WO2020193532A1 - Cleaning composition having amylase enzymes - Google Patents

Cleaning composition having amylase enzymes Download PDF

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Publication number
WO2020193532A1
WO2020193532A1 PCT/EP2020/058127 EP2020058127W WO2020193532A1 WO 2020193532 A1 WO2020193532 A1 WO 2020193532A1 EP 2020058127 W EP2020058127 W EP 2020058127W WO 2020193532 A1 WO2020193532 A1 WO 2020193532A1
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Prior art keywords
seq
polypeptide
cleaning composition
amino acid
acid sequence
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PCT/EP2020/058127
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French (fr)
Inventor
Asfia QURESHI
Stefan Jenewein
Stefan SEEMAYER
Jesper Nielsen
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Basf Se
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Publication of WO2020193532A1 publication Critical patent/WO2020193532A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01001—Alpha-amylase (3.2.1.1)
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C—CHEMISTRY; METALLURGY
    • C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
    • C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
    • C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
    • C11D3/16—Organic compounds
    • C11D3/38—Products with no well-defined composition, e.g. natural products
    • C11D3/386—Preparations containing enzymes, e.g. protease or amylase
    • C11D3/38681—Chemically modified or immobilised enzymes

Abstract

A cleaning composition comprisingamylaseenzyme,which can be used in laundry detergents, dish washing detergents, and cleaning products for homes, industry, or vehicle care.

Description

CLEANING COMPOSITION HAVING AMYLASE ENZYMES
SEQUENCE LISTING
This application includes a nucleotide and amino acid sequence listing in computer readable form (CRF) as an ASC II text (.txt) file according to“Standard for the Presentation of Nucleotide and Amino Acid Sequence Listings in International Patent Applications Under the Patent Cooperation Treaty (PCT)” ST.25. The sequence listing is identified below and is hereby incorporated by reference into the specification of this application in its entirety and for all purposes.
Figure imgf000002_0001
TECHNICAL FIELD
Amylase enzymes are useful in many different applications such as laundry detergents, dish washing detergents, and cleaning products for homes, industry, vehicle care, baking, animal feed, pulp and paper processing, starch processing, and ethanol production. Amylases have been employed in the removal of starch stains and have been added to various compositions such as cleaning products. Current cleaning and/or fabric care compositions comprise formulations of many active ingredients that impact with the ability of amylases to remove starch stains. Thus, the need exists for amylase enzymes that can function in the harsh environment of compositions used for cleaning.
SUMMARY OF THE INVENTION
A cleaning composition comprising a polypeptide having amylase activity, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO: 8.
A cleaning composition comprising a polypeptide having amylase activity, wherein the polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.
The cleaning composition comprising a polypeptide having amylase activity, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO: 8; wherein the polypeptide is a fragment of the full-length amino acid sequence and the fragment has amylase activity. The cleaning composition comprising a polypeptide having amylase activity, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO: 8; wherein the pH of the cleaning composition is from pH7 to pH 10.
The cleaning composition comprising a polypeptide having amylase activity, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO: 8; wherein the pH of the cleaning composition is from pH7.5 to pH8.5.
The cleaning composition comprising a polypeptide having amylase activity, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO: 8; wherein the pH of the cleaning composition is pH8.
The cleaning composition comprising a polypeptide having amylase activity, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO: 8; wherein the pH of the cleaning composition is pH 10.
The cleaning composition comprising a polypeptide having amylase activity, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, wherein the pH of the cleaning composition is pH8 or pH 10.
The cleaning composition comprising a polypeptide having amylase activity, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2, wherein the pH of the cleaning composition is pH8 or pH 10.
The cleaning composition comprising a polypeptide having amylase activity, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO: 8; wherein the amylase is 0.005 to 0.50 ppm of the cleaning composition.
The cleaning composition comprising a polypeptide having amylase activity, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO: 8; wherein the amylase concentration is selected from: 0.005 ppm, 0.1 ppm, 0.2ppm, 0.4ppm, and 0.5 ppm of the cleaning composition. The cleaning composition comprising a polypeptide having amylase activity, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 ; wherein the amylase concentration is selected from: 0.005 ppm, 0.1 ppm, 0.2ppm, 0.4ppm, and 0.5 ppm of the cleaning composition.
The cleaning composition comprising a polypeptide having amylase activity, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2; wherein the amylase concentration is selected from: 0.005 ppm, 0.1 ppm, 0.2ppm, 0.4ppm, and 0.5 ppm of the cleaning composition.
The cleaning composition comprising a polypeptide having amylase activity, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO: 8; wherein the cleaning composition is for cleaning at a temperature from 20 degrees C to 40 degrees C.
The cleaning composition comprising a polypeptide having amylase activity, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO: 8; wherein the cleaning composition is for cleaning at a temperature selected from the group consisting of 20 degrees C, 21 degrees C, 22 degrees C, 23 degrees C, 24 degrees C, 25 degrees C, 26 degrees C, 27 degrees C, 28 degrees C, 29 degrees C, 30 degrees C, 31 degrees C, 32 degrees C, 33 degrees C, 34 degrees C, 35 degrees C, 36 degrees C, 37 degrees C, 38 degrees C, 39 degrees C, 40 degrees C, 41 degrees C, 42 degrees C, 43 degrees C, 44 degrees C, 45 degrees C, 46 degrees C, 47 degrees C, 48 degrees C, 49 degrees C, and 50 degrees C.
The cleaning composition comprising a polypeptide having amylase activity, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO: 8; wherein the cleaning composition is for cleaning at a temperature o 20 degrees C.
The cleaning composition comprising a polypeptide having amylase activity, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO: 8; wherein the cleaning composition is for cleaning at a temperature of 40 degrees C. The cleaning composition comprising a polypeptide having amylase activity, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: l ; wherein the cleaning composition is for cleaning at a temperature of 20 degrees C or 40 degrees C.
The cleaning composition comprising a polypeptide having amylase activity, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2; wherein the cleaning composition is for cleaning at a temperature of 20 degrees C or 40 degrees C.
The cleaning composition comprising a polypeptide having amylase activity, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8; wherein the brightness is from 0.60 to 0.80 RGB.
The cleaning composition comprising a polypeptide having amylase activity, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8; wherein the brightness is from 0.60, 0.61, 0.62, 0.63, 0.64, 0.65, 0.66, 0.67, 0.68, 0.69, 0.70, 0.71, 0.72, 0.73, 0.74, 0.75, 0.76, 0.77, 0.78, 0.79, and 0.80 RGB.
The cleaning composition comprising a polypeptide having amylase activity, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO: 8; and further comprising a second enzyme selected from the group consisting of: a second amylase, a lipase, a protease, a cellulase, a mannanase, a laccase, a pectinase, a nuclease, and any combination thereof.
The cleaning composition comprising a polypeptide having amylase activity, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO: 8; and further comprising a second enzyme, wherein the second enzyme is a second amylase.
The cleaning composition comprising a polypeptide having amylase activity, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO: 8; and further comprising a second enzyme which is a protease.
The cleaning composition comprising a polypeptide having amylase activity, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID N0:4, SEQ ID N0:5, SEQ ID N0:6, SEQ ID N0:7, and SEQ ID NO: 8; and further comprising a second enzyme which is a cellulase.
The cleaning composition comprising a polypeptide having amylase activity, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO: 8; and further comprising a second enzyme which is a mannanase.
The cleaning composition comprising a polypeptide having amylase activity, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO: 8; and further comprising a second enzyme which is a lipase.
The cleaning composition comprising a polypeptide having amylase activity, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO: 8; wherein the cleaning composition is a liquid, a granule, a gel, or any combination thereof.
The cleaning composition comprising a polypeptide having amylase activity, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO: 8; wherein the cleaning composition is a liquid.
The cleaning composition comprising a polypeptide having amylase activity, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1 ; wherein the cleaning composition is a liquid.
The cleaning composition comprising a polypeptide having amylase activity, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2; wherein the cleaning composition is a liquid.
The cleaning composition comprising a polypeptide having amylase activity, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO: 8; wherein the cleaning composition is a laundry detergent.
The cleaning composition comprising a polypeptide having amylase activity, wherein the polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8; wherein the cleaning composition is a laundry detergent. The cleaning composition comprising a polypeptide having amylase activity, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2; wherein the cleaning composition is a laundry detergent.
The cleaning composition comprising a polypeptide having amylase activity, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO:2; wherein the cleaning composition is a laundry detergent.
A polynucleotide comprising a nucleic acid sequence that encodes the polypeptide having amylase activity, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.
A polynucleotide comprising a nucleic acid sequence of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16; wherein the polynucleotide encodes the polypeptide having amylase activity, wherein the polypeptide comprises an amino acid sequence selected from the amino acid sequence of: SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.
A polynucleotide comprising a nucleic acid sequence selected from the group consisting of: SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.
A method of making the polypeptide having amylase activity, wherein the amylase is selected from the ammo acid sequence of: SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8; comprising: providing a nucleic acid sequence that encodes the polypeptide having amylase activity, transforming the nucleic acid sequence into an expression host, cultivating the expression host to produce the polypeptide, and purifying the polypeptide.
The method of making the polypeptide having amylase activity, wherein the amylase is selected from the amino acid sequence of: SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8; wherein the expression host is selected from the group consisting of: a bacterial expression system, a yeast expression system, a fungal expression system, and a synthetic expression system.
The method of making the polypeptide having amylase activity, wherein the amylase is selected from the amino acid sequence of: SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8; wherein the bacterial expression system is selected from an E. coli, a Bacillus, a Pseudomonas, and a Streptomyces, wherein the yeast expression system is selected from a Candida, a Pichia, a Saccharomyces, a Schizosaccharomyces or, wherein the fungal expression system is selected from a Penicillium, an Aspergillus, a Fusarium, a Myceliopthora, a Themothelomyces, a Rhizomucor, a Rhizopus, a Thermomyces, and a Trichoderma.
The method of making the polypeptide having amylase activity, wherein the polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8; wherein the bacterial expression system is a Bacillus host.
The method of making the polypeptide having amylase activity, wherein the polypeptide has an amino acid sequence of SEQ ID NO: l; wherein the bacterial expression system is a Bacillus host.
The method of making the polypeptide having amylase activity, wherein the polypeptide has an amino acid sequence of SEQ ID NO:2; wherein the bacterial expression system is a Bacillus host.
The method of making an amylase, wherein the polypeptide having amylase activity is a variant polypeptide of the amino acid sequence selected from group consisting of: SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO: 8; and the variant polypeptide has amylase activity.
The method of making an amylase, wherein the variant polypeptide having amylase activity is at least 80% identical, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the amino acid sequence selected from group consisting of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO: 8; and the variant polypeptide has amylase activity.
The method of making an amylase, wherein the variant polypeptide having amylase activity is encoded by a polynucleotide comprising a nucleic acid sequence which is at least 80% identical, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the amino acid sequence selected from group consisting of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: l l, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16; and the variant polypeptide has amylase activity. The method of making an amylase, wherein the variant polypeptide having amylase activity is at least 80% identical, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the amino acid sequence of SEQ ID NO: l, and the variant has amylase activity.
The method of making an amylase, wherein the variant polypeptide having amylase activity is at least 80% identical, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the amino acid sequence of SEQ ID NO:2, and the variant has amylase activity.
An amylase that is selected from an amino acid sequence of: SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.
A polypeptide variant having at least 80% identical, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 98.5%, at least 99%, at least 99.5%, or 100% identical to the amino acid sequence selected from the group consisting of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8; wherein the polypeptide variant has amylase activity.
A polypeptide variant having at least 80% identical, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 98.5%, at least 99%, at least 99.5%, or 100% identical to the amino acid sequence of SEQ ID NO: l; wherein the polypeptide variant has amylase activity.
A polypeptide variant having at least 80% identical, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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 98.5%, at least 99%, at least 99.5%, or 100% identical to the amino acid sequence of SEQ ID NO:2; wherein the polypeptide variant has amylase activity.
A method of using the cleaning composition comprising the polypeptide having amylase activity, wherein the polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8; and the composition removes stains from a surface.
The method of using the cleaning composition comprising the polypeptide having amylase activity, wherein the polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8; wherein the surface is a textile.
The method of using the cleaning composition comprising the polypeptide having amylase activity, wherein the polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO: 8; wherein the textile is selected from a yarn, fiber, fabric, garment, cloth, or any combination thereof.
The method of using the cleaning composition comprising the polypeptide having amylase activity, wherein the polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8; wherein the fiber is a natural fiber, synthetic fiber, or a combination thereof.
The method of using the cleaning composition comprising the polypeptide having amylase activity, wherein the polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8; wherein the surface is a hard surface.
The method of using the cleaning composition comprising the polypeptide having amylase activity, wherein the polypeptide has an amino acid sequence selected from the group consisting of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO: 8; wherein the hard surface is a floor, furnishing, wall, sanitary ceramics, glass, metallic surface, or any combination thereof.
DETAILED DESCRIPTION OF THE INVENTION
An enzyme is a biological molecule (polypeptide) comprising a sequence of amino acid residues, wherein the enzyme can catalyze a reaction. Hence, enzymes are catalytically active proteins or polypeptides. Enzyme names are determined based on the recommendations of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (IUBMB). Enzymes are defined by an EC (Enzyme Commission) number, recommended name, alternative names (if any), catalytic activity, and other factors. Enzymes herein may be identified by polypeptide sequences (also called amino acid sequences herein). The polypeptide sequence specifies the three-dimensional structure including the“active site” of an enzyme which in turn determines the catalytic activity of the same. Polypeptide sequences may be identified by a SEQ ID NO.
Enzymes are obtained from or derived from many different sources including: plants; animals; bacteria, archaea, fungi, yeast, environmental samples containing DNA that encodes an enzyme, or enzymes can be synthetic generated in a laboratory. For example, bacterial sources of enzymes include enzymes derived from Bacillus, Streptomyces, E. coli and Pseudomonas, fungal sources of enzymes include enzymes derived from Aspergillus, Fusarium, Thermomyces and Trichoderma yeast sources of enzymes include enzymes derived from Pichia, and Saccharomyces .
Different classes of enzymes are known to be useful in detergents and cleaning products including: lipase, amylase, protease, cellulase, mannanase, pectate lyase, and nuclease; however, there is a need in the industry to provide an amylase that has more activity, temperature profile, pH profile, has improved performance (stain removal), stability in presence of protease, or a combination thereof. The amylase enzymes of the invention address these industrial needs.
The World Intellectual Property Office (WIPO) Standard ST.25 (1998) provides that the amino acid residues should be represented in the sequence listing using the following three-letter symbols with the first letter as a capital. The table below provides an overview of the amino acid identifiers as well as the corresponding DNA codons that encode the amino acid using the standard genetic standard. The DNA codons that encode amino acid residues can be different depending organism that is used and slightly different tables for translation of the genetic code may apply. A compilation of such non standard code translation tables is maintained at the NCBI.
A“parent” polypeptide amino acid sequence is the starting sequence for introduction of mutations (e.g. by introducing one or more amino acid substitutions, insertions, deletions, or a combination thereof) to the sequence, resulting in“variants” of the parent polypeptide amino acid sequences. A parent includes: A wild-type polypeptide amino acid sequence or synthetically generated polypeptide amino acid sequence that is used as starting sequence for introduction of (further) changes.
A“variant polypeptide” refers to an enzyme that differs from its parent in its amino acid sequence. While the definition below describes variants in the context of amino acid changes, nucleic acids may be similarly modified, e.g. by substitutions.
A“mature polypeptide” means an enzyme in its final form including any post-translational modifications, glycosylation, phosphorylation, truncation, N-terminal modifications, C-terminal modifications, signal sequence deletion. A mature polypeptide can vary depending upon the expression system, vector, promoter, and/or production process.
A "synthetic" or“artificial” compound is produced by in vitro chemical or enzymatic synthesis.
The term“non-naturally occurring” refers to a (poly)nucleotide, amino acid, (poly)peptide, enzyme, protein, cell, organism, or other material that is not present in its original naturally occurring environment or source. In one embodiment, the amylase of the present invention is a non-naturally occurring amylase
Variant polynucleotide and variant polypeptide sequences may be defined by their sequence identity when compared to a parent sequence. Sequence identity usually is provided as“% sequence identity” or“% identity”. For calculation of sequence identities, in a first step a sequence alignment has to be produced. According to this invention, a pairwise global alignment has to be produced, meaning that two sequences have to be aligned over their complete length, which is usually produced by using a mathematical approach, called alignment algorithm.
According to the invention, the alignment is generated by using the algorithm of Needleman and Wunsch (J. Mol. Biol. (1979) 48, p. 443-453). Preferably, the program“NEEDLE” (The European Molecular Biology Open Software Suite (EMBOSS)) is used for the purposes of the current invention, with using the programs default parameter (polynucleotides: gap open=10.0, gap extend=0.5 and matrix=EDNAFULL; polypeptides: gap open=10.0, gap extend=0.5 and matrix=EBLOSUM62).
After aligning two sequences, in a second step, an identity value is determined from the alignment produced.
For this purpose, the %-identity is calculated by dividing the number of identical residues by the length of the alignment region which is showing the respective sequence of the present invention over its complete length multiplied with 100: %-identity = (identical residues / length of the alignment region which is showing the respective sequence of the present invention over its complete length) * 100.
For calculating the percent identity of two nucleic acid sequences the same applies as for the calculation of percent identity of two amino acid sequences with some specifications. For nucleic acid sequences encoding for a protein the pairwise alignment shall be made over the complete length of the coding region of the sequence of this invention from start to stop codon excluding introns. Introns present in the other sequence, to which the sequence of this invention is compared, may also be removed for the pairwise alignment. Percent identity is then calculated by %-identity = (identical residues / length of the alignment region which is showing the sequence of the invention from start to stop codon excluding introns over their complete length) * 100.
Moreover, the preferred alignment program for nucleic acid sequences implementing the Needleman and Wunsch algorithm (J. Mol. Biol. (1979) 48, p. 443-453) is“NEEDLE” (The European Molecular Biology Open Software Suite (EMBOSS)) with the programs default parameters (gapopen=10.0, gapextend=0.5 and matrix=EDNAFULL).
Sequences, having identical or similar regions with a sequence of this invention, and which shall be compared with a sequence of this invention to determine % identity, can easily be identified by various ways that are within the skill in the art, for instance, using publicly available computer methods and programs such as BLAST, BLAST-2, available for example at NCBI.
Variant polypeptides may be defined by their sequence similarity when compared to a parent sequence. Sequence similarity usually is provided as“% sequence similarity” or“%-similarity”. % sequence similarity takes into account that defined sets of amino acids share similar properties, e.g. by their size, by their hydrophobicity, by their charge, or by other characteristics. Herein, the exchange of one amino acid with a similar amino acid may be called“conservative mutation”. Similar amino acids according to the invention are defined as follows, which shall also apply for determination of %-similarity according to this invention, which is also in accordance with the BLOSUM62 matrix as for example used by program“NEEDLE”, which is one of the most used amino acids similarity matrix for database searching and sequence alignments:
Amino acid A is similar to amino acids S
Amino acid D is similar to amino acids E; N
Amino acid E is similar to amino acids D; K; Q
Amino acid F is similar to amino acids W; Y
Amino acid H is similar to amino acids N; Y
Amino acid I is similar to amino acids L; M; V
Amino acid K is similar to amino acids E; Q; R
Amino acid L is similar to amino acids I; M; V
Amino acid M is similar to amino acids I; L; V
Amino acid N is similar to amino acids D; H; S
Amino acid Q is similar to amino acids E; K; R
Amino acid R is similar to amino acids K; Q
Amino acid S is similar to amino acids A; N; T
Amino acid T is similar to amino acids S
Amino acid V is similar to amino acids I; L; M
Amino acid W is similar to amino acids F; Y
Amino acid Y is similar to amino acids F; H; W Conservative amino acid substitutions may occur over the full length of the sequence of a polypeptide sequence of a functional protein such as an enzyme. In one embodiment, such mutations are not pertaining the functional domains of an enzyme. In one embodiment, conservative mutations are not pertaining the catalytic centers of an enzyme.
For calculation of sequence similarity, in a first step a sequence alignment has to be produced as described above. After aligning two sequences, in a second step, a similarity value is determined from the alignment produced.
For this purpose, the %-similarity is calculated by dividing the number of identical residues plus the number of similar residues by the length of the alignment region which is showing the sequence of the invention over its complete length multiplied with 100: %-similarity = [(identical residues + similar residues) / length of the alignment region which is showing the sequence of the invention over its complete length] * 100.
The invention relates to a polypeptide having amylase activity comprising an amino acid sequence that is at least 80% identical, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical to the full length amino acid sequence of SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8.
The invention further relates to a polynucleotide encoding a variant polypeptide of the invention. The terms "polynucleotide(s)", "nucleic acid sequence(s)", "nucleotide sequence(s)", “nucleic acid(s)”,“nucleic acid molecule” are used interchangeably herein and refer to nucleotides, either ribonucleotides or deoxyribonucleotides or a combination of both, in a polymeric unbranched form of any length. A“gene” is a DNA segment carrying a certain genetic information.
A“parent” polynucleotide acid sequence is the starting sequence for introduction of mutations to the sequence, resulting in “variants” of said parent polynucleotide sequence. A “variant polynucleotide” refers to a polynucleotide that encodes an enzyme and the variant polynucleotide differs from its parent polynucleotide in its nucleic acid sequence.
The polynucleotide of the invention in one aspect has a nucleic acid sequence which is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 98.5%, at least 99%, at least 99.5%, or 100% identical when compared to the full length polynucleotide sequence of SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, or SEQ ID NO: 16.
Preferably, the polynucleotide is a codon-optimized polynucleotide for improving expression in a specific host cell.
The one or more amino acid substitution of the variant polypeptides can be one or more conservative amino acid substitution. A“conservative amino acid substitution” or“related amino acid” means replacement of one amino acid residue in an amino acid sequence with a different amino acid residue having a similar property at the same position compared to the parent amino acid sequence. Some examples of a conservative amino acid substitution include but are not limited to replacing a positively charged amino acid residue with a different positively charged amino acid residue; replacing a polar amino acid residue with a different polar amino acid residue; replacing a non-polar amino acid residue with a different non-polar amino acid residue, replacing a basic amino acid residue with a different basic amino acid residue, or replacing an aromatic amino acid residue with a different aromatic amino acid residue.
In another embodiment, the variant polypeptide comprises one or more amino acid residue insertion, deletion, or substitution or combinations thereof, and wherein the variant polypeptide comprises amylase activity.
“Enzymatic activity” means at least one catalytic effect exerted by an enzyme. Enzymatic activity is expressed as units per milligram of enzyme (specific activity) or molecules of substrate transformed per minute per molecule of enzyme (molecular activity). Enzymatic activity can be specified by the enzymes actual function, e.g. proteases exerting proteolytic activity by catalyzing hydrolytic cleavage of peptide bonds, lipases exerting lipolytic activity by hydrolytic cleavage of ester bonds, amylases activity involves (endo)hydrolysis of glucosidic linkages in polysaccharides, etc.
Enzymatic activity may change during storage or operational use of the enzyme. The term “enzyme stability” relates to the retention of enzymatic activity as a function of time during storage or operation. The term“storage” herein means to indicate the fact of products or compositions or formulations being stored from the time of being manufactured to the point in time of being used in final application. Retention of enzymatic activity as a function of time during storage may be called “storage stability” herein.
To determine and quantify changes in catalytic activity of enzymes stored or used under certain conditions over time, the“initial enzymatic activity” is measured under defined conditions at time zero (100%) and at a certain point in time later (x%). By comparison of the values measured, a potential loss of enzymatic activity can be determined in its extent. The extent of enzymatic activity loss determines an enzymes stability or non-stability.
Parameters influencing the enzymatic activity of an enzyme and/or storage stability and/or operational stability are for example pH, temperature, chelators, and presence of oxidative substances.
A variant polypeptide may be active over a broad pH at any single point within the range from about pH 4.0 to about pH 12.0. The variant polypeptides enzyme may be active over a range of pH 5.0 to pH 11.0, pH 6.0 to pH 10.0, and pH 7.0 to pH 9.0. In another embodiment, the variant polypeptides enzyme may be active over a pH 7.1 to pH 8.9, pH 7.2 to pH 8.8, pH 7.3 to pH 8.7, pH 7.4 to pH 8.6, pH 7.5 to pH 8.5. The variant polypeptides may be active at pH 4.0, pH 4.1, pH 4.2, pH
4.3, pH 4.4, pH 4.5, pH 4.6, pH 4.7, pH 4.8, pH 4.9, pH 5.0, pH 5.1, pH 5.2, pH 5.3, pH 5.4, pH 5.5, pH 5.6, pH 5.7, pH 5.8, pH 5.9, pH 6.0, pH 6.1, pH 6.2, pH 6.3, pH 6.4, pH 6.5, pH 6.6, pH 6.7, pH 6.8, pH 6.9, pH 7.0, pH 7.1, pH 7.2, pH 7.3, pH 7.4, pH 7.5, pH 7.6, pH 7.7, pH 7.8, pH 7.9, pH 8.0, pH 8.1, pH 8.2, pH 8.3, pH 8.4, pH 8.5, pH 8.6 pH 8.7, pH 8.8 pH 8.9, pH 9.0, pH 9.1, pH 9.2, pH
9.3, pH 9.4, pH 9.5, pH 9.6, pH 9.7, pH 9.8, pH 9.9, pH 10.0, pH 10.1, pH 10.2, pH 10.3, pH 10.4, pH 10.5, pH 10.6, pH 10.7, pH 10.8, pH 10.9, pH 11.0, pH 11.1, pH 11.2, pH 11.3, pH 11.4, pH 11.5, pH 11.6, pH 11.7, pH 11.8, pH 11.9, pH 12.0, pH 12.1, pH 12.2, pH 12.3, pH 12.4, and pH 12.5, pH 12.6, pH 12.7, pH 12.8, pH 12.9, or higher.
A“pH stability”, refers to the ability of an enzyme to exert enzymatic activity at a specific pH range.
The variant polypeptides may be active over a broad temperature, wherein the temperature is any point in the range from about 10 °C to about 95°C. The variant polypeptides may be active at a temperature range from 10 ° C to 55 ° C, 10 ° C to 50 ° C, 10 ° C to 45 ° C, 10 ° C to 40 ° C, 10 ° C to 35 ° C, 10 ° C to 30 ° C, or 10 ° C to 25° C The variant polypeptides may be active at a temperature range from 20 ° C to 55 ° C, 20 ° C to 50 ° C, 20 ° C to 45 ° C, 20 ° C to 40 ° C, 20 ° C to 35 ° C, 20 ° C to 30 ° C, or 20 ° C to 25° C. The variant polypeptides are active at a temperature of at least 10 ° C, 11 ° C, 12 ° C, 13 ° C, 14 ° C, 15 ° C, 16 ° C, 17 ° C, 18 ° C, 19 ° C, 20 ° C, 21 ° C, 22 ° C, 23 °
C, 24 ° C, 25 ° C, 26 ° C, 27 ° C, 28 ° C, 29 ° C, 30 ° C, 31 ° C, 32 ° C, 33 ° C, 34 ° C, 35 ° C, 36 °
C, 37 ° C, 38 ° C, 39 ° C, 40 ° C, 41 ° C, 42 ° C, 43 ° C, 44 ° C, 45 ° C, 46 ° C, 47 ° C, 48 ° C, 49 °
C, 50 ° C, 51 ° C, 52 ° C, 53 ° C, 54 ° C, 55 ° C, 56 ° C, 57 ° C, 58 ° C, 59 ° C, 60 ° C, 61 ° C, 62 °
C, 63 ° C, 64 ° C, 65 ° C, 66 ° C, 67 ° C, 68 ° C, 69 ° C, 70 ° C, 71 ° C, 72 ° C, 73 ° C, 74 ° C, 75 ° C, 76 ° C, 77 ° C, 78 ° C, 79 ° C, 80 ° C, 81 ° C, 82 ° C, 83 ° C, 84 ° C, 85 ° C, 86 ° C, 87 ° C, 88 ° C, 89 ° C, 90 ° C, 91 ° C, 92 ° C, 93 ° C, 94 ° C, 95 ° C, or higher temperatures. The terms“thermal stability” and“thermostability” refer to the ability of a protein to exert catalytic activity at a specific temperature range. Enzymes Thermostability may be characterized by what is known as the T value (also called half-life, see above). The T indicates the temperature at which 50% residual enzymatic activity is still present after thermal inactivation for a certain time when compared with a reference sample which has not undergone thermal treatment.
In one embodiment, the variant polypeptides improve the thermostability compared to the parent molecule. In another embodiment the variant polypeptides improve the thermostability by 3 C, 4 C, 5 C, 6 C, 7 C, 8 C, 9 C, 10 C, 11 C, 12 C, 13 C, 14 C, 15 C, 16 C, 17
C, 18 C, 19 C, 20 C, or more degrees C when compared to the parent polypeptide. In another embodiment, the thermostability increase is measured at a temperature between 70 C and 100° C. The thermostability increase can be measured at 71 C, 72 C, 73 C, 74 C, 75 C, 76 C, 77 C, 78 C, 79 C, 80 C, 81 C, 82 C, 83 C, 84 C, 85 C, 86 C, 87 C, 88 C, 89 C, 90 C,
91 C, 92 C, 93 C, 94 C, 95 C, 96 C, 97 C, 98 C, 99 C, and/or 100 C. In another embodiment, the thermostability increase is measured at a temperature of 80° C. In another embodiment, the thermostability increase is measured a temperature of 90° C.
In one embodiment, the variant polypeptides improve the amylase residual activity in the presence of protease when compared to the parent polypeptide.
In one embodiment, the variant polypeptides improve the residual activity when compared to the parent polypeptide.
In one embodiment, the variant polypeptide is a fragment of the full-length amino acid sequence and the fragment has amylase activity.
A "Fragment", or“subsequence” as used herein are a portion of a polynucleotide or an amino acid sequence.
The term“functional fragment” refers to any nucleic acid or amino acid sequence which comprises merely a part of the full-length amino acid sequence, respectively, but still has the same or similar activity and/or function. The fragment is at least 75% identical, at least 76% identical, at least 77% identical, at least 78% identical, at least 79% identical, at least 80% identical, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, 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%, at least 98.5%, at least 99%, or at least 99.5% identical to the full length amino acid sequence original sequence. The functional fragment comprises contiguous nucleic acids or amino acids compared to the original nucleic acid or original amino acid sequence, respectively. The polypeptide variants can be a hybrid of more than one amylase enzymes. In one embodiment, the polypeptide variant can be a hybrid of the polypeptide variant and at least one additional enzyme selected from the group consisting of: a second amylase, a lipase, a protease, a cellulase, a laccase, a pectinase, and a nuclease, wherein the hybrid has amylase activity and enzymatic activity of the second enzyme.
In one embodiment, the present invention refers to a polypeptide comprising a hybrid of at least one polypeptide as described herein and a second polypeptide having an activity selected from the group consisting of amylase activity, protease activity, lipase activity, cellulase activity, laccase activity, pectinase activity, and nuclease activity, wherein the hybrid also has amylase activity.
In on embodiment the variant polypeptide comprises a hybrid of at least one variant polypeptide as described herein, and a second polypeptide having amylase activity, wherein the hybrid has amylase activity.
A“hybrid” or“chimeric” or“fusion protein” means that a fragment of the amino acid sequence of a first enzyme is combined with a fragment of the amino acid sequence of a second enzyme to form a hybrid enzyme wherein the hybrid has an enzyme activity.
The hybrid enzymes can be engineered with fragments from amino acid sequences of more than two enzymes. The domain of an amylase of this invention may be combined with a domain of a commercially available amylase such as: Duramyl™, Termamyl™, Termamyl SC™ , Termamyl Ultra™, Fungamyl™, Stainzyme™, Stainzyme Plus™, Natalase™, Liquozyme X, Supramyl™ Amplify™, Amplify Prime™ and BAN™ (from Novozymes A/S), and Rapidase™, Purastar™, Purastar OxAm™ PoweraseTM, Effectenz™ (Ml 00 from DuPont), Preferenz™ (SI 000, SI 10 and FI 000; from DuPont), PrimaGreen™ (ALL; DuPont), Optisize™ (DuPont) and Kam™ (Kao) and Kemzyme™ (Biozym), to form a hybrid enzyme and the hybrid has amylase activity.
In another aspect the present invention refers to a composition comprising the polypeptide described herein. A composition may comprise combinations of the polypeptides with another enzyme. The combination of enzymes can be of the same class, for example a composition comprising a first amylase and a second amylase. Combinations of enzymes can be from a different class of enzymes, for example, a composition comprising a lipase and an amylase. Combinations of enzymes can be compositions comprising at least one amylase of the invention and one or more second enzymes. In one embodiment, the composition comprises one second enzyme, two second enzymes, three second enzymes, four second enzymes, or more than four second enzymes. In one particular embodiment, the second enzyme is selected from the group consisting of: a second amylase, a lipase, a protease, a cellulase, a laccase, a pectinase, and a nuclease, or any combination thereof.
Additional enzymes suitable for the hybrid or the composition of the present invention are further described below. In one embodiment, suitable enzymes include enzyme variants having enzymatic activity which are 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 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% identical when compared to the full length polypeptide sequence of the parent enzyme as disclosed below.
Alpha-amylase (E.C. 3.2.1.1) enzymes may perform endohydrolysis of (l->4)-alpha-D- glucosidic linkages in polysaccharides containing three or more (l->4)-alpha-linked D-glucose units. Amylase enzymes act on starch, glycogen and related polysaccharides and oligosaccharides in a random manner; reducing groups are liberated in the alpha-configuration. Other examples of amylase enzymes include: Beta-amylase (E.C. 3.2.1.2), Glucan 1,4-alpha-maltotetraohydrolase (E.C. 3.2.1.60), Isoamylase (E.C. 3.2.1.68), Glucan 1,4-alpha-maltohexaosidase (E.C. 3.2.1.98), and Glucan 1 ,4-alpha-maltohydrolase (E.C. 3.2.1.133).
Many amylase enzymes have been described in patents and published patent applications including, but not limited to: WO 2002/068589, WO 2002/068597, WO 2003/083054, WO
2004/091544, WO 2008/080093, WO 1995/10603; WO 2002/010355; WO 1999/019467; WO
2002/010355; WO 1996/023872; WO 2000/022103; WO 2009/061380; WO 2013/184577; WO
2010/104675; WO 2000/060060; WO 2006/002643; WO 2011/098531 ; WO 2013/001078; WO
2013/001087; and WO 2006/066594.
Commercially available amylase enzymes include: Duramyl™, Termamyl™, Termamyl SC™ , Termamyl Ultra™, Fungamyl™, Stainzyme™, Stainzyme Plus™, Natalase™, Liquozyme X, Supramyl™ Amplify™, Amplify Prime™ and BAN™ (from Novozymes A/S), and Rapidase™, Purastar™, Purastar OxAm™ PoweraseTM, Effectenz™ (Ml 00 from DuPont), Preferenz™ (SI 000, SI 10 and FI 000; from DuPont), PrimaGreen™ (ALL; DuPont), Optisize™ (DuPont) and Kam™ (Kao) and Kemzyme™ (Biozym).
“Lipases”,“lipolytic enzyme”,“lipid esterase”, all refer to an enzyme of EC class 3.1.1 (“carboxylic ester hydrolase”). Lipases (E.C. 3.1.1.3, Triacylglycerol lipase) may hydrolyze triglycerides to more hydrophilic mono- and diglycerides, free fatty acids, and glycerol. Lipase enzymes usually includes also enzymes which are active on substrates different from triglycerides or cleave specific fatty acids, such as Phospholipase A (E.C. 3.1.1.4), Galactolipase (E.C. 3.1.1.26), cutinase (EC 3.1.1.74), and enzymes having sterol esterase activity (EC 3.1.1.13) and/or wax-ester hydrolase activity (EC 3.1.1.50).
Many lipase enzymes have been described in patents and published patent applications including, but not limited to: W02000/032758, W02003/089620, W02005/032496,
W02005/086900, W02006/00976, W02006/031699, W02008/036863, WO2011/046812, and W02014/059360.
Lipases are used in detergent and cleaning products to remove grease, fat, oil, and dairy stains. Commercially available lipases include but are not limited to: Lipolase™, Lipex™, Lipolex™ and Lipoclean™ (Novozymes A/S), Lumafast (originally from Genencor) and Lipomax (Gist-Brocades/ now DSM).
The methods for determining lipolytic activity are well-known in the literature (see e.g. Gupta et al. (2003), Biotechnol. Appl. Biochem. 37, p. 63-71). E.g. the lipase activity may be measured by ester bond hydrolysis in the substrate para-nitrophenyl palmitate (pNP-Palmitate, C: 16) and releases pNP which is yellow and can be detected at 405 nm.
Enzymes having proteolytic activity are called“proteases” or“peptidases”. Proteases are active proteins exerting“protease activity” or“proteolytic activity”.
Proteases are members of class EC 3.4. Proteases include aminopeptidases (EC 3.4.11), dipeptidases (EC 3.4.13), dipeptidyl-peptidases and tripeptidyl-peptidases (EC 3.4.14), peptidyl-dipeptidases (EC 3.4.15), serine-type carboxypeptidases (EC 3.4.16), metallocarboxypeptidases (EC 3.4.17), cysteine- type carboxypeptidases (EC 3.4.18), omega peptidases (EC 3.4.19), serine endopeptidases (EC 3.4.21), cysteine endopeptidases (EC 3.4.22), aspartic endopeptidases (EC 3.4.23), metallo- endopeptidases (EC 3.4.24), threonine endopeptidases (EC 3.4.25), endopeptidases of unknown catalytic mechanism (EC 3.4.99).
Commercially available protease enzymes include but are not limited to Lavergy™ Pro (BASF); Alcalase®, Blaze®, Duralase™, Durazym™, Relase®, Relase® Ultra, Savinase®, Savinase® Ultra, Primase®, Polarzyme®, Kannase®, Liquanase®, Liquanase® Ultra, Ovozyme®, Coronase®, Coronase® Ultra, Neutrase®, Everlase® and Esperase® (Novozymes A/S), those sold under the tradename Maxatase®, Maxacal®, Maxapem®, Purafect®, Purafect® Prime, Purafect MA®, Purafect Ox®, Purafect OxP®, Puramax®, Properase®, FN2®, FN3®, FN4®, Excellase®, Eraser®, Ultimase®, Opticlean®, Effectenz®, Preferenz® and Optimase® (Danisco/DuPont), Axapem™ (Gist-Brocases N. V.), Bacillus lentus Alkaline Protease, and KAP (Bacillus alkalophilus subtilisin) from Kao.
At least one protease may be selected from serine proteases (EC 3.4.21). Serine proteases or serine peptidases (EC 3.4.21) are characterized by having a serine in the catalytically active site, which forms a covalent adduct with the substrate during the catalytic reaction. A serine protease may be selected from the group consisting of chymotrypsin (e.g., EC 3.4.21.1), elastase (e.g., EC 3.4.21.36), elastase (e.g., EC 3.4.21.37 or EC 3.4.21.71), granzyme (e.g., EC 3.4.21.78 or EC 3.4.21.79), kallikrein (e.g., EC 3.4.21.34, EC 3.4.21.35, EC 3.4.21.118, or EC 3.4.21.119,) plasmin (e.g., EC 3.4.21.7), trypsin (e.g., EC 3.4.21.4), thrombin (e.g., EC 3.4.21.5,) and subtilisin (also known as subtilopeptidase, e.g., EC 3.4.21.62), the latter hereinafter also being referred to as“subtilisin”.
Proteases according to the invention have proteolytic activity. The methods for determining proteolytic activity are well-known in the literature (see e.g. Gupta et al. (2002), Appl. Microbiol. Biotechnol. 60: 381-395). Proteolytic activity may be determined by using Succinyl-Ala-Ala-Pro- Phe-p-nitroanilide (Suc-AAPF-pNA, short AAPF; see e.g. DelMar et al. (1979), Analytical Biochem 99, 316-320) as substrate. pNA is cleaved from the substrate molecule by proteolytic cleavage, resulting in release of yellow color of free pNA which can be quantified by measuring OD405.
"Cellulases", “cellulase enzymes” or “cellulolytic enzymes” are enzymes involved in hydrolysis of cellulose. Three major types of cellulases are known, namely endo-ss-l,4-glucanase (endo-l,4-P-D-glucan 4-glucanohydrolase, E.C. 3.2.1.4; hydrolyzing b- 1 ,4-glucosidic bonds in cellulose), cellobiohydrolase (1,4-P-D-glucan cellobiohydrolase, EC 3.2.1.91), and ss-glucosidase (EC 3.2.1.21).
Cellulase enzymes have been described in patents and published patent applications including, but not limited to: WO1997/025417, WO 1998/024799, W02003/068910, W02005/003319, and W02009020459.
Commercially available cellulase enzymes include are Celluzyme™, Endolase™, Carezyme™, Cellusoft™, Renozyme™, Celluclean™ (from Novozymes A/S), Ecostone™, Biotouch™, Econase™, Ecopulp™ (from AB Enzymes Finland), Clazinase™, and Puradax HA™, Genencor detergent cellulase L, IndiAge™ Neutra (from Genencor International Inc./DuPont), Revitalenz™ (2000 from DuPont), Primafast™ (DuPont) and KAC-500™ (from Kao Corporation).
Cellulases according to the invention have“cellulolytic activity” or“cellulase activity”. Assays for measurement of cellulolytic activity are known to those skilled in the art. For example, cellulolytic activity may be determined by virtue of the fact that cellulase hydrolyses carboxymethyl cellulose to reducing carbohydrates, the reducing ability of which is determined colorimetrically by means of the ferricyanide reaction, according to Hoffman, W. S., J. Biol. Chem. 120, 51 (1937).
Mannanase (E.C. 3.2.1.78) enzymes hydrolyze internal b-1,4 beta-D-mannosidic linkages in mannans, galactomannans and glucomannans“Mannanase” may be an alkaline mannanase of Family 5 or 26. Mannanase enzymes are known to be derived from wild-type from Bacillus or Humicola, particularly B. agaradhaerens, B. licheniformis, B. halodurans, B. clausii, or H. insolens. Suitable mannanase are described in WO 99/064619.
Commercially available mannanase enzymes include: Mannaway® (Novozymes AIS).
Pectate lyase (E.C. 4.2.2.2) enzymes eliminative cleavage of (l->4)-alpha-D-galacturonan to give oligosaccharides with 4-deoxy-alpha-D-galact-4-enuronosyl groups at their non-reducing ends.
Pectate lyase enzymes have been described in patents and published patent applications including, but not limited to: W02004/090099. Pectate lyase are known to be derived from Bacillus , particularly B. licheniformis or B. agaradhaerens, or a variant derived of any of these, e. g. as described in US 6,124,127, WO 99/027083, WO 99/027084, WO 2002/006442, WO 2002/092741, WO 2003/095638.
Commercially available pectate lyase enzymes include: Xpect™, Pectawash™ and Pectaway™ (Novozymes A/S); PrimaGreen™, EcoScour (DuPont).
Nuclease (EC 3.1.21.1) also known as Deoxyribonuclease I, or DNase preforms endonucleolytic cleavage to 5'-phosphodinucleotide and 5'-phosphooligonucleotide end-products.
Nuclease enzymes have been described in patents and published patent applications including, but not limited to: US3451935, GB1300596, DE10304331, WO2015155350, WO2015155351, WO2015166075, WO2015181287, and WO2015181286.
In another embodiment, the present invention refers to a method of making the variant polypeptide as described herein, comprising: providing a nucleic acid sequence encoding the polypeptide described herein, transforming the nucleic acid sequence into a host cell, cultivating the host cell to produce the variant polypeptide, and optionally purifying the variant polypeptide from the host cell.
A polynucleotide encoding a polypeptide may be“expressed”. The term“expression” or “gene expression” means the transcription of a specific gene or specific genes or specific nucleic acid construct. The term“expression” or“gene expression” in particular means the transcription of a gene or genes or genetic construct into structural RNA (e.g., rRNA, tRNA) or mRNA with or without subsequent translation of the latter into a protein. The process includes transcription of DNA and processing of the resulting mRNA product.
Industrial production of enzymes usually is done by using expression systems.“Expression system” may mean a host microorganism, expression hosts, host cell, production organism, or production strain and each of these terms can be used interchangeably. In one embodiment, the expression host is selected from the group consisting of: a bacterial expression system, a yeast expression system, a fungal expression system, and a synthetic expression system. The expression host may be a wildtype cell or a recombinant cell.“Wild-type cells” herein means cells prior to a certain modification. The term“recombinant cell” (also called“genetically modified cell” herein) refers to a cell which has been genetically altered, modified or engineered such it that exhibits an altered, modified or different genotype as compared to the wild-type cell which it was derived from. The“recombinant cell” may comprise an exogenous polynucleotide encoding a certain protein or enzyme and therefore may express said protein or enzyme.
Thus, in one embodiment, the invention is directed to a genetic construct comprising a polynucleotide encoding the amylase as described herein.
In one embodiment, the invention is directed to a host cell comprising a polynucleotide encoding the amylase as described herein.
In yet another embodiment, the present invention is directed to a method of expressing a polynucleotide, comprising the steps of
(a) providing a host cell comprising a heterologous nucleic acid construct comprising a polynucleotide encoding the amylase described herein by introducing the nucleic acid construct comprising the polynucleotide encoding the amylase as described herein into the host cell;
(b) cultivating the recombinant host cell of step (a) under conditions conductive for the expression of the polynucleotide; and
(c) optionally, recovering a protein of interest encoded by the polynucleotide.
Examples of expression systems include but are not limited to: Aspergillus niger, Aspergillus oryzae, Hansenula polymorpha, Thermomyces lanuginosus, fusarium oxysporum, Fusarium heterosporum, Escherichia coli, Bacillus, preferably Bacillus subtilis, or Bacillus licheniformis, Pseudomonas, preferably Pseudomonas fluorescens, Pichia pastoris (also known as Komagataella phaffii), Myceliopthora thermophile (Cl), Themothelomyces thermophila, Schizosaccharomyces pombe, Trichoderma, preferably Trichoderma reesei and Saccharomyces, preferably Saccharomyces cerevisiae. The variant polypeptides may be produced using the expression system listed above. In one embodiment, the bacterial expression system is selected from an E. coli, a Bacillus, a Pseudomonas, and a Streptomyces. In one embodiment, the yeast expression system is selected from a Candida, a Pichia, a Saccharomyces, a Schizosaccharomyces. In one embodiment, the fungal expression system is selected from a Penicillium, an Aspergillus, a Fusarium, a Myceliopthora, a Rhizomucor, a Rhizopus, a Thermomyces, and a Trichoderma.
The term "heterologous” (or exogenous or foreign or recombinant) in the context of polynucleotides and polypeptides is defined herein as:
(a) not native to the host cell;
(b) native to the host cell but structural modifications, e.g., deletions, substitutions, and/or insertions, are included as a result of manipulation of the DNA of the host cell by recombinant DNA techniques to alter the native sequence; or
(c) native to the host cell but expression is quantitatively altered, or expression is directed from a genomic location different from the native host cell as a result of manipulation of the DNA of the host cell by recombinant DNA techniques, e.g., a stronger promoter.
With respect to two or more polynucleotide sequences or two or more amino acid sequences, the term "heterologous” is used to characterize that the two or more polynucleotide sequences or two or more amino acid sequences do not occur naturally in the specific combination with each other.
“Genetic Construct” or“expression cassette” as used herein, is a DNA molecule composed of at least one sequence of interest to be expressed, operably linked to one or more control sequences (at least to a promoter) as described herein. Typically, the expression cassette comprises three elements: a promoter sequence, an open reading frame, and a 3' untranslated region that, in eukaryotes, usually contains a polyadenylation site. Additional regulatory elements may include transcriptional as well as translational enhancers. An intron sequence may also be added to the 5' untranslated region (UTR) or in the coding sequence to increase the amount of the mature message that accumulates in the cytosol. The expression cassette may be part of a vector or may be integrated into the genome of a host cell and replicated together with the genome of its host cell. The expression cassette usually is capable of increasing or decreasing expression.
The term“vector” as used herein comprises any kind of construct suitable to carry foreign polynucleotide sequences for transfer to another cell, or for stable or transient expression within a given cell. The term“vector” as used herein encompasses any kind of cloning vehicles, such as but not limited to plasmids, phagemids, viral vectors (e.g., phages), bacteriophage, baculoviruses, cosmids, fosmids, artificial chromosomes, or and any other vectors specific for specific hosts of interest. Low copy number or high copy number vectors are also included. Foreign polynucleotide sequences usually comprise a coding sequence which may be referred to herein as“gene of interest”. The gene of interest may comprise introns and exons, depending on the kind of origin or destination of host cell.
A vector as used herein may provide segments for transcription and translation of a foreign polynucleotide upon transformation into a host cell or host cell organelles. Such additional segments may include regulatory nucleotide sequences, one or more origins of replication that is required for its maintenance and/or replication in a specific cell type, one or more selectable markers, a polyadenylation signal, a suitable site for the insertion of foreign coding sequences such as a multiple cloning site etc. One example is when a vector is required to be maintained in a bacterial cell as an episomal genetic element (e.g. plasmid or cosmid molecule). Non-limiting examples of suitable origins of replication include the fl-ori and colEl.
A vector may replicate without integrating into the genome of a host cell, e.g. as a plasmid in a bacterial host cell, or it may integrate part or all of its DNA into the genome of the host cell and thus lead to replication and expression of its DNA.
Foreign nucleic acid may be introduced into a vector by means of cloning. Cloning may mean that by cleavage of the vector (e.g. within the multiple cloning site) and the foreign polynucleotide by suitable means and methods (e.g., restriction enzymes), fitting structures within the individual nucleic acids may be created that enable the controlled fusion of said foreign nucleic acid and the vector.
Once introduced into the vector, the foreign nucleic acid comprising a coding sequence may be suitable to be introduced (transformed, transduced, transfected, etc.) into a host cell or host cell organelles. A cloning vector may be chosen suitable for expression of the foreign polynucleotide sequence in the host cell or host cell organelles.
The term“introduction” or“transformation” as referred to herein encompasses the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer. That is, the term“transformation” as used herein is independent from vector, shuttle system, or host cell, and it not only relates to the polynucleotide transfer method of transformation as known in the art (cf , for example, Sambrook, J. et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY), but it encompasses any further kind polynucleotide transfer methods such as, but not limited to, transduction or transfection. Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a genetic construct and a whole plant regenerated therefrom. The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed.
In one embodiment of the invention, a vector is used for transformation of a host cell.
The polynucleotide may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid.“Stable transformation” may mean that the transformed cell or cell organelle passes the nucleic acid comprising the foreign coding sequence on to the next generations of the cell or cell organelles. Usually stable transformation is due to integration of nucleic acid comprising a foreign coding sequence into the chromosomes or as an episome (separate piece of nuclear DNA).
“Transient transformation” may mean that the cell or cell organelle once transformed expresses the foreign nucleic acid sequence for a certain time - mostly within one generation. Usually transient transformation is due to nucleic acid comprising a foreign nucleic acid sequence is not integrated into the chromosomes or as an episome.
Alternatively, it may be integrated into the host genome. The resulting transformed plant cell may then be used to regenerate a transformed plant in a manner known to persons skilled in the art.
Recombinant cells may exhibit“increased” or“decreased” expression when compared to the respective wild-type cell.
The term“increased expression”,“enhanced expression” or“overexpression” as used herein means any form of expression that is additional to the original wild-type expression level (which can be absence of expression or immeasurable expression as well). Reference herein to“increased expression”, “enhanced expression” or“overexpression” is taken to mean an increase in gene expression and/or, as far as referring to polypeptides, increased polypeptide levels and/or increased polypeptide activity, relative to control organisms. The increase in expression may be in increasing order of preference at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, or 100% or even more compared to that of control organisms.
Methods for increasing expression of genes or gene products are well documented in the art and include, for example, overexpression driven by appropriate promoters, the use of transcription enhancers or translation enhancers. Isolated nucleic acids which serve as promoter or enhancer elements may be introduced in an appropriate position (typically upstream) of a non-heterologous form of a polynucleotide so as to increase expression of a nucleic acid encoding the polypeptide of interest. For example, endogenous promoters may be altered in vivo by mutation, deletion, and/or substitution (see, Kmiec, US 5,565,350; Zarling et al, WO 93/22443), or isolated promoters may be introduced into an organism in the proper orientation and distance from a gene of the present invention so as to control the expression of the gene.
An intron sequence may also be added to the 5' untranslated region (UTR) or the coding sequence of the partial coding sequence to increase the amount of the mature message that accumulates in the cytosol. Inclusion of a spliceable intron in the transcription unit in expression constructs has been shown to increase gene expression at both the mRNA and protein levels up to 1000-fold (Buchman and Berg (1988) Mol. Cell biol. 8: 4395-4405; Callis et al. (1987) Genes Dev 1 : 1183-1200). Such intron enhancement of gene expression is typically greatest when placed near the 5' end of the transcription unit.
To obtain increased expression or overexpression of a polypeptide most commonly the nucleic acid encoding this polypeptide is overexpressed in sense orientation with a polyadenylation signal. Introns or other enhancing elements may be used in addition to a promoter suitable for driving expression with the intended expression pattern.
Enzymes are generally produced commercially by using recombinant cells which express the desired enzyme by cultivation of the same under conditions suitable for expression of the desired enzyme.
Cultivation normally takes place in a suitable nutrient medium allowing the recombinant cells to grow (this process may be called fermentation) and express the desired protein. At the end of fermentation, fermentation broth is collected and may be further processed, wherein the fermentation broth comprises a liquid fraction and a solid fraction.
The enzyme of interest may be further purified from the fermentation broth. The term “purification” or“purifying” refers to a process in which at least one component, e.g., a protein of interest, is separated from at least another component, e.g., a particulate matter of a fermentation broth, and transferred into a different compartment or phase, wherein the different compartments or phases do not necessarily need to be separated by a physical barrier. Examples of such different compartments are two compartments separated by a filtration membrane or cloth, i.e., filtrate and retentate; examples of such different phases are pellet and supernatant or cake and filtrate, respectively. The resulting solution after purifying the enzyme of interest from the fermentation broth is called herein“purified enzyme solution”.
The desired enzyme may be secreted (into the liquid fraction of the fermentation broth) or may not be secreted from the host cells (and therefore is comprised in the cells of the fermentation broth). Depending on this, the desired enzyme may be recovered from the liquid fraction of the fermentation broth or from cell lysates. Recovery of the desired enzyme uses methods known to those skilled in the art. Suitable methods for recovery of proteins or enzymes from fermentation broth include but are not limited to collection, centrifugation, filtration, extraction, and precipitation. If the enzyme of interest precipitates or crystallizes in the fermentation broth or binds at least in part to the particulate matter of the fermentation broth additional treatment steps might be needed to release the enzyme from the biomass or solubilize enzyme crystals and precipitates. US6316240B1 describes a method for recovering an enzyme, which precipitates and / or crystallizes during fermentation, from the fermentation broth. In case the desired enzyme is comprised in the cells of the fermentation broth release of the enzyme from the cells might be needed. Release from the cells can be achieved for instance, but not being limited thereto, by cell lysis with techniques well known to the skilled person.
The purified enzyme solution may be further processed to form an“enzyme formulation”. “Enzyme formulation” means any non-complex formulation comprising a small number of ingredients, wherein the ingredients serve the purpose of stabilizing the enzymes comprised in the enzyme formulation and/or the stabilization of the enzyme formulation itself. The term“enzyme stability” relates to the retention of enzymatic activity as a function of time during storage or operation. The term“enzyme formulation stability” relates to the maintenance of physical appearance of the enzyme formulation during storage or operation as well as the avoidance of microbial contamination during storage or operation.
An“enzyme formulation” is a composition which is meant to be formulated into a complex formulation which itself may be determined for final use. An“enzyme formulation” according to the invention is not a complex formulation comprising several components, wherein the components are formulated into the complex formulation to exert each individually a specific action in a final application. A complex formulation may be without being limited thereto a detergent formulation, wherein individual detergent components are formulated in amounts effective in the washing performance of the detergent formulation.
In one aspect of the invention, at least one amylase variant of the invention is comprised in an enzyme formulation.
The enzyme formulation can be either solid or liquid. Enzyme formulations can be obtained by using techniques known in the art. For instance, without being limited thereto, solid enzyme formulations can be obtained by extrusion or granulation. Suitable extrusion and granulation techniques are known in the art and are described for instance in W09419444A1 and W09743482A1. “Liquid” in the context of enzyme formulation is related to the physical appearance at 20°C and 101.3 kPa.
Liquid enzyme formulations may comprise amounts of enzyme in the range of 0.1% to 40% by weight, or 0.5% to 30% by weight, or 1% to 25% by weight, or 3% to 10%, all relative to the total weight of the enzyme formulation.
The liquid enzyme formulation may comprise more than one type of enzyme. In one embodiment, the enzyme formulation comprises one or more amylases according to the present invention. In one embodiment, the enzyme formulation comprises one or more amylases according to the present invention and at least one additional enzyme selected from the group consisting of selected from the group consisting of: a second amylase, a lipase, a protease, a cellulase, a laccase, a pectinase, a nuclease, and any combination thereof.
Aqueous enzyme formulations of the invention may comprise water in amounts of more than about 50% by weight, more than about 60% by weight, more than about 70% by weight, or more than about 80% by weight, all relative to the total weight of the enzyme formulation.
Liquid enzyme formulations of the invention may comprise residual components such as salts originating from the fermentation medium, cell debris originating from the production host cells, metabolites produced by the production host cells during fermentation.
In one embodiment, residual components may be comprised in liquid enzyme formulations in amounts less than 30% by weight, less than 20% by weight less, than 10% by weight, or less than 5% by weight, all relative to the total weight of the aqueous enzyme formulation. In one embodiment, the enzyme formulation, in particular the liquid enzyme formulation, comprises in addition to the one or more enzymes one or more additional compounds selected from the group consisting of solvent, salt, pH regulator, preservative, stabilizer, chelators, and thickening agent. The preservative in a liquid enzyme formulation maybe a sorbitol, a benzoate, a proxel, or any combination therefore. The stabilizers in a liquid enzyme formulation maybe an MPG, a glycerol, an acetate, or any combination thereof. The chelators in a liquid enzyme formulation maybe a citrate.
In one embodiment, an enzyme formulation comprises at least one polypeptide variant of the invention and at least one preservative. Non-limiting examples of suitable preservatives include (quaternary) ammonium compounds, isothiazolinones, organic acids, and formaldehyde releasing agents. Non-limiting examples of suitable (quaternary) ammonium compounds include benzalkonium chlorides, polyhexamethylene biguanide (PHMB), Didecyldimethylammonium chloride(DDAC), and N-(3-aminopropyl)-N-dodecylpropane-l, 3-diamine (Diamine). Non-limiting examples of suitable isothiazolinones include 1 ,2-benzisothiazolin-3 -one (BIT), 2-methyl-2H-isothiazol-3-one (MIT), 5- chloro-2-methyl-2H-isothiazol-3-one (CIT), 2-octyl-2H-isothiazol-3-one (OIT), and 2-butyl- benzo[d]isothiazol-3-one (BBIT). Non-limiting examples of suitable organic acids include benzoic acid, sorbic acid, L-(+)-lactic acid, formic acid, and salicylic acid. Non-limiting examples of suitable formaldehyde releasing agent include N,N'-methylenebismorpholine (MBM), 2,2',2''-(hexahydro- l,3,5-triazine-l,3,5- triyl)triethanol (HHT), (ethylenedioxy)dimethanol, .alpha., . alpha. ',.alpha.''- trimethyl-l,3,5-triazine-l,3,5(2H,4H,6H)-triethanol (HPT), 3,3'-methylenebis[5-methyloxazolidine] (MBO), and cis-l-(3-chloroallyl)-3,5,7-triaza-l- azoniaadamantane chloride (CTAC).
Further useful preservatives include iodopropynyl butylcarbamate (IPBC), halogen releasing compounds such as dichloro-dimethyl-hydantoine (DCDMH), bromo-chloro-dimethyl-hydantoine (BCDMH), and dibromo-dimethyl-hydantoine (DBDMH); bromo-nitro compounds such as Bronopol (2-bromo-2-nitropropane-l,3-diol), 2,2-dibromo-2-cyanoacetamide (DBNPA); aldehydes such as glutaraldehyde; phenoxy ethanol; Biphenyl-2-ol; and zinc or sodium pyrithione.
In one embodiment, an enzyme formulation comprises at least one polypeptide variant of the invention and at least one enzyme stabilizer. An enzyme stabilizer is selected from substances which are capable of reducing loss of enzymatic activity during storage of at least one enzyme comprised in a liquid enzyme formulation. Reduced loss of enzymatic activity within this invention may mean that the loss of enzymatic activity is reduced by at least 5%, by at least 10%, by at least 15%, by at least 20%, by at least 25%, by at least 30%, by at least 40%, by at least 50%, by least 60%, at least 65%, at least 70%, at least 75%, 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%, at least 99%, or at least 99.5% when compared to the initial enzymatic activity before storage. Preferred stabilizers are selected from the group consisting of salt (e.g., CaC12), propanediol, polyethylene glycol, an MPG, a glycerol, an acetate, or any combination thereof.
Enzyme applications
In another embodiment, the polypeptide variant as described herein may be used in foods, for example the enzyme can be an additive for baking. The enzymes can be used in feed, for example the enzyme is an animal feed additive. The enzyme can be used in the starch processing industry, for example the amylases are used in the conversion of starch to ethanol or sugars (high fructose corn syrup) and other byproducts such as oil, dry distiller’s grains, etc. The polypeptide variants are used in in pulp and paper processing, for example, the enzymes can be used for improving paper strength. The enzymes can be used for mining and oil well services, for example cellulases can be used for breaking guar during oil well fracturing. In one embodiment, the polypeptide variant as described herein are used in detergent formulations or cleaning formulations.
In one embodiment, the present invention refers to a method of preparing a dough or a baked product prepared from the dough, the method comprising adding one of the variant polypeptides having amylase activity as described herein to the dough and baking it. In one embodiment, the present invention refers to a method of use of the variant polypeptide having amylase activity as described herein for processing starch. In one embodiment, the present invention refers to a method of use of the variant polypeptide having amylase activity as described herein for cleaning or washing textiles, hard surfaces, or dishes. In one embodiment, the present invention refers to a method of use of the variant polypeptide having amylase activity as described herein for making ethanol. In one embodiment, the present invention refers to a method of use of the variant polypeptide having amylase activity as described herein for processing pulp or paper. In one embodiment, the present invention refers to a method of use of the variant polypeptide having amylase activity as described herein for feeding an animal.
In one embodiment, the amylases of the present invention are used in detergent formulations or cleaning formulations.
“Detergent formulation” or “cleaning formulation” means compositions designated for cleaning soiled material. Cleaning includes laundering and hard surface cleaning. Soiled material according to the invention includes textiles and/or hard surfaces.
The term“laundering” relates to both household laundering and industrial laundering and means the process of treating textiles with a solution containing a detergent composition of the present invention. The laundering process may be carried out by using technical devices such as a household or an industrial washing machine. Alternatively, the laundering process may be done by hand.
The term“textile” means any textile material including yarns (thread made of natural or synthetic fibers used for knitting or weaving), yarn intermediates, fibers, non-woven materials, natural materials, synthetic materials, as well as fabrics (a textile made by weaving, knitting or felting fibers) made of these materials such as garments (any article of clothing made of textile), cloths and other articles.
The term“fibers” includes natural fibers, synthetic fibers, and mixtures thereof. Examples of natural fibers are of plant (such as flax, jute and cotton) or animal origin, comprising proteins like collagen, keratin and fibroin (e.g. silk, sheep wool, angora, mohair, cashmere). Examples for fibers of synthetic origin are polyurethane fibers such as Spandex® or Lycra®, polyester fibers, polyolefins such as elastofin, or polyamide fibers such as nylon. Fibers may be single fibers or parts of textiles such as knitwear, woven, or nonwovens.
The term“hard surface cleaning” is defined herein as cleaning of hard surfaces wherein hard surfaces may include any hard surfaces in the household, such as floors, furnishing, walls, sanitary ceramics, glass, metallic surfaces including cutlery or dishes.
The term“dish wash” refers to all forms of washing dishes, e.g. by hand or automatic dish wash. Dish washing includes, but is not limited to, the cleaning of all forms of crockery such as plates, cups, glasses, bowls, all forms of cutlery such as spoons, knives, forks and serving utensils as well as ceramics, plastics such as melamine, metals, china, glass and acrylics.
The detergent formulation of the invention comprises one or more detergent component(s). The component(s) chosen depend(s) on the desired cleaning application and/or physical form of a detergent composition.
The term“detergent component” is defined herein to mean any types of ingredient, which is suitable for detergent compositions, such as surfactants, building agents, polymers, bleaching systems. Any component(s) known in the art acknowledging their known characteristics are suitable detergent component(s) according to the invention. Detergent components in one embodiment means components which provide washing or cleaning performance, or which effectively aid the processing (maintain physical characteristics during processing, storage and use; e.g. rheology modifiers, hydrotropes, desiccants) when present in effective amounts.
Usually, a detergent composition is a complex formulation of more than two detergent components.
Detergent components may have more than one function in the final application of a detergent formulation, therefore any detergent component mentioned in the context of a specific function herein, may also have another function in the final application of a detergent formulation. The function of a specific detergent component in the final application of a detergent formulation usually depends on its amount within the detergent formulation, i.e. the effective amount of a detergent component.
The term“effective amount” includes amounts of certain components to provide effective stain removal and effective cleaning conditions (e.g. pH, quantity of foaming), amounts of certain components to effectively provide optical benefits (e.g. optical brightening, dye transfer inhibition), and amounts of certain components to effectively aid the processing (maintain physical characteristics during processing, storage and use; e.g. rheology modifiers, hydrotropes, desiccants).
In one embodiment, a detergent formulation is a formulation of more than two detergent components, wherein at least one component is effective in stain-removal, at least one component is effective in providing the optimal cleaning conditions, and at least one component is effective in maintaining the physical characteristics of the detergent.
Cleaning performance is evaluated under relevant cleaning conditions. The term "relevant cleaning conditions" herein refers to the conditions, particularly cleaning temperature, time, cleaning mechanics, suds concentration, type of detergent and water hardness, actually used in laundry machines, automatic dish washers or in manual cleaning processes.
Individual detergent components and usage in detergent compositions are known to those skilled in the art. Suitable detergent components comprise inter alia surfactants, builders, polymers, alkaline, bleaching systems, fluorescent whitening agents, suds suppressors and stabilizers, hydrotropes, and corrosion inhibitors. Further examples are described e.g. in“complete Technology Book on Detergents with Formulations (Detergent Cake, Dishwashing Detergents, Liquid & Paste Detergents, Enzyme Detergents, Cleaning Powder & Spray Dried Washing Powder)”, Engineers India Research Institute (EIRI), 6th edition (2015). Another reference book for those skilled in the art may be“Detergent Formulations Encyclopedia”, Solverchem Publications, 2016.
Detergent components vary in type and/or amount in a detergent formulation depending on the desired application such as laundering white textiles, colored textiles, and wool. The component(s) chosen further depend(s) on physical form of a detergent formulation (liquid, solid, gel, provided in pouches or as a tablet, etc.). The component(s) chosen e.g. for laundering formulations further depend on regional conventions which themselves are related to aspects like washing temperatures used, mechanics of laundry machine (vertical vs. horizontal axis machines), water consumption per wash cycle etc. and geographical characteristics like average hardness of water.
For example: A low detergent concentration system includes laundering formulations where less than about 800 ppm of detergent components are present in the wash water. A medium detergent concentration includes laundering formulations where between about 800 ppm and about 2,000 ppm of detergent components are present in the wash water. A high detergent concentration includes laundering formulations where more than about 2,000 ppm of detergent components are present in the wash water.
The numeric ranges recited for the individual detergent components provide amounts comprised in detergent compositions. Such ranges have to be understood to be inclusive of the numbers defining the range and include each integer within the defined range.
If not described otherwise,“% by weight” or“% w/w” is meant to be related to total detergent composition. In this case“% by weight” or“% w/w” is calculated as follows: concentration of a substance as the weight of that substance divided by the total weight of the composition, multiplied by 100.
Detergent formulations of the invention may comprise one or more surfactant(s). "Surfactant" (synonymously used herein with“surface active agent”) means an organic chemical that, when added to a liquid, changes the properties of that liquid at an interface. According to its ionic charge, a surfactant is called non-ionic, anionic, cationic, or amphoteric.
Non-limiting examples of surfactants are disclosed McCutcheon's 2016 Detergents and Emulsifiers, and McCutcheon's 2016 Functional Materials, both North American and International Edition, MC Publishing Co, 2016 edition. Further useful examples are disclosed in earlier editions of the same publications which are known to those skilled in the art.
Non-ionic surfactant means a surfactant that contains neither positively nor negatively charged (i.e. ionic) functional groups. In contrast to anionic and cationic surfactants, non-ionic surfactants do not ionize in solution.
Example 1: Amylase expression
The amylase genes were designed in silico using Geneious software. The genes were synthesized and cloned into Bacillus expression vectors by GenScript (New Jersey, USA). Constructs for each variant were received from GenScript as sequence-confirmed plasmid DNA. Each construct was transformed into Bacillus subtilis. 5 mΐ of plasmid DNA, 20-200 ng/ mΐ was added to 500 mΐ freshly prepared Bacillus subtilis competent cells and incubated at 37°C for 3.5 hours. The NEBuilder assembled amylases were transformed in similar fashion but using 5 mΐ of NEbuilder product directly. Cells were subsequently plated onto LB + 50 ug/mL Kanamycin agar plates and grown overnight at 37°C. To confirm the amylases in Bacillus subtilis, the resulting colonies were screened via colony PCR and sequencing. Prior to PCR, each colony was lysed in buffer containing 20mM DTT and 0.5 mg/mL Proteinase K at 55°C for 5 minutes followed by 95°C for 6 minutes. 20 mΐ PCR reactions using 1 mΐ of lysed cells and TaKara Ex Taq (TaKaRa Cat# RROOl) polymerase were performed as follows: initial denaturation for 3 minutes at 98°C, 30 cycles of denaturation, annealing, and extension for 10 seconds at 95°C, 30 seconds at 55°C, and 2.5 minutes at 72°C, respectively. A final extension for 5 minutes at 72°C completed the PCR reactions. Expression of the amylases was done in 96-deep well plate format, and SDS PAGE carried out to quantify the material. The amylase enzymes that expressed well were sequenced as provided in the table below and tested for wash performance.
Figure imgf000035_0001
Example 2: High throughput wash performance of amylase enzymes
Amylase samples were tested in the 24- well cloth assay for wash performance in ESI/M with pH adjusted to pH 8 and pH 10. All enzymes including benchmarks were dosed at a final concentration of 0.1, 0.2 and 0.4ppm. Amylase benchmarks Stainzyme Plus 12L (pH 10) or Amplify Prime (pH 8) were used as controls. 5g/L surfactant blend was used containing 150ppm CaCb and 2% citrate. Assays were conducted at 40°C on CFT-CS28 (cotton soiled with rice starch), and the activity is measured as a clearing or brightness from the original stained fabric color using a scanner and Digimizer (MedCalc Software) to measure intensity (RGB). Host control (V.cont) was diluted in the same manner as enzymes.
The results, as provided in the tables below, show the amylase variants exhibited cloth performance (removed more statins and became cleaner) at pH 10.0. Moreover, the amylase variants of the invention consistently outperformed all other amylase variants and the benchmarks at both pH8 and pH 10. Amylase_0190 and Amylase_0191, were the best performing variants at both pH values.
Figure imgf000035_0002
Figure imgf000035_0003
Figure imgf000036_0001
Example 3: Full scale washing performance
The amylase enzymes were tested at full scale washing under the following conditions: wash Program Cold Wash (Cotton 20 °C) and warm wash (40 °C); Water hardness 2.5 mmol/L Ca/Mg/HC03; 4: 1 :8; Total washing time 2:00 hours; Amount of water 15 L (Main wash); Fabric/liquor ratio 1 : 4; Detergent dosage ES1M 20%, 3% Citrate, 150 ppm CaC12 (4 g/L); Persil Non Bio (Unilever, UK); Enzyme dosage 0.005 to 0.1 ppm active; Soiled fabric 2 x BASF09 (KC- H004, KC-H057, KC-H102, KC-H118, KC-H097, KC-H009, KC-H040, KC-H033, KC-H041, KC- H179, KC-H056, KC-H059, KC-H076, and KC-H119); and 2 x BASF10 (C-05, C-l l, C-S-25, C-S- 80, C-S-127, C-S-128, C-S-129, C-S-06, C-S-38, W-20 D, E-125, E-160, E-161, E-163, C-S-26, C- S-27, C-S-28, C-S-29, C-S-126, and E-117); Ballast 3,5 kg towels (new ballast soil!) 2 x wfk SBL 2004; Repetition 1 = 2; pH 8.5.
The results, as provided in the tables below, show Amylase_0190, performed better (removed more statins and became cleaner) on all stains for BASF09 and BASF 10, when compared to the other amylases in an ESI and Persil non-bio detergent formulation at 20 degrees C and 40 degrees C.
Figure imgf000036_0002
Figure imgf000037_0001
Figure imgf000037_0002
Figure imgf000037_0003
Figure imgf000038_0001
Figure imgf000038_0002
Figure imgf000039_0001
Figure imgf000039_0002
Figure imgf000039_0003
Figure imgf000040_0001

Claims

CLAIMS: The invention claimed is:
1. A cleaning composition comprising a polypeptide having amylase activity, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.
2. The cleaning composition of claim 1, wherein the polypeptide is a fragment of the full-length amino acid sequence and the fragment has amylase activity.
3. The cleaning composition as in any one of claims 1 or 2, wherein the pH of the cleaning composition is from pH7 to pH 10.
4. The cleaning composition as in any one of claims 1-3, wherein the amylase is 0.005 to 0.50 ppm of the cleaning composition.
5. The cleaning composition as in any one of claims 1-4, wherein the cleaning composition is for cleaning at a temperature from 20 degrees C to 40 degrees C.
6. The cleaning composition as in any one of claims 1-5, wherein the brightness is from 0.60 to 0.80 RGB.
7. The cleaning composition as in any one of claims 1-6, wherein the cleaning composition further comprising a second enzyme selected from the group consisting of: a second amylase, a lipase, a protease, a cellulase, a mannanase, a laccase, a pectinase, a nuclease, and any combination thereof.
8. The cleaning composition as in any one of claims 1-7, wherein the cleaning composition is a liquid, a granule, a gel, or any combination thereof.
9. The cleaning composition as in any one of claims 1-8, wherein the cleaning composition is a laundry detergent.
10. A polynucleotide comprising a nucleic acid sequence that encodes the polypeptide of claim 1.
11. A method of making the polypeptide as in any of claims 1-10, comprising: providing a nucleic acid sequence that encodes the polypeptide having amylase activity, transforming the nucleic acid sequence into an expression host, cultivating the expression host to produce the polypeptide, and purifying the polypeptide.
12. The method of claim 11, wherein the expression host is selected from the group consisting of: a bacterial expression system, a yeast expression system, a fungal expression system, and a synthetic expression system.
13. The method of claim 12, wherein the bacterial expression system is selected from an E. coli, a Bacillus, a Pseudomonas, and a Streptomyces, wherein the yeast expression system is selected from a Candida, a Pichia, a Saccharomyces, a Schizosaccharomyces or, wherein the fungal expression system is selected from a Penicillium, an Aspergillus, a Fusarium, a Myceliopthora, a Themothelomyces, a Rhizomucor, a Rhizopus, a Thermomyces, and a Trichoderma.
14. The method of claim 11, wherein the polypeptide having amylase activity is a variant polypeptide of the amino acid sequence selected from group consisting of: SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8 and the variant polypeptide has amylase activity.
15. The method of claim 14, wherein the variant polypeptide having amylase activity is at least 80% identical to the amino acid sequence selected from group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.
16. An amylase that is selected from an amino acid sequence of: SEQ ID NO: l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, and SEQ ID NO:8.
17. A method of using the cleaning composition as in any of claims 1 -9, wherein the composition removes stains from a surface.
18. The method of claim 17, wherein the surface is a textile.
19. The method of claim 18, wherein the textile is selected from a yarn, fiber, fabric, garment, cloth, or any combination thereof.
20. The method of claim 19, wherein the fiber is a natural fiber, synthetic fiber, or a combination thereof.
21. The method of claim 20, wherein the surface is a hard surface.
22. The method of claim 21, wherein the hard surface is a floor, furnishing, wall, sanitary ceramics, glass, metallic surface, or any combination thereof.
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