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CA-3301529-A1 - LIPASE VARIANTS AND MICROCAPSULE COMPOSITIONS COMPRISING SUCH LIPASE VARIANTS

CA3301529A1CA 3301529 A1CA3301529 A1CA 3301529A1CA-3301529-A1

Abstract

The present invention relates to variants of a parent lipase which has lipase activity and comprise one or more substitutions corresponding to G23S, D27N, A40I, F51I,L, E56R, D57N, V60E,K, K98I, N101D, R118F, G163S, T231R, N233R, Y220F, T244E, and P256T using SEQ ID NO: 2 for numbering. The present invention also relates to compositions or microcapsule compositions comprising a lipase variant of the invention and to liquid products comprising a microcapsule composition of the invention as well as the use of said microcapsule composition for stabilizing lipase variants of the invention.

Inventors

  • Miguel Duarte Guilherme Pereira Toscano
  • Thomas Agersten Poulsen
  • Carsten Hoerslev Hansen
  • Lone Baunsgaard
  • Keith Gibson

Assignees

  • NOVOZYMES A/S

Dates

Publication Date
20260302
Application Date
20180925
Priority Date
20170927

Claims (1)

  1. <pat:ClaimStatement>CLAIMS</pat:ClaimStatement> <pat:Claims com:id="claims"> <pat:Claim com:id="CLM-00001"> <pat:ClaimNumber>1</pat:ClaimNumber> <pat:ClaimText>1. A variant of a parent lipase which variant has lipase activity, has at least 60%, but less than 100% sequence identity with SEQ ID NO: 2 and comprises substitution A40I at a position corresponding to position A40 in SEQ ID NO: 2. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00002"> <pat:ClaimNumber>2</pat:ClaimNumber> <pat:ClaimText>2. The variant of claim 1, wherein the variant further comprises substitutions T231R and/or N233R corresponding to positions T231 and/or N223 in SEQ ID NO: 2. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00003"> <pat:ClaimNumber>3</pat:ClaimNumber> <pat:ClaimText>3. The variant of any of claim 1 or 2, which is a variant of a parent lipase selected from the group consisting of: a) a polypeptide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99% or 100% sequence identity to SEQ ID NO: 2; b) a polypeptide encoded by a polynucleotide that hybridizes under low stringency conditions, medium stringency conditions, medium-high stringency conditions, high stringency conditions, or very high stringency conditions with (i) the polypeptide coding sequence of SEQ ID NO: 1 or (ii) the full-length complement of (i); c) a polypeptide encoded by a polynucleotide having at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to SEQ ID NO: 1; and d) a fragment of the polypeptide of SEQ ID NO: 2. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00004"> <pat:ClaimNumber>4</pat:ClaimNumber> <pat:ClaimText>4. The variant of any of claims 1-3, wherein the variant has at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identity, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to SEQ ID NO: </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00002"> <pat:ClaimNumber>2</pat:ClaimNumber> <pat:ClaimText>2. 5. A composition comprising the variant of any one of claims 1-4, optionally further comprising a surfactant. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00006"> <pat:ClaimNumber>6</pat:ClaimNumber> <pat:ClaimText>6. Use of the variant of any of claims 1-4 for hydrolyzing a lipase substrate. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00007"> <pat:ClaimNumber>7</pat:ClaimNumber> <pat:ClaimText>7. A method for cleaning a surface comprising contacting the surface with the variant of any of claims 1-4. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00008"> <pat:ClaimNumber>8</pat:ClaimNumber> <pat:ClaimText>8. A method of hydrolyzing a lipase substrate, comprising treating the lipase substrate with a lipase variant of any of claims 1-4. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00009"> <pat:ClaimNumber>9</pat:ClaimNumber> <pat:ClaimText>9. A polynucleotide encoding a variant of any of claims 1-4. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00010"> <pat:ClaimNumber>10</pat:ClaimNumber> <pat:ClaimText>10. A nucleic acid construct comprising the polynucleotide of claim 9, wherein the polynucleotide is operably linked to one or more control sequences that direct the production of the lipase variant in a recombinant host cell. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00011"> <pat:ClaimNumber>11</pat:ClaimNumber> <pat:ClaimText>11. An expression vector comprising the polynucleotide of claim 9 or nucleic acid construct of claim 10. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00012"> <pat:ClaimNumber>12</pat:ClaimNumber> <pat:ClaimText>12. A host cell comprising a nucleic acid construct of claim 10 or an expression vector of claim </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00011"> <pat:ClaimNumber>11</pat:ClaimNumber> <pat:ClaimText>11. 13. A method of producing a lipase variant, comprising: a) cultivating the host cell of claim 12 under conditions suitable for expression of the variant; and b) recovering the variant. </pat:ClaimText> </pat:Claim> <pat:Claim com:id="CLM-00014"> <pat:ClaimNumber>14</pat:ClaimNumber> <pat:ClaimText>14. A microcapsule composition, wherein the membrane of the microcapsule is produced by cross-linking of a polybranched polyamine having a molecular weight of more than 1 kDa, wherein the microcapsule comprising a lipase variant of any of claims 1-4. </pat:ClaimText> </pat:Claim> </pat:Claims>

Description

LIPASE VARIANTS AND MICROCAPSULE COMPOSITIONS COMPRISING SUCH LIPASE VARIANTS REFERENCE TO A SEQUENCE LISTING This application contains a Sequence Listing in computer readable form. BACKGROUND OF THE INVENTION Field of the Invention The present invention relates to lipase variants, polynucleotides encoding the variants, as well as methods of producing said variants. The invention also relates to compositions and microcapsule compositions comprising a lipase variant of the invention and liquid products comprising a microcapsule composition of the invention. Description of the Related Art Lipases are important biocatalysts which have shown to be useful for various applications. Wild-type Thermomyces lanuginosus lipase (synonym Humicola lanuginosa) sold under the tradename LIPOLASE™ and variants thereof have been commercialized as active ingredient in detergent compositions for the removal of lipid stains by hydrolyzing triglycerides to generate fatty acids. Detergent, cleaning and/or fabric care compositions comprise active ingredients which interfere with the ability of lipases to remove lipid stains. Many known Thermomyces lanuginosus lipase variants with good wash performance form odor-generating short-chain fatty acids during wash and/or has a short storage stability. Thus, there is still a need and desire for lipases with improved wash performance, reduced odor-generation and/or improved storage stability/longer shelf life/increased thermostability. SUMMARY OF THE INVENTION The present invention relates to variants of a parent lipase which variant has lipase activity, has at least 60%, but less than 100% sequence identity with SEQ ID NO: 2 and comprises one or more (e.g. several) substitutions at positions corresponding to G23S, D27N, A40I, F51I,L, E56R, D57N, V60E,K, K98I, N101D, R118F, G163S, Y220F, T231R, N233R, T244E, and P256T. The present invention furthermore relates to compositions comprising a lipase variant of the invention as well as the use thereof for hydrolyzing a lipid substrate. Furthermore, the invention relates to polynucleotides encoding the variants of the invention; nucleic acid constructs, vectors, and host cells comprising the polynucleotides. In one aspect, the invention relates to microcapsule compositions, wherein the membrane of the microcapsule is produced by cross-linking of a polybranched polyamine having a molecular weight of more than 1 kDa, wherein the microcapsule comprising a lipase variant of the invention. In a furher aspect the invention relates to microcapsule compositions, comprising a lipase variant of the invention entrapped in a compartment formed by a membrane, which membrane is produced by cross-linking of (a) a polybranched polyamine having a molecular weight of more than 800 Da, and (b) an aliphatic or aromatic amine having a molecular weight of less than 300 Da; wherein the weight ratio of (a)/(b) is in the range of <math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><mn>0.1</mn><annotation encoding="application/x-tex">0.1</annotation></semantics></math> to <math xmlns="http://www.w3.org/1998/Math/MathML"><semantics><mn>1000</mn><annotation encoding="application/x-tex">1000</annotation></semantics></math>. Finally, the invention relates to liquid products comprising a microcapsule composition of the invention. DEFINITIONS Lipase: The terms "lipase", "lipase enzyme", "lipolytic enzyme", "lipid esterase", "lipolytic polypeptide", and "lipolytic protein" refers to an enzyme in class EC3.1.1 as defined by Enzyme Nomenclature. It may have lipase activity (triacylglycerol lipase, EC3.1.1.3), cutinase activity (EC3.1.1.74), sterol esterase activity (EC3.1.1.13) and/or wax-ester hydrolase activity (EC3.1.1.50). For purposes of the present invention, lipase activity is determined according to the procedure described in the Examples. In one aspect, the variants of the present invention have at least 20%, e.g., at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% of the lipase activity of the polypeptide of SEQ ID NO: 2. Allelic variant: The term "allelic variant" means any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequences. An allelic variant of a polypeptide is a polypeptide encoded by an allelic variant of a gene. cDNA: The term "cDNA" means a DNA molecule that can be prepared by reverse transcription from a mature, spliced, mRNA molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks intron sequences that may be present in the corresponding genomic DNA. The initial, primary RNA transcript is a precursor to mRNA that is pro