EP4028517A1

EP4028517A1 - Thermostable glucose isomerase variants

Info

Publication number: EP4028517A1
Application number: EP20772507.8A
Authority: EP
Inventors: William A. Cuevas; Carol Marie FIORESI
Original assignee: Danisco US Inc
Current assignee: Danisco US Inc
Priority date: 2019-09-13
Filing date: 2020-09-03
Publication date: 2022-07-20
Also published as: CN114729346B; WO2021050342A1; CN114729346A; BR112022004257A2; MX2022002712A

Abstract

Disclosed are compositions and methods relating to thermostable glucose isomerase variants. The variants are particularly useful for making high fructose corn sugar, in some cases with reduced need for a chromatographic enrichment step.

Description

THERMOSTABLE GLUCOSE ISOMERASE VARIANTS

FIELD OF THE INVENTION

[001] Disclosed are compositions and methods relating to thermostable glucose isomerase variants. The variants are particularly useful for making high fructose com sugar, in some cases with reduced need for a chromatographic enrichment step.

BACKGROUND

[002] Glucose isomerase (GI) 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.

[003] In a typical industrial process, 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). Isomerization equilibrium is a function of temperature, where higher temperatures favor fructose formation. Commercial production is typically performed at 57°C? using 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.

[004] To obtain 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. 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.

SUMMARY

[005] The present compositions and methods 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:

1. In one aspect, a non-naturally-occuring variant of a parent glucose isomerase (GI) is provided, 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. 2. In some embodiments of the variant of paragraph 1, the mutation is selected from 41K, 59V 70K, 70M, 71K, 89M, 212F, 2971 and 314S using SEQ ID NO: 1 for numbering.

3. In some embodiments of the variant of paragraph 1 or 2, the mutation is selected from R41K, I59V E70K, E70M, H71K, A89M, Y212F, D297I and K314S using SEQ ID NO: 1 for numbering.

4. In some embodiments of the variant of any of the preceding paragraphs, the variant comprises at least any two, three, four or more mutations.

5. In some embodiments of the variant of any of the preceding paragraphs, the variant is derived from a GI from Streptomyces rubiginosus.

6. In some embodiments of the variant of any of the preceding paragraphs, 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.

7. In another aspect, a process for producing high-fructose com syrup (HFCS) 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.

8. The process of paragraph 7, wherein the variant GI comprises a mutation selected from 41K, 59V 70K, 70M, 71K, 89M, 212F, 2971 and 314S using SEQ ID NO: 1 for numbering.

9. The method of paragraph 7 or 8, wherein the 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.

10. The process of any of paragraphs 7-9, wherein the variant GI comprises at least any two, three, four or more mutations.

11. The process of any of paragraphs 7-10, wherein the variant GI is derived from a GI from Streptomyces rubiginosus . 12. The process of any of paragraphs 7-11, wherein 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.

13. The process of any of paragraph 7-12, wherein contacting the glucose syrup with the variant glucose isomerase is performed 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.

These and other aspects and embodiments of the compositions and methods/processes will be apparent from the present description and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

[006] Figure 1 is a graph showing residual activity after immobilization versus specific activity of wild-type GI and several SEL variants.

DETAILED DESCRIPTION

I. Definitions and abbreviations

[007] Prior to describing the various aspects and embodiments of the present compositions and methods, the following definitions and abbreviations are described.

[008] The term “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). Formally, 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.

[009] The term “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.

[0010] The terms, “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. Similarly, the terms “wild-type,” “parental,” or “reference,” with respect to a polynucleotide, refer to a naturally-occurring polynucleotide that does not include a man-made nucleoside change. However, note that 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.

[0011] The term “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. Thus, for example, 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.

[0012] 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.

[0013] The term “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.

[0014] The term “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.

[0015] The terms “thermostable” and “thermostability,” with reference to an enzyme, refer 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 (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.

[0016] Reference to the wild-type polypeptide is understood to include the mature form of the polypeptide. 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.

[0017] The term “variant,” with respect to a polypeptide, 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. Similarly, the term “variant,” with respect to 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.

[0018] The term “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.

[0019] A “pH range,” with reference to an enzyme, refers to the range of pH values under which the enzyme exhibits catalytic activity.

[0020] The terms “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).

[0021] The term “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).

[0022] The term “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.

[0023] The terms “transformed,” “stably transformed,” and “transgenic,” used with reference to a cell 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.

[0024] The term “introduced” in the context of inserting a nucleic acid sequence into a cell, means “transfection”, “transformation” or “transduction,” as known in the art. [0025] 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.

[0026] The term “heterologous” with reference to a polynucleotide or protein refers to a polynucleotide or protein that does not naturally occur in a host cell.

[0027] The term “endogenous” with reference to a polynucleotide or protein refers to a polynucleotide or protein that occurs naturally in the host cell.

[0028] The term “expression” refers to the process by which a polypeptide is produced based on a nucleic acid sequence. The process includes both transcription and translation.

[0029] 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. [0030] “Biologically active” refer to a sequence having a specified biological activity, such an enzymatic activity.

[0031] The term “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.

[0032] As used herein, “water hardness” is a measure of the minerals (e.g., calcium and magnesium) present in water.

[0033] “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:

Gap opening penalty: 10.0

Gap extension penalty: 0.05 Protein weight matrix: BLOSUM series

DNA weight matrix: IUB

Delay divergent sequences %: 40 Gap separation distance: 8

DNA transitions weight: 0.50

List hydrophilic residues: GPSNDQEKR

Use negative matrix: OFF Toggle Residue specific penalties: ON Toggle hydrophilic penalties: ON Toggle end gap separation penalty OFF

[0034] Note that the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “an enzyme” includes a plurality of such enzymes, and reference to “the dosage” includes reference to one or more dosages and equivalents thereof known to those skilled in the art, and so forth.

[0035] The present document is organized into a number of sections for ease of reading; however, the reader will appreciate that statements made in one section may apply to other sections. In this manner, the headings used for different sections of the disclosure should not be construed as limiting.

[0036] The following abbreviations/acronyms have the following meanings unless otherwise specified:

°C degrees Celsius dFhO or DI deionized water dlFEO deionized water, Milli-Q filtration

DNA deoxyribonucleic acid

EC Enzyme Commission eq. equivalents g or gm grams GI glucose isomerase

H₂O water

HEPES 2-[4-(2-hydroxyethyl)piperazin-l-yl]ethanesulfonic acid

HFCS high fructose com syrup

HMF hydroxymethylfurfural hr hour

IGI immobilized glucose isomerase kg kilograms

M molar mg milligrams min minute mL and ml milliliters mm millimeters mM millimolar

MOPS 3-(7V-morpholino)propanesul Tonic acid

MTP micro-titer plates

N normal

NCBI National Center for Biotechnology Information

PCR polymerase chain reaction ppm parts per million, e.g., mg protein per gram dry solid sec seconds

SEL site evaluation library sp. species

U units v/v volume/volume w/v weight/volume w/w weight/weight wt wild type

P micrograms pL and pi microliters pm micrometer mM micromolar nm nanometers

OD optical density

GIU glucose isomerase activity units

II. Glucose isomerase variants

[0037] The present compositions and methods relate to variants of glucose isomerase (GI) capable of tolerating higher temperatures compared to the wild-type enzyme. This allows glucose syrup to be contacted with GI, including immobilized GI (IGI) at a higher temperature, shifting the isomerization equilibrium to favor conversion to fructose. The present GI variants allow the production of 55% HFCS (HFCS-55) with reduced need to chromatographically enrich a portion of the HFCS.

[0038] While the engineering of a more thermotolerant GI from 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. [0039] The amino acid sequence of wild-type S. rubiginosus glucose isomerase (referred to as xylose isomerase according to Genbank Accession No. AAA26838) is set forth in SEQ ID NO:

1, below:

1 MNYQPTPEDR FTFGLWTVGW QGRDPFGDAT RRALDPVESV RRLAELGAHG VTFHDDDLIP

61 FGSSDSEREE HVKRFRQALD DTGMKVPMAT TNLFTHPVFK DGGFTANDRD VRRYALRKTI

121 RNIDLAVELG AETYVAWGGR EGAESGGAKD VRDALDRMKE AFDLLGEYVT SQGYDIRFAI

181 EPKPNEPRGD ILLPTVGHAL AFIERLERPE LYGVNPEVGH EQMAGLNFPH GIAQALWAGK

241 LFHIDLNGQN GIKYDQDLRF GAGDLRAAFW LVDLLESAGY SGPRHFDFKP PRTEDFDGVW

301 ASAAGCMRNY LILKERAAAF RADPEVQEAL RASRLDELAR PTAADGLQAL LDDRSAFEEF

361 DVDAAAARGM AFERLDQLAM DHLLGARG

[0040] In some embodiments, 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.

[0041] In some embodiments, 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.

[0042] 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.

[0043] In some embodiments, 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

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% amino acid sequence identity, but less than 100% amino acid sequence identity, to SEQ ID NO: 1, or to an active fragment, thereof. [0044] 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.

III. Nucleotides encoding variant glucose isomerase

[0045] In another aspect, 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.

[0046] In embodiment, 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

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: 1 or 3, or even up to 100% identical to SEQ ID NO: 3.

[0047] In similar embodiment, 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

99% homology/identity to SEQ ID NO: 1 or 3, or even up to 100% identical to SEQ ID NO: 3. [0048] In another embodiment, 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.

It will be appreciated that due to the degeneracy of the genetic code, a plurality of nucleic acids may encode the same polypeptide.

[0049] 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.

IV. Production of variant glucose isomerases

[0050] 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) comprising a variant GI can be obtained following secretion of the variant GI into the cell medium. Optionally, 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 . In a preferred embodiment, the GI variants are expressed in Bacillus subtilis.

V. Compositions and uses of GI variants

[0051] 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. Accordingly, 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.

[0052] 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.

[0053] In order to further illustrate the compositions and methods, and advantages thereof, the following specific examples are given with the understanding that they are illustrative rather than limiting.

EXAMPLES

Example 1 Assays

[0054] Various assays used herein are set forth, below, for ease of reading. Any deviations from the protocols in later Examples are indicated in the relevant sections. In these experiments, a spectrophotometer was used to measure the absorbance of the products formed after the completion of the reactions. A. Glucose isomerase activity determination

[0055] Enzyme activity was determined by mixing 100 mΐ enzyme containing material with 100 mΐ 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 mΐ of arrested reactions were mixed with 50 mΐ 7.5M hydrochloric acid in a PCR style 96-well microtiter plate (BioRad; Cat. No. HSP9621) and incubated for 30 min at 74°C followed by cooling on ice. 10 mΐ were mixed with 100 mΐ water in a UV capable 96-well polypropylene MTP (ThermoFisher; Cat. No. 8404). Optical density at 284 nm (OD284 ) measured for all wells using spectrophotometer was a metric of the amount of hydroxy methylfurfural (HMF) formed by the fructose produced by glucose isomerase from glucose in the first reaction and is proportional to glucose isomerase activity. GI Units (GIU) are defined as the change in OD284 nm multiplied by the inverse of the time of the reaction (i.e. , min ¹), which can be abbreviated as AOD284 n * min ¹ or AOD284 nm / min. All liquid handling steps were carried out using a Biomek FX “robot” (Beckman Coulter). A SpectraMAX MTP Reader (type 340-Molecular Devices) spectrophotometer was used to measure OD values. A thermocycler PCR machine (Biometra), and an incubator/shaker (Kuhner) were used for all incubation steps. Replicate values were averaged. When appropriate, error bars showing standard deviation within replicates are displayed.

B. Protein expression determination

[0056] 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 mΐ was injected onto aZorbax 300 SB-C3 column (Agilent, P/N 858750-909) equilibrated with 30% acetonitrile in 0.1% trifluoracetic acid.

[0057] 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 CHEMSTATION™ software (Agilent Technologies). Protein concentration was determined based on a standard curve generated using the commercial product GENSWEET™, having a known concentration, and reported as ppm.

C. Specific activity determination

[0058] Specific activity is reported as GIU of lysed broth of glucose isomerase / pg glucose isomerase (GIU/pg). D. Immobilization and immobilization yield

[0059] 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 505™ 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.

[0060] Secondary additions (10 mΐ each) of PEI and GA were added and mixed on an external MTP mixer for 5 min and the particles were washed with 10 mM HEPES pH 7.6 containing 1 mM magnesium sulfate and 1 mM sodium metabilsulfite by having the robot add the buffer to the particles and then spinning them down in a centrifuge and using the robot to remove most of the liquid. This procedure was performed three times. The final volume remaining was 100 mΐ. [0061] Immobilization yield was calculated as the ratio of activity present in the immoblized materials versus the activity of the lysate used in their preparation.

E. Thermal stability determination

[0062] The 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. For each variant, the ratio of the initial and residual amylase activities was used to calculate the thermostability as follows: Thermostability (in this limited case abbreviated “t”) = [t residual value] / [t initial value], such that heat stability activity ratio was calculated based on enzyme activity after heat incubation divided by enzyme activity before heat incubation.

Example 2

Generation of 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.

[0064] 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.

GTGAACTACCAACCTACTCCGGAAGACAGATTCACATTCGGACTGTGGACAGTCGGCTGGCAGG

GACGCGACCCTTTCGGTGATGCTACAAGACGCGCGCTTGACCCGGTCGAATCAGTACGCAGATT

GGCTGAACTTGGCGCACACGGAGTCACATTCCACGATGACGACCTTATCCCTTTCGGCTCTAGC

GATAGCGAACGCGAAGAACACGTCAAACGCTTCAGACAAGCGCTGGATGACACTGGCATGAAAG

TACCTATGGCTACAACTAACCTTTTCACACACCCGGTATTCAAAGATGGCGGATTCACTGCTAA

CGATCGCGACGTTAGACGCTACGCACTTAGAAAAACAATCCGCAACATTGATTTGGCGGTCGAA

CTTGGCGCTGAAACTTATGTAGCTTGGGGAGGCCGCGAAGGTGCAGAATCAGGAGGCGCTAAAG

ATGTTAGAGATGCTTTAGATCGCATGAAAGAAGCATTCGACCTTTTGGGCGAATACGTCACATC

TCAAGGATACGATATCAGATTCGCAATTGAACCTAAACCGAACGAACCTCGCGGCGACATCTTG

CTTCCTACTGTCGGCCACGCATTGGCGTTCATCGAAAGACTTGAACGTCCTGAATTATACGGCG

TGAACCCTGAAGTCGGCCACGAACAAATGGCTGGACTTAACTTCCCGCACGGCATCGCGCAGGC

TTTGTGGGCGGGCAAACTTTTCCACATCGATTTAAACGGCCAAAACGGAATCAAATACGATCAA

GACCTTCGCTTCGGCGCGGGAGATTTGAGAGCTGCGTTCTGGCTTGTAGATTTGTTAGAATCTG

CTGGCTACAGCGGCCCGAGACACTTCGATTTCAAACCTCCTCGTACAGAAGATTTCGATGGAGT

ATGGGCTTCAGCGGCTGGCTGCATGAGAAACTACCTTATCTTAAAAGAACGTGCGGCGGCTTTC

CGCGCTGATCCTGAAGTACAAGAAGCGCTTAGAGCTTCTCGTTTGGATGAACTTGCTCGCCCTA

CAGCTGCGGATGGTCTTCAGGCTTTGTTAGATGACAGATCTGCTTTTGAAGAGTTCGATGTCGA

CGCGGCAGCGGCTCGTGGAATGGCTTTTGAAAGACTTGATCAGTTGGCGATGGATCATCTTTTA

GGTGCCCGCGGCTAA

[0065] The wild-type GI polypeptide encoded by the polynucleotide of SEQ ID NO: 2 is shown, below, as SEQ ID NO: 3. 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.

VNYQPTPEDRFTFGLWTVGWQGRDPFGDATRRALDPVESVRRLAELGAHGVTFHDDDLIPFGSS

DSEREEHVKRFRQALDDTGMKVPMATTNLFTHPVFKDGGFTANDRDVRRYALRKTIRNIDLAVE

LGAETYVAWGGREGAESGGAKDVRDALDRMKEAFDLLGEYVTSQGYDIRFAIEPKPNEPRGDIL

LPTVGHALAFIERLERPELYGVNPEVGHEQMAGLNFPHGIAQALWAGKLFHIDLNGQNGIKYDQ

DLRFGAGDLRAAFWLVDLLESAGYSGPRHFDFKPPRTEDFDGVWASAAGCMRNYLILKERAAAF

RADPEVQEALRASRLDELARPTAADGLQALLDDRSAFEEFDVDAAAARGMAFERLDQLAMDHLL

GARG

[0066] 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).

[0067] 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% SOYTONE™ (BD Biosciences)) for robust cell growth) containing 10 ppm neomycin. Bacteral growth resulted in the production of intracellular GI.

Example 3

Generation of a glucose isomerase site evaluation library [0068] 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.

[0069] 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. Example 4

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.

Table 1. Performance of GI SEL variants relative to wild-type

[0071] Only variants that had overall beneficial mutations are shown in Table 1. It will be understood that many variants exibited poor expression and/or other detrimental properties that rendered them of no interest commercially. In general, the major factors for selecting certain mutations for further study were thermostability and/or specific activity coupled with no major decrease in expression. Several muations ( e.g ., E70K/M, A89M and Y212F) significantly increased specific activity, and about half the mutations, including A364N, R41K, I59V, S66P, H71K, D297I and K314S improved immobilization yield, although usually at the expense of expression.

[0072] 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.

[0073] Although the foregoing compositions and methods have been described in some detail by way of illustration and examples for purposes of clarity of understanding, it will be apparent to those skilled in the art that certain changes and modifications may be made. Therefore, the description should not be construed as limiting the scope of the invention, which is delineated by the appended claims.

[0074] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entireties for all purposes and to the same extent as if each individual publication, patent, or patent application were specifically and individually indicated to be so incorporated by reference.

Claims

CLAIMS What is claimed is:

1. A non-naturally-occuring variant of a parent glucose isomerase (GI) 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.

2. The variant of claim 1, wherein the mutation is selected from 41K, 59V 70K, 70M,

7 IK, 89M, 212F, 2971 and 314S using SEQ ID NO: 1 for numbering.

3. The variant of claim 1 or 2, wherein the mutation is selected from R41K, I59V E70K, E70M, H71K, A89M, Y212F, D297I and K314S using SEQ ID NO: 1 for numbering.

4. The variant of any of the preceding claims, comprising at least any two, three, four or more mutations.

5. The variant of any of the preceding claims, wherein the variant is derived from a GI from Streptomyces rubiginosus.

6. The variant of any of the preceding claims, wherein 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.

7. A process for producing high-fructose com syrup (HFCS) 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.

8. The process of claim 7, wherein the variant GI comprises a mutation selected from 41K, 59V 70K, 70M, 71K, 89M, 212F, 2971 and 314S using SEQ ID NO: 1 for numbering.

9. The process of claim 7 or 8, wherein the 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.

10. The process of any of claims 7-9, wherein the variant GI comprises at least any two, three, four or more mutations.

11. The process of any of claims 7-10, wherein the variant GI is derived from a GI from Streptomyces rubiginosus.

12. The process of any of claims 7-11, wherein 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.

13. The process of any of claim 7-12, wherein contacting the glucose syrup with the variant glucose isomerase is performed 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.