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CN-122012456-A - Cutinase variants and polynucleotides encoding same

CN122012456ACN 122012456 ACN122012456 ACN 122012456ACN-122012456-A

Abstract

Variants having cutinase activity of a parent cutinase, which variants comprise a change at one or more (e.g., several) positions corresponding to positions 181, 182, 115, 161, 1,2, 43, 55, 79, or 5 of SEQ ID No. 2, wherein the change is a substitution for positions 181, 115, 161, 43, 55, 79, and 5, and a deletion for positions 1,2, and 182, and wherein the variant has at least 75% but less than 100% sequence identity with the mature polypeptide of SEQ ID No. 2. Polynucleotides encoding these variants, nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for obtaining the variants and methods for producing the variants. Compositions comprising variants and methods for using variants.

Inventors

  • SUN TING
  • L. de Maria
  • SAIKIA RAKHI
  • S. Mei Muwasu Lampard

Assignees

  • 诺维信公司

Dates

Publication Date
20260512
Application Date
20141210
Priority Date
20131211

Claims (19)

  1. 1. A variant having cutinase activity of a parent cutinase, the variant comprising a change at one or more (e.g., several) positions corresponding to positions 181, 182, 115, 161, 1, 2, 43, 55, 79, or 5 of SEQ ID No. 2, wherein the change is to a substitution at positions 181, 115, 161, 43, 55, 79, and 5, and to a deletion at positions 1, 2, and 182, and wherein the variant has at least 75% but less than 100% sequence identity to the mature polypeptide of SEQ ID No. 2.
  2. 2. The variant of claim 1, wherein the parent cutinase is selected from the group consisting of: a. a polypeptide having at least 75% sequence identity to the mature polypeptide of SEQ ID NO. 2; b. A polypeptide encoded by a polynucleotide that hybridizes under low stringency conditions to the mature polypeptide coding sequence of SEQ ID No. 1, or to the full-length complement thereof; c. a polypeptide encoded by a polynucleotide having at least 75% identity to the mature polypeptide coding sequence of SEQ ID NO. 1, and Fragments of the mature polypeptide of SEQ ID NO. 2.
  3. 3. The variant of any of claims 1-2, wherein the variant comprises at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% of the number of amino acids of SEQ ID No. 2.
  4. 4. A variant according to any one of claims 1-3, wherein the number of changes is 1-20, such as 1-10 and 1-5, such as1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 changes.
  5. 5. The variant of any of claims 1-4, comprising one or more (e.g., several) changes selected from the group consisting of R181P, G182 x, V115I, A161L, Q x, L2 x, a43C, I55C, N a, and I5V.
  6. 6. The variant of any of claims 1-5, which comprises or consists of a change selected from the group consisting of: a. V115I +R181P +G182* b. A161L +R181P +G182* c. Q1* +L2* +I5V +A43C +I55C +N79A +V115I +R181P +G182* d. Q1* +L2* +A43C +I55C +N79A +V115I +R181P +G182* e. I5V +A43C +I55C +N79A +V115I f. A43C +I55C +N79A g. I5V +A43C +I55C +N79A +V115I +R181P +G182* h. Q1* +L2* i. I5V j. A43C +I55C k. N79A l. V115I m. A161L n.R181P +G182*。
  7. 7. the variant of any of claims 1-6, further comprising an N-terminal extension.
  8. 8. The variant of claim 7, wherein the N-terminal extension is selected from the group consisting of: a. AAVDSNHTPAVPELVAR b. AVDSNHTPAVPELVAR c. VDSNHTPAVPELVAR d. DSNHTPAVPELVAR e. SNHTPAVPELVAR f. NHTPAVPELVAR g. HTPAVPELVAR h. TPAVPELVAR i. PAVPELVAR j. AVPELVAR k. VPELVAR l. PELVAR m. ELVAR n. LVAR o. VAR p. AR q.R。
  9. 9. The variant of any of claims 1-8, which has an improved property relative to the parent, wherein the improved property is selected from the group consisting of specific activity, substrate binding, substrate cleavage, substrate specificity, thermostability, and reduced tendency to pill.
  10. 10. A composition comprising the variant of any one of claims 1-9.
  11. 11. A method for modifying a polyester, the method comprising using the variant of any one of claims 1-9.
  12. 12. A process for hydrolyzing a cyclic oligomer of poly (ethylene terephthalate), the process comprising using the variant of any one of claims 1-9.
  13. 13. A method for reducing the pilling tendency of a fabric comprising or consisting of a polyester using the variant according to any one of claims 1 to 9.
  14. 14. A polynucleotide encoding the variant of any one of claims 1-9.
  15. 15. A nucleic acid construct comprising the polynucleotide of claim 14.
  16. 16. An expression vector comprising the polynucleotide of claim 14.
  17. 17. A host cell comprising the polynucleotide of claim 14.
  18. 18. A method of producing a cutinase variant, the method comprising: a. culturing a host cell according to claim 17 under conditions suitable for expression of the variant, and B. Recovering the variant.
  19. 19. A method for obtaining a cutinase variant, the method comprising introducing a change in a parent cutinase at one or more of positions 181, 182, 115, 161, 1, 2, 43, 55, 79, 5 and 71 corresponding to the mature polypeptide of SEQ ID No. 2, wherein the change is a substitution at positions 181, 115, 161, 43, 55, 79 and 5 and a deletion at positions 1, 2 and 182, and the variant has cutinase activity. And recovering the variant.

Description

Cutinase variants and polynucleotides encoding same The present application is a divisional application of the application patent application of which the application date is 2014, 12, 10, and the application number is 201480065727.6, and the application name is "cutinase variant and polynucleotide for encoding the same". Reference to sequence Listing The present application comprises a sequence listing in computer readable form, which is incorporated herein by reference. Background Technical Field The present invention relates to cutinase variants, polynucleotides encoding these variants, methods of producing these variants, and methods of using these variants. Description of related Art Poly (ethylene terephthalate) (abbreviated PET) fibers account for a major portion of polyesters used in the textile industry. These fibers are produced by the polycondensation of terephthalic acid and ethylene glycol and drawing the fibers from the melt. Polyesters have certain core advantages including high strength, soft hand, stretch resistance, stain resistance, machine washability, wrinkle resistance, and abrasion resistance. However, polyesters are less than optimal with respect to their hydrophobicity, pilling, static electricity, dyeability, blunt surfaces as adhesion medium, i.e. softening or wettability enhancing compounds, lack of breathability, and undesirable luminous or glossy appearance. Because of its strength, polyester fabrics and/or garments are susceptible to spherulitic formation, and the process of finishing cloths applied to polyester staple fiber materials is probably most important to those designed for pilling control. All staple fiber materials tend to form globules or "pellets" with entangled fibers on the surface of the cloth when subjected to slight abrasion during washing and wear. If the fabric contains a substantial proportion of fibers that are highly resistant to bending abrasion, the pellets will remain on the surface of the fabric in an amount insufficient to produce an unpleasant hand and appearance. Another problem with polyesters is the formation of cyclic or linear oligomers of poly (ethylene terephthalate), such as bis-2-benzoyloxy-ethyl terephthalate (BETEB) and/or cyclic tris (ethylene terephthalate), during the synthesis of PET. These oligomers are partially deposited on the machine and partially remain on/within the fibers. The oligomers tend to give the fabric a light grey appearance. This is due to the deposition of oligomers on the fabric surface, in particular, the deposition is in tank terms after high temperature wet processing like high temperature dyeing. These oligomers can be removed by a severe alkali treatment, which results in a significant loss of fibrous material. The organic extraction of these oligomers is a technical possibility but is not industrially viable. Among other things, the industry has made great efforts to improve the characteristics of polyesters by using cutinases. Cutinases are known from different fungi, for example a filamentous fungal cutinase, for example a strain produced by humicola or fusarium, in particular humicola specifically such as humicola insolens strain DSM1800 (US 5827719), or fusarium solani. Methods of reducing the pilling propensity of polyester fabrics and/or garments with a diethyl terephthalate hydrolase (ETE hydrolase) and/or an ethyleneglycol dibenzyl ester hydrolase (BEB hydrolase) (WO 99/001604), methods comprising treating polyesters with an esterase enzyme to modify the polyesters (WO 2001/34899), and enzymatic hydrolysis of cyclic oligomers comprising poly (ethylene terephthalate) that subject the cyclic oligomers to the action of one or more carboxylic ester hydrolases (WO 97/27237) have been disclosed. Cutinase variants have been described in, for example, WO 0192502, wherein the use of specific humicola variants for treating polyester textiles has been disclosed. However, there is a continuing need for improved benefits of enzymatic polyester fabric and/or garment treatments, including enhancing the efficiency of these enzymes on their substrates. It is therefore desirable to identify such enzymes with improved properties for use in a method of treating fabrics. Summary of The Invention The present invention relates to variants having cutinase activity of a parent cutinase, which variants comprise a change at one or more (e.g., several) positions corresponding to positions 181, 182, 115, 161, 1,2, 43, 55, 79, or 5 of SEQ ID No. 2, wherein the change is to a substitution at positions 181, 115, 161, 43, 55, 79, and 5, and wherein the variant has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% but less than 100% sequence identity to the mature polypeptide of SEQ ID No. 2. The invention also relates to polynucleotides encoding these variants, nucleic acid constructs, vectors, and host cells comprising the polynucleotides, as well as meth