EP2064326A1 - Lipolytic enzyme variants - Google Patents
Lipolytic enzyme variantsInfo
- Publication number
- EP2064326A1 EP2064326A1 EP07803617A EP07803617A EP2064326A1 EP 2064326 A1 EP2064326 A1 EP 2064326A1 EP 07803617 A EP07803617 A EP 07803617A EP 07803617 A EP07803617 A EP 07803617A EP 2064326 A1 EP2064326 A1 EP 2064326A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- seq
- residue
- polypeptide
- amino acid
- substitution
- Prior art date
- Legal status (The legal status 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 status listed.)
- Withdrawn
Links
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 11
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 11
- 230000002366 lipolytic effect Effects 0.000 title claims abstract description 11
- 230000000694 effects Effects 0.000 claims abstract description 14
- 108010055297 Sterol Esterase Proteins 0.000 claims abstract description 9
- 102000000019 Sterol Esterase Human genes 0.000 claims abstract description 9
- 229920001184 polypeptide Polymers 0.000 claims description 24
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 24
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 24
- 238000006467 substitution reaction Methods 0.000 claims description 16
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 11
- 238000000034 method Methods 0.000 claims description 9
- 125000000539 amino acid group Chemical group 0.000 claims description 6
- 238000012217 deletion Methods 0.000 claims description 6
- 230000037430 deletion Effects 0.000 claims description 6
- 238000003780 insertion Methods 0.000 claims description 5
- 230000037431 insertion Effects 0.000 claims description 5
- 102220578197 Rho-related GTP-binding protein RhoJ_Y83F_mutation Human genes 0.000 claims description 2
- 230000002209 hydrophobic effect Effects 0.000 claims description 2
- 108090001060 Lipase Proteins 0.000 abstract description 8
- 102000004882 Lipase Human genes 0.000 abstract description 8
- 239000004367 Lipase Substances 0.000 abstract description 8
- 235000019421 lipase Nutrition 0.000 abstract description 8
- 101710098556 Lipase A Proteins 0.000 abstract description 7
- 101710099648 Lysosomal acid lipase/cholesteryl ester hydrolase Proteins 0.000 abstract description 7
- 102100026001 Lysosomal acid lipase/cholesteryl ester hydrolase Human genes 0.000 abstract description 7
- 150000001413 amino acids Chemical class 0.000 abstract description 7
- 230000004075 alteration Effects 0.000 abstract description 5
- 241000222173 Candida parapsilosis Species 0.000 abstract description 4
- 241000242346 Constrictibacter antarcticus Species 0.000 abstract 1
- 241001661345 Moesziomyces antarcticus Species 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 4
- 150000002148 esters Chemical class 0.000 description 4
- 230000007062 hydrolysis Effects 0.000 description 4
- 238000006460 hydrolysis reaction Methods 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 125000002252 acyl group Chemical group 0.000 description 3
- TWJNQYPJQDRXPH-UHFFFAOYSA-N 2-cyanobenzohydrazide Chemical compound NNC(=O)C1=CC=CC=C1C#N TWJNQYPJQDRXPH-UHFFFAOYSA-N 0.000 description 2
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 2
- 241001225321 Aspergillus fumigatus Species 0.000 description 2
- TUNFSRHWOTWDNC-UHFFFAOYSA-N Myristic acid Natural products CCCCCCCCCCCCCC(O)=O TUNFSRHWOTWDNC-UHFFFAOYSA-N 0.000 description 2
- 235000021360 Myristic acid Nutrition 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 102000057234 Acyl transferases Human genes 0.000 description 1
- 108700016155 Acyl transferases Proteins 0.000 description 1
- 241000222122 Candida albicans Species 0.000 description 1
- 241000257303 Hymenoptera Species 0.000 description 1
- 241000893045 Pseudozyma Species 0.000 description 1
- 241000556225 Pseudozyma sp. Species 0.000 description 1
- 229930182558 Sterol Natural products 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 150000001298 alcohols Chemical class 0.000 description 1
- 229940091771 aspergillus fumigatus Drugs 0.000 description 1
- 238000011914 asymmetric synthesis Methods 0.000 description 1
- 229940095731 candida albicans Drugs 0.000 description 1
- 229940055022 candida parapsilosis Drugs 0.000 description 1
- 150000001735 carboxylic acids Chemical class 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 125000001165 hydrophobic group Chemical group 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 150000003432 sterols Chemical class 0.000 description 1
- 235000003702 sterols Nutrition 0.000 description 1
- 235000010692 trans-unsaturated fatty acids Nutrition 0.000 description 1
- 150000003626 triacylglycerols Chemical group 0.000 description 1
Classifications
-
- 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
Definitions
- Fig. 1 shows a three-dimensional model of Candida antarctica lipase A (CALA, SEQ ID NO: 1 ) with a substrate analog (myristic acid).
- CALA Candida antarctica lipase A
- substrate analog myristic acid
- CpLIPI or CpLIP2 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.
- 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 posi- tion.
- 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.
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
Variants with increased cholesterol esterase activity can be designed on the basis of a three-dimensional model by making amino acid alterations in the lid region of of lipolytic enzymes such as C. antarctica lipase A or the lipase/cholesterol esterase from C. parapsilosis.
Description
LIPOLYTIC ENZYME VARIANTS
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
V Neugnot et al., European Journal of Biochemistry, Vol. 269 (6) pp. 1734-1745 (2002) March, describes a lipase/acyltransferase from Candida parapsilosis.
Candida antarctica lipase A is desacribed in WO8802775A1 and buy de Maria PD, Jopurnal of Molecular Catalysis B: Enzymatic, 37 (2005) 36-46. WO9401541A1 discloses vari- ants of C. antarctica lipase A. WQOI 535.1.1 Al describes the use of Candida antarctica lipase A for ester synthesis from a sterol and a carboxylic acid.
SUMMARY OF THE INVENTION
The inventors have found that variants with increased cholesterol esterase activity can be designed on the basis of a three-dimensional model by making amino acid alterations in the lid region of lipolytic enzymes such as C. antarctica lipase A or the lipase/cholesterol esterase from C. parapsilosis.
Accordingly, the invention provides a method of preparing a polypeptide, comprising: a) providing a parent polypeptide having lipolytic enzyme activity and an amino acid sequence having at least 80 % identity to any of SEQ ID NOS: 1-5, b) selecting an amino acid residue in the parent polypeptide at a position corresponding to any of 413-431 of SEQ ID NO: 1 , 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 se- lected residue, d) preparing an altered polypeptide having the altered amino acid sequence, e) determining the cholesterol esterase activity of the altered polypeptide, and f) selecting an altered polypeptide which has an increased cholesterol esterase activity compared to the parent polypeptide. The invention also provides 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 the difference from SEQ ID NO: 1 comprises an amino acid substitution, deletion or in-sertion at a position corresponding to any of residues 413-431.
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 (CaLIPI , ADY62089) SEQ ID NO: 6 (ADY62090, CaLI P2) 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 (CpLIPI , 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 CpLIPI (SEQ ID NO: 3) or CpLIP2 (SEQ ID NO: 4), any of CaLIP1-10 from Candida albicans, e.g. CaLIPI or CaLIP2 shown as SEQ ID NOS: 5-6, or Aspergillus fumigatus lipase (SEQ ID NO: 7).
Three-dimensional model and selection of amino acid residue
The invention is based on 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 lid region is found at positions 413-431.
An amino acid residue is selected at a position corresponding to the lid region in the 3D model for SEQ ID NO: 1.
Altered amino acid sequence
The selected residue may be substituted with a different residue, particularly with a smaller residue. Amino acid residues are ranked as follows from smallest to largest: (an equal sign indicates residues with sizes that are practically indistinguishable): G < A < S=C < V = T = P < L = I = N = D = M < E =Q < K < H < R = 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.
The selected residue may particularly be substituted 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
CpLIPI 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 posi- tion. 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 amino acid alterations
The variant may comprise one or more of the following substitutions: F223A,I,L, F421V,P,L, Y83F, L357I, A420G, V268G, V267G, E360S, E355G, E325A, K423V,N, Q418D, D1 12S or V110G or a deletion corresponding to Delta(11 1-1 12). More particularly, the variant may comprise a combination of substitutions F223A F421 V or F223A F421 V 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 T21 1W 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.
Use of lipolytic enzyme variant
The variants of the invention have increased cholesterol esterase 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:
• Pitch control in papermaking, e.g. as described in Calero-Rueda O et al., International Journal of Biotechnology, Vol. 6 (4) pp. 367-375 (2004).
• Biosensor as described in Singh S et al., Analytica Chimica Acta, Vol. 568 (1-2) pp. 126-132 (2006).
• Cleaning of lipid-stained contact lenses. Sugihara et al., Bioscience, Biotechnology and Biochemistry, Vol. 66 (1 1 ) pp. 2347-2355 (2002) Nov.
• Hydrolysis or synthesis of esters of tertiary or sterically hindered alcohols. Hydrolysis at the sn-2 position of triglycerides. Hydrolysis or synthesis of esters of trans-fatty acids. Ester syn- thesis or hydrolysis at high temperature and/or acidic pH. Asymmetric synthesis of amino
acids/amino esters, de Maria PD et al., Journal of Molecular Catalysis B: Enzymatic 37 (2005) 36-46.
EXAMPLES
Claims
1. A method of preparing a polypeptide, comprising a) providing a parent polypeptide having lipolytic enzyme activity and an amino acid sequence having at least 80 % identity to any of SEQ ID NOS: 1-5, b) selecting an amino acid residue in the parent polypeptide at a position corresponding to any of 413-431 of SEQ ID NO: 1 , 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 adja- cent to the selected residue, d) preparing an altered polypeptide having the altered amino acid sequence, e) determining the cholesterol esterase activity of the altered polypeptide, and f) selecting an altered polypeptide which has an increased cholesterol esterase activity compared to the parent polypeptide.
2. The method of the preceding claim wherein the selected residue corresponds to any of 416- 425 Of SEQ ID NO: 1.
3. The method of any preceding claim wherein the substitution is made with a residue which is smaller.
4. The method of any preceding claim wherein the substitution is made with another residue of the same type where the type is negative, positive, hydrophobic or hydrophilic.
5. The method of any preceding claim wherein the substitution is made with a residue found at the corresponding position in SEQ ID NO: 3 or 4.
6. The method of any preceding claim wherein a residue is deleted at a position where SEQ ID NO: 3 or 4 has a gap in alignment with the parent polypeptide.
7. 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 the difference from SEQ ID NO: 1 comprises an amino acid substitution, deletion or insertion at a position corresponding to any of residues 413-431.
8. The polypeptide of either preceding claim wherein the difference from SEQ ID NO: 1 comprises a substitution corresponding to any of F223A,I,L, F421V,P,L, Y83F, L357I, A420G, V268G, V267G, E360S, E355G, E325A, K423V,N, Q418D, D112S or V1 10G or a deletion corresponding to Delta(1 11-1 12).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP07803617A EP2064326A1 (en) | 2006-10-03 | 2007-10-02 | Lipolytic enzyme variants |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP06121680 | 2006-10-03 | ||
| PCT/EP2007/060474 WO2008040740A1 (en) | 2006-10-03 | 2007-10-02 | Lipolytic enzyme variants |
| EP07803617A EP2064326A1 (en) | 2006-10-03 | 2007-10-02 | Lipolytic enzyme variants |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| EP2064326A1 true EP2064326A1 (en) | 2009-06-03 |
Family
ID=38050245
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| EP07803617A Withdrawn EP2064326A1 (en) | 2006-10-03 | 2007-10-02 | Lipolytic enzyme variants |
Country Status (2)
| Country | Link |
|---|---|
| EP (1) | EP2064326A1 (en) |
| WO (1) | WO2008040740A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102321595B (en) * | 2011-08-31 | 2013-01-09 | 北京利德曼生化股份有限公司 | Cholesterol esterase and its nucleotide sequence, recombinant vector, recombinant host cell, preparation method and kit |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DK88892D0 (en) * | 1992-07-06 | 1992-07-06 | Novo Nordisk As | CONNECTION |
-
2007
- 2007-10-02 WO PCT/EP2007/060474 patent/WO2008040740A1/en not_active Ceased
- 2007-10-02 EP EP07803617A patent/EP2064326A1/en not_active Withdrawn
Non-Patent Citations (1)
| Title |
|---|
| See references of WO2008040740A1 * |
Also Published As
| Publication number | Publication date |
|---|---|
| WO2008040740A1 (en) | 2008-04-10 |
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