# Patent

## Title
Lipolytic enzymes

## Abstract

The inventors have developed a method using protein engineering to produce lipolytic enzymes having a relatively high activity for one ester bond in an amphiphilic substrate with two lipophilic groups) and a relatively low activity for the ester bond in an amphiphilic substrate with one lipophilic group, e.g. a relatively high phospholipase activity and a relatively low lysophospholipase activity.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. application Ser. No. 10/495,597 filed on May 14, 2004 (now abandoned), which claims priority of 35 U.S.C. 371 national application of PCT/DK03/00028 filed Jan. 16, 2003, which claims priority or the benefit under 35 U.S.C. 119 of Danish application no. PA 2002 00074 filed Jan. 16, 2002 and U.S. provisional application No. 60/353,557 filed Feb. 4, 2002, the contents of which are fully incorporated herein by reference.


FIELD OF THE INVENTION
The present invention relates to a lipolytic enzyme variant and a method of producing such a variant. More particularly, the variant has a relatively high activity for one ester bond in an amphiphilic substrate with two lipophilic groups and a relatively low activity for the ester bond in an amphiphilic substrate with one lipophilic group, e.g. a relatively high phospholipase activity and a relatively low lysophospholipase activity.
BACKGROUND OF THE INVENTION
EP 870840, JP-A 10-42884, JP-A 4-135456 or JP-A 2-49593 describe the use of a phospholipase to hydrolyze a phospholipid to produce lysophospholipid.
U.S. Pat. No. 4,567,046, WO 94/04035, EP 109244, EP 585988, WO 98/26057, WO 98/45453, WO 99/53769, WO 00/32758, WO 0139602 and EP 575133 describe the addition of various lipolytic enzymes to dough in the preparation of baked products and the preparation of lipolytic enzyme variants.
WO 00/32758 discloses that the substrate specificity of a lipolytic enzyme can be modified by making alterations to the amino acid sequence.
SUMMARY OF THE INVENTION
The inventors have developed a method using protein engineering to produce lipolytic enzymes having a relatively high activity for one ester bond in an amphiphilic substrate with two lipophilic groups and a relatively low activity for the ester bond in an amphiphilic substrate with one lipophilic group, e.g. a relatively high phospholipase activity and a relatively low lysophospholipase activity.
Accordingly, the invention provides a method of producing a lipolytic enzyme variant comprising:

    
    
        a) selecting a parent fungal lipolytic enzyme,
        b) in the parent lipolytic enzyme altering at least one specified amino acid residue,
        c) optionally, altering one or more amino acid residues other than b),
        d) preparing the variant resulting from steps a)-c),
        e) testing hydrolytic activities of the variant towards a first substrate and a second substrate,
        f) selecting a variant having a ratio of hydrolytic activities towards the first substrate and the second substrate which is lower than the parent lipolytic enzyme, and
        g) producing the selected variant.
    
    


The first substrate is a molecule comprising one fatty acyl group linked through an ester or thioester bond to a hydrophilic group. The second substrate is a molecule comprising a first lipophilic group which is a fatty acyl group linked through an ester or thioester bond to a hydrophilic group, and a second lipophilic group linked to the hydrophilic group, where the second lipolhilic group may be a second fatty acyl group linked through an ester, thioester or amide bond, or it may be a fatty alcohol linked through an ether or thioether bond.
Each amino acid alteration may be an amino acid substitution, deletion or insertion. The amino acid residue to be altered may be determined from a three-dimensional model of a phospholipid docked with the parent lipolytic enzyme as a residue which comprises an atom (excluding H atoms) which lies within 10 Å (particularly 7 Å or 5 Å) of an atom (excluding H atoms) of the lyso-phospholipid, or it may be the C-terminal amino acid.
Alternatively, the amino acid residue to be altered may be determined by aligning the amino acid sequence of the parent lipolytic enzyme with the T. lanuginosus lipase and selecting a residue corresponding to any of residues 17-18, 20-23, 26, 37, 39, 62, 64, 80-96, 110-113, 144-151, 171-177, 200-211, 213, 215, 227, 253-261 or 263-269.
The invention also provides a 1. lipolytic enzyme having an amino acid sequence derived from the T. lanuginosus lipase (SEQ ID NO: 14) comprising the following amino acid alterations:

    
    
        a) R84W+G91A+D96F+E99K+G263Q+L264A+I265T+G266D+T267A+L269N,
        b) G91A+D96W+E99K+L227G+G263Q+L264A+I265T+G266D+T267A+L269N,
        c) R84W+G91A:D96F+E99K+G263Q+L264A+I265T+G266S+T267A+L269N+270A+271G+272G+273F+274S,
        d) SPPCGRRP(-E) (SEQ ID NO: 17)+Y21K+E99N+N101S+E239C+Q249R,
        e) G91A+D96K+E99K+G263Q+L264A+I265T+G266D+T267A+L269N,
        f) Y21V+R84G+G91A:D96F+E99K+G263Q+L264A+I265T+G266D+T267A+L269N+270A+271G+272G+273F+274S,
        g) V60G+D62W+R84W+G91A+D96F+E99K+G263Q+L264A+I265T+G266D+T267A+L269N,
        h) V60A+D62S+G91A+D96W+E99K+W221R+G263Q+L264A+I265T+G266D+T267A+L269N+270A+271G+272G+273F+274S,
        i) R84A+S85D+E87A+G91A+D96G+K98E+E99D,
        j) G91A+D96W+E99K+P250N+G263Q+L264A+I265T+G266D+T267A+L269N+270A+271G+272G+273F+274S,
        k) G91A+D96W+E99K+P256N+G263Q+L264A+I265T+G266D+T267A+L269N+270A+271G+272G+273F+274S,
        l) R84W+G91A+D96W+E99K+Y261Q+G263Q+L264A+I265T+G266D+T267A+L269N+270A+271G+272G+273F+274S,
        m) G91A+D96W+E99K+P250L+P253Q+D254DEL+P257S+G263Q+L264A+I265T+G266D+T267A+L269N+270A+271G+272G+273F+274S,
        n) R84Y+G91A+D96F+E99K+G263Q+L264A+I265T+G266D+T267A+L269N, or
        o) R84S+G91A+D96F+E99K+E129A+V203i+L206F+G263Q+L264A+I265T+G266D+T267A+L269N.
    
    


The invention also provides a lipolytic enzyme having an amino acid sequence derived from a Fusarium lipase comprising at least one amino acid alteration corresponding to the following in the Fusarium oxysporum lipase (phospholipase (SEQ ID NO: 7):

    
    
        a) H257W,
        b) S142A,
        c) V157D,
        d) S271P,
        e) S80T,
        f) A127T,
        g) D263G,
        h) Y21W,
        i) S80T,
        j) R274A,
        k) 275YRSAESVDKR (SEQ ID NO:15) or
        l) 275YRSAESVDKAATMTDAELEKKLNSYVQMDKEYVKNNQARS (SEQ ID NO:16).

Further, the invention provides a lipolytic enzyme which:

        a) has an amino acid sequence having at least 90% identity to that of the Thermomyces lanuginosus lipase or the Fusarium oxysporum lipase/phospholipase,
        b) has phospholipase activity, and
        c) has a lysophospholipase to phospholipase ratio below the limit indicated below.
    
    





BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 gives the coordinates of a three-dimensional model of the T. lanuginosus lipase (SEQ ID NO: 14) docked with a phospholipid as substrate: 1-palmetoyl-2-oleylglycero-sn-3-phosphocholine (POPC).
FIG. 2 shows an alignment of the amino acid sequences of the above fungal lipolytic enzymes, based on a comparison of the available 3-dimensional structures, wherein the following amino acid sequences are aligned:

    
    
        SEQ ID NO: 1: Absidia reflexa 
        SEQ ID NO: 2: Absidia corymbefera 
        SEQ ID NO: 3: Rhizmucor miehei 
        SEQ ID NO: 4: Rhizopus delemar (oryzae)
        SEQ ID NO: 5: Aspergillus niger 
        SEQ ID NO: 6: Aspergillus tubingensis 
        SEQ ID NO: 7: Fusarium oxysporum 
        SEQ ID NO: 8: Fusarium heterosporum 
        SEQ ID NO: 9: Aspergillus oryzae 
        SEQ ID NO: 10: Penicilium camembertii 
        SEQ ID NO: 11: Aspergillus foetidus 
        SEQ ID NO: 12: Aspergillus niger 
        SEQ ID NO: 13: Aspergillus oryzae 
        SEQ ID NO: 14: Thermomyces lanuginosus (Humicola lanuginosa).
    
    





DETAILED DESCRIPTION OF THE INVENTION
Test Substrates
The invention uses two different polar lipids as test substrates. Both are amphiphilic (amphipolaric), having a hydrophilic part and one or two lipophilic groups, respectively.
First Substrate
The first substrate has the general formula A-B—C where:

    
    
        A is an acyl group, particularly straight-chain and unsubstituted. It may be saturated or may have one or more double bonds. It may have an even number of carbon atoms, e.g. from 12 to 24
        B is O (oxygen) or S (sulfur) forming an ester or thioester bond between A and C.
        C is a polyol having B attached to an OH group, optionally having other functional groups and/or a hydrophilic group attached to an OH group. The polyol may be a sugar alcohol such as glycerol, e.g. having B attached in the sn1 or sn2 position of glycerol. The other functional groups may be one or more aldehyde, keto or carboxyl groups; thus, the polyol may be a monosaccharide or a corresponding uronic acid. The hydrophilic group linked to an OH group, e.g. in the sn3 position of glycerol, may be:
        
            A phosphate group, optionally linked to an alcohol such as choline, ethanolamine, serine or inositol.
            Mono- or digalactosyl link to C through a glycosidic bond.
        
        
    
    


The first substrate may be a lysophospholipid such as lyso-lecithin or a lysogalactolipid such as digalactosyl monoglyceride (DGMG) or monogalactosyl monoglyceride (MGMG). The lyso-phospholipid may be a 1-lyso-phospholipid with an acyl group at the sn1-position or a 2-lyso-phospholipid with an acyl group at the sn2-position.
The activity of interest is a hydrolytic activity towards the bond B—C.
Second Substrate
The second substrate has the general formula A′-B′-(A′′-B′′—)C where:

    
    
        A′ is an acyl group defined as for A above.
        B′ is O (oxygen) or S (sulfur) forming an ester or thioester bond between A′ and C.
        A′′ is an acyl or alkyl group particularly straight-chain and unsubstituted. It may be saturated or may have one or more double bonds. It may have an even number of carbon atoms, e.g. from 12 to 24
        B′′ is O (oxygen), S (sulfur) or NH forming an ester, thioester, amide, ether or thioether bond between A′′ and C.
        C is the same as for the first substrate.
    
    


A′′ and B′′ of the second substrate may be chosen identical to A and B of the second substrate and attached in the same position of C, or they may be chosen independently.
The second substrate may be a phospholipid such as lecithin or a galactolipid such as digalactosyl diglyceride (DGDG) or monogalactosyl diglyceride (MGDG), or it may be prepared synthetically by attaching a fatty alcohol through an ether bond or thioether bond to a lysophospholipid or a lyso-galactolipid.
The activity of interest is a hydrolytic activity towards the B′—C bond.
Lipolytic Enzyme Activities
The lipolytic enzyme of the invention has a low ratio of activity for the first substrate compared to activity for the second substrate. Thus, it has a relatively low hydrolytic activity towards the B—C (thio)ester bond of the first substrate and a relatively high hydrolytic activity towards the B′—C (thio)ester bond of the second substrate. The activity towards the second substrate may be phospholipase A1 (EC 3.1.1.32) or A2 (EC 3.1.1.4), or it may be a galactolipase activity.
The activity ratio may be found by contacting the lipolytic enzyme with each substrate separately or by contacting it with a mixture including both substrates. The activity ratio may be measured by the PLARN assay, the RLPLA assay or a plate assay described below. The lipolytic enzyme may have a ratio of lysophospholipase activity to phospholipase activity corresponding to PLARN below 1000 (particularly 500, below 200 or below 50) or RLPLA (0.1/2.5) below 2 (particularly below 1 or below 0.5).
The lipolytic enzyme of the invention may have phospholipase (PL) activity with a relatively low lysophospholipase (LPL) activity. The lyso-phospholipid may be a 1-lyso-phospholipid with an acyl group at the sn1-position or a 2-lyso-phospholipid with an acyl group at the sn2-position. The lipolytic enzyme may in particular have phospholipase A1 activity with low 1-lysophospholipase activity.
The lipolytic enzyme of the invention may have hydrolytic activity towards a carboxylic ester bond in DGDG (digalactosyl diglyceride) with a relatively low hydrolytic activity towards the ester bond in DGMG (digalactosyl monoglyceride).
Optionally, the lipolytic enzyme may also have triacylglycerol lipase activity (EC 3.1.1.3), i.e. hydrolytic activity for carboxylic ester bonds in triglycerides, e.g. 1,3-specific activity, particularly on long-chain triglycerides such as olive oil. The enzyme may have a substrate specificity for hydrolyzing long-chain fatty acyl groups rather than short-chain groups, e.g. expressed as a high ratio of activities on olive oil and tributyrin, e.g. a ratio SLU/LU>3 as described in WO 0032758.
LPL/PL Ratio (PLARN)
Phospholipase activity is determined at 30° C. using 4% (w/v) lecithin (phosphatidyl choline) in 50 mM sodium acetate, 5 mM CaCl2, pH 5.0. One unit of phospholipase activity is defined as 1 mmol free fatty acids released per minute per mg enzyme using the above conditions.
Lysophospholipase activity is determined at 30° C. using 1% (w/v) lysolechitin in 50 mM sodium acetate, 5 mM CaCl2, pH 5.0. One unit of lysophospholipase activity is defined as 1 mmol free fatty acids released per minute per mg enzyme using the above conditions. The lysolechitin may be an equilibrium mixture or pure 1-lysolechitin in the case of a phospholipase A1 and pure 2-lysolechitin in the case of a phospholipase A2.
The PLARN ratio is defined as the lysophospholipase activity divided by the phospholipase activity, both determined by the above methods.
Relative Activity on Lysopholipids and Phospholipids (RLPLA)
This activity measurement expresses the relative activity on lysophosphatidyl choline and phosphatidyl choline in an equimolar mixture.
More specifically the assay is carried out as follows: The activity at different concentrations of phospholipase is determined at 30° C. blending 1:1 solutions of phosphatidyl choline (25 mM) and lysophosphatidyl choline (25 mM) in 50 mM NaOAc buffer (pH 5). 50 μl enzyme solution (e.g. having 0.1 or 2.5 mg enzyme protein per ml) is added to the substrate solution and allowed to react for 30 minutes. 100 μl of the sample is inactivated at 95° C. for 5 minutes and dissolved in 900 μl CHCl3/MeOH 50%/50%. The sample is centrifuged at 14000 rpm for 2 minutes. The supernatant is analyzed by HPLC after filtering through a 0.45 μm filter. Column: Microsorb-MV 100Si 250 mm column (analytical instruments). Mobile phases: A: 80% CHCl3, 19.5% MeOH, 0.5% NH4OH; B: 60% CHCl3, 34% MeOH, 0.5% NH4OH, 5.5% H2O. Gradient: 0-3 minutes 100% A, 3-23 minutes 100% B, 23-45 minutes 100% A. Injection volume 20 μl Detector: Sedere, Sedex 75 light scattering, Temp 40° C., pressure 3.5 Bar. The RLPLA is then measured as the depletion of phosphatidyl choline relative to lysophosphatidyl choline. A variant with a lower RLPLA value indicates a higher accumulation of lysophospholipid under the conditions of analysis.
The relative activity can be expressed as “RLPLA ratio” by measuring lysolecithin hydrolysis at an enzyme dosage of 0.1 mg/ml and lecithin hydrolysis at a dosage of 2.5 mg/ml, and taking the ratio of the two.
Plate Assay
Plates including each of the substrates may be prepared in analogy with WO 0032758 using suitable pH and substrate concentration. Optionally, other ingredients such as flour may be included. A suitably diluted enzyme solution is applied to holes in the plates, and clearing zones are read after incubation for a suitable time at a suitable temperature.
Preparation of Lipolytic Enzyme
The lipolytic enzyme may be obtained by preparing variants of a parent lipolytic enzyme by altering its amino acid sequence and screening for a variant with an improved activity ratio. The parent lipolytic enzyme may have phospholipase activity, DGDG hydrolytic activity and/or triacylglycerol lipase activity. Variants may be prepared from the parent lipolytic enzyme by known methods, e.g. by subjecting a DNA sequence encoding the parent lipolytic enzyme to site-directed mutagenesis, localized random mutagenesis or site-saturation mutagenesis, e.g. using methods described in WO 0032758. Resulting DNA sequences may be further modified by gene shuffling and directed evolution.
Parent Lipolytic Enzyme
The lipolytic enzyme to be used in the present invention is one that can hydrolyze ester bonds. Such enzymes include, for example, lipases, such as triacylglycerol lipase (EC 3.1.1.3), lipoprotein lipase (EC 3.1.1.34), monoglyceride lipase (EC 3.1.1.23), phospholipase A1 or A2 (EC 3.1.1.26, 3.1.1.4), lysophospholipase (EC 3.1.1.5), galactolipase (EC 3.1.1.26), ferulic acid esterase and esterase (EC 3.1.1.1, EC 3.1.1.2).
The parent lipolytic enzyme may be the Thermomyces lanuginosus lipase (Humicola lanuginosa lipase) (EP 305216) or it may have an amino acid sequence with at least 50% identity (e.g. at least 90% identity). The amino acid sequence of the T. lanuginosus lipase is shown in U.S. Pat. No. 5,869,438 and as seq14 in FIG. 2 of this application.
Thus, the parent lipolytic enzyme may be a naturally occurring enzyme as described at pages 5-6 of WO 0032758. Examples are the lipolytic enzymes from the following organisms. They are known in the prior art; and their amino acid sequences are given in the attached sequence listing.

    
    
        1. Absidia reflexa 
        2. Absidia corymbefera 
        3. Rhizmucor miehei 
        4. Rhizopus delemar (oryzae)
        5. Aspergillus niger 
        6. Aspergillus tubingensis 
        7. Fusarium oxysporum 
        8. Fusarium heterosporum 
        9. Aspergillus oryzae 
        10. Penicilium camembertii 
        11. Aspergillus foetidus 
        12. Aspergillus niger 
        13. Aspergillus oryzae 
        14. Thermomyces lanuginosus (Humicola lanuginosa)
    
    


As indicated above, the amino acid to be altered may be determined on the basis of an alignment of the parent lipolytic enzyme with the T. lanuginosus lipase. FIG. 2 shows an alignment of the amino acid sequences of the above fungal lipolytic enzymes, based on a comparison of the available 3-dimensional structures:
Other amino acid sequences may be aligned with those shown in FIG. 2 by using the GAP alignment to the most homologous sequence found by the GAP program. GAP is 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). The following settings are used for polypeptide sequence comparison: GAP creation penalty of 3.0 and GAP extension penalty of 0.1.
Alternatively, the parent lipolytic used in the present invention may be a variant of the above, e.g. a variant of the T. lanuginosus lipase (SEQ ID NO: 14) or the F. oxysporum lipase/phospholipase (SEQ ID NO: 7). A variant with phospholipase activity or galactolipase activity may be used, e.g. as described in Example 5, 6 or 13 of WO 0032758. Particular examples are variants of SEQ ID NO: 14 with the following amino acid alterations:














L259S



G266D



G91A + D96W + E99K + G263Q + L264A + I265T + G266D +



T267A + L269N + 270A + 271G + 272G + 273F + 274S



G266E



G263A + G266A



E1SPCRPRP + E239C + Q249R + G266A



E1SPCRPRP + E239C + Q249R + G266S



D96S + G266A



D96S + G266S



D96S + G266W



E1SPPCGRRP (SEQ ID NO: 17) + D96S + E239C + Q249R +



G263D + L264I + I265N + G266E + T267GS









Three-Dimensional Model

FIG. 1 gives the coordinates of a three-dimensional model of the T. lanuginosus lipase docked with a phospholipid as substrate: 1-palmetoyl-2-oleylglycero-sn-3-phosphocholine (POPC). This may be used as a starting point for building a similar model for any given fungal lipolytic enzyme. Using this model, the following amino acid residues are found to be within 10 Å, 7 Å and 5 Å of an atom of the substrate (LIP1 in the pdb structure shown in FIG. 1):

    
    
        10 Å: 17-18, 20-23, 26, 37, 39, 62, 64, 80-96, 110-113, 144-151, 171-177, 200-211, 213, 215, 227, 253-261, 263-269.
        7 Å: 21, 81-95, 110, 113, 145-148, 150, 172-175, 201-208, 213, 254-256, 258-259, 264-269.
        5 Å: 21, 82-86, 89-90, 92-93, 95, 110, 113, 145-147, 174, 202-203, 206-208, 255, 258-259, 265-268.
    
    


More particularly, amino acid alterations may be made at one or more positions corresponding to the following amino acids in the T. lanuginosus lipase (SEQ ID NO: 14): Y212, R84, S85, E87, D96, V203, L206, L227, P253, D254, P256, P257 and/or Y261, particularly one or more alterations corresponding to Y21V/K, R84W/A/G/Y/S, S85D, E87A, D96F/K/G, V203I, L206F, P253Q, D254*, P256N, P257S and/or Y261Q.
Further, amino acid alterations may be made at one or more positions corresponding to H257, S142, S80, D263 and/or Y21 of the F. oxysporum lipase/phospholipase (SEQ ID NO: 7), particularly one or more corresponding to H257W, S142A, 580T, D263G and/or Y21W.
Also, amino acid alterations may be made at the C-terminal or at any position down-stream of L269 of SEQ ID NO: 14. Such alteration may be addition or deletion of a peptide extension or deletion of a peptide extension of one or more amino acids (e.g. 1-50 amino acids such as 2-15) at the C-terminal.
Amino Acid Alteration
The amino acid alteration may be substitution with a larger amino acid. The amino acid residues are ranked by size as follows from smallest to largest:

    
    
        G, A, S, C, V, T, P, L, I, N, D, M, E, Q, K, H, R, F, Y, W
    
    


An amino acid residue within 10 Å (or 7 Å or 5 Å) of a C atom in the alkyl group R of R—COO attached to sn1 of the lyso-phospholipid may be substituted with an amino acid residue which is larger or more hydrophilic.
An amino acid residue within 10 Å (or 7 Å or 5 Å) of an atom (other than H) of the phosphate group attached to sn3 (i.e. the O atom at sn3 or any atom beyond that) may be substituted with an amino acid which is larger or more hydrophobic.
Amino acid residues are ranked as follows from most hydrophilic to most hydrophobic:

    
    
        R, K, E, D, N, Q, H, S, T, Y, C, M, G, A, V, P, L, I, F, W

Lipolytic Enzyme Variant

    
    


Starting from a variant having phospholipase activity derived from the T. lanuginosus lipase, and improvement regarding increased ratio of lecithin/lysolecithin (sn1) activity and lowered activity against lysolecithin (sn1) of lysolecithin in general, or/and with improved sn1 lecithase activity may be achieved by use of the following concepts.
One concept is to lower the binding energy of sn1 acyl chain and in this way increase the ratio of activity lecithin/lysolecithin (sn1). Secondary to increase sn2 binding to favour lecithin rather than lysolecithin.
Thus, 206, 95, 203 and 93 may be made smaller and more hydrophilic. Also, positions 253, 255 and 256 may be made bigger. Some particular examples of such variants are:

    
    
        L206V/S/T/A
        V203T/S/A
        F95I/L/Y
        L93V/I/A/T
    
    


A doped library 206/203 and 95/93. appr. 90% wt and appr 10% variant may be used.
A second concept is to make libraries in the contacts to both acyl chains found docked structures, and in the regions close to the substrate found by comparison of good and bad lysolecithase active homologous enzymes. The regions will be doped according to the homologous enzymes sequence.
Thus, region 265-269 is of interest, e.g. P253. Some particular examples of such variants are:
A doped library 247-260 may be used, optionally together with doped 1202P and L206V. The doping may be appr. 90% wt and appr. 10% variants.

    
    
        P253TG/L
        D254S/L
        I255
        P256L/A
        A257D/A
        L259
        W260H
        Proline removal(s):
        P256X
        P253X
        together with 1202P
        L227D
        Library 247-260 and 206, 95, 203 and 93 may be combined.

Amino Acid Identity

    
    


The lipolytic enzyme of the invention and the parent lipolytic enzyme may have an amino acid identity of at least 50% (particularly at least 90%, e.g. more than 95% or more than 98%) with the T. lanuginosus lipase (SEQ ID NO: 14).
The degree of identity 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.
Use of Lipolytic Enzyme Variant
Hydrolysis of Phospholipid
A variant with phospholipase activity can be used to prepare lysophospholipid (e.g. lyso-lecithin) by treating the corresponding phospholipid with the variant, e.g. as described in EP 870840, JP-A 10-42884, JP-A 4-135456 or JP-A 2-49593. The variant can also be used to make mayonnaise, e.g. as described in EP 628256, EP 398666 or EP 319064.
Advantageously, a low ratio of lysophospholipase/phospholipase activity can lead to a high degree of phospholipid hydrolysis with a low degree of lysophospholipid hydrolysis. This may allow the use of long reaction time and the use of phospholipid with a high lysophospholipid content, e.g. from cereals such as oats.
Baking
Lipolytic enzymes according to the invention have improved baking performance, e.g. a lower dough stickiness, a better dough extensibility and elasticity, a better dough stability, a better crumb structure of the baked product, a larger loaf volume and/or improved resistance to over-proofing or other abuse.
The invention provides a baking additive in the form of a granulate, an agglomerated powder or a stabilized liquid, comprising a lipolytic enzyme which:

    
    
        a) has phospholipase activity, and
        b) has a lysophospholipase to phospholipase ratio corresponding to PLARN below 500 or RLPLA (0.1/2,5). below 1.0.
    
    


The baking additive may have a narrow particle size distribution with more than 95% (by weight) of the particles in the range from 25 to 500 p.m.
Granulates and agglomerated powders may be prepared by conventional methods, e.g. by spraying the lipolytic enzyme onto a carrier in a fluid-bed granulator. The carrier may consist of particulate cores having a suitable particle size. The carrier may be soluble or insoluble, e.g. a salt (such as NaCl or sodium sulfate), a sugar (such as sucrose or lactose), a sugar alcohol (such as sorbitol), starch, rice, corn grits, or soy. Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods.
The invention further relates to a pre-mix comprising flour and the lipolytic enzyme described above. The pre-mix may contain other dough-improving and/or bread-improving additives, e.g. any of the additives, including enzymes, mentioned above.
The invention also provides a method of preparing a dough or a baked product prepared from dough. The method may comprise preparing a variant by the above method and adding it to the dough. Alternatively, the method may comprise:

    
    
        a) testing at least one lipolytic enzyme for its hydrolytic activities towards intact phospholipid (PL) and lyso-phospholipid (LPL),
        b) selecting a lipolytic enzyme having a hydrolytic activity ratio for LPL/PL corresponding to PLARN below 500, and
        c) adding the selected lipolytic enzyme to the dough.

Dough

    
    


The dough generally comprises wheat meal or wheat flour and/or other types of meal, flour or starch such as corn flour, corn starch, rye meal, rye flour, oat flour, oat meal, soy flour, sorghum meal, sorghum flour, rice starch, rice flour, potato meal, potato flour or potato starch. The dough may be fresh, frozen or par-baked. It may particularly be a leavened dough.
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.
The dough may comprise fat (triglyceride) such as granulated fat or shortening, but the invention is particularly applicable to a dough where less than 1% by weight of fat (triglyceride) is added, and particularly to a dough which is made without addition of fat.
The dough may further comprise an emulsifier such as mono- or diglycerides, diacetyl tartaric acid esters of mono- or diglycerides, sugar esters of fatty acids, polyglycerol esters of fatty acids, lactic acid esters of monoglycerides, acetic acid esters of monoglycerides, polyoxyethylene stearates, or lysolecithin, but the invention is particularly applicable to a dough which is made without addition of emulsifiers (other than optionally phospholipid).
Baked Product
The process of the invention may be used for any kind of baked product prepared from dough, either of a soft or a crisp character, either of a white, light or dark type. Examples are bread (in particular white, whole-meal or rye bread), typically in the form of loaves or rolls, French baguette-type bread, pita bread, tortillas, cakes, pancakes, biscuits, cookies, muffins, pie crusts, crisp bread, steamed bread, pizza and the like.
Additional Enzyme
Optionally, an additional enzyme may be used together with the lipolytic enzyme. The additional enzyme may be a second lipolytic enzyme (e.g. as described in PCT/DK01/00472), an amylase, particularly an anti-staling amylase, an amyloglucosidase, a cyclodextrin glucanotransferase, or the additional enzyme may be a peptidase, in particular an exopeptidase, a transglutaminase, a cellulase, a hemicellulase, in particular a pentosanase such as xylanase, a protease, a protein disulfide isomerase, e.g., a protein disulfide isomerase as disclosed in WO 95/00636, a glycosyltransferase, a branching enzyme (1,4-α-glucan branching enzyme), a 4-α-glucanotransferase (dextrin glycosyltransferase), a lactase (galactosidase), or an oxidoreductase, e.g., a peroxidase, a laccase, a glucose oxidase, a pyranose oxidase, a lipoxygenase, an L-amino acid oxidase or a carbohydrate oxidase.
The amylase may be a fungal or bacterial alpha-amylase, e.g. from Bacillus, particularly B. licheniformis or B. amyloliquefaciens, or from Aspergillus, particularly A. oryzae, a beta-amylase, e.g. from plant (e.g. soy bean) or from microbial sources (e.g. Bacillus). The amylase may be an anti-staling amylase, as described in WO 99/53769, i.e. an amylase that is effective in retarding the staling (crumb firming) of baked products, particularly a maltogenic alpha-amylase, e.g. from Bacillus stearothermophilus strain NCIB 11837.
Other Uses
The lipolytic enzyme variant may also be used in the production of pasta and noodles in analogy with EP 1057415.
A lipolytic enzyme variant with phospholipase activity may be used in cheese production as described in WO 00/54601.
EXAMPLES
Example 1
Variants Based on a T. lanuginosus Lipase Variant
A prior-art variant of the T. lanuginosus lipase with phospholipase activity was chosen as the starting point (parent lipolytic enzyme), and variants were prepared by introducing further amino acid alterations into the prior-art variant.
Experiment A
Activities of the new variants were determined with lecithin and lysolecithin (pure 1-phosphatidyl choline, 1-lysolecithin) as substrates by the methods described above. More specifically, 1.7 mL of the reaction mixture was shaken for between 15 and 90 min in an Eppendorf tube shaken at 1300 rpm by an “Eppendorf Thermomixer comfort”. The enzyme was inactivated at 95 C for 5 min, and centrifuged at 14000 rpm by Eppendorf centrifuge 5417R. The liberated fatty acids were determined (relative to a control sample where the enzyme was inactivated before it was added to the substrate) by NEFA C test from Wako following the ACS-ACOD method described for the NEFA-C test.
Three variants were found to have phospholipase activity and a to have a lower ratio of lysophospholipase to phospholipase than the prior-art variant.

    
    
        R84W+G91A+D96F+E99K+G263Q+L264A+I265T+G266D+T267A+L269N
        G91A+D96W+E99K+L227G+G263Q+L264A+I265T+G266D+T267A+L269N
        R84W+G91A+D96F+E99K+G263Q+L264A+I265T+G266S+T267A+L269N+270A+271G+272G+273F+274S
    
    


Experiment B
Activities of further variants were determined with lecithin and lysolecithin (mixture of 1- and 2-lysolecithin) as substrates at 0.1 and 2.5 mg/ml by the RLPLA method described above. All variants were found to have a high activity on lysolecithin compared to lecithin.
Variants having the following amino acid alterations compared to SEQ ID NO: 14 were found to have phospholipase activity and a to have a lower ratio of lysophospholipase to phospholipase than the prior-art variant.












SPPCGRRP(-E) (SEQ ID NO: 17) + Y21K + E99N + N101S + E239C + Q249R


G91A + D96K + E99K + G263Q + L264A + I265T + G266D + T267A + L269N


Y21V + R84G + G91A + D96F + E99K + G263Q + L264A + I265T + G266D + T267A +


L269N + 270A + 271G + 272G + 273F + 274S


V60G + D62W + R84W + G91A + D96F + E99K + G263Q + L264A + I265T + G266D +


T267A + L269N


V60A + D62S + G91A + D96W + E99K + W221R + G263Q + L264A + I265T + G266D +


T267A + L269N + 270A + 271G + 272G + 273F + 274S


R84A + S85D + E87A + G91A + D96G + K98E + E99D


G91A + D96W + E99K + P250N + G263Q + L264A + I265T + G266D + T267A + L269N +


270A + 271G + 272G + 273F + 274S


G91A + D96W + E99K + P256N + G263Q + L264A + I265T + G266D + T267A + L269N +


270A + 271G + 272G + 273F + 274S


R84W + G91A + D96W + E99K + Y261Q + G263Q + L264A + I265T + G266D + T267A +


L269N + 270A + 271G + 272G + 273F + 274S


G91A + D96W + E99K + P250L + P253Q + D254DEL + P257S + G263Q + L264A + I265T +


G266D + T267A + L269N + 270A + 271G + 272G + 273F + 274S


R84Y + G91A + D96F + E99K + G263Q + L264A + I265T + G266D + T267A +


L269N


R84S + G91A + D96F + E99K + E129A + V203i + L206F + G263Q + L264A + I265T +


G266D + T267A + L269N









Example 2
Variants of F. oxysporum Lipase/Phospholipase
Variants of the F. oxysporum lipase/phospholipase were prepared, having the following amino acid alterations compared to SEQ ID NO: 7:

    
    
        H257W
        S142A
        V157D+5271P
        S80T+A127T
        D263G
        Y21W
        S80T
    
    


The following variants with modified C-terminal sequences were prepared by making the substitutions R274A and/or R284A to remove one or two cleavage points for the Kex-2 protease:

    
    
        R274A+275YRSAESVDKR (SEQ ID NO:15)
        R274A+275YRSAESVDKAATMTDAELEKKLNSYVQMDKEYVKNNQARS (SEQ ID NO:16)
    
    


Activities of the variants and the parent enzyme were determined as in Example 2. Compared to the parent enzyme, the variants were found to have lower activity on lysolecithin and a lower ratio of lysolecithin activity to lecithin activity.
Example 3
Preparation of Dough
Doughs were prepared from Meneba flour according to the European straight dough method (ABF-SP 1201.1) with 40 ppm Fungamyl Super MA (Novozymes), 40 ppm ascorbic acid, and different dosages of lipolytic enzyme.
The stickiness of the dough was evaluated an a 1-10 scale by the baker as the degree to which a dough adheres to ones hands or other surfaces, where 5 is identical to a control without addition of lipolytic enzyme, 1 is the lowest degree of stickiness and 10 is the highest degree of stickiness.
The extensibility of the dough was evaluated an a 1-10 scale by the baker as the degree to which a dough can be stretched without tearing, where 5 is identical to a control without addition of lipolytic enzyme, 1 indicates the lowest (shortest) extensibility and 10 indicates the highest (longest) extensibility
The elasticity of the dough was evaluated an a 1-10 scale by the baker as the degree to which a dough tends to recover its original shape after release from a deforming force, where 5 is identical to a control without addition of lipolytic enzyme, 1 indicates the lowest (weakest) elasticity and 10 indicates the highest (strongest) elasticity.
A lipolytic enzymes prepared in Example 1 was tested, and the prior-art variant was tested for comparison. The results were as follows
Experiment A















Lipolytic

PLARN





enzyme
Dosage
ratio
Stickiness
Extensibility
Elasticity




















Invention
 0.2 mg/kg
690
5
6
4



dough






Prior art
250 LU/kg
5800
5
7
3



dough









Experiment B















Lipolytic

RLPLA





enzyme
Dosage
ratio
Stickiness
Extensibility
Elasticity







Invention
 0.2 mg/kg
1.06
4
4
6



flour






Prior art
250 LU/kg
1.58
5
5
5



dough









The results show that lipolytic enzymes with a lower ratio of lysolecithin activity to lecithin activity make doughs with a desirable combination of lower extensibility and higher elasticity than the prior-art lipolytic enzymes, and they furthermore tend to make a less sticky dough.

## Claims

1. An isolated lipolytic enzyme variant, comprising a polypeptide having at least 90% identity to the amino acid sequence of SEQ ID NO: 14 and comprises an amino acid substitution R84G, R84A, R84Y, R84S, or L206F of SEQ ID NO: 14, and wherein the variant has phospholipase activity and lysophospholipase activity, and has a lysophospholipase to phospholipase activity ratio (PLARN) below 1000, wherein phospholipase activity is determined at 30° C. using 4% (w/v) lecithin (phosphatidyl choline) in 50 mM sodium acetate, 5 mM CaCl2, pH 5.0, and lysophospholipase activity is determined at 30° C. using 1% (w/v) lysolechitin in 50 mM sodium acetate, 5 mM CaCl2, pH 5.0.

2. The lipolytic enzyme variant of claim 1, comprising the following amino acid alterations:
a) Y21V+R84G+G91A+D96F+E99K+G263Q+L264A+1265T+G266D+T267A+L269N+270A+271G+272G+273F+274S,
b) R84A+S85D+E87A+G91A+D96G+K98E+E99D,
c) R84Y+G91A+D96F+E99K+G263Q+L264A+1265T+G266D+T267A+L269N, or
d) R84S+G91A+D96F+E99K+E129A+V203i+L206F+G263Q+L264A+1265T+G266D+T267A+L269N.

3. The lipolytic enzyme variant of claim 1, further comprising the following amino acid alterations:
a) R84W+G91A+D96F+E99K+G263Q+L264A+1265T+G266D+T267A+L269N,
b) G91A+D96W+E99K+L227G+G263Q+L264A+1265T+G266D+T267A+L269N,
c) R84W+G91A+D96F+E99K+G263Q+L264A+1265T+G266S+T267A+L269N+270A+271 G+272G+273F+274S,
d) SPPCGRRP(-E)+Y21K+E99N+N101S+E239C+Q249R,
e) G91A+D96K+E99K+G263Q+L264A+1265T+G266D+T267A+L269N,
f) V60G+D62W+R84W+G91A+D96F+E99K+G263Q+L264A+1265T+G266D+T267A+L269N,
g) V60A+D62S+G91A+D96W+E99K+W221R+G263Q+L264A+1265T+G266D+T267A+L269N+270A+271G+272G+273F+274S,
h) G91A+D96W+E99K+P250N+G263Q+L264A+1265T+G266D+T267A+L269N+270A+271 G+272G+273F+274S,
i) G91A+D96W+E99K+P256N+G263Q+L264A+I265T+G266D+T267A+L269N+270A+271G+272G+273F+274S,
j) R84W+G91A+D96W+E99K+Y261Q+G263Q+L264A+I265T+G266D+T267A+L269N+270A+271G+272G+273F+274S, or
k) G91A+D96W+E99K+P250L+P253Q+D254DEL+P257S+G263Q+L264A+I265T+G266D+T267A+L269N+270A+271G+272G+273F+274S.

4. The lipolytic enzyme variant of claim 1, which comprises an amino acid substitution R84G.

5. The lipolytic enzyme variant of claim 1, which comprises an amino acid substitution R84A.

6. The lipolytic enzyme variant of claim 1, which comprises an amino acid substitution R84Y.

7. The lipolytic enzyme variant of claim 1, which comprises an amino acid substitution R84S.

8. The lipolytic enzyme variant of claim 1, which comprises an amino acid substitution L206F.

9. A composition comprising the lipolytic enzyme variant of claim 1.

10. The composition of claim 9, further comprising a carrier selected from a salt, a sugar, a sugar alcohol, starch, rice, corn grits, or soy.

11. The composition of claim 9, wherein the composition is in the form of a granulate or agglomerated powder.

12. The composition of claim 11, wherein more than 95 weight % of the granulate or agglomerated powder has a particle size between 25 and 500 μm.