WO2004099400A2 - Variant lipolytic ensymes - Google Patents

Variant lipolytic ensymes Download PDF

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Publication number
WO2004099400A2
WO2004099400A2 PCT/DK2004/000292 DK2004000292W WO2004099400A2 WO 2004099400 A2 WO2004099400 A2 WO 2004099400A2 DK 2004000292 W DK2004000292 W DK 2004000292W WO 2004099400 A2 WO2004099400 A2 WO 2004099400A2
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WO
WIPO (PCT)
Prior art keywords
polypeptide
amino acid
dough
seq
activity
Prior art date
Application number
PCT/DK2004/000292
Other languages
French (fr)
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WO2004099400A3 (en
Inventor
Kim Borch
Luise Erlandsen
Jesper Vind
Allan Svendsen
Christel Thea JØRGENSEN
Original Assignee
Novozymes A/S
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novozymes A/S filed Critical Novozymes A/S
Priority to DK04730201.3T priority Critical patent/DK1625216T3/en
Priority to AU2004236305A priority patent/AU2004236305B2/en
Priority to US10/556,511 priority patent/US7465570B2/en
Priority to CA2523400A priority patent/CA2523400C/en
Priority to EP04730201A priority patent/EP1625216B1/en
Publication of WO2004099400A2 publication Critical patent/WO2004099400A2/en
Publication of WO2004099400A3 publication Critical patent/WO2004099400A3/en
Priority to US12/169,679 priority patent/US7638317B2/en
Priority to US12/612,398 priority patent/US8298800B2/en
Priority to AU2010241524A priority patent/AU2010241524B2/en
Priority to AU2010246333A priority patent/AU2010246333B2/en

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Classifications

    • A—HUMAN NECESSITIES
    • A21—BAKING; EDIBLE DOUGHS
    • A21D—TREATMENT, e.g. PRESERVATION, OF FLOUR OR DOUGH, e.g. BY ADDITION OF MATERIALS; BAKING; BAKERY PRODUCTS; PRESERVATION THEREOF
    • A21D8/00—Methods for preparing or baking dough
    • A21D8/02—Methods for preparing dough; Treating dough prior to baking
    • A21D8/04—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes
    • A21D8/042—Methods for preparing dough; Treating dough prior to baking treating dough with microorganisms or enzymes with enzymes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/18—Carboxylic ester hydrolases (3.1.1)
    • C12N9/20—Triglyceride splitting, e.g. by means of lipase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
    • C12Y301/01032—Phospholipase A1 (3.1.1.32)

Definitions

  • 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.
  • Phospholipases and galactolipases are known as enzymes with hydrolytic activity towards ester bonds in polar lipids such as phospholipids and gaiactolipids.
  • 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.
  • 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.
  • the invention provides a method of producing a polypeptide, comprising: a) selecting an amino acid sequence for a parent polypeptide which is a fungal lipolytic enzyme, b) selecting an amino acid residue in the sequence which corresponds to A29, K33, 183 or A255 of SEQ ID NO: 1 (corresponding to P29, N33, R84 or P256 of SEQ ID NO: 2), c) modifying the amino acid sequence by substituting or deleting the selected residue, d) preparing a variant polypeptide having the modified amino acid sequence, and e) adding the polypeptide to a dough and testing dough stickiness.
  • the invention also provides a variant polypeptide which: a) has hydrolytic activity towards an ester bonds in a polar lipid, and b) has an amino acid sequence which 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, 183 or A255, or 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.
  • Figure 1 shows an alignment of amino acid sequences of fungal lipolytic enzymes to identify corresponding amino acids in SEQ ID NO: 1 to 15.
  • SEQ ID NO: 1 is the lipase/phospholipase from Fusarium oxysporum (WO 9826057).
  • SEQ ID NO: 2 is a variant with pohospholipase and galactolipase activity disclosed in WO 0032758.
  • SEQ ID NO: 3 to 15 are known lipolytic enzymes from the following organisms: Absidia reflexa, Absidia corymbefera, Rhizomucor miehei, Rhizopus delemar (oryzae), Aspergillus niger, Aspergillus tubingensis, Fusarium heterosporum, Aspergillus oryzae, Penicilium camembertii, Aspergillus foetidus, Aspergillus niger, Aspergillus oryzae and Thermomyces lanuginosus.
  • 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.
  • the parent polypeptide has lipolytic enzyme activity, e.g. hydrolytic activity towards an ester bond in a polar lipid.
  • 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/N.
  • 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/WY/V.
  • 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/YV.
  • 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.
  • 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 ⁇ 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.
  • the polypeptide may additionally comprise amino acid modifications such as insertions or deletions.
  • 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.
  • 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.
  • 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.
  • 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, Wisconsin, USA 53711) (Needleman, S.B. and Wunsch, CD., (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.
  • 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 -10mg 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 conveyer 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.
  • 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.
  • 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 http://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).
  • 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.
  • 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/orlower extensibility.
  • the process may be conducted in analogy with US 5578489 or US 6077k336.
  • the polypeptides of the invention perform better than known lipolytic enzyme variants in terms of volume and crumb structure.
  • 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 US 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).
  • composition of a typical dough can be found in WO 99/53769.
  • 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).
  • a fungal or bacterial ⁇ -amylase may be added, e.g. from Aspergillus or Bacillus, particularly A. oryzae, B. licheniformis or ⁇ . amyloliquefaciens.
  • 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. 7. reesei, or from a strain of Humicula, e.g. H. insolens (WO 92/17573), a proteiase and/or a glucose oxidase.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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:
  • a dough is prepared by mixing the below ingredients for 3 minutes slow and 7 minutes fast.
  • 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.
  • 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 3hours at 27C, 86%RH.
  • the sponge is mixed with the dough ingredients listed above and with enzymes for 1 minutes slow and 18 minutes fast.
  • 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.
  • 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.
  • Example 2 Baking evaluation of a polypeptide according to the invention.
  • 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
  • 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.
  • Variant polypeptides with the following amino acid alterations compared SEQ ID NO: 2 (variant of 7. 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.

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Abstract

The inventors have developed improved polypeptides by substituting or deleting specified amino acids in fungal lipolytic enzymes. More particularly, the polypeptides result in a reduction of dough stickiness when they are added to a dough. The polypeptides may particularly have activity on polar lipids.

Description

VARIANT LIPOLYTIC ENZYMES
FIELD OF INVENTION
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.
BACKGROUND OF THE INVENTION
Phospholipases and galactolipases are known as enzymes with hydrolytic activity towards ester bonds in polar lipids such as phospholipids and gaiactolipids. 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.
SUMMARY OF THE INVENTION
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.
Accordingly, the invention provides a method of producing a polypeptide, comprising: a) selecting an amino acid sequence for a parent polypeptide which is a fungal lipolytic enzyme, b) selecting an amino acid residue in the sequence which corresponds to A29, K33, 183 or A255 of SEQ ID NO: 1 (corresponding to P29, N33, R84 or P256 of SEQ ID NO: 2), c) modifying the amino acid sequence by substituting or deleting the selected residue, d) preparing a variant polypeptide having the modified amino acid sequence, and e) adding the polypeptide to a dough and testing dough stickiness.
The invention also provides a variant polypeptide which: a) has hydrolytic activity towards an ester bonds in a polar lipid, and b) has an amino acid sequence which 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, 183 or A255, or 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. BRIEF DESCRIPTION OF DRAWINGS
Figure 1 shows an alignment of amino acid sequences of fungal lipolytic enzymes to identify corresponding amino acids in SEQ ID NO: 1 to 15. SEQ ID NO: 1 is the lipase/phospholipase from Fusarium oxysporum (WO 9826057). SEQ ID NO: 2 is a variant with pohospholipase and galactolipase activity disclosed in WO 0032758. SEQ ID NO: 3 to 15 are known lipolytic enzymes from the following organisms: Absidia reflexa, Absidia corymbefera, Rhizomucor miehei, Rhizopus delemar (oryzae), Aspergillus niger, Aspergillus tubingensis, Fusarium heterosporum, Aspergillus oryzae, Penicilium camembertii, Aspergillus foetidus, Aspergillus niger, Aspergillus oryzae and Thermomyces lanuginosus.
DETAILED DESCRIPTION OF THE INVENTION
Parent polypeptide
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.
The parent polypeptide has lipolytic enzyme activity, e.g. hydrolytic activity towards an ester bond in a polar lipid.
Variant polypeptide
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/N. 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/WY/V.
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/YV. 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, SΛ 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.
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.
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. 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
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, Wisconsin, USA 53711) (Needleman, S.B. and Wunsch, CD., (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
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 -10mg 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 conveyer 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 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 http://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).
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
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/orlower extensibility. The process may be conducted in analogy with US 5578489 or US 6077k336. 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.
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 US 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).
The composition of a typical dough can be found in WO 99/53769.
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 β. 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. 7. reesei, or from a strain of Humicula, e.g. H. insolens (WO 92/17573), a proteiase and/or a glucose oxidase.
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.
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.
EXAMPLES
Baking evaluation of polypeptides with phospholipase activity
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:
A dough is prepared by mixing the below ingredients for 3 minutes slow and 7 minutes fast.
Figure imgf000006_0001
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:
Figure imgf000007_0001
A liquid sponge is 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 3hours at 27C, 86%RH. The sponge is mixed with the dough ingredients listed above and with enzymes for 1 minutes slow and 18 minutes fast.
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.
Example 1. Construction of polypeptides.
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.
Figure imgf000007_0002
Figure imgf000008_0001
Figure imgf000009_0001
Example 2. Baking evaluation of a polypeptide according to the invention.
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.
Figure imgf000009_0002
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
Figure imgf000010_0001
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
Figure imgf000010_0002
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
Figure imgf000010_0003
Example 3. Variant polypeptides derived from SEQ ID NO: 1
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.
Figure imgf000011_0001
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.
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.
Example 4. Variant polypeptides derived from SEQ ID NO: 2
Variant polypeptides with the following amino acid alterations compared SEQ ID NO: 2 (variant of 7. 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.
R84D
R84I
R84M R84Q
P256A
P256D
P256I
P256L
P256Q
P256S
P256V
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.
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.

Claims

1. A method of producing a polypeptide, comprising: a) selecting an amino acid sequence for a fungal lipolytic enzyme, b) selecting an amino acid residue in the sequence which corresponds to A29, K33, I83 or A255 of SEQ ID NO: 1 , c) modifying the amino acid sequence by substituting or deleting the selected residue, d) preparing a polypeptide having the modified amino acid sequence, and e) adding the polypeptide to a dough and testing dough stickiness.
2. The method of claim 1 which further comprises testing hydrolytic activity of the polypeptide towards ester bonds in polar lipids and selecting a polypeptide which has such activity.
3. A polypeptide which: a) has hydrolytic activity towards an ester bond in a polar lipid, and b) has an amino acid sequence which 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, 183 or A255, or 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.
4. The polypeptide of claims 3 which has phospholipase activity or galactolipase activity
(EC 3.1.1.26), particularly phospholipase A1 activity (EC 3.1.1.32).
5. The polypeptide of claim 3 or 4 wherein the amino acid at the position corresponding to A29 of SEQ ID NO: 1 is P.
6. The polypeptide of any of claims 3-5 wherein the amino acid at the position corresponding to K33 of SEQ ID NO: 1 is N.
7. The polypeptide of any of claims 3-6 wherein the amino acid at the position corresponding to I83 of SEQ ID NO: 1 is N/C/W.
8. The polypeptide of any of claims 3-7 which consists of amino acids 1-272, 1-273, 1-
274 or 1-286 of SEQ ID NO: 1 with the following substitutions: A29P
K33N
A29P +I83T
A29P +I83N
A29P + I83C
A29P +I83F
A29P +I83L
K33N +I83W
K33N +I83L
K33N + I83Q
K33N +I83S
K33N +I83N
K33N +I83R
K33N +I83L
K33N +270VASLGDDTEAPRASTRGPP
A29P +I83N +A255V
9. The polypeptide of claim 3 or 4 wherein the amino acid at the position corresponding to R84 of SEQ ID NO: 2 is L/M/Q/l/D.
10. The polypeptide of claim 3, 4 or 9 wherein the amino acid at the position corresponding to P256 of SEQ ID NO: 2 is V/Q/A/D/S/l.
11. The polypeptide of any of claims 3, 4, 9 or 10 which consists of SEQ ID NO: 2 with the following substitutions:
Figure imgf000014_0001
12. The polypeptide of any of claims 3, 4 and 9-10 which compared to SEQ ID NO: 2 has 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 is truncated after N269.
13. A polynucleotide encoding the polypeptide of any of claims 3-12.
14. A method of preparing a dough or an edible product made from dough, comprising adding the polypeptide of any of claims 3-12 to the dough.
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EP04730201A EP1625216B1 (en) 2003-05-09 2004-04-29 Variant lipolytic enzymes
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