WO2010117511A1 - Halomonas strain wdg195-related alpha-amylases, and methods of use, thereof - Google Patents
Halomonas strain wdg195-related alpha-amylases, and methods of use, thereof Download PDFInfo
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- WO2010117511A1 WO2010117511A1 PCT/US2010/025368 US2010025368W WO2010117511A1 WO 2010117511 A1 WO2010117511 A1 WO 2010117511A1 US 2010025368 W US2010025368 W US 2010025368W WO 2010117511 A1 WO2010117511 A1 WO 2010117511A1
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- 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
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- 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
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2408—Glucanases acting on alpha -1,4-glucosidic bonds
- C12N9/2411—Amylases
- C12N9/2414—Alpha-amylase (3.2.1.1.)
- C12N9/2417—Alpha-amylase (3.2.1.1.) from microbiological source
Definitions
- compositions and methods relating to ⁇ -amylase enzymes obtained from Halomonas strain WDG195, and to structurally related amylases are disclosed.
- Starch consists of a mixture of amylose (15-30% w/w) and amylopectin (70-85% w/w).
- Amylose consists of linear chains of ⁇ -l,4-linked glucose units having a molecular weight (MW) from about 60,000 to about 800,000.
- MW molecular weight
- Amylopectin is a branched polymer containing ⁇ -1,6 branch points every 24-30 glucose units; its MW may be as high as 100 million.
- Sugars from starch in the form of concentrated dextrose syrups, are currently produced by an enzyme catalyzed process involving: (1) liquefaction (or viscosity reduction) of solid starch with an ⁇ -amylase into dextrins having an average degree of polymerization of about 7- 10, and (2) saccharification of the resulting liquefied starch (i.e. starch hydrolysate) with amyloglucosidase (also called glucoamylase or GA).
- amyloglucosidase also called glucoamylase or GA
- the resulting syrup has a high glucose content.
- Much of the glucose syrup that is commercially produced is subsequently enzymatically isomerized to a dextrose/fructose mixture known as isosyrup.
- ⁇ -amylases (EC 3.2.1.1) hydrolyze starch, glycogen, and related polysaccharides by cleaving internal ⁇ -l,4-glucosidic bonds at random.
- ⁇ -amylase enzymes have been used for a variety of different purposes, including starch liquefaction, textile desizing, starch modification in the paper and pulp industry, and for brewing. These enzymes can also be used to remove starchy stains during dishwashing and laundry washing. Laundry and dish soils vary greatly in composition and therefore also in their ability to be removed and few amylases in the market place can be used for both laundry and dish applications.
- Halomonas species are Gram-negative, moderately halophilic bacteria that grow optimally in media containing 3-15% NaCl, although most will grow over a very wide range of salinities. Some species are alkaliphilic.
- the only described amylase from Halomonas sp. is from Halomonas meridiana DSM5425 (Coronado, MJ. et al. (2000) FEMS Microbiology Letters 183:67-71). This ⁇ -amylase (i.e., amyH) has been cloned and a deduced amino acid sequence (AmyH) predicted (Coronado, MJ. et al, (2000) Microbiology 146:861-868).
- amyH The nucleotide sequence for the amyH gene (AJ239061) and the predicted amino acid sequence for the AmyH polypeptide (CAB92963) are available in GenBank.
- the amino acid sequence of AmyH is also set forth in SEQ ID NO: 1.
- AmyH is predicted to be a polypeptide of 457 amino acids, including a signal sequence of 20 amino acids and a mature polypeptide sequence of 437 amino acids.
- AmyH appears to belong to be a family 13 glycosyl hydrolases, it has low sequence identity with its closest homologues, as shown in the following Table:
- compositions and methods relate to an ⁇ -amylase from Halomonas strain WDG195 and related ⁇ -amylases, which represent a unique family of amylases useful for industrial applications.
- the amylases are referred to collectively as AmyWDG195-related polypeptides or AmyWDG195-related amylases.
- an isolated polypeptide having at least 80% amino acid sequence identity to AmyWDG195 and including at least one of the following amino acid residues at the indicated amino acid position relative to AmyWDG195, as determined by aligning the amino acid sequence of the polypeptide with the amino acid sequence of AmyWDG195: E at position 3, E at position 20, M at position 25, N at position 26, L at position 40, D at position 68, E at position 82, Q at position 91, G at position 94, L at position 177, H at position 179, G at position 181, N at position 224, L at position 267, D at position 278, D at position 280, V at position 281, E at position 294, R at position 297, T at position 298, L at position 307, D at position 340, V at position 350, S at position 360, L at position 381, E at position 386, D at position 390, G at position 418, M at position 449, H at position 453, D at position 470, and Q at position 479.
- the polypeptide includes at least one of the following amino acid residues at the indicated amino acid position relative to AmyWDG195: E at position 3, E at position 20, M at position 25, N at position 26, D at position 68, E at position 82, Q at position 91, G at position 94, L at position 177, H at position 179, G at position 181, N at position 224, D at position 278, D at position 280, V at position 281, E at position 294, R at position 297, T at position 298, D at position 340, V at position 350, S at position 360, E at position 386, D at position 390, G at position 418, M at position 449, H at position 453, D at position 470, and Q at position 479.
- the AmyWDG195-related polypeptide may have any subset of these amino acid residues at the indicated positions, or all of these amino acid residues at the indicated positions.
- the polypeptide includes at least one of the following amino acid residues at the indicated amino acid position relative to AmyWDG195: M at position 25, N at position 26, L at position 177, H at position 179, E at position 294, T at position 298, and M at position 449.
- the polypeptide includes at least one of the following combinations of amino acid residues at the indicated amino acid position relative to
- AmyWDG195 M at position 25 and N at position 26, optionally with E at position 20; Q at position 91 and G at position 94; L at position 177, H at position 179, and G at position 181; D at position 280 and V at position 281, optionally with D at position 278; R at position 297 and T at position 298, optionally with E at position 294; E at position 386 and D at position 390, optionally with L at position 381; and M at position 449 and H at position 453.
- the polypeptide includes at least 30 glutamate residues.
- the polypeptide includes at least one of the following amino acid residues at the indicated amino acid position relative to AmyWDG195: N at position 104, D at position 194, N at position 200, and H at position 235.
- the polypeptide includes at least one of the following amino acid residues at the indicated amino acid position relative to AmyWDG195: Y at position 302, G at position 300, N at position 427, D at position 404, and W at position 403
- the polypeptide includes at least one of the following amino acid residues at the indicated amino acid position relative to AmyWDG195: N at position 289, V at position 324, D at position 325, and S at position 337.
- the polypeptide includes the following amino acid residue at the indicated amino acid position relative to AmyWDG195: L at position 197.
- AmyWDG195 has the amino acid sequence of SEQ ID NO: 12.
- the polypeptide has at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the polypeptide has at least 95% amino acid sequence identity to the amino acid sequence of SEQ ID NO: 12. In particular embodiments, the polypeptide has the amino acid sequence of SEQ ID NO: 12 (AmyWDG195).
- the polypeptide has the amino acid sequence of SEQ ID NO: 23
- the polypeptide has ⁇ - amylase activity. In some embodiments, the activity is independent of calcium.
- a cultured cell material comprising one or more of any of the aforementioned polypeptides is provided.
- composition comprising one or more of any of the aforementioned polypeptides.
- the composition is a cleaning composition.
- the composition is effective for removing starchy stains from laundry, dishes, or textiles.
- a method for removing a starchy stain from a surface comprising incubating the surface in the presence of a aqueous composition comprising an effective amount of a polypeptide of claim 1, allowing the polypeptides to hydrolyse starch components present in the starchy stain to produce smaller starch-derived molecules that dissolve in the aqueous composition, thereby removing the starchy stain from the surface.
- the incubating is in the absence of calcium. In some embodiments, the incubating is in the presence of a calcium chelating agent. In some embodiments, the surface is a textile surface.
- a method for expressing a polypeptide having amylase activity comprising: constructing an expression vector comprising a polynucleotide encoding a signal sequence linked to a polynucleotide encoding a polypeptide of claim 1 ; introducing the expression vector into a host cell, expressing the polypeptides in the presence of at least about 1% NaCl, recovering the polypeptide expressed.
- expressing the polypeptide is performed in the presence of at least about 2% NaCl. In some embodiments, expressing the polypeptide is performed in the presence of at least about 3% NaCl. [0027] In some embodiments, the amount of polypeptide expressed in the presence of 1% NaCl is at least 5 -fold the amount expressed in minimal media without added NaCl. In some embodiments, the amount of polypeptide expressed in the presence of 2% NaCl is at least 20- fold the amount expressed in minimal media without added NaCl. In some embodiments, the amount of polypeptide expressed in the presence of 1% NaCl is at least 20-fold the amount expressed in minimal media without added NaCl.
- a vector comprising a polynucleotide sequence encoding an AmyWDG 195 -related amylase.
- the AmyWDG195-related amylase coding sequence may be operably linked to a promoter or to other control elements.
- a host cell comprising a polynucleotide encoding an AmyWDG195-related amylase, or vector comprising such a polynucleotide, is provided.
- the isolated host cell can be a prokaryote or eukaryote.
- the isolated host cell can be a bacterium (e.g., B. subtilis, B. licheniformis, B. lentus, B.
- Geobacillus (formerly Bacillus) stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. coagulans, B. circulans, B. lautus, B. thuringiensis, Streptomyces lividans, S. murinus, or Escherichia col ⁇ ).
- Another aspect contemplates a detergent additive comprising an AmyWDG 195 -related amylase, wherein the detergent additive is optionally in the form of a non-dusting granulate, microgranulate, stabilized liquid, gel, or protected enzyme.
- the polypeptide in the detergent additive can be a truncated polypeptide as described above.
- the detergent additive can contain about 0.02 mg to about 200 mg of polypeptide per gram of the detergent additive.
- the detergent additive can further comprise an enzyme selected from the group consisting of a protease, a lipase, a peroxidase, an oxidase, an amylolytic enzyme, a cellulase, a polyesterase, and any combination thereof.
- a detergent composition comprising any of the described detergent additives.
- a detergent composition can optionally comprise one or more of: a surfactant, a bleaching system or bleach, a detergent builder, a polymer, a stabilizing agent, a fabric conditioner, a foam booster, a suds suppressor, an anti-corrosion agent, a dye, a perfume, a soil suspending agent, a tarnish inhibitor, an optical brightener, or a bacteriocide.
- a detergent composition can comprise or further comprise an additional enzyme, wherein the enzyme is a protease, a lipase, a peroxidase, an oxidase, an amylolytic enzyme, a cellulase, a polyesterase, or any combination thereof.
- the enzyme is a protease, a lipase, a peroxidase, an oxidase, an amylolytic enzyme, a cellulase, a polyesterase, or any combination thereof.
- Another aspect contemplates a manual or automatic dishwashing detergent composition comprising an AmyWDG 195 -related amylase.
- Yet a further aspect contemplates a method of washing dishes comprising applying a manual or automatic dishwashing detergent described herein to a dish or dishes in need thereof. The method of washing the dishes contemplates adding the dishwashing detergent in an amount such that the wash liquor contains a polypeptide described herein in the amount of about 0.01 ppm to about 4 ppm.
- Another aspect contemplates a laundry detergent composition comprising a detergent additive described herein. Yet a further aspect contemplates a method of cleaning a textile comprising washing a soiled textile in solution with a detergent composition described herein. The method further contemplates having the polypeptide described herein in an amount in the solution of about 0.01 to about 2 ppm in the solution.
- Figure 1 shows a diagram of the vector pRANGER-BTB3-Cat.
- Figure 2 shows the results of a BLAST search showing that AmyWDG195 belongs to the ⁇ -amylase superfamily.
- Figure 3 shows a diagram of the expression vector pHPLT.
- Figures 4 and 5 show the results of assays to evaluate cleaning performance.
- Figure 6 is an image of a Coomassie-stained gel showing expression of AmySWT282.
- Figure 7 shows and alignment of AmyWDG195, AmySWT282, and Amy K38, emphasizing residues conserved between AmyWDG195 and AmySWT282.
- Figure 8 shows and alignment of AmyWDG195, AmySWT282, and Amy K38, showing conserved residues and a consensus sequence.
- Figure 9 lists the polypeptide and nucleotide sequences referred to in the description.
- SIiQ ID NO: i is the amino acid sequence of the AmyH precursor (i.e., immature or full- length) polypeptide from ⁇ alomcmas merhli ⁇ ia.
- the signal sequence is the firs* about 20 residues.
- SEQ ID NO: 2 is a partial nucleotide sequence of the 16S rRNA gene of Halomonas strain WDG 195
- SEQ ID NOs: 3-8 are the nucleotide sequences of primers described in the Examples.
- SEQ ID NO: 9 is the nucleotide sequence of the amyWDG195 gene.
- SEQ ID NO: 10 is the amino acid sequence of the AmyWDG195 precursor (i.e., immature or full-length) polypeptide.
- the signal sequence is the first about 22 residues, with the remaining residue representing the mature polypeptide.
- SEQ ID NO: 11 is the amino acid sequence of the AmyWDG195 signal peptide.
- SEQ ID NO: 12 is the amino acid sequence of the AmyWDG195 mature polypeptide.
- SEQ ID Nos: 13-17 are the nucleotide sequences of primers described in the Examples.
- SEQ ID NO: 18 is the partial nucleotide sequence of the AmySWT282 gene.
- SEQ ID NO: 19 is the nucleotide sequence encoding the AmySWT282 precursor (i.e., immature or full-length) polypeptide.
- SEQ ID NO: 20 is the nucleotide sequence encoding the AmySWT282 mature polypeptide.
- SEQ ID NO: 21 is nucleotide sequence encoding the AmySWT282 signal peptide.
- SEQ ID NO: 22 is the amino acid sequence of the AmySWT282 precursor (i.e., immature or full-length) polypeptide.
- the signal sequence is the first about 22 residues, with the remaining residue representing the mature polypeptide.
- SEQ ID NO: 23 is the amino acid sequence of the AmySWT282 mature polypeptide.
- SEQ ID NO: 24 is the amino acid sequence of the AmySWT282 signal peptide.
- SEQ ID Nos: 25-33 are the nucleotide sequences of primers described in the Examples.
- SEQ ID NO: 34 is the amino acid sequence of the AmyK38 precursor (i.e., immature or full-length) polypeptide (gil6874476).
- the signal sequence is the first about 21 residues, with the remaining residue representing the mature polypeptide
- SEQ ID NO: 35 is the amino acid sequence of the AmyK38 mature polypeptide.
- SEQ ID Nos: 36-41 are the nucleotide sequences of primers described in the Examples.
- compositions and methods relating to an ⁇ -amylase isolated from Halomonas strain WDG195 herein called "AmyWDG195" (SEQ ID NO: 12). While this amylase belongs to the ⁇ -amylase super-family, it is unrelated to AmyH (SEQ ID NO: 1), the amylase previously isolated from Halomonas meridiana strain DSM5425, which is assigned to the family 13 glycosyl hydrolases. In particular, AmyWDG195 and AmyH share less than 11% sequence identity. [0063] AmyWDG195 shares significant sequence identity with another heretofore unknown amylase from Bacillus sp.
- AmySWT282 (SEQ ID NO: 23)
- AmyK38 a known amylase from Bacillus sp. strain K38
- AmyWDG195 shares about 78.1% sequence identity with AmySWT282 and 81.5% sequence identity with AmyK38.
- AmySWT282 and AmyK38 share 77.9% sequence identity; therefore, these three amylases are approximately equidistant in evolutionary distance.
- AmyWDG195 and AmySWT282 include key residues that are associated with binding to three sodium ions in AmyK38 (Hagihari, H. et al. (2001) Eur. J. Biochem. 268:3974-82; Nonaka, T. et al. (2003) /. Biol. Chem. 278:24818-25).
- N at position 104, D at position 194, N at position 200, and H at position 235 which is associated with the binding of AmyK38 to a first sodium
- N at position 289, V at position 324, D at position 325, and S at position 337 which is associated with the binding of AmyK38 to a third sodium, is conserved in both AmyWDG195 and AmySWT282.
- AmyWDG195 and AmySWT282 are expected to share certain halophilic properties with AmyK38, which is confirmed by experiments described herein.
- AmyWDG195, AmySWT282, and AmyK38 all share an L at position 197, unlike many other Bacillus amylases which have an M at this position. This makes these amylases less susceptible to chemical oxidation than other Bacillus amylases.
- AmyWDG195 and AmySWT282 can readily be distinguished from AmyK38 based on amino acid residues present at each of 32 different positions, at which AmyWDG195 and AmySWT282 share the same amino acid residue, which is different from that present in AmyK38.
- AmyWDG195 and AmySWT282 also contain a significantly larger number of glutamate residues and have lower predicted isoelectric points (pi) than AmyK38.
- AmyWDG195 and AmySWT282 represent related, heretofore unknown, calcium- independent amylases, which are resistant to chelating reagents and chemical oxidants, making them very attractive for cleaning applications.
- AmyWDG195, AmySWT282, and related polypeptides are referred to collectively as AmyWDG195-related polypeptides or AmyWDG 195 -related amylases.
- AmyWDG195 and AmyK38 amino acid position numbering of AmyWDG195 and AmyK38 is used with reference to these polypeptides (see, e.g., Figures 7 and 8).
- AmySWT28 is one amino acid shorter at the N-terminus than AmyWDG195 and AmyK38, thereof the actual amino acid positions of AmySWT28 are one less (i.e., -1) with respect to AmyWDG 195 and AmyK38.
- IPTG isopropyl ⁇ -D-thiogalactoside kDa kiloDalton
- amlase or "amylolytic enzyme” refer to an enzyme that is, among other things, capable of catalyzing the degradation of starch.
- Amylases are hydrolases that cleave the ⁇ -D-(l ⁇ 4) O-glycosidic linkages in starch.
- ⁇ -amylases (EC 3.2.1.1; ⁇ -D-(l ⁇ 4)- glucan glucanohydrolase) are defined as endo-acting enzymes cleaving ⁇ -D-(l ⁇ 4) O-glycosidic linkages within the starch molecule in a random fashion.
- exo-acting amylolytic enzymes such as ⁇ -amylases (EC 3.2.1.2; ⁇ -D-(l— >4)-glucan maltohydrolase) and some product-specific amylases like maltogenic ⁇ -amylase (EC 3.2.1.133) cleave the starch molecule from the non-reducing end of the substrate.
- starch refers to any material comprised of the complex polysaccharide carbohydrates of plants, comprised of amylose and amylopectin with the formula (C 6 Hio ⁇ 5 ) x , wherein X can be any number.
- the term includes plant-based materials such as grains, grasses, tubers and roots, and more specifically materials obtained from wheat, barley, corn, rye, rice, sorghum, brans, cassava, millet, potato, sweet potato, and tapioca.
- wild-type “parental,” or “reference,” with respect to a polypeptide, refer to a naturally-occurring polypeptide that does not include a man-made substitution, insertion, or deletion at one or more amino acid positions.
- wild-type refers to a naturally-occurring polynucleotide that does not include a man-made nucleoside change.
- a polynucleotide encoding a wild-type, parental, or reference polypeptide is not limited to a naturally-occurring polynucleotide, and encompasses any polynucleotide encoding the wild-type, parental, or reference polypeptide.
- variant refers to a polypeptide that differs from a specified wild-type, parental, or reference polypeptide in that it includes a man-made substitution, insertion, or deletion at one or more amino acid positions.
- a polynucleotide refers to a polynucleotide that differs in nucleotide sequence from a specified wild-type, parental, or reference polynucleotide. The identity of the wild-type, parental, or reference polypeptide or polynucleotide will be apparent from context.
- recombinant when used in reference to a subject cell, nucleic acid, protein or vector, indicates that the subject has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified.
- recombinant cells express genes that are not found within the native (non-recombinant) form of the cell, or express native genes at different levels or under different conditions than found in nature.
- polypeptides cell, nucleic acid, amino acid, or other specified material or component that is removed from at least one other material or component with which it is naturally associated as found in nature.
- purified refers to material (e.g., an isolated polypeptide or polynucleotide) that is in a relatively pure state, e.g., at least about 90% pure, at least about 95% pure, at least about 98% pure, or even at least about 99% pure.
- thermostability refers to the ability of the enzyme to retain activity after exposure to an elevated temperature.
- the thermostability of an enzyme such as an amylase enzyme, is measured by its half- life (ty 2 ) given in minutes, hours, or days, during which half the enzyme activity is lost under defined conditions.
- the half-life may be calculated by measuring residual ⁇ -amylase activity following exposure to (i.e., challenge by) an elevated temperature.
- a "pH range,” with reference to an enzyme, refers to the range of pH values under which the enzyme exhibits catalytic activity.
- pH stable and “pH stability,” with reference to an enzyme, relate to the ability of the enzyme to retain activity over a wide range of pH values for a predetermined period of time (e.g., 15 min., 30 min., 1 hour).
- amino acid sequence is synonymous with the terms
- polypeptide polypeptide
- protein protein
- peptide polypeptide
- amino acid sequences exhibit activity, they may be referred to as an "enzyme.”
- the conventional one-letter or three- letter codes for amino acid residues are used, with amino acid sequences being presented in the standard amino-to-carboxy terminal orientation (i.e., N ⁇ C).
- nucleic acid encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. Nucleic acids may be single stranded or double stranded, and may be chemical modifications. The terms “nucleic acid” and “polynucleotide” are used interchangeably. Because the genetic code is degenerate, more than one codon may be used to encode a particular amino acid, and the present compositions and methods encompass nucleotide sequences which encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in a 5'-to-3' orientation.
- homologue shall mean an entity having a specified degree of identity with the subject amino acid sequences and the subject nucleotide sequences.
- a homologous sequence is taken to include an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%,
- homologues will include the same active site residues as the subject amino acid sequence, unless otherwise specified.
- hybridization refers to the process by which one strand of nucleic acid base pairs with a complementary strand, as occurs during blot hybridization techniques and PCR techniques. Stringent hybridization conditions are exemplified by the following: 50 0 C and 0.2X
- a "synthetic" molecule is produced by in vitro chemical or enzymatic synthesis rather than by an organism.
- transformed As used herein, the terms “transformed,” “stably transformed,” and “transgenic,” used with reference to a cell means that the cell contains a non-native (e.g., heterologous) nucleic acid sequence integrated into its genome or carried as an episome that is maintained through multiple generations.
- a non-native nucleic acid sequence integrated into its genome or carried as an episome that is maintained through multiple generations.
- a "host strain” or “host cell” is an organism into which an expression vector, phage, virus, or other DNA construct, including a polynucleotide encoding a polypeptide of interest
- host strains are bacterial cells.
- host cell includes protoplasts created from cells, such as those of a
- heterologous with reference to a polynucleotide or protein refers to a polynucleotide or protein that does not naturally occur in a host cell.
- endogenous with reference to a polynucleotide or protein refers to a polynucleotide or protein that occurs naturally in the host cell.
- expression refers to the process by which a polypeptide is produced based on a nucleic acid sequence. The process includes both transcription and translation.
- a "selective marker” or “selectable marker” refers to a gene capable of being expressed in a host to facilitate selection of host cells carrying the gene.
- selectable markers include but are not limited to antimicrobials (e.g., hygromycin, bleomycin, or chloramphenicol) and/or genes that confer a metabolic advantage, such as a nutritional advantage on the host cell.
- a "vector” refers to a polynucleotide sequence designed to introduce nucleic acids into one or more cell types.
- Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes and the like.
- An "expression vector” refers to a DNA construct comprising a DNA sequence encoding a polypeptide of interest, which coding sequence is operably linked to a suitable control sequence capable of effecting expression of the DNA in a suitable host.
- control sequences may include a promoter to effect transcription, an optional operator sequence to control transcription, a sequence encoding suitable ribosome binding sites on the mRNA, enhancers and sequences which control termination of transcription and translation.
- operably linked means that specified components are in a relationship
- a regulatory sequence is operably linked to a coding sequence such that expression of the coding sequence is under control of the regulatory sequences.
- a "signal sequence” is a sequence of amino acids attached to the N-terminal portion of a protein, which facilitates the secretion of the protein outside the cell.
- the mature form of an extracellular protein lacks the signal sequence, which is cleaved off during the secretion process.
- biologically active refers to a sequence having a specified biological activity, such an enzymatic activity.
- Water hardness is a measure of the minerals (e.g. , calcium and magnesium) present in water.
- a cultured cell material comprising an AmyWDG195-related amylase refers to a cell lysate or supernatant that includes an AmyWDG195-related amylase as a component.
- the cell material is preferably from a heterologous host that is grown in culture for the purpose of producing the AmyWDG195-related amylase.
- Amy WDG 195 -related amylase Nucleic Acids and Polypeptides One aspect of the present compositions and methods is an AmyWDG 195 -related polypeptide.
- the polypeptide may correspond to AmyWDG195, AmySWT282, an amylase having a specified degree of identity to AmyWDG195 or AmySWT282, variants of AmyWDG195 or AmySWT282 that include man-made substitutions, insertions, or deletions, or chimeras, thereof.
- Exemplary AmyWDG195 and AmySWT282 polypeptides have the amino acid sequences of SEQ ID NO: 12 and SEQ ID NO: 23, respectively.
- Additional polypeptides have at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even 99% homology/identity to an AmyWDG195 or AmySWT282 polypeptide, such as the polypeptides of SEQ ID NO: 12 and SEQ ID NO: 23.
- the present compositions and methods exclude an Amy K38 polypeptide, such as a polypeptide having the amino acid sequence of SEQ ID NO: 35.
- AmyWDG195 and AmySWT282 can be distinguished from AmyK38 based on the amino acid residue that is present at each of 32 different positions, at which AmyWDG195 and AmySWT282 share the same amino acid residue, which is different from that present in AmyK38. Positions occupied by the same amino acid residues in AmyWDG 195 and AmySWT282 (using AmyWDG 195 numbering) but different in Amy K38 are summarized in the following Table:
- the AmyWDG 195 -related polypeptide has one of more of the following amino acid residues at the indicated positions,: E at position 3, E at position 20, M at position 25, N at position 26, L at position 40, D at position 68, E at position 82, Q at position 91, G at position 94, L at position 177, H at position 179, G at position 181, N at position 224, L at position 267, D at position 278, D at position 280, V at position 281, E at position 294, R at position 297, T at position 298, L at position 307, D at position 340, V at position 350, S at position 360, L at position 381, E at position 386, D at position 390, G at position 418, M at position 449, H at position 453, D at position 470, and Q at position 479.
- the AmyWDG195-related polypeptide may have any subset of these amino acid residues at the indicated positions, or
- the AmyWDG195-related polypeptide has one of more of the following amino acid residues: E at position 3, E at position 20, M at position 25, N at position 26, D at position 68, E at position 82, Q at position 91, G at position 94, L at position 177, H at position 179, G at position 181, N at position 224, D at position 278, D at position 280, V at position 281, E at position 294, R at position 297, T at position 298, D at position 340, V at position 350, S at position 360, E at position 386, D at position 390, G at position 418, M at position 449, H at position 453, D at position 470, and Q at position 479.
- the AmyWDG 195 -related polypeptide may have any subset of these amino acid residues at the indicated positions, or all of these amino acid residues at the indicated positions.
- the AmyWDG195-related polypeptide has one of more of the following amino acid residues: M at position 25, N at position 26, L at position 177, H at position 179, E at position 294, T at position 298, and M at position 449.
- the AmyWDG 195 -related polypeptide may have any subset of these amino acid residues at the indicated positions, or all of these amino acid residues at the indicated positions.
- the AmyWDG 195 -related polypeptide has one of more of the following combinations of amino acid residues at the indicated positions: M at position 25 and N at position 26, optionally with E at position 20; Q at position 91 and G at position 94; L at position 177, H at position 179, and G at position 181; D at position 280 and V at position 281, optionally with D at position 278; R at position 297 and T at position 298, optionally with E at position 294; E at position 386 and D at position 390, optionally with L at position 381; and M at position 449 and H at position 453.
- the AmyWDG195-related polypeptide may have any subset of these amino acid residue combination, or all of these amino acid residue combinations.
- the AmyWDG195-related polypeptide retains N at position 104, D at position 194, N at position 200, and/or H at position 235, which is believed to be associated with binding to a first sodium, Y at position 302, G at position 300, N at position 427, D at position 404, and/or W at position 403, which is believed to be associated with binding to a second sodium, and/or N at position 289, V at position 324, D at position 325, and S at position 337, which is believed to be associated with binding to a third sodium.
- the AmyWDG 195 -related polypeptide retains L at position 197, believed to associated with reduced susceptibility to chemical oxidation.
- AmyWDG195 and AmySWT282 contain a significantly larger number of glutamate residues and have lower predicted isoelectric points (Pi) than AmyK38. These differences are summarized in the following Table:
- the AmyWDG 195 -related polypeptide has at least 25, at least 26, at least 27, at least 28, at least 29, at least 30, at least 31, at least 32, at least 33, at least 34, at least 35, or even at least 36 glutamate residues. In some embodiments, the AmyWDG195- related polypeptide has a pi of less than 4.7, less than 4.6, or even less than 4.5.
- AmyWDG 195 -related polypeptide is a variant having a specified degree of homology to AmyWDG195 or AmySWT282
- one or more of the specified amino acid positions (or combinations, thereof) may be fixed, such that they do not change (i.e., are conserved or retained) in the variant, while the remaining residues are subject to variation. Alternatively or additionally, the number of glutamate residues in the polypeptide is maintained.
- the polypeptides may be a "full-length" AmyWDG195-related amylase, which includes a signal sequence, or the mature form of an AmyWDG195-related amylase, which lacks the signal sequence.
- Exemplary immature forms of the AmyWDG195 and AmySWT282 polypeptides have the amino acid sequences of SEQ ID NO: 10 and SEQ ID NO: 22, respectively, while exemplary mature forms of the AmyWDG195 and AmySWT282 polypeptides have the amino acid sequences of SEQ ID NO: 12 and SEQ ID NO: 23, respectively. Mature forms of the polypeptides are most useful for use in cleaning compositions.
- the polypeptides may also be a truncated form of an AmyWDG195-related amylase, which lacks the N or C-terminus of the mature form, or fragments of an AmyWDG195-related amylase that retain at least a portion of the ⁇ -amylase activity characteristic of the parental Amy WDG 195 -related amylase.
- polypeptides include variant polypeptides, such as those that include man-made substitutions.
- exemplary substitutions are conservative amino acid substitutions, such as those listed in the following table.
- Preferred polypeptides retain ⁇ -amylase activity but may have altered biochemical properties with reference to a naturally-occurring parental polypeptide.
- the parental polypeptide is that of SEQ ID NOs: 12 or 23.
- the polypeptide may also be a chimeric polypeptide that includes at least a portion of an AmyWDG195-related amylase as described, and at least a portion of a second polypeptide.
- the second polypeptide may be, for example, a second amylase, a heterologous signal sequence, an epitope to allow tracking or purification, or the like.
- Exemplary heterologous signal sequences are from B. subtilis amylase (AmyE) or AprE, and Streptomyces CeIA.
- Another aspect of the present compositions and methods is a nucleic acid encoding an AmyWDG 195 -related polypeptide.
- the nucleic acid may encode AmyWDG195, AmySWT282, an amylase having a specified degree of identity to AmyWDG195 or AmySWT282, variants of AmyWDG195 or AmySWT282 that include man-made substitutions, insertions, or deletions, or chimeras, thereof.
- the nucleic acid encodes an amylase having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even 99% homology/identity to an AmyWDG195 or AmySWT282 polypeptide, such as the polypeptides of SEQ ID NO: 12 and SEQ ID NO: 20, respectively. Due to the degeneracy of the genetic code, it is understood that a plurality of nucleic acids may encode the same polypeptide.
- the nucleic acid may also have a specified degree of homology to an exemplary polynucleotide encoding an AmyWDG195-related amylase, such as SEQ ID NO: 9, which encodes AmyWDG195 or SEQ ID NO: 20, which encodes AmySWT282.
- the nucleic acid has at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or even 99% identity to one or both of these exemplary sequences.
- the nucleic acid hybridizes under stringent or very stringent conditions to one of these exemplary sequences.
- Nucleic acids may encode a "full-length" or "fl" AmyWDG195-related amylase, which includes a signal sequence, only the mature form of an AmyWDG195-related amylase, which lacks the signal sequence, a truncated form of Amy WDG 195 -related amylase, which lacks the N or C-terminus of the mature form, or fragments, thereof that retain at least a portion of the ⁇ -amylase activity characteristic of the AmyWDG195-related amylase.
- a nucleic acid that encodes anAmyWDG195-related amylase can be operably linked to various promoters and regulators in a vector for expression in various host cells.
- a nucleic acid that encodes anAmyWDG195-related amylase can also be linked to other coding sequences, e.g., to encode a chimeric polypeptide.
- Exemplary promoters are the B. subtilis Amy E and AprE promoters, and the Streptomyces CeIA promoters.
- AmyWDG 195 -related amylase can be expressed, in enzyme form, using an expression vector which typically includes control sequences encoding a suitable promoter, operator, ribosome binding site, translation initiation signal, and, optionally, a repressor gene or various activator genes.
- an expression vector typically includes control sequences encoding a suitable promoter, operator, ribosome binding site, translation initiation signal, and, optionally, a repressor gene or various activator genes.
- a large number of vectors are commercially available for use with recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced.
- the vector may be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid, a bacteriophage or an extrachromosomal element, mini-chromosome or an artificial chromosome.
- the vector may be one which, when introduced into an isolated host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated.
- the integrated gene may also be amplified to create multiple copies of the gene in the chromosome by use of an amplifiable construct driven by antibiotic selection or other selective pressure, such as an essential regulatory gene or by complementation through dose effect of an essential metabolic pathway gene.
- the DNA sequence should be operably linked to a suitable promoter sequence.
- the promoter may be any DNA sequence that shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell.
- Exemplary promoters for directing the transcription of the DNA sequence encoding an AmyWDG195-related amylase, especially in a bacterial host, are the promoter of the lac operon of E.
- the Streptomyces coelicolor agarase gene dagA or celA promoters the promoters of the Bacillus licheniformis ⁇ -amylase gene (amyL), the promoters of the Geobacillus (formerly Bacillus) stearothermophilus maltogenic amylase gene (amyM), the promoters of the Bacillus amyloliquefaciens ⁇ -amylase (amyQ), the promoters of the Bacillus subtilis xylA and xylB genes etc.
- useful promoters are those derived from the gene encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral ⁇ -amylase, A. niger acid stable ⁇ -amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A. oryzae triose phosphate isomerase, or A. nidulans acetamidase.
- a suitable promoter can be selected, for example, from a bacteriophage promoter including a T7 promoter and a phage lambda promoter.
- suitable promoters for the expression in a yeast species include but are not limited to the Gal 1 and Gal 10 promoters of Saccharomyces cerevisiae and the Pichia pastoris AOXl or A0X2 promoters.
- the CBHII (cellobiohydrolase II) promoter may be used.
- An expression vector may also comprise a suitable transcription terminator and, in eukaryotes, polyadenylation sequences operably linked to the DNA sequence encoding an AmyWDG 195 -related amylase. Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter.
- the vector may further comprise a DNA sequence enabling the vector to replicate in the host cell.
- a DNA sequence enabling the vector to replicate in the host cell. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUBllO, pE194, pAMBl, and pIJ702.
- the vector may also comprise a selectable marker, e.g., a gene the product of which complements a defect in the isolated host cell, such as the dal genes from B. subtilis or B. licheniformis, or a gene that confers antibiotic resistance such as, e.g., ampicillin, kanamycin, chloramphenicol or tetracycline resistance.
- a selectable marker e.g., a gene the product of which complements a defect in the isolated host cell, such as the dal genes from B. subtilis or B. licheniformis, or a gene that confers antibiotic resistance such as, e.g., ampicillin, kanamycin, chloramphenicol or tetracycline resistance.
- the vector may comprise Aspergillus selection markers such as amdS, argB, niaD and xxsC, a marker giving rise to hygromycin resistance, or the selection may be accomplished by co-transformation, such as known in the art.
- AmyWDG 195 -related amylase comprises a signal sequence at the amino terminus that permits secretion into the culture medium. If desirable, this signal peptide may be replaced by a different sequence, conveniently accomplished by substitution of the DNA sequences encoding the respective signal polypeptide.
- the expression vector typically includes the components of a cloning vector, such as, for example, an element that permits autonomous replication of the vector in the selected host organism and one or more phenotypically detectable markers for selection purposes.
- the expression vector normally comprises control nucleotide sequences such as a promoter, operator, ribosome binding site, translation initiation signal and optionally, a repressor gene or one or more activator genes.
- the expression vector may comprise a sequence coding for an amino acid sequence capable of targeting the Amy WDG 195 -related polypeptide to a host cell organelle such as a peroxisome, or to a particular host cell compartment.
- a targeting sequence includes but is not limited to the sequence, SKL.
- the nucleic acid sequence of the AmyWDG 195 -related polypeptide is operably linked to the control sequences in proper manner with respect to expression.
- a portion of an exemplary vector is depicted in Figure 3.
- An isolated cell is advantageously used as a host cell in the recombinant production of an AmyWDG195-related amylase.
- the cell may be transformed with the DNA construct encoding the enzyme, conveniently by integrating the DNA construct (in one or more copies) in the host chromosome. This integration is generally considered to be an advantage, as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromosome may be performed according to conventional methods, e.g., by homologous or heterologous recombination. Alternatively, the cell may be transformed with an expression vector as described above in connection with the different types of host cells.
- suitable bacterial host organisms are Gram positive bacterial species such as Bacillaceae including Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Geobacillus (formerly Bacillus) stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans, Bacillus lautus, Bacillus megaterium, and Bacillus thuringiensis; Streptomyces species such as Streptomyces murinus; lactic acid bacterial species including Lactococcus spp. such as Lactococcus lactis; Lactobacillus spp.
- Bacillaceae including Bacillus subtilis, Bacillus licheniformis, Bacillus lentus, Bacillus brevis, Geobacillus (formerly Bacillus) stearothermophilus, Bacillus alkalophilus, Bacillus amyloliquefaciens, Bacillus coagulans,
- strains of a Gram negative bacterial species belonging to Enterobacteriaceae including E. coli, or to Pseudomonadaceae can be selected as the host organism.
- a suitable yeast host organism can be selected from the biotechnologically relevant yeasts species such as but not limited to yeast species such as Pichia sp., Hansenula sp., or Kluyveromyces, Yarrowinia, Schizosaccharomyces species or a species of Saccharomyces, including Saccharomyces cerevisiae or a species belonging to Schizosaccharomyces such as, for example, S. pombe species.
- a strain of the methylotrophic yeast species, Pichia pastoris can be used as the host organism.
- the host organism can be a Hansenula species.
- Suitable host organisms among filamentous fungi include species of Aspergillus, e.g., Aspergillus niger, Aspergillus oryzae, Aspergillus tubigensis, Aspergillus awamori, or Aspergillus nidulans.
- strains of a Fusarium species e.g., Fusarium oxysporum or of a Rhizomucor species such as Rhizomucor miehei can be used as the host organism.
- Other suitable strains include Thermomyces and Mucor species.
- Trichoderma reesei can be used as a host.
- a suitable procedure for transformation of Aspergillus host cells includes, for example, that described in EP 238023.
- a method of producing an AmyWDG195-related amylase comprising cultivating a host cell as described above under conditions conducive to the production of the enzyme and recovering the enzyme from the cells and/or culture medium.
- the medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in question and obtaining expression of an AmyWDG195-related amylase. Suitable media and media components are available from commercial suppliers or may be prepared according to published recipes (e.g., as described in catalogues of the American Type Culture Collection).
- an enzyme secreted from the host cells is used in a whole broth preparation.
- the preparation of a spent whole fermentation broth of a recombinant microorganism can be achieved using any cultivation method known in the art resulting in the expression of an alpha-amylase. Fermentation may, therefore, be understood as comprising shake flask cultivation, small- or large-scale fermentation (including continuous, batch, fed-batch, or solid state fermentations) in laboratory or industrial fermenters performed in a suitable medium and under conditions allowing the amylase to be expressed or isolated.
- the term "spent whole fermentation broth” is defined herein as unfractionated contents of fermentation material that includes culture medium, extracellular proteins (e.g., enzymes), and cellular biomass. It is understood that the term "spent whole fermentation broth” also encompasses cellular biomass that has been lysed or permeabilized using methods well known in the art.
- An enzyme secreted from the host cells may conveniently be recovered from the culture medium by well-known procedures, including separating the cells from the medium by centrifugation or filtration, and precipitating proteinaceous components of the medium by means of a salt such as ammonium sulfate, followed by the use of chromatographic procedures such as ion exchange chromatography, affinity chromatography, or the like.
- An aspect contemplates the polynucleotide in a vector is operably linked to a control sequence that is capable of providing for the expression of the coding sequence by the host cell, i.e. the vector is an expression vector.
- the control sequences may be modified, for example by the addition of further transcriptional regulatory elements to make the level of transcription directed by the control sequences more responsive to transcriptional modulators.
- the control sequences may in particular comprise promoters.
- Host cells may be cultured under suitable conditions that allow expression of an Amy WDG 195 -related amylase.
- Expression of the enzymes may be constitutive such that they are continually produced, or inducible, requiring a stimulus to initiate expression.
- protein production can be initiated when required by, for example, addition of an inducer substance to the culture medium, for example dexamethasone or IPTG or Sopharose.
- Polypeptides can also be produced recombinantly in an in vitro cell-free system, such as the TnTTM (Promega) rabbit reticulocyte system.
- An AmyWDG 195 -related amylase-expressing host also can be cultured in the appropriate medium for the host, under aerobic conditions. Shaking or a combination of agitation and aeration can be provided, with production occurring at the appropriate temperature for that host, e.g., from about 25°C to about 75°C (e.g., 30 0 C to 45°C), depending on the needs of the host and production of the desired AmyWDG 195 -related polypeptide. Culturing can occur from about 12 to about 100 hours or greater (and any hour value there between, e.g., from 24 to 72 hours).
- the culture broth is at a pH of about 5.5 to about 8.0, again depending on the culture conditions needed for the host relative to production of an AmyWDG 195 -related amylase.
- an AmyWDG195-related amylase is expressed in the presence of additional NaCl added to the media, as compared to, for example, minimal media.
- the minimal media used herein included about 50 mM NaCl in addition to a small amount contributed by other component.
- the addition of 1%, 2%, or even 3% salt resulted in a significant increase in the amount of Amy WDG 195 -related amylase produced. Previous studies have suggested that NaCl was important for AmyK38 activity but not expression.
- An aspect of the composition and method is the expression of an AmyWDG195- related polypeptide in the presence of additional NaCl, which increases the yield of polypeptide that is expressed in host cells.
- the amount of NaCl is about 1% and the increase in yield is at least 3, at least 6, and even at least 9-fold.
- the amount of NaCl is about 2% and the increase in yield is at least 4, at least 8, at least 12, and even at least 16-fold.
- the amount of NaCl is about 3% and the increase in yield is at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, and even at least 35- fold.
- an expression vector containing a polynucleotide encoding a signal sequence linked to a polynucleotide encoding an AmyWDG195-related polypeptide is constructed and introduced into a host cell.
- the AmyWDG195-related polypeptide is then expressed in host cells using medium enriched for NaCl as described, wherein the signal peptide directs secretion of the AmyWDG195-related polypeptide and the additional salt increases the yield.
- the AmyWDG195-related polypeptide is recovered and purified as desired.
- Fermentation, separation, and concentration techniques are well known in the art and conventional methods can be used in order to prepare a concentrated AmyWDG195-related amylase-containing solution.
- a fermentation broth is obtained, the microbial cells and various suspended solids, including residual raw fermentation materials, are removed by conventional separation techniques in order to obtain an amylase solution. Filtration, centrifugation, microfiltration, rotary vacuum drum filtration, ultrafiltration, centrifugation followed by ultrafiltration, extraction, or chromatography, or the like, are generally used.
- the enzyme containing solution is concentrated using conventional concentration techniques until the desired enzyme level is obtained. Concentration of the enzyme containing solution may be achieved using any of the techniques discussed herein or otherwise known in the art. Exemplary methods of concentration include but are not limited to rotary vacuum filtration and/or ultrafiltration.
- Concentration may be performed using e.g. , a precipitation agent, such as a metal halide precipitation agent.
- a precipitation agent such as a metal halide precipitation agent.
- Metal halide precipitation agents include but are not limited to alkali metal chlorides, alkali metal bromides and blends of two or more of these metal halides.
- Exemplary metal halides include sodium chloride, potassium chloride, sodium bromide, potassium bromide and blends of two or more of these metal halides.
- the metal halide precipitation agent, sodium chloride can also be used as a preservative.
- the metal halide precipitation agent is used in an amount effective to precipitate the Amy WDG 195 -related polypeptide.
- the selection of at least an effective amount and an optimum amount of metal halide effective to cause precipitation of the enzyme, as well as the conditions of the precipitation for maximum recovery including incubation time, pH, temperature and concentration of enzyme, will be readily apparent to one of ordinary skill in the art, after routine testing.
- the concentration of the metal halide precipitation agent will depend, among others, on the nature of the specific AmyWDG 195 -related amylase and on its concentration in the concentrated enzyme solution.
- organic compounds include: 4-hydroxybenzoic acid, alkali metal salts of 4-hydroxybenzoic acid, alkyl esters of 4-hydroxybenzoic acid, and blends of two or more of these organic compounds.
- the addition of said organic compound precipitation agents can take place prior to, simultaneously with or subsequent to the addition of the metal halide precipitation agent, and the addition of both precipitation agents, organic compound and metal halide, may be carried out sequentially or simultaneously.
- the organic precipitation agents are selected from the group consisting of alkali metal salts of 4-hydroxybenzoic acid, such as sodium or potassium salts, and linear or branched alkyl esters of 4-hydroxybenzoic acid, wherein the alkyl group contains from 1 to 12 carbon atoms, and blends of two or more of these organic compounds.
- the organic compound precipitation agents can be, for example, linear or branched alkyl esters of 4-hydroxybenzoic acid, wherein the alkyl group contains from 1 to 10 carbon atoms, and blends of two or more of these organic compounds.
- Exemplary organic compounds are linear alkyl esters of 4- hydroxybenzoic acid, wherein the alkyl group contains from 1 to 6 carbon atoms, and blends of two or more of these organic compounds.
- Methyl esters of 4-hydroxybenzoic acid, propyl esters of 4-hydroxybenzoic acid, butyl ester of 4-hydroxybenzoic acid, ethyl ester of 4-hydroxybenzoic acid and blends of two or more of these organic compounds can also be used.
- Additional organic compounds also include but are not limited to 4-hydroxybenzoic acid methyl ester (named methyl PARABEN), 4-hydroxybenzoic acid propyl ester (named propyl PARABEN), which also are both amylase preservative agents.
- methyl PARABEN 4-hydroxybenzoic acid methyl ester
- propyl PARABEN 4-hydroxybenzoic acid propyl ester
- Addition of the organic compound precipitation agent provides the advantage of high flexibility of the precipitation conditions with respect to pH, temperature, AmyWDG195-related polypeptide concentration, precipitation agent concentration, and time of incubation.
- the organic compound precipitation agent is used in an amount effective to improve precipitation of the enzyme by means of the metal halide precipitation agent. The selection of at least an effective amount and an optimum amount of organic compound precipitation agent, as well as the conditions of the precipitation for maximum recovery including incubation time, pH, temperature and concentration of enzyme, will be readily apparent to one of ordinary skill in the art, in light of the present disclosure, after routine testing.
- the concentrated enzyme solution containing the metal halide precipitation agent, and the organic compound precipitation agent, can be adjusted to a pH, which will, of necessity, depend on the enzyme to be purified. Generally, the pH is adjusted at a level near the isoelectric point of the amylase. The pH can be adjusted at a pH in a range from about 2.5 pH units below the isoelectric point (pi) up to about 2.5 pH units above the isoelectric point.
- the incubation time necessary to obtain a purified enzyme precipitate depends on the nature of the specific enzyme, the concentration of enzyme, and the specific precipitation agent(s) and its (their) concentration.
- the time effective to precipitate the enzyme is between about 1 to about 30 hours; usually it does not exceed about 25 hours. In the presence of the organic compound precipitation agent, the time of incubation can still be reduced to less about 10 hours and in most cases even about 6 hours.
- the temperature during incubation is between about 4°C and about 50 0 C.
- the method is carried out at a temperature between about 10 0 C and about 45°C (e.g., between about 20 0 C and about 40 0 C).
- the optimal temperature for inducing precipitation varies according to the solution conditions and the enzyme or precipitation agent(s) used.
- the overall recovery of purified enzyme precipitate, and the efficiency with which the process is conducted, is improved by agitating the solution comprising the enzyme, the added metal halide and the added organic compound.
- the agitation step is done both during addition of the metal halide and the organic compound, and during the subsequent incubation period. Suitable agitation methods include mechanical stirring or shaking, vigorous aeration, or any similar technique.
- the purified enzyme is then separated from the dissociated pigment and other impurities and collected by conventional separation techniques, such as filtration, centrifugation, microfiltration, rotary vacuum filtration, ultrafiltration, press filtration, cross membrane microfiltration, cross flow membrane microfiltration, or the like. Further purification of the purified enzyme precipitate can be obtained by washing the precipitate with water. For example, the purified enzyme precipitate is washed with water containing the metal halide precipitation agent, or with water containing the metal halide and the organic compound precipitation agents.
- an Amy WDG 195 -related amylase accumulates in the culture broth.
- the culture broth is centrifuged or filtered to eliminate cells, and the resulting cell-free liquid is used for enzyme purification.
- the cell-free broth is subjected to salting out using ammonium sulfate at about 70% saturation; the 70% saturation-precipitation fraction is then dissolved in a buffer and applied to a column such as a Sephadex G-100 column, and eluted to recover the enzyme-active fraction.
- a conventional procedure such as ion exchange chromatography may be used.
- Purified enzymes are useful for laundry and cleaning applications. For example, they can be used in laundry detergents and spot removers. They can be made into a final product that is either liquid (solution, slurry) or solid (granular, powder).
- the dialyzed sample was then applied to a Sephacryl S-200 column, which had previously been equilibrated with 20 mM Tris/HCl buffer, (pH 7.0), 5 mM CaCl 2 , and eluted at a linear flow rate of 7 cm/hr with the same buffer. Fractions from the column were collected and assessed for activity as judged by enzyme assay and SDS-PAGE. The protein was further purified as follows. A Toyopearl HW55 column (Tosoh Bioscience, Montgomeryville, PA; Cat. No. 19812) was equilibrated with 20 mM Tris/HCl buffer (pH 7.0) containing 5 mM CaCl 2 and 1.5 M (NH 4 ) 2 SO 4 .
- the enzyme was eluted with a linear gradient of 1.5 to 0 M (NH 4 ) 2 SO 4 in 20 mM Tris/HCL buffer, pH 7.0 containing 5 mM CaCl 2 .
- the active fractions were collected, and the enzyme precipitated with (NH 4 ) 2 SO 4 at 80% saturation.
- the precipitate was recovered, re-dissolved, and dialyzed as described above.
- the dialyzed sample was then applied to a Mono Q HR5/5 column (Amersham Pharmacia; Cat. No. 17-5167-01) previously equilibrated with 20 mM Tris/HCl buffer (pH 7.0) containing 5 mM CaCl 2 , at a flow rate of 60 mL/hour.
- the active fractions are collected and added to a 1.5 M (NH 4 ) 2 SO 4 solution.
- the active enzyme fractions were re-chromatographed on a Toyopearl HW55 column, as before, to yield a homogeneous enzyme as determined by SDS-PAGE. See J. Sumitani et al. , "New type of starch-binding domain: the direct repeat motif in the C-terminal region of Bacillus sp. no. 195 ⁇ -amylase contributes to starch binding and raw starch degrading," Biochem. J. 350: 477-484 (2000) for general discussion of the method and variations thereon.
- an AmyWDG195-related amylase can be partially purified as generally described above by removing cells via flocculation with polymers.
- the enzyme can be purified by microfiltration followed by concentration by ultrafiltration using available membranes and equipment.
- the enzyme does not need to be purified, and whole broth culture can be lysed and used without further treatment.
- the enzyme can then be processed, for example, into granules.
- An aspect of the present compositions and methods is a cleaning composition that includes an Amy WDG 195 -related amylase as a component.
- An Amy WDG 195 -related amylase can be used as a component in detergent compositions for hand washing, laundry washing, dishwashing, and other hard-surface cleaning.
- an AmyWDG195-related amylase is incorporated into detergents at or near a concentration conventionally used for amylase in detergents.
- an AmyWDG195-related amylase may be added in amount corresponding to 0.00001 - 1 mg (calculated as pure enzyme protein) of ⁇ -amylase per liter of wash/diswash liquor.
- Exemplary formulations are provided herein, as exemplified by the following:
- An AmyWDG195-related amylase may typically be a component of a detergent composition, as the only enzyme or with other enzymes including other amylolytic enzymes. As such, it may be included in the detergent composition in the form of a non-dusting granulate, a stabilized liquid, or a protected enzyme. Non-dusting granulates may be produced, e.g., as disclosed in U.S. Patent Nos. 4,106,991 and 4,661,452 and may optionally be coated by methods known in the art.
- waxy coating materials are poly(ethylene oxide) products (polyethyleneglycol, PEG) with mean molar weights of 1,000 to 20,000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids.
- PEG poly(ethylene oxide) products
- PEG polyethyleneglycol
- Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods.
- a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid
- Other enzyme stabilizers are well known in the art.
- Protected enzymes may be prepared according to the method disclosed in for example EP 238,216. Polyols have long been recognized as stabilizers of proteins, as well as improving protein solubility. See, e.g., Kaushik, J.K. et al. (2003) /. Biol. Chem. 278: 26458-65 and references cited therein; and Monica Conti, M. et al. (1997) /. Chromatography A 757: 237-245.
- the detergent composition may be in any useful form, e.g., as powders, granules, pastes, or liquid.
- a liquid detergent may be aqueous, typically containing up to about 70% of water and 0% to about 30% of organic solvent. It may also be in the form of a compact gel type containing only about 30% water.
- the detergent composition comprises one or more surfactants, each of which may be anionic, nonionic, cationic, or zwitterionic.
- the detergent will usually contain 0% to about 50% of anionic surfactant, such as linear alkylbenzenesulfonate (LAS); ⁇ -olefinsulfonate (AOS); alkyl sulfate (fatty alcohol sulfate) (AS); alcohol ethoxysulfate (AEOS or AES); secondary alkanesulfonates (SAS); ⁇ -sulfo fatty acid methyl esters; alkyl- or alkenylsuccinic acid; or soap.
- anionic surfactant such as linear alkylbenzenesulfonate (LAS); ⁇ -olefinsulfonate (AOS); alkyl sulfate (fatty alcohol sulfate) (AS); alcohol ethoxysulfate (AEOS or AES); secondary alkanesulfonates (SAS);
- the composition may also contain 0% to about 40% of nonionic surfactant such as alcohol ethoxylate (AEO or AE), carboxylated alcohol ethoxylates, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, or polyhydroxy alkyl fatty acid amide (as described for example in WO 92/06154).
- nonionic surfactant such as alcohol ethoxylate (AEO or AE), carboxylated alcohol ethoxylates, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, or polyhydroxy alkyl fatty acid amide (as described for example in WO 92/06154).
- the detergent composition may additionally comprise one or more other enzymes, such as lipase, another amylolytic enzyme, cutinase, protease, cellulase, peroxidase, and/or laccase in any combination.
- enzymes such as lipase, another amylolytic enzyme, cutinase, protease, cellulase, peroxidase, and/or laccase in any combination.
- the detergent may contain about 1% to about 65% of a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, citrate, nitrilotriacetic acid (NTA), ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DTMPA), alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g., SKS-6 from Hoechst).
- the detergent may also be unbuilt, i.e. essentially free of detergent builder.
- the enzymes can be used in any composition compatible with the stability of the enzyme.
- Enzymes generally can be protected against deleterious components by known forms of encapsulation, for example, by granulation or sequestration in hydro gels. Enzymes, and specifically ⁇ -amylases, such as AmyWDG195 molecules, either with or without starch binding domains, can be used in a variety of compositions including laundry and dishwashing applications, surface cleaners, as well as in compositions for ethanol production from starch or biomass.
- the detergent may comprise one or more polymers.
- the detergent may contain a bleaching system, which may comprise a H 2 O 2 source such as perborate or percarbonate, which may be combined with a peracid-forming bleach activator such as tetraacetylethylenediamine (TAED) or nonanoyloxybenzenesulfonate (NOBS).
- a bleaching system which may comprise a H 2 O 2 source such as perborate or percarbonate, which may be combined with a peracid-forming bleach activator such as tetraacetylethylenediamine (TAED) or nonanoyloxybenzenesulfonate (NOBS).
- the bleaching system may comprise peroxyacids (e.g., the amide, imide, or sulfone type peroxyacids).
- the bleaching system can also be an enzymatic bleaching system, for example, perhydrolase, such as that described in International PCT Application WO 2005/056783.
- the enzymes of the detergent composition may be stabilized using conventional stabilizing agents, e.g., a polyol such as propylene glycol or glycerol; a sugar or sugar alcohol; lactic acid; boric acid or a boric acid derivative such as, e.g., an aromatic borate ester; and the composition may be formulated as described in, e.g., WO 92/19709 and WO 92/19708.
- stabilizing agents e.g., a polyol such as propylene glycol or glycerol
- a sugar or sugar alcohol lactic acid
- boric acid or a boric acid derivative such as, e.g., an aromatic borate ester
- the composition may be formulated as described in, e.g., WO 92/19709 and WO 92/19708.
- the detergent may also contain other conventional detergent ingredients such as e.g., fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil- suspending agents, anti-soil redeposition agents, dyes, bactericides, tarnish inhibiters, optical brighteners, or perfumes.
- fabric conditioners including clays, foam boosters, suds suppressors, anti-corrosion agents, soil- suspending agents, anti-soil redeposition agents, dyes, bactericides, tarnish inhibiters, optical brighteners, or perfumes.
- the pH (measured in aqueous solution at use concentration) is usually neutral or alkaline, e.g., pH about 7.0 to about 11.0.
- compositions comprising the Amy WDG 195 -related polypeptides can be formulated to include:
- a detergent composition formulated as a granulate having a bulk density of at least 600 g/L comprising linear alkylbenzenesulfonate (calculated as acid) about 7% to about 12%; alcohol ethoxysulfate (e.g., Ci 2-I8 alcohol, 1-2 ethylene oxide (EO)) or alkyl sulfate (e.g., Ci 6 -i 8 ) about 1% to about 4%; alcohol ethoxylate (e.g., C 14 _ 15 alcohol, 7 EO) about 5% to about 9%; sodium carbonate (e.g., Na 2 COs) about 14% to about 20%; soluble silicate (e.g., Na 2 O, 2SiO 2 ) about 2 to about 6%; zeolite (e.g., NaAlSiO 4 ) about 15% to about 22%; sodium sulfate (e.g., Na 2 SO 4 ) 0% to about 6%; sodium citrate/c
- a detergent composition formulated as a granulate having a bulk density of at least 600 g/L comprising linear alkylbenzenesulfonate (calculated as acid) about 6% to about 11%; alcohol ethoxysulfate (e.g., Cn-n alcohol, 1-2 EO) or alkyl sulfate (e.g., Ci 6 - I8 ) about 1% to about 3%; alcohol ethoxylate (e.g., Ci 4-I5 alcohol, 7 EO) about 5% to about 9%; sodium carbonate (e.g., Na 2 CO 3 ) about 15% to about 21%; soluble silicate (e.g., Na 2 O, 2SiO 2 ) about 1% to about 4%; zeolite (e.g., NaAlSiO 4 ) about 24% to about 34%; sodium sulfate (e.g,.
- Na 2 SO 4 about 4% to about 10%
- sodium citrate/citric acid e.g., C 6 H 5 Na 3 O 7 / C 6 H 8 O 7
- carboxymethylcellulose (CMC) 0% to about 2%
- polymers e.g., maleic/acrylic acid copolymer, PVP, PEG
- enzymes calculated as pure enzyme protein
- minor ingredients e.g., suds suppressors, perfume
- a detergent composition formulated as a granulate having a bulk density of at least 600 g/L comprising linear alkylbenzenesulfonate (calculated as acid) about 8% to about 12%; alcohol ethoxylate (e.g., Cn-is alcohol, 7 EO) about 10% to about 25%; sodium carbonate (as Na 2 CO 3 ) about 14% to about 22%; soluble silicate (e.g., Na 2 O, 2SiO 2 ) about 1% to about 5%; zeolite (e.g., NaAlSiO 4 ) about 25% to about 35%; sodium sulfate (e.g., Na 2 SO 4 ) 0% to about 10%; carboxymethylcellulose (CMC) 0% to about 2%; polymers (e.g., maleic/acrylic acid copolymer, PVP, PEG) 1-3%; enzymes (calculated as pure enzyme protein) 0.0001-0.1%; and minor ingredients (e.g.,
- An aqueous liquid detergent composition comprising linear alkylbenzenesulfonate (calculated as acid) about 15% to about 21%; alcohol ethoxylate (e.g., Cn-is alcohol, 7 EO or Ci 2 - I5 alcohol, 5 EO) about 12% to about 18%; soap as fatty acid (e.g., oleic acid) about 3% to about 13%; alkenylsuccinic acid (Ci 2-I4 ) 0% to about 13%; aminoethanol about 8% to about 18%; citric acid about 2% to about 8%; phosphonate 0% to about 3%; polymers (e.g., PVP, PEG) 0% to about 3%; borate (e.g., B 4 O 7 ) 0% to about 2%; ethanol 0% to about 3%; propylene glycol about 8% to about 14%; enzymes (calculated as pure enzyme protein) 0.0001-0.1%; and minor ingredients (e.g.,
- An aqueous structured liquid detergent composition comprising linear alkylbenzenesulfonate (calculated as acid) about 15% to about 21%; alcohol ethoxylate (e.g., Ci 2 -i5 alcohol, 7 EO, or Ci 2-I5 alcohol, 5 EO) 3-9%; soap as fatty acid (e.g., oleic acid) about 3% to about 10%; zeolite (as NaAlSiO 4 ) about 14% to about 22%; potassium citrate about 9% to about 18%; borate (e.g., B 4 O 7 ) 0% to about 2%; carboxymethylcellulose (CMC) 0% to about 2%; polymers (e.g., PEG, PVP) 0% to about 3%; anchoring polymers such as, e.g., lauryl methacrylate/acrylic acid copolymer; molar ratio 25:1, MW 3800) 0% to about 3%;glycerol 0% to about 5%
- a detergent composition formulated as a granulate having a bulk density of at least 600 g/L comprising fatty alcohol sulfate about 5% to about 10%; ethoxylated fatty acid monoethanolamide about 3% to about 9%; soap as fatty acid 0-3%; sodium carbonate (e.g., Na 2 CO 3 ) about 5% to about 10%; Soluble silicate (e.g., Na 2 O, 2SiO 2 ) about 1% to about 4%; zeolite (e.g., NaAlSiO 4 ) about 20% to about 40%; Sodium sulfate (e.g., Na 2 SO 4 ) about 2% to about 8%; sodium perborate (e.g., NaBO 3 H 2 O) about 12% to about 18%; TAED about 2% to about 7%; polymers (e.g., maleic/acrylic acid copolymer, PEG) about 1% to about 5%; enzymes (calculated as pure enzyme
- a detergent composition formulated as a granulate comprising linear alkylbenzenesulfonate (calculated as acid) about 8% to about 14%; ethoxylated fatty acid monoethanolamide about 5% to about 11%; soap as fatty acid 0% to about 3%; sodium carbonate (e.g., Na 2 CO 3 ) about 4% to about 10%; soluble silicate (Na 2 O, 2SiO 2 ) about 1% to about 4%; zeolite (e.g., NaAlSiO 4 ) about 30% to about 50%; sodium sulfate (e.g., Na 2 SO 4 ) about 3% to about 11%; sodium citrate (e.g., CeHsNa 3 O 7 ) about 5% to about 12%; polymers (e.g., PVP, maleic/acrylic acid copolymer, PEG) about 1% to about 5%; enzymes (calculated as pure enzyme protein) 0.0001-0.1%; and minor
- a detergent composition formulated as a granulate comprising linear alkylbenzenesulfonate (calculated as acid) about 6% to about 12%; nonionic surfactant about 1% to about 4%; soap as fatty acid about 2% to about 6%; sodium carbonate (e.g., Na 2 CO 3 ) about 14% to about 22%; zeolite (e.g., NaAlSiO 4 ) about 18% to about 32%; sodium sulfate (e.g., Na 2 SO 4 ) about 5% to about 20%; sodium citrate (e.g., C 6 H 5 Na 3 O 7 ) about 3% to about 8%; sodium perborate (e.g., NaBO 3 H 2 O) about 4% to about 9%; bleach activator (e.g., NOBS or TAED) about 1% to about 5%; carboxymethylcellulose (CMC) 0% to about 2%; polymers (e.g., polycarboxylate or PEG) about 1% to
- An aqueous liquid detergent composition comprising linear alkylbenzenesulfonate (calculated as acid) about 15% to about 23%; alcohol ethoxysulfate (e.g., Ci 2 -i5 alcohol, 2-3 EO) about 8% to about 15%; alcohol ethoxylate (e.g., Ci 2-I5 alcohol, 7 EO, or Ci 2-I5 alcohol, 5 EO) about 3% to about 9%; soap as fatty acid (e.g., lauric acid) 0% to about 3%; aminoethanol about 1% to about 5%; sodium citrate about 5% to about 10%; hydrotrope (e.g., sodium toluensulfonate) about 2% to about 6%; borate (e.g., B 4 O 7 ) 0% to about 2%; carboxymethylcellulose 0% to about 1%; ethanol about 1% to about 3%; propylene glycol about 2% to about 5%; enzymes (calculated as pure enzyme protein)
- An aqueous liquid detergent composition comprising linear alkylbenzenesulfonate (calculated as acid) about 20% to about 32%; alcohol ethoxylate (e.g., Ci 2-I5 alcohol, 7 EO, or Ci 2-I5 alcohol, 5 EO) 6-12%; aminoethanol about 2% to about 6%; citric acid about 8% to about 14%; borate (e.g., B 4 O 7 ) about 1% to about 3%; polymer (e.g., maleic/acrylic acid copolymer, anchoring polymer such as, e.g., lauryl methacrylate/acrylic acid copolymer) 0% to about 3%; glycerol about 3% to about 8%; enzymes (calculated as pure enzyme protein) 0.0001-0.1%; and minor ingredients (e.g., hydrotropes, dispersants, perfume, optical brighteners) 0-5%.
- alcohol ethoxylate e.g., Ci 2-I5 alcohol, 7 EO, or
- a detergent composition formulated as a granulate having a bulk density of at least 600 g/L comprising anionic surfactant (linear alkylbenzenesulfonate, alkyl sulfate, ⁇ - olefinsulfonate, ⁇ -sulfo fatty acid methyl esters, alkanesulfonates, soap) about 25% to about 40%; nonionic surfactant (e.g., alcohol ethoxylate) about 1% to about 10%; sodium carbonate (e.g., Na 2 CO 3 ) about 8% to about 25%; soluble silicates (e.g., Na 2 O, 2SiO 2 ) about 5% to about 15%; sodium sulfate (e.g., Na 2 SO 4 ) 0% to about 5%; zeolite (NaAlSiO 4 ) about 15% to about 28%; sodium perborate (e.g., NaBO 3 4H 2 O) 0% to about 20%; bleach activ
- compositions 1-12) supra wherein all or part of the linear alkylbenzenesulfonate is replaced by (Ci 2 -Ci 8 ) alkyl sulfate.
- NaAlSiO 4 about 10% to about 20%
- layered disilicate e.g., SK56 from Hoechst
- sodium carbonate e.g., Na 2 COs
- soluble silicate e.g.,
- Na 2 O, 2SiO 2 0% to about 6%; sodium citrate about 4% to about 8%; sodium percarbonate about
- enzymes calculated as pure enzyme protein
- minor ingredients e.g., optical brightener, photobleach, perfume, suds suppressors
- a detergent composition formulated as a granulate having a bulk density of at least 600 g/L comprising (Ci 2 -Ci 8 ) alkyl sulfate about 4% to about 8%; alcohol ethoxylate about
- sodium carbonate (as Na 2 CO 3 ) about 2% to about 8%; soluble silicate (e.g., Na 2 O, 2SiO 2 )
- the manganese catalyst for example is one of the compounds described in Rage et al. (1994) Nature 369:637-639.
- Detergent composition formulated as a non-aqueous detergent liquid comprising a liquid nonionic surfactant such as, e.g., linear alkoxylated primary alcohol, a builder system
- a liquid nonionic surfactant such as, e.g., linear alkoxylated primary alcohol, a builder system
- the detergent may also comprise anionic surfactant and/or a bleach system.
- Amy WDG 195 -related amylase may be incorporated in concentrations conventionally employed in detergents. It is at present contemplated that, in the detergent composition, the enzyme may be added in an amount corresponding to 0.00001-1.0 mg
- the detergent composition may for example be formulated as a hand (manual) or machine (automatic) laundry detergent composition, including a laundry additive composition suitable for pre-treatment of stained fabrics and a rinse added fabric softener composition, or be formulated as a detergent composition for use in general household hard surface cleaning operations, or be formulated for manual or automatic dishwashing operations.
- the detergent composition can comprise 2,6- ⁇ -D-fructan hydrolase in addition to an Amy WDG 195 -related amylase, and one or more other cleaning enzymes, such as a protease, a lipase, a cutinase, a carbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, a galactanase, another amylolytic enzyme, a xylanase, an oxidase, a laccase, and/or a peroxidase, and/or combinations thereof.
- cleaning enzymes such as a protease, a lipase, a cutinase, a carbohydrase, a cellulase, a pectinase, a mannanase, an arabinase, a galactanase, another amylolytic enzyme
- proteases include those of animal, vegetable or microbial origin. Chemically modified or protein engineered mutants are included, as well as naturally processed proteins.
- the protease may be a serine protease or a metalloprotease, such as an alkaline microbial protease, a trypsin-like protease, or a chymotrypsin-like protease.
- alkaline proteases are subtilisins, especially those derived from Bacillus, e.g., subtilisin Novo, subtilisin Carlsberg, subtilisin 309, subtilisin 147, and subtilisin 168 (see, e.g., WO 89/06279).
- trypsin-like proteases are trypsin (e.g., of porcine or bovine origin), and Fusarium proteases (see, e.g., WO 89/06270 and WO 94/25583).
- useful proteases also include but are not limited to the variants described in WO 92/19729, WO 98/20115, WO 98/20116, and WO 98/34946.
- protease enzymes include but are not limited to: ALCALASE®, SAVINASE®, PRIMASETM, DURALASETM, ESPERASE®, and KANNASETM (Novo Nordisk A/S); MAXATASE®, MAXACALTM, MAXAPEMTM, PROPERASE®, PURAFECT®, PURAFECT OXPTM, FN2TM, and FN3TM (Genencor International, Inc.).
- Lipases include those of bacterial or fungal origin. Chemically modified, proteolytically modified, or protein engineered mutants are included. Examples of useful lipases include but are not limited to lipases from Humicola (synonym Thermomyces), e.g., from H. lanuginosa (T. lanuginosus) (see e.g., EP 258068 and EP 305216), from H. insolens (see e.g., WO 96/13580); a Pseudomonas lipase (e.g., from P. alcaligenes or P. pseudoalcaligenes; see, e.g., EP 218 272), P.
- cepacia see e.g., EP 331 376
- P. stutzeri see e.g., GB 1,372,034
- P. fluorescens Pseudomonas sp. strain SD 705 (see e.g., WO 95/06720 and WO 96/27002)
- P. wisconsinensis see e.g., WO 96/12012
- Bacillus lipase e.g., from B. subtilis; see e.g., Dartois et al.
- Additional lipase variants contemplated for use in the formulations include those described for example in: WO 92/05249, WO 94/01541, WO 95/35381, WO 96/00292, WO 95/30744, WO 94/25578, WO 95/14783, WO 95/22615, WO 97/04079, WO 97/07202, EP 407225, and EP 260105.
- Some commercially available lipase enzymes include LIPOLASE® and LIPOLASE ULTRATM (Novo Nordisk A/S).
- Polyesterases Suitable polyesterases can be included in the composition, such as those described in, for example, WO 01/34899 and WO 01/14629.
- Amylases The compositions can be combined with other amylases, such as non- production enhanced ⁇ -amylase. These can include commercially available amylases, such as but not limited to DURAMYL®, TERMAMYL®, FUNGAMYL® and BANTM (Novo Nordisk A/S); RAPIDASE® and PURASTAR® (from Genencor International, Inc.).
- Cellulases Cellulases can be added to the compositions. Suitable cellulases include those of bacterial or fungal origin. Chemically modified or protein engineered mutants are included.
- Suitable cellulases include cellulases from the genera Bacillus, Pseudomonas, Humicola, Fusarium, Thielavia, Acremonium, e.g., the fungal cellulases produced from Humicola insolens, Myceliophthora thermophila and Fusarium oxysporum disclosed for example in U.S. Patent Nos. 4,435,307; 5,648,263; 5,691,178; 5,776,757; and WO 89/09259.
- Exemplary cellulases contemplated for use are those having color care benefit for the textile.
- cellulases examples include cellulases described in for example EP 0495257, EP 0531372, WO 96/11262, WO 96/29397, and WO 98/08940.
- cellulase variants such as those described in WO 94/07998; WO 98/12307; WO 95/24471; PCT/DK98/00299; EP 531315; U.S. Patent Nos. 5,457,046; 5,686,593; and 5,763,254.
- Peroxidases/Oxidases Suitable peroxidases/oxidases contemplated for use in the compositions include those of plant, bacterial or fungal origin. Chemically modified or protein engineered mutants are included. Examples of useful peroxidases include peroxidases from Coprinus, e.g., from C. cinereus, and variants thereof as those described in WO 93/24618, WO 95/10602, and WO 98/15257. Commercially available peroxidases include for example GUARDZYMETM (Novo Nordisk A/S).
- the detergent enzyme(s) may be included in a detergent composition by adding separate additives containing one or more enzymes, or by adding a combined additive comprising all of these enzymes.
- a detergent additive i.e. a separate additive or a combined additive, can be formulated e.g., as a granulate, a liquid, a slurry, etc.
- Exemplary detergent additive formulations include but are not limited to granulates, in particular non-dusting granulates, liquids, in particular stabilized liquids or slurries.
- Non-dusting granulates may be produced, e.g., as disclosed in U.S. Patent Nos. 4,106,991 and 4,661,452 and may optionally be coated by methods known in the art.
- waxy coating materials are poly(ethylene oxide) products ⁇ e.g., polyethyleneglycol, PEG) with mean molar weights of 1,000 to 20,000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12 to 20 carbon atoms and in which there are 15 to 80 ethylene oxide units; fatty alcohols; fatty acids; and mono- and di- and triglycerides of fatty acids.
- poly(ethylene oxide) products ⁇ e.g., polyethyleneglycol, PEG) with mean molar weights of 1,000 to 20,000; ethoxylated nonylphenols having from 16 to 50 ethylene oxide units; ethoxylated fatty alcohols in which the alcohol contains from 12
- Liquid enzyme preparations may, for instance, be stabilized by adding a polyol such as propylene glycol, a sugar or sugar alcohol, lactic acid or boric acid according to established methods.
- Protected enzymes may be prepared according to the method disclosed in EP 238,216.
- the detergent composition may be in any convenient form, e.g. , a bar, a tablet, a powder, a granule, a paste, or a liquid.
- a liquid detergent may be aqueous, typically containing up to about 70% water, and 0% to about 30% organic solvent.
- Compact detergent gels containing about 30% or less water are also contemplated.
- the detergent composition can optionally comprise one or more surfactants, which may be non-ionic, including semi-polar and/or anionic and/or cationic and/or zwitterionic.
- the surfactants can be present in a wide range, from about 0.1% to about 60% by weight.
- the detergent When included therein the detergent will typically contain from about 1% to about 40% of an anionic surfactant, such as linear alkylbenzenesulfonate, ⁇ -olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate, ⁇ -sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid, or soap.
- an anionic surfactant such as linear alkylbenzenesulfonate, ⁇ -olefinsulfonate, alkyl sulfate (fatty alcohol sulfate), alcohol ethoxysulfate, secondary alkanesulfonate, ⁇ -sulfo fatty acid methyl ester, alkyl- or alkenylsuccinic acid, or soap.
- the detergent will usually contain from about 0.2% to about 40% of a non-ionic surfactant such as alcohol ethoxylate, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or N-acyl-N-alkyl derivatives of glucosamine ("glucamides").
- a non-ionic surfactant such as alcohol ethoxylate, nonylphenol ethoxylate, alkylpolyglycoside, alkyldimethylamineoxide, ethoxylated fatty acid monoethanolamide, fatty acid monoethanolamide, polyhydroxy alkyl fatty acid amide, or N-acyl-N-alkyl derivatives of glucosamine (“glucamides”).
- glucamides N-acyl-N-alkyl derivatives of glucosamine
- the detergent may contain 0% to about 65% of a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g.,SKS-6 from Hoechst).
- a detergent builder or complexing agent such as zeolite, diphosphate, triphosphate, phosphonate, carbonate, citrate, nitrilotriacetic acid, ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid, alkyl- or alkenylsuccinic acid, soluble silicates or layered silicates (e.g.,SKS-6 from Hoechst).
- a detergent builder or complexing agent such as ze
- Exemplary polymers include carboxymethylcellulose (CMC), poly(vinylpyrrolidone) (PVP), poly(ethylene glycol) (PEG), poly( vinyl alcohol) (PVA), poly(vinylpyridine-N-oxide), poly(vinylimidazole), polycarboxylates e.g., polyacrylates, maleic/acrylic acid copolymers), and lauryl methacrylate/acrylic acid copolymers.
- CMC carboxymethylcellulose
- PVP poly(vinylpyrrolidone)
- PEG poly(ethylene glycol)
- PVA poly( vinyl alcohol)
- PVA poly(vinylpyridine-N-oxide)
- poly(vinylimidazole) polycarboxylates e.g., polyacrylates, maleic/acrylic acid copolymers), and lauryl methacrylate/acrylic acid copolymers.
- the enzyme(s) of the detergent composition may be stabilized using conventional stabilizing agents, e.g., as polyol (e.g., propylene glycol or glycerol), a sugar or sugar alcohol, lactic acid, boric acid, or a boric acid derivative (e.g., an aromatic borate ester), or a phenyl boronic acid derivative (e.g., 4-formylphenyl boronic acid).
- polyol e.g., propylene glycol or glycerol
- a sugar or sugar alcohol lactic acid, boric acid, or a boric acid derivative (e.g., an aromatic borate ester), or a phenyl boronic acid derivative (e.g., 4-formylphenyl boronic acid).
- the composition may be formulated as described in WO 92/19709 and WO 92/19708.
- the AmyWDG195- related polypeptides may be added in an amount corresponding to about 0.01 to about 100 mg of enzyme protein per liter of wash liquor (e.g., about 0.05 to about 5.0 mg of enzyme protein per liter of wash liquor or 0.1 to about 1.0 mg of enzyme protein per liter of wash liquor).
- an AmyWDG195-related amylase is usually used in a liquid composition containing propylene glycol.
- the enzyme is solubilized in for example in propylene glycol by mixing in a 25% volume/volume propylene glycol solution containing 10% calcium chloride.
- An AmyWDG 195 -related amylase thereof discussed herein can be formulated in detergent compositions for use in cleaning dishes or other cleaning compositions. These can be powders, gels, or liquids.
- the compositions can comprise the enzyme alone, or with other amylolytic enzymes and/or with other cleaning enzymes or bleach activating enzymes, and other components common to cleaning compositions.
- a dishwashing detergent composition can comprise a surfactant.
- the surfactant may be anionic, non-ionic, cationic, amphoteric or a mixture of these types.
- the detergent can contain 0% to about 90% by weight of a non-ionic surfactant, such as low- to non-foaming ethoxylated propoxylated straight-chain alcohols.
- the detergent composition may contain detergent builder salts of inorganic and/or organic types.
- the detergent builders may be subdivided into phosphorus-containing and non- phosphorus -containing types.
- the detergent composition usually contains about 1% to about 90% of detergent builders.
- Examples of phosphorus-containing inorganic alkaline detergent builders, when present, include the water-soluble salts, especially alkali metal pyrophosphates, orthophosphates, and polyphosphates.
- An example of phosphorus -containing organic alkaline detergent builder, when present, includes the water-soluble salts of phosphonates.
- non-phosphorus-containing inorganic builders when present, include water-soluble alkali metal carbonates, borates, and silicates, as well as the various types of water-insoluble crystalline or amorphous alumino silicates, of which zeolites are the best-known representatives.
- suitable organic builders include the alkali metal; ammonium and substituted ammonium; citrates; succinates; malonates; fatty acid sulphonates; carboxymethoxy succinates; ammonium poly acetates; carboxylates; polycarboxylates; aminopolycarboxylates; polyacetyl carboxylates; and polyhydroxysulphonates.
- Suitable organic builders include the higher molecular weight polymers and copolymers known to have builder properties, for example appropriate polyacrylic acid, polymaleic and polyacrylic/polymaleic acid copolymers, and their salts.
- the cleaning composition may contain bleaching agents of the chlorine/bromine-type or the oxygen-type.
- inorganic chlorine/bromine-type bleaches are lithium, sodium or calcium hypochlorite, and hypobromite, as well as chlorinated trisodium phosphate.
- organic chlorine/bromine-type bleaches are heterocyclic N-bromo-and N-chloro- imides such as trichloroisocyanuric, tribromoisocyanuric, dibromoisocyanuric, and dichloroisocyanuric acids, and salts thereof with water-solubilizing cations such as potassium and sodium. Hydantoin compounds are also suitable.
- the cleaning composition may contain oxygen bleaches, for example in the form of an inorganic persalt, optionally with a bleach precursor or as a peroxy acid compound.
- oxygen bleaches for example in the form of an inorganic persalt, optionally with a bleach precursor or as a peroxy acid compound.
- suitable peroxy bleach compounds are alkali metal perborates, both tetrahydrates and monohydrates, alkali metal percarbonates, persilicates, and perphosphates.
- exemplary activator materials are TAED, and glycerol triacetate.
- Enzymatic bleach activation systems may also be present in the formulation, e.g., such as perborate or percarbonate, glycerol triacetate and perhydrolase ⁇ see, e.g., WO 2005/056783).
- the cleaning composition may be stabilized using conventional stabilizing agents for the enzyme(s), e.g., a polyol such as, e.g., propylene glycol, a sugar or a sugar alcohol, lactic acid, boric acid, or a boric acid derivative ⁇ e.g., an aromatic borate ester).
- a polyol such as, e.g., propylene glycol, a sugar or a sugar alcohol, lactic acid, boric acid, or a boric acid derivative ⁇ e.g., an aromatic borate ester.
- the cleaning composition may also contain other conventional detergent ingredients, e.g., deflocculant material, filler material, foam depressors, anti-corrosion agents, soil- suspending agents, sequestering agents, anti-soil redeposition agents, dehydrating agents, dyes, bactericides, fluorescers, thickeners, and perfumes.
- a "swatch" is a piece of material such as a fabric that has a stain applied thereto.
- the material can be, for example, fabrics made of cotton, polyester or mixtures of natural and synthetic fibers.
- the swatch can further be paper, such as filter paper or nitrocellulose, or a piece of a hard material such as ceramic, metal, or glass.
- the stain is starch based, but can include blood, milk, ink, grass, tea, wine, spinach, gravy, chocolate, egg, cheese, clay, pigment, oil, or mixtures of these compounds.
- a "smaller swatch” is a section of the swatch that has been cut with a single hole punch device, or has been cut with a custom manufactured 96-hole punch device, where the pattern of the multi-hole punch is matched to standard 96-well microtiter plates, or the section has been otherwise removed from the swatch.
- the swatch can be of textile, paper, metal, or other suitable material.
- the smaller swatch can have the stain affixed either before or after it is placed into the well of a 24-, 48- or 96-well microtiter plate.
- the "smaller swatch” can also be made by applying a stain to a small piece of material.
- the smaller swatch can be a stained piece of fabric 5/8" or 0.25" in diameter.
- the custom manufactured punch is designed in such a manner that it delivers 96 swatches simultaneously to all wells of a 96-well plate.
- the device allows delivery of more than one swatch per well by simply loading the same 96-well plate multiple times.
- Multi-hole punch devices can be conceived of to deliver simultaneously swatches to any format plate, including but not limited to 24-well, 48-well, and 96-well plates.
- the soiled test platform can be a bead made of either metal, plastic, glass, ceramic, or other suitable material that is coated with the soil substrate.
- the one or more coated beads are then placed into wells of 96-, 48-, or 24- well plates or larger formats, containing suitable buffer and enzyme.
- supernatant can be examined for released soil either by direct absorbance measurement or after a secondary color development reaction. Analysis of the released soil might also be taken by mass spectral analysis.
- a further microscreening assay can be to deliver and secure a swatch, for example an indigo dyed denim, to a well of a multi-well plate, and add particles such as sand or larger particles such as for example garnet sieved to include particle 6 to 8, or 9 gauge, and agitate the plate so as to cause abrasion of the swatch by the added particles.
- This assay has found use in the assessment of cellulases in stone washing applications.
- the effectiveness of the enzyme can be judged by either color release (e.g., released indigo is dissolved in dimethylsulfoxide and absorbance at A ⁇ oo nm is measured) to the reaction buffer or by reflectance measurements of the abraded swatch.
- Swatches having stains of known "strength" on various types of material are commercially available (EMPA, St. Gallen, Switzerland; wfk— Testgewebe GmbH, Krefeld Germany; or Center for Test Materials, Vlaardingen, The Netherlands) and/or can be made by the practitioner (Morris and Prato, Textile Research Journal 52(4): 280 286 (1982)).
- test swatches include but are not limited to blood/milk/ink (BMI) stain(s) on a cotton-containing fabric, a spinach stain on a cotton-containing fabric, or grass on a cotton-containing fabric, and chocolate/milk/soot on a cotton-containing fabric.
- BMI blood/milk/ink
- a BMI stain can be fixed to cotton with 0.0003% to 0.3% hydrogen peroxide.
- Other combinations include grass or spinach fixed with 0.001% to 1% glutaraldehyde, gelatin and Coomassie stain fixed with 0.001% to 1% glutaraldehyde, or chocolate, milk and soot fixed with 0.001% to 1% glutaraldehyde.
- the swatch can also be agitated during incubation with the enzyme and/or detergent formulation. Wash performance data is dependent on the orientation of the swatches in the wells (horizontal versus vertical), particularly in the 96-well plate. This would indicate that mixing was insufficient during the incubation period.
- a plate holder in which the microtiter plate is sandwiched between two plates of aluminum can be constructed. This can be as simple as placing, for example, an adhesive plate sealer over the wells then clamping the two aluminum plates to the 96-well plate with any type of appropriate, commercially available clamps. It can then be mounted in a commercial incubator shaker.
- Trinitrobenzenesulfonic acid can be used to quantify the concentration of amino groups in the wash liquor. This can serve as a measure of the amount of protein that was removed from the swatch ⁇ see e.g., Cayot and Tainturier, Anal. Biochem. 249: 184-200 (1997)). However, if a detergent or an enzyme sample leads to the formation of unusually small peptide fragments (for example, from the presence of peptidases in the sample), then one will obtain a larger TNBS signal, i.e., more "noise".
- Another means of measuring wash performance of blood/milk/ink or other stain is based on ink release. Proteolysis of protein on the swatches leads to the release of ink particles which can be quantified by measuring the absorbance of the wash liquor. The absorbance can be measured at any wavelength between 350 and 800 nm. The absorbance is measured at 410 nm or 620 nm. The wash liquor can also be examined to determine the wash performance on stains containing grass, spinach, gelatin or Coomassie stain. Exemplary wavelengths for these stains include 670 nm for spinach or grass and 620 nm for gelatin or Coomassie.
- an aliquot of the wash liquor (typically 100-150 ⁇ L from a 96-well microplate, for example) is removed and placed in a cuvette or multiwell microplate. This is then placed in a spectrophotometer and the absorbance is read at an appropriate wavelength.
- the system can also be used to determine an enhanced enzyme and/or detergent composition for dish washing, for example, using a blood/milk/ink stain on a suitable substrate such as cloth, plastic or ceramic.
- the BMI stain is fixed to cotton by applying 0.3% hydrogen peroxide to the BMI/cotton swatch for 30 minutes at 25°C or by applying 0.03% hydrogen peroxide to the BMI/cotton swatch for 30 minutes at 60 0 C.
- Smaller swatches of approximately 0.25" are cut from the BMI/cotton swatch and placed in the wells of a 96-well microtiter plate.
- a known mixture of a detergent composition and an enzyme is placed into each well. After placing an adhesive plate sealer onto the top of the microtiter plate, the microtiter plate is clamped to an aluminum plate and agitated on an orbital shaker at approximately 250 rpm for about 10 to 60 minutes.
- the supernatants are transferred to wells in a new microtiter plate and the absorbance of the ink at 620 nm is measured.
- This can be similarly tested with spinach stains or grass stains fixed to cotton by applying 0.01% glutaraldehyde to the spinach/cotton swatch or grass/cotton swatch for 30 minutes at 25 0 C. The same can be done with chocolate, milk, and/or soot stains.
- Strain WDG195 was obtained from Prof. Grant, University of Leicester, UK. This strain was isolated from soda soil on the shore of Mono Lake, California, USA. Partial sequencing of the 16S rRNA gene (429 bp) indicated that the strain belongs to the species Halomonas variabilis. The partial 16S rRNA gene sequence is shown in SEQ ID NO: 2. [00238] Cells of Halomonas variabilis WDG 195 were cultivated on an alkaline medium, for example using methods according to Duckworth et ah, FEMS Microbiology Letters 19 (1996) 181-191.
- the resulting genomic DNA was used by Eurofins Medigenomix GmbH (Martinsried, Germany) for the construction of an expression library. Fragments of 3-5 kb were obtained by random shearing and the fragments were cloned into low copy number vector pRANGER-BTB3-Cat (Lucigen Corporation, Middleton, WI USA, DQ058731) ( Figure 1).
- the vector was first modified for TA shotgun cloning by Medigenomix.
- Ligation mixtures were transformed into electrocompetent TOPlO E. coli cells (Invitrogen Carlsbad, CA, USA) and plated on RBB plates. These agar plates contain 10 g yeast extract, 16 g tryptone, 5 g NaCl, 15 g agar, 25 mg chloramphenicol, and 5 g starch dyed with Remazol brilliant blue R (Fluka / Sigma-Aldrich Chemie B.V., Zwijndrecht, The Netherlands) per litter. An E. coli clone that formed a halo on the RBB plate, indicating the presence of an amylase gene that originates from Halomonas variabilis WG 195, was selected for further analysis.
- the insert was sequenced by Baseclear (Leiden, The Netherlands) using the primers pRANGER-FW (SEQ ID NO: 3) and pRANGER-RV (SEQ ID NO: 4), which hybridize to the pRANGER vector. Based on this initial sequence analysis, the complete sequence of the amyWDG195 gene (SEQ ID NO: 9) was determined using four internal primers (SEQ ID NO: 5, 6, 7 and 8).
- the amyWDG195 gene encodes a precursor protein of 502 amino acids (SEQ ID NO: 10).
- the precursor protein consist of a signal peptide of 22 amino acids (SEQ ID NO: 11) and a mature portion of 480 amino acids (AmyWDG195, SEQ ID NO: 12).
- the amyWDG195 amylase gene was amplified from the genomic DNA sample by PCR performed on a MJ Research PTC-200 thermocycler with Phusion High Fidelity DNA polymerase (Finnzymes OY, Espoo, Finland) according to the instructions of the manufacturer (annealing temperature of 55°C).
- the two primers used were: Pstl-Amyl95-I-Fw (SEQ ID NO: 13) and Hpal-Amyl95- Rv (SEQ ID NO: 14).
- the resulting PCR fragment was digested with restriction enzymes Pstl and Hpal, and incubated with pHPLT DNA (50 ng/ ⁇ l, also digested with Pstl and Hpa ⁇ ) in the presence of T4 DNA ligase according to the instructions of the supplier (Roche Applied Science, Indianapolis, IN, USA). The ligation mixture was transformed into B.
- subtilis strain BG6006 which is deleted for nine proteases ⁇ i.e., degUHy32,oppA,DspoII3501, amyE::xylRPxylAcomK- ermC, DaprE, DnprE, Depr, DispA, Dbpr, Dvpr, DwprA, Dmpr-ybfJ, DnprB) [see, e.g., U.S. Pat. Pub Nos. 20050202535 and 20020182734 (WO 02/14490)].
- the transformation mixture was plated on DifcoTM Heart Infusion Agar plates containing 10 mg/L neomycin and 0.5% starch dyed with Remazol brilliant blue R.
- a starch-halo-positive colony was selected and the presence of the AmyWDG195 amylase gene in pHPLT was confirmed using colony PCR and sequencing.
- colony PCR the transformed bacteria was resuspended in 100 ⁇ L of sterile water of which 1 ⁇ L was used in a PCR reaction containing Invitrogen Platinum Taq DNA Polymerase and two primers: pHPLT-Fl (SEQ ID NO: 15) and pHPLT-Rl (SEQ ID NO: 16). [00243] PCR was performed at 95 0 C for 2 minutes followed by 25 cycles at 95 0 C for 0.5 minute, 55°C for 1 minute, and 68 0 C for 2 minutes.
- the reaction was stopped after a final incubation at 68 0 C for 5 minutes.
- the PCR product was sequenced at BaseClear (Leiden, The Netherlands) using primers pHPLT-Fl (SEQ ID NO: 15), pHPLT-Rl (SEQ ID NO: 16) and Amyl95.int.-Rv (SEQ ID NO: 17).
- AmySWT282 An amylase (AmySWT282) similar to AmyWDG195 (-78% amino acid identity) was identified from the alkaliphilic Bacillus strain SWT282 (isolated on alkaline tomato puree agar from a sample provided by Dr. Dimitri Sorokin (Inst, of Microbiology, Moscow, Russia), which was collected from the Kalunda Steppe (north) around Karasuk in the Novosibirsk region of Russia).
- the cycling conditions were as follows: 95 0 C, 4'; 95 0 C 1'; 50 0 C, 1'; 72°C, T30"; 72°C, 5'; 4°C.
- a short DNA fragment of roughly 700 bp was amplified (as visualized on a 1.2% agarase gel) and sequenced.
- the DNA sequence of this fragment is shown in Figure 9 as SEQ ID NO: 18.
- a BlastX search proved that this fragment indeed encodes an alpha-amylase fragment.
- PCR reactions were set up using the MMQFEW forward primer separately in combination with each of the reverse primers listed above.
- Two ⁇ L of genomic DNA template and 1 ⁇ M of each primer (final concentration) were combined with water to a final volume of 25 ⁇ l with a PCR bead (GE Healthcare).
- the cycling conditions were as follows: 95°C, 4', 95°C, 1'; 50 0 C, 1'; 72°C, l'3O"; 72°C, 5'; 4°C.
- a 2.5 ⁇ l sample of the PCR reaction was analyzed on a 1.2% agarose gel.
- the forward MMQFEW and reverse VTINGDGWGEF primers produced a 1.4kb band on the gel.
- the band was gel purified using Qiagen gel purification kit and the DNA was eluted in 35 ⁇ l of buffer EB. 4 ⁇ l of the purified fragment was cloned into the PCR2.1 TOPO ® vector using a 30 min TOPO ® cloning reaction (Invitrogen Corp., Carlsbad, CA) at room temp. 2.5 ⁇ l of the reaction was transformed into 50 ⁇ l of Top 10 chemically competent cells. After ice and heat shock, 250 ⁇ l of SOC media was added to the cells and sample shaken at 37°C for 1 hour.
- AmySWT282 genomic DNA was digested with Xbal and ligated to the Xbal cassette.
- the first PCR reaction for the N terminal portion of the gene were carried out with primers 282 N3 (5'-
- the resulting PCR products were gel purified and ligated into the Topo TA vector, then transformed into 50 ⁇ l of Top 10 chemically competent cells. 4 colonies each were picked for PCR with primers Topo F and TOPO R (described, above) and the PCR products were sequenced. This resulted in new sequence information of the SWT282 amylase gene.
- amySWT282 The last piece of sequence information of the amySWT282 gene was retrieved by directly sequencing genomic DNA of Bacillus strain SWT282 (Fidelity systems, Cessna Avenue, Gaithersburg, MD), thereby completing the sequence of the amySWT282 gene.
- the full-length amySWT282 gene and AmySWT282 (precursor) protein sequences are shown as SEQ ID NOs: 19 and NO: 22, respectively.
- AmySWT282 has a signal peptide (SEQ ID NO: 21 and NO: 24), and is thus an extracellular protein.
- the mature AmySWT282 polypeptide has 479 amino acid residues (SEQ ID NO: 23) and is encoded by the polynucleotide of SEQ ID NO: 20.
- AmySWT282 polypeptides showed moderate homology ( ⁇ 61% identity) to the ⁇ -amylases from Bacillus licheniformis, Geobacillus (formerly Bacillus) stearothermophilus , and Bacillus amyloliquefaciens.
- amySWT282 gene was cloned into the pHPLT B. subtilis expression vector as described for amyWDG195 in Example 2.
- a B. subtilis transformant harboring pHPLT- AmySWT282 was grown in shake flasks containing MBD medium with 10 mg/L neomycin, 20 mM extra CaCl 2 , and with and without additional NaCl (3%). After 72 h growth, culture supernatants were analyzed by SDS-PAGE ( Figure 6). The addition of extra NaCl to the growth medium was necessary for production of AmySWT282, as observed for AmyWDG195. In contrast, the addition of CaCl 2 showed no beneficial affect on the heterologous expression of AmySWT282.
- ⁇ -amylase cleaning assay For the ⁇ -amylase cleaning assay, a preselected buffer was added to the wells and equilibrated to a preselected temperature. For initial testing the buffers were 25 mM HEPES (pH 8.0) or 25 mM CAPS (pH 10.0), additionally containing 2 mM CaCl 2 and 0.0005% TWEEN 80. About 200 ⁇ L of buffer was added to different wells and allowed to equilibrate at 40 0 C. 10-20 ⁇ L of diluted culture supernatant solution (final amylase concentration of 0 ppm to 2 ppm) were added to selected wells and the plates incubated with shaking at 1150 rpm for 1 hour.
- the buffers were 25 mM HEPES (pH 8.0) or 25 mM CAPS (pH 10.0), additionally containing 2 mM CaCl 2 and 0.0005% TWEEN 80.
- About 200 ⁇ L of buffer was added to different wells and allowed to
- Enzyme cleaning performance was based on the amount of enzyme-dependent color released into the wash liquor. Color release was quantified spectro-photometrically at 488 nm by the transfer of 100 ⁇ L final wash liquor to a fresh microtiter plate. The data were plotted with the aid of GRAFIT from Erithicus Software. The data points were fitted with the Langmuir isotherm- fitting algorithm, which takes the same form as the Michaelis-Menten fitting algorithm. [00255] The results of this assay demonstrated that AmyWDG195 is highly efficient at removing starchy stains from textile swatches at 40 0 C at pH 8.0, but slightly less efficient at pH 10.0 ( Figures 4 and 5).
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EP10705768.9A EP2417254B1 (en) | 2009-04-08 | 2010-02-25 | Halomonas strain wdg195-related alpha-amylases, and methods of use, thereof |
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EP2417254A1 (en) | 2012-02-15 |
US20120171748A1 (en) | 2012-07-05 |
CN102388131B (en) | 2014-04-30 |
CN102388131A (en) | 2012-03-21 |
BRPI1012590A2 (en) | 2015-09-22 |
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US8877479B2 (en) | 2014-11-04 |
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