EP4028517A1 - Variants thermostables de glucose isomérase - Google Patents

Variants thermostables de glucose isomérase

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Publication number
EP4028517A1
EP4028517A1 EP20772507.8A EP20772507A EP4028517A1 EP 4028517 A1 EP4028517 A1 EP 4028517A1 EP 20772507 A EP20772507 A EP 20772507A EP 4028517 A1 EP4028517 A1 EP 4028517A1
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EP
European Patent Office
Prior art keywords
variant
seq
amino acid
polypeptide
numbering
Prior art date
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Pending
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EP20772507.8A
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German (de)
English (en)
Inventor
William A. Cuevas
Carol Marie FIORESI
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Danisco US Inc
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Danisco US Inc
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Publication date
Application filed by Danisco US Inc filed Critical Danisco US Inc
Publication of EP4028517A1 publication Critical patent/EP4028517A1/fr
Pending legal-status Critical Current

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Classifications

    • 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
    • C12N9/90—Isomerases (5.)
    • C12N9/92—Glucose isomerase (5.3.1.5; 5.3.1.9; 5.3.1.18)
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L29/00—Foods or foodstuffs containing additives; Preparation or treatment thereof
    • A23L29/30—Foods or foodstuffs containing additives; Preparation or treatment thereof containing carbohydrate syrups; containing sugars; containing sugar alcohols, e.g. xylitol; containing starch hydrolysates, e.g. dextrin
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
    • 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
    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/24—Preparation of compounds containing saccharide radicals produced by the action of an isomerase, e.g. fructose
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y503/00—Intramolecular oxidoreductases (5.3)
    • C12Y503/01—Intramolecular oxidoreductases (5.3) interconverting aldoses and ketoses (5.3.1)
    • C12Y503/01005—Xylose isomerase (5.3.1.5)

Definitions

  • compositions and methods relating to thermostable glucose isomerase variants are particularly useful for making high fructose com sugar, in some cases with reduced need for a chromatographic enrichment step.
  • Glucose isomerase is an enzyme used to convert glucose, typically obtained from the hydrolysis of com starch, to fructose, which is much sweeter to the taste and of higher value to the food and beverage industry than glucose.
  • the global market for GI is about 30 million USD/year.
  • D-glucose syrup obtained from wet milling com is introduced into a series of reactors (i.e., columns) containing immobilized glucose isomerase (IGI), in a down-fed manner, to obtain high fructose com syrup (HFCS).
  • IGI immobilized glucose isomerase
  • HFCS high fructose com syrup
  • IGI immobilized glucose isomerase
  • HFCS-42 immobilized wild-type GI from Streptomycese rubiginosus
  • Conversion is typically to standard target fructose concentrations, such as about 42% HFCS (HFCS-42), which is used for food and beverages, and 55% HFCS (HFCS-55), which is preferred for beverages.
  • HFCS-55 a portion of HFCS-42 is typically subjected to chromatographic enrichment to produce 90% HFCS (HFCS-90), which is then blended with HFCS-42 to produce HFCS-55.
  • HFCS-90 90% HFCS
  • IGI The process of making HFCS using IGI is described in, for example, U.S. Pat. Nos. 5,177,005, 5,437,993, 5,811,280, 5,916,789 and 7,297,510.
  • thermostable variant glucose isomerase polypeptides relate to thermostable variant glucose isomerase polypeptides, and methods of use, thereof. Aspects and embodiments of the present compositions and methods are summarized in the following separately-numbered paragraphs:
  • a non-naturally-occuring variant of a parent glucose isomerase comprising a mutation at an amino acid residue corresponding to a position selected from 41, 59, 70, 71, 89, 212, 297 and 314, using SEQ ID NO: 1 for numbering.
  • the mutation is selected from 41K, 59V 70K, 70M, 71K, 89M, 212F, 2971 and 314S using SEQ ID NO: 1 for numbering.
  • the mutation is selected from R41K, I59V E70K, E70M, H71K, A89M, Y212F, D297I and K314S using SEQ ID NO: 1 for numbering.
  • the variant comprises at least any two, three, four or more mutations.
  • the variant is derived from a GI from Streptomyces rubiginosus.
  • the variant has at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% but less than 100% amino acid sequence identity to the polypeptide of SEQ ID NO: 1 or wherein the variant has at least 60%, at least 70%, at least 80%, at least 90% or at least 95% amino acid sequence identity to the polypeptide of SEQ ID NO: 3.
  • a process for producing high-fructose com syrup comprising greater than about 42% fructose, comprising contacting a glucose syrup with a variant of a parent glucose isomerase (GI) comprising one or more mutations at an amino acid residue corresponding to a position selected from 41, 59, 70, 70, 71, 89, 212, 297 and 314, using SEQ ID NO: 1 for numbering, wherein the variant has altered GI activity and/or increased thermal stability compared to the GI of the parent, wherein the process involving contacting a glucose syrup with the variant GI requires reduced chromatographic enrichment to produce a subsequent HFCS comprising a total of greater than 55% fructose compared to the amount of chromatographic enrichment required to obtain 55% fructose prepared by an equivalent method utilizing the parent GI.
  • GI glucose isomerase
  • variant GI comprises a mutation selected from 41K, 59V 70K, 70M, 71K, 89M, 212F, 2971 and 314S using SEQ ID NO: 1 for numbering.
  • variant GI comprises a mutation selected from R41K, I59V E70K, E70M, H71K, A89M, Y212F, D297I and K314S 212F using SEQ ID NO: 1 for numbering.
  • variant GI comprises at least any two, three, four or more mutations.
  • the variant GI is derived from a GI from Streptomyces rubiginosus .
  • the variant has at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% but less than 100% amino acid sequence identity to the polypeptide of SEQ ID NO: 1 or wherein the variant has at least 60%, at least 70%, at least 80%, at least 90% or at least 95% amino acid sequence identity to the polypeptide of SEQ ID NO: 3.
  • Figure 1 is a graph showing residual activity after immobilization versus specific activity of wild-type GI and several SEL variants.
  • glucose isomerase is a name commonly used in the sweetener industry to refer to a xylose isomerase (EC 5.3.1.5) for use in converting glucose to fructose for the production of high fructose com syrup (HFCS).
  • a xylose isomerase catalyzes the interconversion of D-xylose and D-xylulose.
  • the systematic name of the enzyme class is D- xylose aldose-ketose-isomerase.
  • Other names in common use include D-xylose isomerase, D- xylose ketoisomerase, and D-xylose ketol-isomerase.
  • starch refers to any material comprised of the complex polysaccharide carbohydrates of plants, comprised of amylose and amylopectin with the formula (C6HIO05) X , wherein X can be any number.
  • the term includes plant-based materials such as grains, cereal, grasses, tubers and roots, and more specifically materials obtained from wheat, barley, com, rye, rice, sorghum, brans, cassava, millet, milo, potato, sweet potato, and tapioca.
  • wild-type refers 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.
  • recombinant when used in reference to a subject cell, nucleic acid, protein or vector, indicates that the subject has been modified from its native state.
  • 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.
  • Recombinant nucleic acids differ from a native sequence by one or more nucleotides and/or are operably linked to heterologous sequences, e.g., a heterologous promoter in an expression vector.
  • Recombinant proteins may differ from a native sequence by one or more amino acids and/or are fused with heterologous sequences.
  • a vector comprising a nucleic acid encoding an amylase is a recombinant vector.
  • the terms “recovered,” “isolated,” and “separated,” refer to a compound, protein (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.
  • An “isolated” polypeptides, thereof, includes, but is not limited to, a culture broth containing secreted polypeptide expressed in a heterologous host cell.
  • 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.
  • enriched refers to material (e.g., an isolated polypeptide or polynucleotide) that is in about 50% pure, at least about 60% pure, at least about 70% pure, or even at least about 70% pure.
  • thermostability refers to the ability of the enzyme to retain activity after exposure to an elevated temperature.
  • the thermostability of an enzyme is measured by its half-life (tl/2) given in min, hours, or days, during which half the enzyme activity is lost under defined conditions.
  • the half-life may be calculated by measuring residual a-amylase activity following exposure to (i.e., challenge by) an elevated temperature.
  • a “mature” polypeptide or variant, thereof, is one in which a signal sequence is absent, for example, cleaved from an immature form of the polypeptide during or following expression of the polypeptide.
  • variant refers to a polypeptide that differs from a specified wild-type, parental, or reference polypeptide in that it includes one or more naturally-occurring or man-made substitutions, insertions, or deletions of an amino acid.
  • variant 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.
  • combinatorial variant refers to variants comprising two or more mutations, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more, substitutions, deletions, and/or insertions.
  • 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,” “protein,” and “peptide,” and are used interchangeably. Where such 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 that encode a particular amino acid sequence. Unless otherwise indicated, nucleic acid sequences are presented in 5'-to-3' orientation.
  • transformed means that the cell contains anon-native (e.g., heterologous) nucleic acid sequence integrated into its genome or carried as an episome that is maintained through multiple generations.
  • anon-native e.g., heterologous
  • 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 (e.g., an amylase) has been introduced.
  • Exemplary host strains are microorganism cells (e.g, bacteria, filamentous fungi, and yeast) capable of expressing the polypeptide of interest and/or fermenting saccharides.
  • the term “host cell” includes protoplasts created from cells.
  • 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 “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” refer to a sequence having a specified biological activity, such an enzymatic activity.
  • specific activity refers to the number of moles of substrate that can be converted to product by an enzyme or enzyme preparation per unit time under specific conditions. Specific activity is generally expressed as units (U)/mg of protein.
  • water hardness is a measure of the minerals (e.g., calcium and magnesium) present in water.
  • Percent sequence identity means that a particular sequence has at least a certain percentage of amino acid residues identical to those in a specified reference sequence, when aligned using the CLUSTAL W algorithm with default parameters. See Thompson et al. (1994) Nucleic Acids Res. 22:4673-80. Default parameters for the CLUSTAL W algorithm are:
  • PCR polymerase chain reaction ppm parts per million, e.g., mg protein per gram dry solid sec seconds
  • the present compositions and methods relate to variants of glucose isomerase (GI) capable of tolerating higher temperatures compared to the wild-type enzyme.
  • GI glucose isomerase
  • IGI immobilized GI
  • the present GI variants allow the production of 55% HFCS (HFCS-55) with reduced need to chromatographically enrich a portion of the HFCS.
  • thermotolerant GI from Thermoanaerobacterium saccharolyticum
  • Thermoanaerobacterium saccharolyticum has been described (e.g., U.S. Pat. Nos. 7,919,300)
  • the present description involves the engineering of a more thermotolerant GI from Streptomycese rubiginosus, which is the most robust, and most preferred GI, on the market.
  • S. rubiginosus glucose isomerase referred to as xylose isomerase according to Genbank Accession No. AAA26838
  • the thermotolerant variant GI has one or more mutations at a position selected from 41, 5970, 71, 89, 212, 297 and 314, using SEQ ID NO: 1 for numbering. In some embodiments, the thermotolerant variant GI has one or more mutations selected from 41K, 59V 70K, 70M, 71K, 89M, 212F, 2971 and 314S using SEQ ID NO: 1 for numbering. In some embodiments, the thermotolerant variant GI has one or more mutations selected from R41K, I59V E70K/M, H71K, A89M, Y212F, D297I and K314S using SEQ ID NO: 1 for numbering.
  • the described variant thermotolerant GI further includes an additional substitution, a deletion and/or an insertion at one a few positions. In some embodiments, the described variant thermotolerant GI further includes an N-terminal and/or C- terminal extension of one or a few amino acid residues. In some embodiments, the described variant thermotolerant GI further includes an N-terminal and/or C-terminal truncation of one or a few amino acid residues. Furthermore, the present variant GI may include any number of conservative amino acid substitutions, which are well-known in the art.
  • the present variant GI may be a “chimeric” or “hybrid” polypeptide, in that it includes at least a portion of a first GI polypeptide, and at least a portion of a second GI polypeptide.
  • the present variant GI may further include heterologous signal sequence, an epitope to allow tracking or purification, or the like.
  • the described variant thermotolerant GI has one or more mutations with respect to SEQ ID NO: 1, and at least 60%, at least 65%, at least 70%, at least
  • the present variant GI may, of course, be immobilized, e.g., as described in for example, U.S. Pat. Nos. 5,177,005, 5,437,993, 5,811,280, 5,916,789 and 7,297,510.
  • nucleic acids encoding a variant GI are provided.
  • the nucleic acid may encode a particular GI, or a GI having a specified degree of amino acid sequence identity to the particular described GI.
  • the nucleic acid encodes a GI having at least 60%, at least 65%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least
  • the nucleic acid is codon optimized and encodes a GI having at least 60%, at least 65%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or even at least
  • the nucleic acid has at least 60%, at least 65%, at least 70%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or even at least 99% homology/identity to SEQ ID NO: 2.
  • nucleic acids may encode the same polypeptide.
  • Nucleic acids that encodes GI can be operably linked to various promoters and regulators in a vector suitable for expressing the GI in host cells.
  • the present GI variants can be produced in homologous or heterologous host cells, for example, by secretion or intracellular expression.
  • a cultured cell material e.g., a whole-cell broth
  • the variant GI can be isolated from the host cells, or even isolated from the cell broth, depending on the desired purity of the final variant GI.
  • Suitable host cells include bacterial, fungal (including yeast and filamentous fungi), and plant cells (including algae).
  • Particularly useful host cells include Aspergillus niger , Aspergillus oryzae, Trichoderma reesei, Bacillus subtilis, Bacillus licheniformis and Streptomyces spp., including, in some embodiments, homologous host, Streptomycese rubiginosus .
  • the GI variants are expressed in Bacillus subtilis.
  • compositions and uses of GI variants V. Compositions and uses of GI variants
  • Thermostable GI variants are useful for a variety of industrial applications, including the conversion of starch-derived glucose to high fructose com syrup.
  • the increased thermostability of the GI variant shifts the isomerization equilibrium to favor conversion to fructose, in some cases, allowing the production of a much higher fructose-percent HFCS directly, without the need to chromatographically enrich a portion of the HFCS.
  • the compositons and methods include the ability to produce HFCS-55 (or HFCS having an even higher fraction of fructose) directly, by way of contacting glucose with the present GI variants at a temperature of at least 48°C, at least, 49°C, at least 50°C, at least 51°C, at least 52°C, at least 53°C, at least 54°C, or even at least 55°C, with reduced need to enrich a portion of the HFCS for fructose chromatograpically and then adding the enriched HFCS back to the HFCS contacted directly with GI.
  • the compositons and methods include IGI and columns containing IGI, as disclosed in the aforementioned patent documents, and as used in the HFCS industry for decades.
  • Thermostable variant GI can be immobilized using known processes as described in, e.g., U.S. Pat. Nos. 5,177,005, 5,437,993, 5,811,280, 5,916,789 and 7,297,510.
  • Enzyme activity was determined by mixing 100 m ⁇ enzyme containing material with 100 m ⁇ 1 M glucose in wells of a polypropylene 96-well microtiter plate (MTP; ThermoFisher Scientific; Cat. No. 267245), sealed with adhesive sealing film (BioRad; Cat. No. MSB1001), followed by 60 min reaction incubation at 50°C with 150 rpm in shaking incubator. Reactions were arrested by immediately placing sealed plates on ice. 10 m ⁇ of arrested reactions were mixed with 50 m ⁇ 7.5M hydrochloric acid in a PCR style 96-well microtiter plate (BioRad; Cat. No.
  • GI Units are defined as the change in OD284 nm multiplied by the inverse of the time of the reaction (i.e.
  • Protein determination was carried out using ultra performance liquid chromatography. Clarified culture lysates from cells grown in 96-well MTP for 68 hours at 37°C with shaking at 270 rpm and 70% humidity were prepared by 10X dilution in ultrapure water and filtration (0.45 pm). 10 m ⁇ was injected onto aZorbax 300 SB-C3 column (Agilent, P/N 858750-909) equilibrated with 30% acetonitrile in 0.1% trifluoracetic acid.
  • Chromatography was performed at a flow rate of 1 ml/min, at 80°C, using a 2-min, 30- 95% acetonitrile gradient, in 0.1% trifluoracetic acid. Absorbance was monitored at 220 nm and peaks corresponding to GI were integrated using CHEMSTATIONTM software (Agilent Technologies). Protein concentration was determined based on a standard curve generated using the commercial product GENSWEETTM, having a known concentration, and reported as ppm.
  • Gl-containing lysed broth was immobilized in microtiter plates using a known crosslinking method (Lantero; USPN 4,355,105). Bentonite (Cholino, Patagonia, Argentine;
  • P/N F30 was hydrated in water overnight with stirring.
  • Polyethyleneimine (PEI; Sigma Aldrich P/N 181978), gluteraldehyde GA (Sigma Aldrich), and CELITE 505TM diatomaceous earth (Imerys) was added to the bentonite and mixed for 30 min to create a master mix. 20 pi of this mix was dispersed into MTP containing 1 :4-diluted GI or GI variant cultures using a Biomek FX Robot (Beckman Coulter) and mixed in the pipet tips.
  • thermostability of GI variants was reported as the ratio of the activity of stressed immobilized samples versus the activity of unstressed immobilized samples (residual activity). Immobilized samples were incubated at 65°C for 24 hours (without shaking) in a Kuhner incubator/shaker. Replicate immobilized samples were simultaneously stored at 4°C (representing unstressed materials). Activities of stressed and unstressed materials were determined as described above.
  • thermostability [t residual value] / [t initial value]
  • heat stability activity ratio was calculated based on enzyme activity after heat incubation divided by enzyme activity before heat incubation.
  • B. subtilis strains expressing glucose isomerase and variants thereof [0063] In this example, the construction of Bacillus subtilis strains expressing GI and variants, thereof, is described.
  • a synthetic, codon-optimized DNA encoding wild-type GI from Streptomyces rubiginosus was produced by GeneArt AG (Regensburg, Germany) and served as template DNA for the construction of plasmids for expressing wild-type GI and variants, thereof.
  • the synthetic DNA is represented, below as SEQ ID NO: 2.
  • the valine start codon (underlined), which is preferred in B. subtilis.
  • the stop codon is also underlined.
  • SEQ ID NO: 3 differs from aforementioned SEQ ID NO: 1 only by the presence of valine (underlined), rather than methionine, as the first amino acid residue.
  • the codon-optimized wild-type GI gene was cloned into the pSB expression vector (Babe, L.M. etal. (1998) Biotechnol. Appl. Biochem. 27:117-24) by GeneArt and fused to the B. subtilis aprE promoter using unique restriction sites, resulting in plasmid pSB-GI.
  • This plasmid includes elements from pUBl 10 (McKenzie et al. (1986) Plasmid 15: 93-103) including a neomycin/kanamycin resistance gene (neo) and a bleomycin resistance marker (bleo).
  • a suitable B. subtilis strain was transformed with pSB-GI plasmid DNA using a previosuly descibed method (WO 2002/14490).
  • B. subtilis transformants were selected on Luria agar plates (Teknova) with 10 mg/L neomycin sulfate (Sigma; Cat. No. N-1876), which contains 732 mg neomycin per mg. Selective growth of B.
  • subtilis transformants harboring the pSB-GI plasmid was performed in MTP at 37°C for ⁇ 68 hr in MBD medium (enriched, semi-defined medium, based on MOPS buffer, with urea as the major nitrogen source, glucose as the main carbon source, and supplemented with 1% SOYTONETM (BD Biosciences)) for robust cell growth) containing 10 ppm neomycin. Bacteral growth resulted in the production of intracellular GI.
  • MBD medium enriched, semi-defined medium, based on MOPS buffer, with urea as the major nitrogen source, glucose as the main carbon source, and supplemented with 1% SOYTONETM (BD Biosciences)
  • SEL glucose isomerase site evaluation library
  • the construction of a glucose isomerase site evaluation library (SEL) was performed by GeneArt using its technology platform for gene optimization, gene synthesis, and library generation (see, e.g., European Patent Nos. 0200362 and 0201 184, US Patent Nos. 4,683,195, 4,683,202, and 6,472,184, and international patent application number WO 2004/059556A3).
  • the pSB- GI plasmid DNA served as template to produce a SEL having mutations at 160 amino acid positions in the wild-type GI of SEQ ID NO: 3.
  • the corresponding codons for each site were changed to those encoding each of the different 19 amino acids.
  • the pSB- GI plasmids were sequenced and delivered in a standardized format.
  • the codon-mutagenized pSB- GI plasmids were used to transform competent B. subtilis cells, as described (WO 2002/014490) to generate the GI variant library.
  • Transformation mixtures were plated on Luria agar plates containing 10 mg/L neomycin sulfate. For each library, single bacterial colonies were picked and grown in Luria broth (tryptone and soy-based broth) liquid medium with 10 mg/ml neomycin selection. To generate samples of wild-type GI and variants, thereof, for biochemical characterization, selective growth of the variants was performed in 96-well MTP at 37°C at 270 RPM for ⁇ 68 hours with 70% humidity in MBD medium.
  • results obtained from evaluation the GI SEL library [0070] Expression, specific activity, immobilization yield and stability of the variants as well as the wild-type parental GI were determined as described in Example 1. The results obtained for variants are reported relative to those for the wild-type GI in Table 1.
  • the graph in Figure 1 shows the relationship between thermostability and specific activity. Variants that plot in the upper-right quadrant of the graph are most desirable, but there appears to be a trade-off between stability and activity. Overall, the most ideal variants apear to be R41K, I59V, E70K, E70M, H71K, A89M, Y212F, D297I and K314S. The selection of a particular variant (or several variants for immobilization on a single columns) depends on the desire for speed of fructose conversion versus the desire for the highest possible level of conversion to fructose to reduce the need for chromatographic enrichment.

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Abstract

La présente invention concerne des compositions et des méthodes associées à des variants thermostables de glucose isomérase. Les variants sont particulièrement utiles pour la fabrication de sucre de maïs à haute teneur en fructose, dans certains cas avec un besoin réduit d'une étape d'enrichissement chromatographique.
EP20772507.8A 2019-09-13 2020-09-03 Variants thermostables de glucose isomérase Pending EP4028517A1 (fr)

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