added to dough to improve the texture, volume, flavor, and / or freshness of baked products, as well as to improve the machinability and stability of the dough. Typically, bread improvers include one or more enzymes (e.g., amylase (α-amylase, β-amylase, glucoamylase, raw starch-degrading amylase), xylanase (hemicellulase), cellulase, pectinase, protease, pectinate lyase, oxidase (peroxidase, glucose oxidase, pyranose oxidase, hexose oxidase, L-amino acid oxidase, carbohydrate oxidase, sulfurhydryl oxidase), lipoxygenase, dehydrogenase, laccase, transglutaminase, acyltransferase, protein disulfide isomerase), and one or more oxidizing or reducing agents (e.g., ascorbyl). A product comprising, essentially consisting of, or consisting of, bic acid, glutathione, cysteine, etc., one or more emulsifiers (e.g., diacetyl tartrate of monoglycerides (DATEM), sodium stearoyl lactylate (SSL), calcium stearoyl lactylate (CSL), glycerol monostearate (GMS), rhamnolipid, lecithin, sucrose ester, bile salt, etc.), one or more lipid materials (e.g., margarine, butter, oil, shortening, etc.), one or more vitamins (e.g., pantothenic acid and vitamin E, etc.), one or more gums, (dried) sourdough, and / or one or more fiber sources (e.g., oat fiber, etc.).

[0065] A further aspect of the present invention is to provide a method for producing a food product, preferably a baked product, which includes the step of mixing a polypeptide having the galactolipase activity taught herein with a food material.

[0066] In certain embodiments, if the method is a method for producing a baked product, the method may include the step of adding a polypeptide having galactolipase activity as taught herein to a dough or batter. Preferably, the polypeptide having galactolipase activity as taught herein is added to the dough or batter before baking. Preferably, in the method taught herein, the dough or batter has improved resistance.

[0067] In certain embodiments, the dough or batter taught herein has improved dough tolerance compared to a reference dough or batter, wherein the reference dough or batter is prepared in the absence (i.e., without containing) a polypeptide having galactolipase activity as taught herein. In even more specific embodiments, the dough or batter taught herein has at least 1.0%, at least 2.0%, at least 3.0%, at least 4.0%, at least 5.0%, at least 10.0%, or at least 15.0% higher dough tolerance compared to a reference dough or batter, wherein the reference dough or batter is prepared in the absence (i.e., without containing) a polypeptide having galactolipase activity as taught herein.

[0068] In this regard, a further aspect is the use of polypeptides having galactolipase activity taught herein in the production of food products having improved dough tolerance, preferably wherein the dough tolerance is at least 1.0%, at least 2.0%, at least 3.0%, at least 4.0%, at least 5.0%, at least 10.0%, or at least 15.0% higher than the dough tolerance of a reference dough or batter, wherein the reference dough or batter is a dough or batter produced in the absence of (i.e., without) the polypeptide having galactolipase activity taught herein.

[0069] Preferably, the baked product is a bakery or patisserie product. Bakery or patisserie products known in the art include, but are not limited to, bread, soft rolls, bagels, donuts, Danish pastries, hamburger rolls, pizzas, pita bread, ciabatta, sponge cakes, cream cakes, pound cakes, muffins, cupcakes, steamed cakes, waffles, brownies, cake donuts, yeast-leavened donuts, baguettes, rolls, crackers, cookies, pie crusts, rusks, and / or other baked products. More preferably, the baked product is bread, a baguette, and / or a roll.

[0070] In certain embodiments, the food product contains whole grain flour. In even more specific embodiments, the food contains at least 50.0% (w / w), at least 55.0% (w / w), at least 60.0% (w / w), at least 65.0% (w / w), at least 70.0% (w / w), or at least 75.0% (w / w) whole grain flour.

[0071] Whole grain flour (i.e., whole grain flour) is flour made using whole grains of wheat. In a further aspect, the present invention relates to the use of polypeptides having galactolipase activity as taught herein in bakery and pastry applications. It has been found that the use of polypeptides having galactolipase activity as taught herein can reduce or even suppress the use of undesirable dough or batter components, such as emulsifiers. In certain embodiments, the use taught herein can improve the resilience of dough or batter compared to a reference dough or batter, where the reference dough or batter is a dough or batter prepared in the absence of (i.e., without) the polypeptide having galactolipase activity as taught herein. In the context of the present invention, “dough or batter resilience” means the ability of dough or batter, preferably bakery dough or batter, to maintain its shape under stress conditions such as long fermentation times or mechanical shocks during or after fermentation, and to provide a baked product after baking that has properties (e.g., volume) equivalent to or better than a baked product obtained from an unstressed dough.

[0072] Further aspects include providing food products (e.g., bread improvers), dough products, batter products, or bakery products (e.g., baked products) comprising polypeptides having the galactlipase activity taught herein.

[0073] Further aspects provide food products, dough products, batter products, or bakery products that can be obtained or made available by the uses or methods taught herein. In a particular embodiment, the food product is a dough or batter having a polypeptide having galactolipase activity as taught herein, comprising wheat flour, preferably whole wheat flour, in an amount of 10,000 to 100,000 SDGU units / kg (wheat flour), 12,000 to 100,000 SDGU units / kg (wheat flour), 20,000 to 100,000 SDGU units / kg (wheat flour), or 30,000 to 90,000 SDGU units / kg (wheat flour).

[0074] A further aspect of this invention provides a method for identifying polypeptides having galactolipase activity for improving dough tolerance, the method comprising the step of determining whether the polypeptide has at least 90.0%, more preferably at least 91.0%, at least 92.0%, at least 93.0%, at least 94.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 99.0%, or 100.0% structural similarity to the structure of the polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. Structural similarity may be defined as described elsewhere in this specification.

[0075] In a particular embodiment, the method includes the steps of: comparing the structure of a test polypeptide with a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4; and determining that the test polypeptide is a polypeptide having galactolipase activity for improving dough tolerance if the structure of the test polypeptide has at least 90.0%, more preferably at least 91.0%, at least 92.0%, at least 93.0%, at least 94.0%, at least 95.0%, at least 96.0%, at least 97.0%, at least 98.0%, at least 99.0%, or 100.0% structural similarity to the structure of the polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4.

[0076] In a particular embodiment, the method includes the steps of: comparing the structure of a test polypeptide with the structure of a polypeptide containing the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4; and determining that the test polypeptide is a polypeptide having galactolipase activity to improve dough tolerance if, when compared with the structure of SEQ ID NO: 2 (coordinates shown in Figure 1) or the structure of SEQ ID NO: 4, the structure of the test polypeptide has a TM score of at least 0.90, more preferably at least 0.91, at least 0.92, at least 0.93, at least 0.94, at least 0.95, at least 0.96, at least 0.97, at least 0.98, at least 0.99, or 1.0.

[0077] A further aspect is provided of a method for identifying polypeptides having galactolipase activity for improving dough tolerance, comprising the step of determining whether the three-dimensional protein coordinates of atoms in the amino acid sequence of the polypeptide have a root mean square deviation from the set of atomic structure coordinates shown in Figure 1, which are 1.8 Å or less, 1.7 Å or less, 1.6 Å or less, 1.5 Å or less, 1.0 Å or less, or 0.5 Å or less. The three-dimensional protein coordinates of atoms in the amino acid sequence of a polypeptide, protein, or peptide can be determined by methods described elsewhere in this specification.

[0078] In a particular embodiment, the method includes the steps of: comparing the three-dimensional protein coordinates of atoms in the amino acid sequence of a test polypeptide with the set of atomic structure coordinates shown in Figure 1; and determining that the test polypeptide is a polypeptide having galactolipase activity to improve dough tolerance if the three-dimensional protein coordinates of atoms in the amino acid sequence of the test polypeptide have a root mean square deviation from the set of atomic structure coordinates shown in Figure 1 of 1.8 Å or less, 1.7 Å or less, 1.6 Å or less, 1.5 Å or less, 1.0 Å or less, or 0.5 Å or less.

[0079] Those skilled in the art will understand that certain embodiments of the products taught herein also apply to the methods and uses taught herein, and vice versa.

[0080] Although the present invention has been described in relation to specific embodiments, it will be obvious to those skilled in the art, based on the foregoing description, that many alternatives, modifications, and variations are apparent. Accordingly, in accordance with the spirit and broad scope of the appended claims, it is intended to encompass all such alternatives, modifications, and variations.

[0081] Aspects and embodiments of the present invention disclosed herein are further supported by the following non-limiting embodiments. [Examples]

[0082] Example 1: Determination of enzyme activity Lipase Lipase activity is measured using p-nitrophenyl palmitate (pNPP) as the substrate. The release of yellow p-nitrophenol by lipase is measured at 414 nm by spectrophotometric method. 120 μl of 1 mM pNPP solution (dissolved in 0.05 M sodium phosphate buffer (pH 7.5, containing 0.69 M acetone and 0.0049 M Triton X-100)) is mixed with 60 μl of enzyme sample and incubated at 45°C for 30 minutes. The absorbance at 414 nm is measured in a 96-well microplate using the substrate blank as a control.

[0083] One lipase milliunit (LmU) is defined as the amount of enzyme required to release 1 nanomolar (nmole) of p-nitrophenol from p-nitrophenyl palmitate per minute at 45°C and pH 7.5. Phospholipase Phospholipase activity is measured using 1,2-dioleoyl-sn-glycero-3-phospho-rac-1-glycerol (DOPG) as a substrate. The release of free fatty acids by phospholipase is measured at 550 nm by spectrophotometric method. 10 μL of 1% DOPG substrate solution (dissolved in 0.1 M sodium phosphate buffer (pH 7.5, containing 5% sodium deoxycholate)) is mixed with 10 μL of enzyme sample and incubated at 30°C for 15 minutes. Then, 2 μL of 1 M orthophosphate is added to stop the reaction. The released fatty acids are measured using the Fujifilm NEFA-HR(2) kit according to the manufacturer's instructions (add 200 μL of reagent R1 and incubate at 37°C for 10 minutes; add 100 μL of reagent R2 and incubate at 37°C for 10 minutes). A calibration curve is established using different concentrations of oleic acid provided in the kit. The absorbance at 550 nm is measured for a blank sample in a 96-well microplate.

[0084] One phospholipase active unit (NefU) is defined as the amount of enzyme required to release 1 nmol of free fatty acid per minute at 30°C and pH 7.5. Galactolipase Galactolipase activity is measured using digalactosyldiacylglycerol (DGDG) as the substrate. The release of free fatty acids by galactolipase is measured at 550 nm by spectrophotometric method. 10 μL of 1% DGDG substrate solution (dissolved in 0.1 M Na-phosphate buffer (pH 7.5, containing 5% Na-deoxycholate)) is mixed with 10 μL of enzyme sample and incubated at 30°C for 15 minutes. Then, 2 μL of 1 M orthophosphate solution is added to stop the reaction. The released fatty acids are measured using the Fujifilm NEFA-HR(2) kit in the manner described above.

[0085] One galactolipase active unit (SDGU) is defined as the amount of enzyme required to release 1 nmol of free fatty acid per minute at 30°C and pH 7.5. Example 2: Cloning, expression, and fermentation of galatripase as taught herein. Cloning of the galactolipase enzyme gene Based on bioinformatics studies (sequence and structure-based approaches), Sequence ID No. 4 was predicted to be a promising galactolipase enzyme. This sequence is derived from the DNA-to-protein translation of the whole genome sequence of Clostridium saccharobutylicum DSM 13864 (GenBank accession number: CP006721.1 (http: / / www.ncbi.nlm.nih.gov / )).

[0086] DNA sequences encoding a polypeptide with the sequence of SEQ ID NO: 4, and DNA sequences encoding a polypeptide with the sequence of SEQ ID NO: 6 (a variant with 17 amino acid substitutions), were synthesized using a standard protocol for expression in the pET 28a(+) plasmid.

[0087] The synthesized complete DNA fragments were subcloned into pUC19-derived plasmids. These plasmids were used to transform *E. coli* DH5α® ultracompetent cells. Purified plasmid preparations, prepared using the Pure Yield Midiprep System (Promega), were digested with appropriate restriction enzymes to isolate DNA fragments containing the coding sequences for galactolipase enzymes. These fragments were subcloned into pET 28a(+) cloning vectors (Novagen), and the resulting recombinant plasmids were used to transform *E. coli* BL21(DE3) cells (Agilent Technologies). Purified plasmid preparations, prepared using the Pure Yield Midiprep System (Promega), were sequenced using an ABI3700 DNA sequencer (Applied Biosystems). Sequences of the inserted fragments were performed using universal primers, the T7 promoter and T7 terminator, as well as primers corresponding to the internal DNA sequences. The resulting sequences were consistent with the expected sequences. Culture of recombinant strains and production of NGL (corresponding to SEQ ID NO: 2 and NGLv2 (corresponding to SEQ ID NO: 6)) 15 ml of pre-cultured Escherichia coli (E. coli) BL21 (DE3) cells containing the galactolipase enzyme gene (5 hours at 37°C) was centrifuged at 10,000 g for 1 minute. The precipitate was resuspended in a 2 L shaking flask in 500 ml of Terrific broth containing 200 μg / ml ampicillin (12 g / l bactotryptone (Difco), 24 g / l yeast extract (Difco), 4 ml / l glycerol, 12.54 g / l K2HPO4, 2.31 g / l KH2PO4). The culture was incubated at 37°C at 250 rpm until the absorbance at 550 nm reached 3-4, at which point enzyme expression was induced with 1 mM isopropyl-1-thio-β-galactopyranoside. Recovery of NGL and NGLv2 After incubation at 37°C for 15 hours, the cells were recovered by centrifugation at 18,000 g for 30 minutes at 4°C, resuspended in 50 mM BICINE (containing 10 mM NaCl), and lysed at 1500 bar in a pre-cooled cell disruptor (Panda 2K, Niro Soavi, GEA Process Engineering Division), followed by centrifugation at 40,000 g for 30 minutes. Chromosomal DNA was removed from the crude cell lysate by treatment with 0.2% protamine sulfate (Calbiochem) and centrifugation at 40,000 g for 30 minutes. Then, 25 units of benzonase (Merck, Darmstadt, Germany) were added to the solution.

[0088] The enzyme preparations were clarified by end filtration with a cutoff range of 0.05–1 μm using a Millipore POD system, and then concentrated by ultrafiltration on a cross-flow filtration system (Sartocon-Sartorius) with a 5 kDa cutoff. The concentrated enzyme solutions were filtered on a sterile filtration system including 0.8 μm and 0.22 μm end filters (absolute filters).

[0089] Example 3: Characterization of the enzyme NGL and NGLv2 were evaluated for lipase (LIP), phospholipase (PL), and galactolipase (GL) activity using the method described in Example 1, and compared with commercially available baking enzymes listed below, along with the manufacturer and stated activity: - Noopazyme (Novozymes): Lipase - Lipopan Max (Novozymes): Lipase - Lipase F (Novozymes): lipase - Panamore Golden (DSM): Lipase - Powerbake 4080 (DuPont / Danisco): Glycolipase

[0090] [Table 1]

[0091] Example 4: Whole Wheat Bread The bread dough was prepared using the ingredients listed in Table 2. The control test was based on the addition of an emulsifier (MD HP20, Puratos, Belgium) known to have a positive effect on dough tolerance.

[0092] [Table 2]

[0093] process All ingredients were mixed in a Diosna SP24 mixer at low speed for 2 minutes and at high speed for 8 minutes. The final dough temperature was 27°C. The dough was shaped by hand (600g dough pieces) and allowed to undergo intermediate fermentation for 20 minutes at 21°C and 50-55% relative humidity in the bakery laboratory. The dough pieces (600g each) were prepared and shaped using the Quality & Ethics-Jac Unic line set to R6 / L16. The dough pieces that were not subjected to impact were given a final fermentation in a Koma SunRiser at 35°C and 95% relative humidity for 50 minutes. The dough pieces that were subjected to impact were given a final fermentation in a Koma SunRiser at 35°C and 95% relative humidity for 70 minutes. Impact was applied by dropping a baking pan from a height of 5.5 cm. Next, the dough was baked in a Miwe Condo oven 2 (Michael Wenz - Arnstein - Germany) at 230°C for 35 minutes, with steam injected (0.1L before baking, 0.2L after baking). The baked bread was allowed to cool for 180 minutes before volume measurement. Bread analysis The volume of the bread was measured using the commonly used rapeseed displacement method. The average volume of four loaves was calculated. The results are shown in Table 3. The values ​​are expressed as a percentage increase / decrease in volume compared to a reference loaf that was not subjected to impact.

[0094] [Table 3]

[0095] Example 5: Whole Wheat Bread Using the ingredients listed in Table 4, bread was prepared and evaluated in the same manner as described in Example 4.

[0096] [Table 4]

[0097] evaluation

[0098] [Table 5]

[0099] Example 6: Croissant For the dough, we used the ingredients listed in Table 6, and prepared the croissants using Aristo croissant (Puratos, Belgium) as the lamination fat.

[0100] [Table 6]

[0101] process All ingredients except for the lamination oil were mixed in a Diosna SP24 mixer at low speed for 4 minutes and at high speed for 3 minutes to obtain the dough. The final dough temperature was 20°C. The dough was cooled at -18°C for 4 minutes and allowed to rest. After the first lamination (two single folds) using the lamination oil, the dough was cooled in a KOMA freezer at -18°C for 30 minutes. The second lamination was performed using the lamination oil (one single fold), followed by cooling in a KOMA freezer at -18°C for 25 minutes. The laminated dough pieces (60g each) were shaped and rolled out. Final fermentation was carried out in a Koma fermentation box at 28°C and 80% relative humidity for 90 minutes. After fermentation, the dough pieces were frozen in a Koma rapid freezer at -30°C for 35 minutes until the core temperature reached approximately -7°C. The frozen croissant dough was stored at -18°C for 3 months. After thawing at room temperature for 30 minutes, the croissant dough was baked in a Miwe Roll-In oven for 19 minutes using the following temperature profile: 225°C for 1 minute (with steam (0.7L)) followed by 195°C for 13 minutes. After baking, the croissants were allowed to cool to room temperature, and their volume was measured using a 3D scanner. evaluation The total volume of five croissants (croissant "1") prepared using DATEM was 1775 ml. The total volume of five croissants (croissant "2") prepared using the enzyme taught herein was 1900 ml.

[0102] Example 7: Partially baked, fully frozen baguette Partially baked and fully frozen baguette using the dough composition shown in Table 7.

[0103] [Table 7]

[0104] process All ingredients were mixed in a Diosna SP24 mixer in two stages: low speed for 2 minutes and high speed for 7 minutes. The final dough temperature was 26°C. The dough was then allowed to rest at 25°C for 5 minutes. After that, dough pieces (160g each) were formed and allowed to rise for 10 minutes at 25°C. Shaping was done in a Bertrand T4.5 / L3 (±28cm), and then the dough pieces were allowed to rise for 75-90 minutes at 28°C in a Koma oven until they reached a height of 6.5cm. Three cuts were then made in each dough piece. The first (pre-)baking was done in a Miwe Roll In oven with the following temperature profile: 190°C for 1 minute (steam (1L)), then 175°C for 15 minutes. The baked products were cooled to 25°C over 30-40 minutes and then cooled in a Koma rapid freezer at -30°C for 45-60 minutes until the core temperature reached approximately -8°C. The pre-baked baguettes were stored in a Koma freezer at -18°C for 3 weeks. Before final baking, the baguettes were thawed at 25°C for 40 minutes. Baking was performed in a Miwe Econo oven at 180°C for 8 minutes. After baking, the baguettes were allowed to cool to room temperature and their volume was measured using the commonly used rapeseed method. evaluation The volume of the baguette prepared using the enzyme taught herein (baguette "2") was 8% larger than the volume of the control baguette prepared without the enzyme (baguette "1").

[0105] Example 8: Crustyroll Crusty rolls were prepared using the dough composition shown in Table 8.

[0106] [Table 8]

[0107] process All ingredients were mixed in an Eberhardt N24 mixer at low speed for 2 minutes and at high speed for 5 minutes. The final dough temperature, as well as the resting and fermentation temperatures, was 25°C. After resting at 25°C for 15 minutes, the dough was reworked by hand and rested for another 10 minutes. Then, a 2kg piece of dough was made and fermented for 10 minutes. The 2kg piece of dough was divided and prepared using a Rotamat. Round pieces of dough (50g each) were obtained. After resting for another 5 minutes, the dough was cut by pressing, half of the pieces were subjected to a final fermentation stage at 35°C for 120 minutes, and the other half was subjected to an impact test (dropping a tray of dough onto a shelf from a height of 10cm) before being subjected to a final fermentation stage at 35°C for 120 minutes. The pieces of dough were baked with steam in a MIWE Roll-In oven (Michael Wenz-Arnstein-Germany) at 230°C. The volume of the six rolls was measured using the commonly used rapeseed method.

[0108] The results are shown in Table 9.

[0109] [Table 9]

[0110] Example 9: Modeling of the SEQ ID NO: 4 structure Using Alphafold (https: / / github.com / deepmind / alphafold), the structure of sequence number 4 was predicted using the protein sequence as input. The algorithm's default parameters (CASP14 model) were used. The generated output is a file containing atomic coordinates, as shown in Figure 1.

[0111] Example 10: Identification of similar structures using the SEQ ID NO: 4 structure (Figure 1) Using the Foldseek tool (available at https: / / github.com / steineggerlab / foldseek), we calculated the TM score between the SEQ ID NO: 4 structure (Figure 1) and the Alphafold protein structure database (available at https: / / alphafold.ebi.ac.uk / ), which contains all available structures (e-value = 0.001, alignment type: TM-align, mode: easy-search). Using Foldseek, as described in Zhang and Skolnick, 2004 (PROTEINS:Structure, Function, and Bioinformatics 57:702-710), the TM score and root mean square deviation between the SEQ ID NO: 4 structure and the 200 million structures in the Alphafold protein structure database can be calculated without the need for a supercomputer. Structures with a TM score of 0.9 to 1 (RMSD of 0 to 1.8 Å) are considered to be galactolipases identical to the SEQ ID NO: 4 structure.

Claims

1. A polypeptide having galactolipase activity, characterized in that the ratio of galactolipase activity to phospholipase activity is 100.0 or more, and / or the ratio of galactolipase activity to lipase activity is 150.0 or more.

2. The polypeptide according to claim 1, wherein the ratio of galactolipase activity to phospholipase activity is 1000.0 or more.

3. The polypeptide according to claim 1 or 2, wherein the ratio of galactolipase activity to lipase activity is 300.0 or more, preferably 500.0 or more, and more preferably 1000.0 or more.

4. below: (a) A polypeptide comprising a sequence having at least 80.0% sequence identity with the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, preferably SEQ ID NO: 4; (b) A polypeptide comprising a sequence encoded by a polynucleotide having at least 80.0% sequence identity with the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 3, preferably SEQ ID NO: 3; (c) A fragment of the polypeptide of (a) or (b) having galactolipase activity, or; (d) A polypeptide having at least 90.0% structural similarity to the polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, preferably SEQ ID NO:

4. A polypeptide according to any one of claims 1 to 3, selected from the above.

5. The polypeptide according to any one of claims 1 to 4, wherein the three-dimensional protein coordinates of the atoms in the amino acid sequence are defined by the set of atomic structure coordinates shown in Figure 1, or have a root mean square deviation of 1.8 Å or less obtained from the set of atomic structure coordinates shown in Figure 1.

6. The polypeptide according to any one of claims 1 to 5, wherein the polypeptide is Clostridium galactolipase.

7. Use of the polypeptide according to any one of claims 1 to 6 as a food additive or in the manufacture of a food product.

8. The use according to claim 7, wherein the food product is (a) dough or dough product; (b) batter or batter product; or (c) bakery or patisserie product.

9. The use according to claim 7, wherein the food additive is a bread improver.

10. The use according to claim 8 or 9, wherein the food product further comprises whole grain flour.

11. Use according to any one of claims 7 to 10 for increasing the resistance of a fabric or batter.

12. A method for producing a food product, comprising the step of mixing a polypeptide having galactolipase activity as described in any one of claims 1 to 6 with a food material.

13. The method according to claim 12, wherein the food product is a bakery or patisserie product, and the polypeptide having galactlipase activity according to any one of claims 1 to 6 is added to the dough or batter.

14. The method according to claim 12 or 13, wherein the food product further comprises whole grain flour.

15. Food products, dough products, batter products, or bakery products obtained by the use or method described in any one of claims 7 to 14.

16. An isolated nucleic acid encoding a polypeptide having galactolipase activity according to any one of claims 1 to 6, preferably comprising a sequence having at least 80.0% sequence identity with SEQ ID NO: 1 or SEQ ID NO: 3, and more preferably comprising a sequence having at least 80.0% sequence identity with SEQ ID NO:

3.

17. An expression vector comprising the nucleic acid described in claim 16.

18. A host cell comprising the nucleic acid described in claim 16 or the expression vector described in claim 17, preferably a bacterial, fungal, or yeast cell.

19. A method for identifying polypeptides having galactolipase activity to improve dough tolerance, comprising the step of determining whether the polypeptide has at least 90.0% structural similarity to a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4, preferably SEQ ID NO:

4.

20. A method for identifying polypeptides having galactolipase activity to improve dough tolerance, comprising the step of determining whether the three-dimensional protein coordinates of atoms in the amino acid sequence of the polypeptide have a root mean square deviation obtained from a set of atomic structure coordinates shown in Figure 1, of 1.8 Å or less.

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