# Patent

## Title
Lipolytic enzyme variants

## Abstract

Variants with increased acyl transferase activity can be designed on the basis of a three-dimensional model by making amino acid alterations near the active Ser of lipolytic enzymes such as C. antarctica lipase A or the lipase/acyl transferase from C. parapsilosis.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a 35 U.S.C. 371 national application of PCT/EP2007/060473 filed Oct. 2, 2007, which claims priority or the benefit under 35 U.S.C. 119 of European application no. 06121678.4 filed Oct. 3, 2006 and U.S. provisional application no. 60/849,072 filed Oct. 3, 2006, the contents of which are fully incorporated herein by reference.


FIELD OF THE INVENTION
The present invention relates to a polypeptide with lipolytic enzyme activity and to a method of preparing it.
BACKGROUND OF THE INVENTION
WO2004064537 discloses a method for the in-situ production of an emulsifier in a foodstuff, wherein a lipid acyl transferase is added to the foodstuff. WO2005066347 discloses a method of producing a variant glycolipid acyl transferase enzyme.
V Neugnot et al., European Journal of Biochemistry, Vol. 269 (6) pp. 1734-1745 (2002) March, describes a lipase/acyl transferase from Candida parapsilosis. 
WO8802775A1 describes Candida antarctica lipase A. WO9401541A1 discloses variants of C. antarctica lipase A.
SUMMARY OF THE INVENTION
The inventors have found that variants with increased acyl transferase activity can be designed on the basis of a three-dimensional model by making amino acid alterations near the active Ser of lipolytic enzymes such as C. antarctica lipase A or the lipase/acyl transferase from C. parapsilosis. 
Accordingly, the invention provides a method of preparing a polypeptide, comprising:
a) providing a three dimensional model of a parent polypeptide having lipolytic enzyme activity and an amino acid sequence with an active Ser having at least 80% identity to any of SEQ ID NOS: 1-5,
b) selecting an amino acid residue in the parent polypeptide which has a non-hydrogen atom within 10 Å of the a non-hydrogen atom in the active Ser in the model,
c) providing an altered amino acid sequence which is at least 80% identical to any of SEQ ID NOS: 1-5, and wherein the difference from the parent polypeptide comprises substitution or deletion of the selected residue or insertion of at least one residue adjacent to the selected residue,
d) preparing an altered polypeptide having the altered amino acid sequence,
e) determining the acyl transferase activity of the altered polypeptide, and
f) selecting an altered polypeptide which has an increased acyl transferase activity compared to the parent polypeptide.
The invention also provides a polypeptide which:
has lipolytic enzyme activity, and
has an amino acid sequence which has at least 80% identity to SEQ ID NO: 1 and has a different residue at a position or has an insertion adjacent to a position corresponding to any of residues 82-87, 108, 132-133, 138, 140-142, 145, 172-179, 182, 202-216, 220-232, 235, 238, 241-242, 257,264, 267-268, 275-277, 280, 282-288, 290-296, 298-299, 304, 320, 324-328, 356-357, 360 and 420-421 of SEQ ID NO: 1.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows a three-dimensional model of Candida antarctica lipase A (CALA, SEQ ID NO: 1) with a substrate analog (myristic acid).
FIG. 2 shows an alignment based on the three-dimensional structure of the following sequences in full-length or truncated form:
SEQ ID NO: 1 (CAND_A)
SEQ ID NO: 5 (CaLIP1, ADY62089)
SEQ ID NO: 6 (ADY62090, CaLIP2)
SEQ ID NO: 7 (ABJ25493, A. fumigatus lipase)
SEQ ID NO: 2 (Pseudozyma lipase, ADZ72148)
SEQ ID NO: 4 (CpLIP2, Q8NIN8)
SEQ ID NO: 3 (CpLIP1, Q8NJ51)



DETAILED DESCRIPTION OF THE INVENTION
Parent Polypeptide
The invention uses a parent polypeptide with lipolytic enzyme activity. It may be Candida antarctica lipase A (CALA, SEQ ID NO: 1), Pseudozyma sp. lipase (SEQ ID NO: 2) described in WO2005040334, C. parapsilosis lipase CpLIP1 (SEQ ID NO: 3) or CpLIP2 (SEQ ID NO: 4), any of CaLIP1-10 from Candida albicans, e.g. CaLIP1 or CaLIP2 shown as SEQ ID NOS: 5-6, or Aspergillus fumigatus lipase (SEQ ID NO: 7).
Three-Dimensional Model
The invention uses a 3D model of the parent polypeptide. FIG. 1 gives the coordinates for a 3D model of CALA with myristic acid as a substrate analogue. The active Ser is in position 174.
Selection of Amino Acid Residue
An amino acid residue is selected in the 3D model having a non-hydrogen atom within 10 Å of a non-H atom of the active Ser.
In the model in FIG. 1, the following residues have a non-H atom within 10 Å of a non-H atom of the active Ser (position 174 of SEQ ID NO: 1): 80-85, 108, 112, 116, 132-133, 139-140, 145, 171-182, 200-207, 211, 215, 220, 223, 264, 268, 318-321, 324-328, 332, 355-357, 359-361, 419-421, 425.
Altered Amino Acid Sequence
The selected residue may be substituted with a different residue, particularly with a more efficient pi electron donor residue. Amino acid residues are ranked as follows from least efficient to most efficient pi donors (an equal sign indicates residues with practically indistinguishable efficiency). Other residues are not considered to be pi electron donors:

T<N<H<F<Y<W

The substitution may particularly be conservative, i.e. substitution with another residue of the same type (negative, positive, hydrophobic or hydrophilic). The negative residues are D,E, the positive residues are K,R, the hydrophobic residues are A,C,F,G,I,L,M,P,V,W,Y, and the hydrophilic residues are H,N,Q,S,T.
Alternatively, an amino acid insertion may be made at the N- or C-terminal side of the selected residue, particularly an insertion of 1-2 residues.
CpLIP1 or CpLIP2 (SEQ ID NO: 3 or 4) may be used as a template for the amino acid alteration by referring to an alignment as shown in FIG. 3 for SEQ ID NOS: 1-5. Thus, the selected residue may be substituted with the residue found in the corresponding position for SEQ ID NO: 3 or 4. The selected residue may be deleted if SEQ ID NO: 3 or 4 has a gap at that position. An insertion may be made adjacent to the selected residue if SEQ ID NO: 3 or 4 has an additional residue at that position; the insertion may in particular be the same residue found in SEQ ID NO: 3 or 4.
Particular Substitutions
The variant may particularly comprise one or more of the following substitutions: Y83W, V103T and/or H132Y. More particularly, it may comprise the combination F223A F421V Y83W.
Nomenclature for Amino Acid Alterations
In this specification, an amino acid substitution is described by use of one-letter codes, e.g. P205W. Multiple substitutions are concatenated, e.g. P205F T211W to indicate a variant with two substitutions. P205W, Y, F is used to indicate alternatives, i.e. substitution of P205 with W, Y or F.
Assay for Acyl Transferase Activity
In general, the activity may be determined by incubating the polypeptide with an acyl ester as acyl donor and an alcohol as acyl acceptor in an aqueous system and analyzing the mixture after the incubation to determine the transfer of the acyl group. This may be done, e.g. as described in WO2004064537.
Use of Lipolytic Enzyme Variant
The variants of the invention have increased acyl transferase activity. They may be used in various processes where they are mixed with an acyl donor and an acyl acceptor in an aqueous system to effect acyl transfer, e.g. as described in the indicated publications:

    
    
        In-situ production of an emulsifier in a foodstuff, such as baked goods made from dough, e.g. as described in WO2004064537.
        Production of a carbohydrate ester, a protein ester or a hydroxyl acid ester, WO2004064987.
        Reducing or removing diglyceride from edible oil, WO2005066351.
        Enzymatic degumming of edible oil, WO2005066351.

## Claims

1. A polypeptide which:
a) has lipolytic enzyme activity, and
b) has an amino acid sequence which has at least 80% identity to SEQ ID NO: 1 and comprises an amino acid difference compared to SEQ ID NO: 1 at a position corresponding to any one of positions 83, 103, 112, 116, 132, 215, 223, 355, 357 and/or 425 in SEQ ID NO: 1.

2. The polypeptide of claim 1, which comprises a substitution at a position corresponding to position 83.

3. The polypeptide of claim 1, which comprises a substitution at a position corresponding to position 103.

4. The polypeptide of claim 1, which comprises a substitution at a position corresponding to position 112.

5. The polypeptide of claim 1, which comprises a substitution at a position corresponding to position 116.

6. The polypeptide of claim 1, which comprises a substitution at a position corresponding to position 132.

7. The polypeptide of claim 1, which comprises a substitution at a position corresponding to position 215.

8. The polypeptide of claim 1, which comprises a substitution at a position corresponding to position 223.

9. The polypeptide of claim 1, which comprises a substitution at a position corresponding to position 355.

10. The polypeptide of claim 1, which comprises a substitution at a position corresponding to position 357.

11. The polypeptide of claim 1, which comprises a substitution at a position corresponding to position 425.

12. The polypeptide of claim 1, which comprises at least one of the substitutions: Y83W, V103T and/or H132Y.

13. The polypeptide of claim 1, which further comprises 1-10 amino acid differences compared to SEQ ID NO: 1 at a position corresponding to any of positions 80-82, 84-85, 108, 133, 139-140, 145, 171-182, 200-207, 211, 220, 264, 268, 318-321, 324-328, 332, 356, 359-361 or 419-421 of SEQ ID NO: 1.

14. The polypeptide of claim 1, which comprises the substitutions F223A and F421V and Y83W.

15. A method of producing an emulsifier in a foodstuff, comprising adding the polypeptide of claim 1 to a dough.