# US 8,541,220 B2 · Isomerases, nucleic acids encoding them and methods for making and using them

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- Number: US 8,541,220 B2
- Document ID: US8541220B2
- Status: Granted
- Kind: B2
- Application number: 12/810,067
- Filed: 2008-12-22
- Published: 2011-03-03
- Granted: 2013-09-24
- Earliest claimed: 2008-01-03
- Assignees: Verenium Corporation
- Applicants: David P. Weiner; Ellen G. Burke; Peter Luginbuhl; Analia Bueno; Joslin M. Cuenca; Mervyn L. De Souza; Sherry Kollmann
- Inventors: David P. Weiner; Ellen G. Burke; Peter Luginbuhl; Analia Bueno; Joslin M. Cuenca; Mervyn L. De Souza; Sherry Kollmann
- Web page: https://patentgrep.com/patent/US8541220B2

## CPC

- C12N 9/90: Isomerases (5.)
- A01K 67/0275: Genetically modified vertebrates, e.g. transgenic
- A23G 3/366: {containing microorganisms, enzymes}
- A23G 4/123: {containing microorganisms, enzymes}
- A23G 4/14: containing peptides or proteins (containing dairy products A23G4/16)
- A23K 20/189: Enzymes
- A23L 29/06: {Enzymes}
- A61K 38/52: Isomerases (5)
- C12Q 1/533: involving isomerase
- A01K 2217/05: Animals comprising random inserted nucleic acids (transgenic)
- A01K 2227/10: Mammal
- A23V 2002/00: Food compositions, function of food ingredients or processes for food or foodstuffs
- A61K 38/00: Medicinal preparations containing peptides (peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00)
- C12Y 501/00: Racemaces and epimerases (5.1)
- C12Y 501/01: acting on amino acids and derivatives (5.1.1)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A23G: COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
- A23K: FODDER
- A23L: FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF (preservation thereof A23B)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- A23V: INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- C12Y: ENZYMES

## Abstract

This invention relates generally to enzymes, polynucleotides encoding the enzymes, the use of such polynucleotides and polypeptides and more specifically to enzymes having isomerase activity, e.g., racemase activity, e.g., amino acid racemase activity, alanine racemase activity, and/or epimerase activity, and/or catalyze the re-arrangement of atoms within a molecule, catalyze the conversion of one isomer into another, catalyze the conversion of an optically active substrate into a raceme, which is optically inactive, catalyze the interconversion of substrate enantiomers, catalyze the stereochemical inversion around the asymmetric carbon atom in a substrate having only one center of asymmetry, catalyze the stereochemical inversion of the configuration around an asymmetric carbon atom in a substrate having more than one asymmetric center, and/or catalyze the racemization of amino acids. Thus, the invention provides enzymes, compositions, methods for production of pharmaceutical compositions, pharmaceutical intermediates, antibiotics, sweeteners, peptide enzymes, peptide hormones, fuel and fuel additive compositions, foods and food additives, beverage and beverage additives, feeds and feed additives, drugs and drug additives, dietary supplements, textiles, wood, paper, pulp, and detergents comprising the polypeptides or polynucleotides in accordance with the invention.

## Claims

### Claim 1

An isolated, synthetic, or recombinant polypeptide or peptide having a racemase activity, selected from a group consisting of:
(a) full length amino acid sequence having at least 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the full length amino acid sequence of SEQ ID NO:412,
(b) an amino acid sequence encoded by a full length nucleic acid sequence of SEQ ID NO:411;
(c) the amino acid sequence of any of (a) to (b), and comprising at least one amino acid residue conservative substitution, and the polypeptide or peptide retains racemase activity; wherein the conservative substitution comprises replacement of an aliphatic amino acid with another aliphatic amino acid; replacement of a serine with a threonine or vice versa; replacement of an acidic residue with another acidic residue; replacement of a residue bearing an amide group with another residue bearing amide group; exchange of a basic residue with another basic residue; or, replacement of an aromatic residue with another aromatic residue, or a combination thereof,
(d) the amino acid sequence of (c), wherein the aliphatic residue comprises Alanine, Valine, Leucine, Isoleucine or a synthetic equivalent thereof; the acidic residue comprises Aspartic acid, Glutamic acid or a synthetic equivalent thereof; the residue comprising an amide group comprises Aspartic acid, Glutamic acid or a synthetic equivalent thereof; the basic residue comprises Lysine, Arginine or a synthetic equivalent thereof; or, the aromatic residue comprises Phenylalanine, Tyrosine or a synthetic equivalent thereof;
(e) the polypeptide of any of (a) to (d) but lacking a signal sequence, a prepro domain, or other domain;
(f) the polypeptide of any of (a) to (e) and further comprising a heterologous sequence;
(g) the polypeptide of (f), wherein the heterologous sequence comprises: (A) a heterologous signal sequence, a heterologous domain, a heterologous dockerin domain, a heterologous catalytic domain (CD), or a combination thereof; (B) the sequence of (A), wherein the heterologous signal sequence, domain or catalytic domain (CD) is derived from a heterologous enzyme; or, (C) a tag, an epitope, a targeting peptide, a cleavable sequence, a detectable moiety or an enzyme; or
(h) the polypeptide of any of (a) to (g), wherein the polypeptide is glycosylated or comprises at least one glycosylation site or further comprises a polysaccharide, wherein optionally the glycosylation is an N-linked glycosylation, and optionally the polypeptide is glycosylated after being expressed in a P. pastoris or a S. pombe.

### Claim 2 (depends on 1)

A beverage, feed, food, food or feed additive, food or feed supplement, or dietary aid comprising the amino acid sequence of claim 1, wherein optionally, the feed, food, food or feed additive, food or feed supplement, or dietary aid further comprises a carrier selected from the group consisting of a grain germ, a grain germ that is spent of oil, a hay, an alfalfa, a timothy, a soy hull, a sunflower seed meal and a wheat midd, and optionally the carrier comprises grain germ that is spent of oil, or optionally the racemase, is glycosylated to provide thermostability at pelletizing conditions, and optionally the delivery matrix is formed by pelletizing a mixture comprising a grain germ and an isomerase, a racemase, an amino acid racemase, an alanine racemase, or an epimerase, and optionally the pelletizing conditions include application of steam, and optionally the pelletizing conditions comprise application of a temperature in excess of about 80° C. for about 5 minutes and the enzyme retains a specific activity of at least 350 to about 900 units per milligram of enzyme.

### Claim 3 (depends on 1)

A composition comprising an organic material comprising a polypeptide having the amino acid sequence of claim 1, wherein optionally the organic material is a biomass, wood, a wood pulp, a Kraft pulp, a paper, a paper product or a paper pulp, and wherein optionally, the biomass, wood, wood pulp, Kraft pulp, paper, paper product or paper pulp comprises a softwood and hardwood, or the wood, wood pulp, Kraft pulp, paper or paper pulp is derived from a softwood and hardwood.

### Claim 4 (depends on 1)

A waste treatment solution comprising a polypeptide having the amino acid sequence of claim 1.

### Claim 5 (depends on 1)

An enzyme mixture or cocktail comprising (a) the enzyme of claim 1, and one or more other enzyme(s); (b) the mixture or cocktail of (a), wherein the one or more other enzyme(s) is an isomerase, racemase, amino acid racemase, alanine racemase, epimerase, a mannanase, a glucanase, cellulases, lipases, esterases, proteases, or endoglycosidases, endo-beta.-1,4-glucanases, beta-glucanases, endo-beta-1,3(4)-glucanases, cutinases, peroxidases, catalases, laccases, amylases, glucoamylases, pectinases, transferases, transaminases, amino transferases, dehydrogenases, oxidoreductases, reductases, oxidases, phenoloxidases, ligninases, pullulanases, arabinanases, hemicellulases, xyloglucanases, xylanase, pectin acetyl esterases, rhamnogalacturonan acetyl esterases, polygalacturonases, rhamnogalacturonases, galactanases, pectin lyases, pectin methylesterases, cellobiohydrolases or transglutaminases.

### Claim 6 (depends on 1)

A composition comprising the polypeptide of claim 1.

### Claim 7

An isolated, synthetic, or recombinant polypeptide having a racemase activity, wherein the polypeptide is a full length amino acid sequence of SEQ ID NO:412.