Variant lipolytic enzymes

[0001] This application is a 35 U.S.C. 371 national application of PCT/DK2004/000292 filed Apr. 29, 2004, which claims priority or the benefit under 35 U.S.C. 119 of Danish application nos. PA 2003 00709 filed May 9, 2003 and PA 2003 00811 filed May 30, 2003 and U.S. Provisional application Nos. 60/469,228, 60/474,881 and 60/479,647 filed May 9, 2003, May 30, 2003, and Jun. 19, 2003, respectively, the contents of which are fully incorporated herein by reference.

[0002] The present invention relates to variant polypeptides made by altering the amino acid sequence of a fungal lipolytic enzyme, particularly to such polypeptides with improved properties for use in a dough, e.g. for making bread and other baked products, and more particularly to such polypeptides having hydrolytic activity towards ester bonds in polar lipids.

[0003] Phospholipases and galactolipases are known as enzymes with hydrolytic activity towards ester bonds in polar lipids such as phospholipids and galactolipids. WO 0032758 discloses lipolytic enzyme variants having phospholipase and galactolipase activity and their use in baking. WO 9826057 discloses a lipase/phospholipase from Fusarium oxysporum and its use in baking. WO 0183770 describes variants of a fungal lipase.

[0004] The inventors have developed variant polypeptides by modifying the amino acid sequence of a parent polypeptide which is a fungal lipolytic enzymes. The variant polypeptides result in a reduced dough stickiness, compared to the parent polypeptide, when they are added to a dough.

[0005] Accordingly, the invention provides a method of producing a polypeptide, comprising:

[0006] a) selecting an amino acid sequence for a parent polypeptide which is a fungal lipolytic enzyme,

[0007] b) selecting an amino acid residue in the sequence which corresponds to A29, K33, I83 or A255 of SEQ ID NO: 1 (corresponding to P29, N33, R84 or P256 of SEQ ID NO: 2),

[0008] c) modifying the amino acid sequence by substituting or deleting the selected residue,

[0009] d) preparing a variant polypeptide having the modified amino acid sequence, and

[0010] e) adding the polypeptide to a dough and testing dough stickiness.

[0011] The invention also provides a variant polypeptide which:

[0012] a) has hydrolytic activity towards an ester bonds in a polar lipid, and

[0013] b) has an amino acid sequence which

• • [0014] i) has at least 80% identity to SEQ ID NO: 1 and has a different amino acid or an amino acid deletion at a position corresponding to A29, K33, I83 or A255, or
  • [0015] ii) has at least 80% identity to SEQ ID NO: 2 and has a different amino acid or an amino acid deletion at a position corresponding to R84 or P256.

Parent Polypeptide

[0017] The parent polypeptide may have the sequence SEQ ID NO: 1 or 2 or one which can be aligned with SEQ ID NO: 1 or 2. It may have at least 50% amino acid identity to SEQ ID NO: 1 or 2, e.g. at least 60%, at least 70% or at least 80%. Examples are the polypeptides having the sequences SEQ ID NO: 1 to 14 or a variant disclosed in WO 0032758.

[0018] The parent polypeptide has lipolytic enzyme activity, e.g. hydrolytic activity towards an ester bond in a polar lipid.

Variant Polypeptide

[0019] The amino acid at the position corresponding to A29 in SEQ ID NO: 1 may be P. The amino acid at the position corresponding to K33 in SEQ ID NO: 1 may be N. The amino acid at the position corresponding to I83 of SEQ ID NO: 1 may be A/R/N/D/C/Q/E/G/H/L/K/M/F/P/S/T/Y/V. The amino acid at the position corresponding to A255 in SEQ ID NO: 1 may be R/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/W/Y/V.

[0020] The amino acid at the position corresponding to R84 of SEQ ID NO: 2 may be A/N/D/C/Q/E/G/H/I/L/K/M/F/P/S/T/Y/V. The amino acid at the position corresponding to P256 in SEQ ID NO: 2 may be A/R/N/D/C/Q/E/G/H/I/L/K/M/F/S/T/W/Y/V. The polypeptide may comprise further modifications compared to SEQ ID NO: 2., e.g. as disclosed in WO 0032758. Thus, it may have the amino acid A/T at position D62, G/T at position A91, D/F/S/G at position W96, E at position K99, G at position S158, D at position G240, S at position N247, D at position N248, K/R at position Q249, K/T at position P250, T at position N251, F at position I252, M/R at position P253, S/Y/W at position D254, L at position I255, G at position A257, H/C at position W260, G at position Q263, L at position A264, I at position T265, G/S/A at position D266, T at position A267, L at position N269 and/or truncation after N269.

[0021] The polypeptide may additionally comprise amino acid modifications such as insertions or deletions. Also, the N- or C-terminus may be modified, e.g. by truncating residues in SEQ ID NO: 2 after position 269 or by extending the C-terminal of SEQ ID NO: 2 with WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS. The C-terminal may be truncated after position 272, 273, 274 or 286 in SEQ ID NO: 1. The N-terminal may have a peptide extension, e.g. as described in WO 0032758 or WO 9704079, such as the addition of the amino acid residues SPIRR.

[0022] A similar amino acid substitution or deletion may be made in other fungal lipolytic enzymes, e.g. SEQ ID NO: 3-14 at a corresponding position. The corresponding positions may be found by aligning a given sequence with SEQ ID NO: 1 or 2, e.g. as shown in FIG. 1. The alignment may be done by use of the GAP program as described below.

[0023] The variant polypeptide may have improved thermostability compared to the parent polypeptide, particularly a variant polypeptide having a substitution at a position corresponding to A29 or K33 of SEQ ID NO: 1, e.g. the substitution A29P or K33N.

Sequence Identity

[0024] The variant polypeptide has at least 80% identity to SEQ ID NO: 1 or 2, particularly at least 85%, at least 90%, at least 95%, or at least 98%. The degree of identity between two sequences may be suitably determined by means of computer programs known in the art, such as GAP provided in the GCG program package (Program Manual for the Wisconsin Package, Version 8, August 1994, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711) (Needleman, S. B. and Wunsch, C. D., (1970), Journal of Molecular Biology, 48, 443-45), using GAP with the following settings for polypeptide sequence comparison: GAP creation penalty of 3.0 and GAP extension penalty of 0.1.

Dough Stickiness

[0025] The variant polypeptide may be tested by adding it to a dough and evaluating the dough stickiness. The dough may be generated according to a typical European straight dough procedure, a typical American sponge & dough procedure or any other bread making procedures. The polypeptide may be added at a dosage of 0.01-10 mg enzyme protein per kg flour, and the dough stickiness may be evaluated directly after mixing or at any point during processing. Of particular importance is the dough stickiness of the finally mixed dough, i.e. at the time where the dough runs through processing equipment such as divider, molder, sheeter and conveyor belts. The mixing time varies depending on procedure. For a typical European straight dough procedure, the mixing time can e.g. be in the range of 6-10 minutes. For a typical American Sponge & dough procedure the mixing time can e.g. be in the range of 6-20 minutes (on final dough). The dough may have a resting period of 5-20 min before further processing, e.g. at 20-35° C. The dough stickiness may be evaluated by hand by trained bakers, by a sensory panel or by instrumental measurements e.g. by the Chen-Hoseney dough stickiness rig developed for Stable Micro Systems TA-XT2 texture analyser, commercially available from Brookfield Engineering Laboratories, Inc.

Hydrolytic Activity towards Ester Bonds in Polar Lipids

[0026] The parent and variant polypeptides have lipolytic enzyme activity, i.e. they have hydrolytic activity towards an ester bond and are classified in EC 3.1.1 Carboxylic Ester Hydrolases according to Enzyme Nomenclature (available at www.chem.qmw.ac.uk/iubmb/enzyme). More specifically, they have hydrolytic activity towards ester bonds in polar lipids so as to split off acyl groups at the sn-1 and/or sn-2 position of polar lipids such as phospholipids and galactolipids. Accordingly, they may have phospholipase activity or galactolipase activity (EC 3.1.1.26), e.g. phospholipase A1 activity (EC 3.1.1.32).

[0027] Phospholipase activity may be determined by known methods, e.g. the “monolayer phospholipase assay” or the plate assay described in WO 0032758. Galactolipase activity may be determined with digalactosyl diglyceride as substrate, e.g. as described in WO 0032758.

Use of Polypeptide

[0028] The polypeptide may be added to a dough, and the dough may be used to prepare a steamed bread, a baked product (particularly bread), pasta or noodles. The addition of the polypeptide may lead to improved dough stabilization, i.e. a larger loaf volume of the baked product and/or a better shape retention and volume during processing and baking, particularly in a stressed system, e.g. in the case of over-proofing or over-mixing. It may also lead to a lower initial firmness and/or a more uniform and fine crumb, improved crumb structure (finer crumb, thinner cell walls, more rounded cells), of the baked product, and it may further improve dough properties, e.g. a less soft dough, higher elasticity and/or lower extensibility.

[0029] The process may be conducted in analogy with U.S. Pat. No. 5,578,489 or U.S. Pat. No. 6,077,336. In the case of un-proofed frozen dough the polypeptides of the invention perform better than known lipolytic enzyme variants in terms of volume and crumb structure.

[0030] The polypeptide can be used in a process for making bread, comprising adding the polypeptide to the ingredients of a dough, kneading the dough and baking the dough to make the bread. This can be done in analogy with U.S. Pat. No. 4,567,046 (Kyowa Hakko), JP-A 60-78529 (QP Corp.), JP-A 62-111629 (QP Corp.), JP-A 63-258528 (QP Corp.), EP 426211 (Unilever) or WO 99/53769 (Novozymes).

[0031] The composition of a typical dough can be found in WO 99/53769.

[0032] The polypeptide of the invention may be added together with an anti-staling amylase and optionally also a phospholipid as described in WO 9953769, particularly a maltogenic alpha-amylase (e.g. from Bacillus sp., such as Novamyl® from Novo Nordisk). Also, a fungal or bacterial α-amylase may be added, e.g. from Aspergillus or Bacillus, particularly A. oryzae, B. licheniformis or B. amyloliquefaciens. Optionally an additional enzyme may be added, e.g. an amyloglucosidase, a beta-amylase, a pentosanase such as a xylanase as described in WO 99/53769, e.g. derived from Aspergillus, in particular of A. aculeatus, A. niger (cf. WO 91/19782), A. awamori (WO 91/18977), or A. tubigensis (WO 92/01793), from a strain of Trichoderma, e.g. T. reesei, or from a strain of Humicula, e.g. H. insolens (WO 92/17573), a proteiase and/or a glucose oxidase.

[0033] The dough may further comprise an emulsifier such as mono- or diglycerides, diacyl tartaric acid esters of mono- or diglycerides, sugar esters of fatty acids, polyglycerol esters of fatty acids, lactic esters of monoglycerides, acetic acid esters of monoglycerides, polyoxyethylene stearates, polysorbates or lysolecithin.

[0034] The dough may also comprise other conventional dough ingredients, e.g.: proteins, such as milk powder, gluten, and soy; eggs (either whole eggs, egg yolks or egg whites); an oxidant such as ascorbic acid, potassium bromate, potassium iodate, azodicarbonamide (ADA) or ammonium persulfate; an amino acid such as L-cysteine; a sugar; a salt such as sodium chloride, calcium acetate, sodium sulfate or calcium sulfate.

Baking Evaluation of Polypeptides with Phospholipase Activity

[0035] In the examples, polypeptides according to the invention were tested together with the corresponding parent polypeptide in a baking evaluation experiment by using conventional baking protocols for European straight dough procedure and US sponge & dough procedure, as follows:

European Straight Dough Procedure:

[0036] A dough is prepared by mixing the below ingredients for 3 minutes slow and 7 minutes fast.

[0037]

	% (baker's - by weight)
	Flour	100
	Compressed yeast	4
	Salt	1.5
	Sugar	1.5
	Water	62
	Ascorbic acid	40	ppm

[0038] Dough stickiness is evaluated right after mixing and again after a resting period of 15 minutes. Dough stickiness is evaluated by a trained and experienced bakers by sensory evaluation by hand. Dough stickiness is a measure of how sticky the dough feels and is expressed on a scale from 0 (little stickiness) to 10 (very sticky). The dough with the variant is compared to a reference dough, which is always given the score 5.

Sponge & Dough Procedure:

[0039]

	Sponge	Dough
	% (baker's - by weight)	% (baker's - by weight)
Flour	60	40
Compressed yeast	7.5
Oil	2.5
Salt		2
High fructose syrup		12
Water	34.4	20.4
Ascorbicc acid		50

[0040] A liquid sponge is prepared by mixing a sponge consisting of the above listed sponge ingredients for 1 minute slow and 4 minutes fast. The sponge is fermented for 3 hours at 27 C, 86% RH. The sponge is mixed with the dough ingredients listed above and with enzymes for 1 minutes slow and 18 minutes fast.

[0041] Dough stickiness is evaluated right after mixing, whereafter the dough is extruded on a rebuild pasta-machine to simulate the dough extrusion used for dough dividing in US. Dough stickiness is evaluated again after extrusion. Dough stickiness is evaluated by a trained and experienced bakers by sensory evaluation by hand. Dough stickiness is a measure of how sticky the dough feels and is expressed on a scale from 0 (little stickiness) to 10 (very sticky). The dough with the variant polypeptide is compared to a reference dough made with the parent polypeptide, which is always given the score 5.

[0042] Polypeptides according to the invention were prepared as described in WO 00/32758. The polypeptides were derived from SEQ ID NO: 15 by making the following amino acid modifications.

[0043]

Polypeptide	Amino acid alterations compared to SEQ ID NO: 15
1	G91A +D96W +E99K +P256M +G263Q +L264A +I265T +G266D +T267A +
	L269N +270A +271G +272G +273F +274S +
	275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS
2	G91A +D96W +E99K +P256N +G263Q +L264A +I265T +G266D +T267A +
	L269N +270A +271G +272G +273F +274S +
	275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS
3	G91A +D96W +E99K +P256V +G263Q +L264A +I265T +G266D +T267A +
	L269N +270A +271G +272G +273F +274S +
	275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS
4	G91A +D96W +E99K +N247S +N248D +Q249K +N251T +P253M +D254S +
	P256L +A257A +G263Q +L264A +I265T +G266D +T267A +L269N
5	G91A +D96W +E99K +N247S +N248D +Q249R +P250T +N251T +P253M +
	D254W +P256V +A257G +G263Q +L264A +I265T +G266D +T267A +
	L269N
6	G91A +D96W +E99K +P256T +G263Q +L264A +I265T +G266D +T267A +
	L269N +270A +271G +272G +273F +274S +
	275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS
7	G91A +D96W +E99K +P256A +G263Q +L264A +I265T +G266D +T267A +
	L269N +270A +271G +272G +273F +274S +
	275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS
8	G91A +D96W +E99K +G240D +P256C +G263Q +L264A +I265T +G266D +
	T267A +L269N +270A +271G +272G +273F +274S +
	275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS
9	G91A +D96W +E99K +P256G +G263Q +L264A +I265T +G266D +T267A +
	L269N +270A +271G +272G +273F +274S +
	275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS
10	G91A +D96W +E99K +P256R +G263Q +L264A +I265T +G266D +T267A +
	L269N +270A +271G +272G +273F +274S +
	275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS
11	G91A +D96W +E99K +P256Q +G263Q +L264A +I265T +G266D +T267A +
	L269N +270A +271G +272G +273F +274S +
	275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS
12	G91A +D96W +E99K +P256K +G263Q +L264A +I265T +G266D +T267A +
	L269N +270A +271G +272G +273F +274S +
	275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS
13	G91A +D96W +E99K +P256L +G263Q +L264A +I265T +G266D +T267A +
	L269N +270A +271G +272G +273F +274S +
	275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS
14	G91A +D96W +E99K +P256D +G263Q +L264A +I265T +G266D +T267A +
	L269N +270A +271G +272G +273F +274S +
	275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS
15	R84E +G91A +D96W +E99K +P256V +G263Q +L264A +I265T +G266D +
	T267A +L269N +270A +271G +272G +273F +274S +
	275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS
16	R84M +G91A +D96W +E99K +P256V +G263Q +L264A +I265T +G266D +
	T267A +L269N +270A +271G +272G +273F +274S +
	275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS
17	R84P +G91A +D96W +E99K +P256V +G263Q +L264A +I265T +G266D +
	T267A +L269N +270A +271G +272G +273F +274S +
	275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS
18	R84S +G91A +D96W +E99K +P256V +G263Q +L264A +I265T +G266D +
	T267A +L269N +270A +271G +272G +273F +274S +
	275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS
19	G91A +D96W +E99K +P250K +N251T +I252F +P253R +D254Y +I255L +
	P256del +G263Q +L264A +I265T +G266D +T267A +L269N +270A +271G +
	272G +273F +274S +
	275WRRYRSAESVDKRATMTDAELEKKLNSYVQMDKEYVKNNQARS

[0044] 5 variant polypeptides according to the invention were compared to the parent polypeptide (SEQ ID NO: 2) in the European straight dough procedure described above. 40 ppm Fungamyl Super MA (a blend of fungal alpha-amylase and xylanase) was added as background to all doughs. The parent enzyme and the variants were dosed at their optimal level, i.e. the level giving best volume and dough stabilising effect. The below results show that all 5 variants give reduced dough stickiness compared to the parent polypeptide.

[0045]

Polypeptide	Parent	P256V	P256A	P256Q	P256L	P256W
Dough	6	5	5	5	5	5
stickiness after
mixing
Dough	6	5	5	5	5	5
stickiness after
15 min table
time

[0046] 3 variant polypeptides according to the invention were compared to the parent polypeptide (SEQ ID NO: 1) in the European straight dough procedure described above. 40 ppm Fungamyl Super MA (a blend of fungal alpha-amylase and xylanase) was added as background to all doughs. The parent enzyme and the variants were dosed at their optimal level, i.e. the level giving best volume and dough stabilising effect. The below results show that all 4 variants give reduced dough stickiness compared to the parent enzyme

[0047]

Polypeptide	Parent	A29P+K33N+I83T	A29P+K33N+I83H	A29P+K33N+I83Q
Dough stickiness	5	4	4	4
after mixing
Dough stickiness	5	4	4	4
after 15 min table
time

[0048] 4 variant polypeptides according to the invention were compared to the parent enzyme (SEQ ID NO: 1) in the European straight dough procedure described above. 10FAU Fungamyl/kg was added as background to all doughs. The parent enzyme and the variants were dosed at their optimal level, i.e. the level giving best volume and dough stabilising effect. The below results show that all 4 variants give reduced dough stickiness compared to the parent enzyme

[0049]

Polypeptide	Parent	A29P + K33N	A29P + I83N	K33N + I83E	K33N + I83K
Dough stickiness	7	6	6	6	6
after mixing
Dough stickiness	7	6	6	6	6
after 15 min table
time

[0050] A variant polypeptide according to the invention was compared to its parent enzyme (SEQ ID NO: 1) in the US sponge & dough procedure described above. 40 ppm Fungamyl Super MA (a blend of fungal alpha-amylase and xylanase) was added as background to all doughs. The parent enzyme and the variant were dosed at their optimal level, i.e. the level giving best volume and dough stabilising effect. The below results show that the variant gives reduced dough stickiness compared to the parent enzyme

[0051]

	Polypeptide	Parent	A29P+I83N
	Dough stickiness after	6	5
	mixing
	Dough stickiness after	6.5	5
	extrusion

[0052] Variant polypeptides with the following amino acid alterations compared SEQ ID NO: 1 (lipase/phosapholipase from F. oxysporum) were prepared and tested by adding each polypeptide to a dough. The polypeptide with unmodified SEQ ID NO: 1 was also tested, for comparison.

[0053]

	A29P
	K33N
	A29P +I83T
	A29P +I83N
	A29P +I83C
	A29P +I83F
	A29P +I83L
	K33N +I83W
	K33N +I83L
	K33N +I83Q
	K33N +I83S
	K33N +I83N
	K33N +I83N
	K33N +I83R
	K33N +I83L
	K33N +270VASLGDDTEAPRASTRGPP
	A29P +I83N +A255V

[0054] The results were that with each of the above polypeptides, dough stickiness was better than with the polypeptide with the unmodified sequence of SEQ ID NO: 1.

[0055] Baking tests with each dough showed that all polypeptides improved the crumb structure, the loaf volume and the dough stability, both for the modified and unmodified sequences.

[0056] Variant polypeptides with the following amino acid alterations compared SEQ ID NO: 2 (variant of T. lanuginosus lipase) were prepared and tested by adding each polypeptide to a dough. The polypeptide with unmodified SEQ ID NO: 2 was also tested for comparison.

[0057]

	R84D
	R84I
	R84M
	R84Q
	P256A
	P256D
	P256I
	P256L
	P256Q
	P256S
	P256V

[0058] The results were that with each of the above polypeptides, dough stickiness was better than with the polypeptide with the unmodified sequence of SEQ ID NO: 2.

[0059] Baking tests with each dough showed that all polypeptides improved the crumb structure, the loaf volume and the dough stability, both for the modified and unmodified sequences.