Thermostable glucose isomerase variants

Abstract

Compositions and methods involving thermostable glucose isomerase variants are disclosed. These variants are particularly useful for the preparation of high fructose corn syrup and, in some cases, reduce the need for chromatographic enrichment steps.

Description

Technical Field

[0001] Compositions and methods are disclosed that relate to thermostable glucose isomerase variants. These variants are particularly useful for preparing high fructose corn syrup and, in some cases, reduce the need for chromatographic enrichment steps. Background Art

[0002] Glucose isomerase (GI) is an enzyme used to convert glucose (typically obtained from the hydrolysis of corn starch) into fructose, which is sweeter than glucose and has higher value for the food and beverage industry. The global market for GI is approximately $30 million USD per year.

[0003] In a typical industrial process, D-glucose syrup obtained from wet-milled corn is introduced in a down-fed manner into a series of reactors (i.e., columns) containing immobilized glucose isomerase (IGI) to obtain high fructose corn syrup (HFCS). The isomerization equilibrium is a function of temperature, where higher temperatures are more favorable for fructose formation. Typically, commercial production is carried out at 57 °C using immobilized wild-type GI from Streptomyces rubiginosus. Typically, conversion is to standard target fructose concentrations, such as approximately 42% HFCS (HFCS-42) for food and beverage and preferably 55% HFCS (HFCS-55) for beverages.

[0004] To obtain HFCS-55, typically a portion of HFCS-42 is chromatographically enriched to produce 90% HFCS (HFCS-90), and then HFCS-90 is blended with HFCS-42 to produce HFCS-55. Methods for preparing HFCS using IGI are described, for example, in U.S. Patent Nos. 5,177,005, 5,437,993, 5,811,280, 5,916,789, and 7,297,510. Summary of the Invention

[0005] The compositions and methods of the present invention relate to thermostable variant glucose isomerase polypeptides and methods of using them. Aspects and embodiments of the compositions and methods of the present invention are summarized in the following separately numbered paragraphs:

[0006] 1. In one aspect, there is provided a non-naturally occurring variant of a parental glucose isomerase (GI) that contains a mutation at an amino acid residue corresponding to a position selected from 41, 59, 70, 71, 89, 212, 297, and 314 numbered using SEQ ID NO:1.

[0007] 2. In some embodiments of the variant as described in paragraph 1, the mutation is selected from 41K, 59V, 70K, 70M, 71K, 89M, 212F, 297I, and 314S numbered using SEQ ID NO:1.

[0008] 3. In some embodiments of the variant as described in paragraph 1 or 2, the mutation is selected from R41K, I59V, E70K, E70M, H71K, A89M, Y212F, D297I, and K314S numbered using SEQ ID NO:1.

[0009] 4. In some embodiments of the variant as described in any of the preceding paragraphs, the variant comprises at least any two, three, four, or more mutations.

[0010] 5. In some embodiments of the variant as described in any of the preceding paragraphs, the variant is derived from a GI from Streptomyces rubiginosus.

[0011] 6. In some embodiments of the variant as described in 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 with 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 with the polypeptide of SEQ ID NO:3.

[0012] 7. In another aspect, a method for producing high fructose corn syrup (HFCS) containing greater than about 42% fructose, the method comprising contacting a glucose syrup with a variant of a parent glucose isomerase (GI) that contains one or more mutations at amino acid residues corresponding to positions selected from 41, 59, 70, 71, 89, 212, 297, and 314 numbered using SEQ ID NO:1, wherein the variant has an altered GI activity and / or increased thermal stability compared to the parent GI, and wherein the method involving contacting the glucose syrup with the variant GI requires a reduced chromatographic enrichment to produce a subsequent HFCS containing a total of greater than 55% fructose compared to the amount of chromatographic enrichment required to obtain 55% fructose by an equivalent method using the parent GI.

[0013] 8. The method as described in paragraph 7, wherein the variant GI comprises mutations selected from 41K, 59V, 70K, 70M, 71K, 89M, 212F, 297I, and 314S numbered using SEQ ID NO:1.

[0014] 9. The method as described in paragraph 7 or 8, wherein the variant GI comprises mutations selected from R41K, I59V, E70K, E70M, H71K, A89M, Y212F, D297I, and K314S numbered using SEQ ID NO:1.

[0015] 10. The method as described in any one of paragraphs 7 - 9, wherein the variant GI comprises at least any two, three, four, or more mutations.

[0016] 11. The method as described in any one of paragraphs 7 - 10, wherein the variant GI is derived from a GI from Streptomyces rubiginosus.

[0017] 12. The method as described in any one 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 with 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 with the polypeptide of SEQ ID NO:3.

[0018] 13. The method as described in any one of paragraphs 7 - 12, wherein contacting the glucose syrup with the variant glucose isomerase is carried out 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.

[0019] These and other aspects, as well as embodiments, of these compositions and methods (methods / processes) will be apparent from the present specification and the drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 is a graph showing the residual activity relative to the specific activity of wild - type GI and several SEL variants after immobilization. DETAILED DESCRIPTION

[0021] I. DEFINITIONS AND ABBREVIATIONS

[0022] Before describing the various aspects and embodiments of the compositions and methods of the present invention, the following definitions and abbreviations are described.

[0023] The term "glucose isomerase" is a commonly used name in the sweetener industry and refers to xylose isomerase (EC 5.3.1.5) which is used to convert glucose into fructose to produce high fructose corn syrup (HFCS). Formally, xylose isomerase catalyzes the interconversion of D-xylose and D-xylulose. The systematic name of the enzyme is D-xylose aldose-ketose-isomerase. Other commonly used names include D-xylose isomerase, D-xylulose isomerase, and D-xylulitol-isomerase.

[0024] The term "starch" refers to any material composed of complex polysaccharide carbohydrates of plants, which are composed of amylose and amylopectin having the formula (C6H 10 O5)x where "X" can be any number. The term includes plant-based materials such as grains, cereals, grasses, tubers, and roots, and more specifically, materials obtained from wheat, barley, corn, rye, rice, sorghum, bran, cassava, millet, milo, potatoes, sweet potatoes, and tapioca starch.

[0025] Regarding polypeptides, the terms "wild-type", "parent", or "reference" refer to a naturally occurring polypeptide that does not contain artificial substitutions, insertions, or deletions at one or more amino acid positions. Similarly, regarding polynucleotides, the terms "wild-type", "parent", or "reference" refer to a naturally occurring polynucleotide that does not contain artificial nucleoside changes. However, note that the polynucleotides encoding wild-type, parent, or reference polypeptides are not limited to naturally occurring polynucleotides and encompass any polynucleotide encoding wild-type, parent, or reference polypeptides.

[0026] 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 natural state. Thus, for example, a recombinant cell expresses a gene not found in the cell in its natural (non-recombinant) form, or expresses a native gene at a level different from that found in nature or under conditions different from those found in nature. A recombinant nucleic acid differs from the native sequence by one or more nucleotides, and / or is operably linked to a heterologous sequence, such as a heterologous promoter in an expression vector. A recombinant protein can differ from the native sequence by one or more amino acids, and / or is fused to a heterologous sequence. A vector containing a nucleic acid encoding amylase is a recombinant vector.

[0027] The terms "recovered", "isolated", and "separated" refer to a compound, protein (polypeptide), cell, nucleic acid, amino acid, or other specified material or component that has been removed from at least one other material or component with which it is naturally associated as it occurs in nature. Its "isolated" polypeptides include, but are not limited to, culture broths containing secreted polypeptides expressed in heterologous host cells.

[0028] The term "purified" refers to a material (e.g., an isolated polypeptide or polynucleotide) 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.

[0029] The term "enriched" refers to a material (e.g., an isolated polypeptide or polynucleotide) that is about 50% pure, at least about 60% pure, at least about 70% pure, or even at least about 70% pure.

[0030] The terms "thermostable" and "thermostability" with respect to an enzyme refer to the ability of the enzyme to retain its activity after exposure to elevated temperatures. The thermostability of an enzyme (e.g., amylase) is measured by its half-life (t1 / 2) given in minutes, hours, or days, during which half of the enzyme activity is lost under defined conditions. The half-life can be calculated by measuring the residual α-amylase activity after exposure (i.e., challenged with) to elevated temperatures.

[0031] References to wild-type polypeptides are to be understood as including the mature forms of the polypeptides. A "mature" polypeptide or variant thereof is a polypeptide or variant in which the signal sequence is absent, e.g., cleaved from the polypeptide in its immature form during or after polypeptide expression.

[0032] The term "variant" with respect to a polypeptide refers to a polypeptide that is different from a designated wild-type, parental, or reference polypeptide in that it includes one or more naturally occurring or man-made amino acid substitutions, insertions, or deletions. Similarly, the term "variant" with respect to a polynucleotide refers to a polynucleotide that is different from a designated wild-type, parental, or reference polynucleotide in its nucleotide sequence. The characteristics of the wild-type, parental, or reference polypeptide or polynucleotide will be apparent from the context.

[0033] The term "combinatorial variant" refers to a variant that includes two or more mutations, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more substitutions, deletions, and / or insertions.

[0034] The "pH range" with respect to an enzyme refers to the range of pH values under which the enzyme exhibits catalytic activity.

[0035] The terms "pH stable" and "pH stability" with respect to an enzyme relate to the ability of the enzyme to retain its activity over a wide range of pH values for a predetermined period (e.g., 15 min., 30 min., 1 hour).

[0036] The term "amino acid sequence" is synonymous with the terms "polypeptide", "protein", and "peptide" and is used interchangeably. When such amino acid sequences exhibit activity, they can be referred to as "enzymes". Amino acid sequences are represented in the standard amino-terminal-to-carboxyl-terminal orientation (i.e., N→C) using the conventional single-letter or three-letter codes for amino acid residues.

[0037] The term "nucleic acid" encompasses DNA, RNA, heteroduplexes, and synthetic molecules capable of encoding a polypeptide. The nucleic acid can be single-stranded or double-stranded and can be chemically modified. The terms "nucleic acid" and "polynucleotide" are used interchangeably. Since the genetic code is degenerate, more than one codon can be used to encode a particular amino acid, and the compositions and methods of the present invention encompass nucleotide sequences encoding a particular amino acid sequence. Unless otherwise specified, nucleic acid sequences are presented in a 5′-to-3′ orientation.

[0038] The terms "transformation", "stable transformation", and "transgenic" as used with respect to a cell mean that the cell contains a non-native (e.g., heterologous) nucleic acid sequence integrated into its genome or carried as an episome maintained through multiple generations.

[0039] In the context of inserting a nucleic acid sequence into a cell, the term "introducing" means "transfection", "transformation", or "transduction" as known in the art.

[0040] A "host strain" or "host cell" is an organism that has been introduced with an expression vector, phage, virus, or other DNA construct, including a polynucleotide encoding a polypeptide of interest (e.g., amylase). Exemplary host strains are microbial cells (e.g., bacteria, filamentous fungi, and yeast) capable of expressing a polypeptide of interest and / or fermenting sugars. The term "host cell" includes protoplasts produced from the cell.

[0041] The term "heterologous" with respect to a polynucleotide or protein means a polynucleotide or protein that is not naturally present in the host cell.

[0042] The term "endogenous" with respect to a polynucleotide or protein means a polynucleotide or protein that is naturally present in the host cell.

[0043] The term "expression" refers to the process of producing a polypeptide based on a nucleic acid sequence. The process includes both transcription and translation.

[0044] A "signal sequence" is an amino acid sequence attached to the N-terminal portion of a protein that facilitates the secretion of the protein outside the cell. The mature form of an extracellular protein lacks the signal sequence that is excised during the secretion process.

[0045] "Biologically active" refers to a sequence having a specified biological activity, such as enzyme activity.

[0046] The term "specific activity" refers to the number of moles of substrate that can be converted to product per unit time by an enzyme or enzyme preparation under specific conditions. Specific activity is usually expressed as units (U) / mg protein.

[0047] As used herein, "water hardness" is a measure of the minerals (e.g., calcium and magnesium) present in water.

[0048] "Percent sequence identity" means that when aligned using the CLUSTAL W algorithm with default parameters, a particular sequence has at least a certain percentage of amino acid residues that are identical to the amino acid residues in a specified reference sequence. See Thompson et al. (1994) Nucleic Acids Res. 22:4673-80. The default parameters of the CLUSTAL W algorithm are:

[0049]

[0050] It should be noted that the singular forms "a / an" and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, reference to "an enzyme" includes a plurality of such enzymes, and reference to "a dose" includes reference to one or more doses and equivalents thereof known to those of ordinary skill in the art, and so on.

[0051] This document is organized into several sections for ease of reading; however, the reader will appreciate that statements made in one section may apply to other sections. In this way, the headings for the different sections of this disclosure should not be construed as limiting.

[0052] Unless otherwise indicated, the following abbreviations / acronyms have the following meanings:

[0053] °C degrees Celsius

[0054] dH2O or DI deionized water

[0055] dIH2O deionized water, Milli-Q filtered

[0056] DNA deoxyribonucleic acid

[0057] EC Enzyme Commission

[0058] eq. equivalent

[0059] g or gm gram

[0060] GI glucose isomerase

[0061] H2O water

[0062] HEPES 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid

[0063] HFCS high fructose corn syrup

[0064] HMF hydroxymethylfurfural

[0065] hr hour

[0066] IGI Immobilized Glucose Isomerase

[0067] kg Kilogram

[0068] M Mole

[0069] mg Milligram

[0070] min Minute

[0071] mL and ml Milliliter

[0072] mm Millimeter

[0073] mM Millimole

[0074] MOPS 3-(N-Morpholino)Propanesulfonic Acid

[0075] MTP Microtiter Plate

[0076] N Normal Concentration

[0077] NCBI National Center for Biotechnology Information

[0078] PCR Polymerase Chain Reaction

[0079] ppm Parts Per Million, e.g., μg Protein / gram dry solid

[0080] sec Second

[0081] SEL Site Evaluation Library

[0082] sp. Species

[0083] U Unit

[0084] v / v Volume / Volume

[0085] w / v Weight / Volume

[0086] w / w Weight / Weight

[0087] wt Wild Type

[0088] μg Microgram

[0089] μL and μl Microliter

[0090] μm Micrometer

[0091] μM Micromole

[0092] nm Nanometer

[0093] OD Optical Density

[0094] GIU Glucose Isomerase Activity Unit

[0095] II. Glucose Isomerase Variants

[0096] The compositions and methods of the present invention relate to variants of glucose isomerase (GI) that are 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 higher temperatures, thereby shifting the isomerization equilibrium in favor of conversion to fructose. The GI variants of the present invention permit the production of 55% HFCS (HFCS-55) while reducing the need for chromatographic enrichment of a portion of the HFCS.

[0097] Although the engineering of more heat-tolerant GI from Thermoanaerobacterium saccharolyticum has been described (e.g., U.S. Patent No. 7,919,300), this specification relates to the engineering of more heat-tolerant GI from Streptomyces rubiginosus, which is the most robust and preferred GI on the market.

[0098] The amino acid sequence of wild-type Streptomyces rubiginosus glucose isomerase (referred to as xylose isomerase according to Genbank accession number AAA26838) is shown as SEQ ID NO:1 below:

[0099]

[0100] In some embodiments, the heat-tolerant variant GI has one or more mutations at positions selected from 41, 59, 70, 71, 89, 212, 297, and 314 numbered using SEQ ID NO:1. In some embodiments, the heat-tolerant variant GI has one or more mutations selected from 41K, 59V, 70K, 70M, 71K, 89M, 212F, 297I, and 314S numbered using SEQ ID NO:1. In some embodiments, the heat-tolerant variant GI has one or more mutations selected from R41K, I59V, E70K / M, H71K, A89M, Y212F, D297I, and K314S numbered using SEQ ID NO:1.

[0101] In some embodiments, the variant heat-tolerant GI further contains additional substitutions, deletions, and / or insertions at one or several positions. In some embodiments, the variant heat-tolerant GI further contains an N-terminal and / or C-terminal extension of one or several amino acid residues. In some embodiments, the variant heat-tolerant GI further contains an N-terminal and / or C-terminal truncation of one or several amino acid residues. In addition, the variant GI of the present invention may contain any number of conservative amino acid substitutions well known in the art.

[0102] Variants GI of the present invention can be "chimeric" or "hybrid" polypeptides as they include at least a portion of a first GI polypeptide and at least a portion of a second GI polypeptide. Variants GI of the present invention can further include a heterologous signal sequence, i.e., an epitope that allows for tracking or purification, etc.

[0103] In some embodiments, the variant thermostable GI has one or more mutations relative to SEQ ID NO:1 and 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% amino acid sequence identity with SEQ ID NO:1 or an active fragment thereof, but less than 100% amino acid sequence identity.

[0104] Of course, variants GI of the present invention can be immobilized, e.g., as described in, for example, U.S. Patent Nos. 5,177,005, 5,437,993, 5,811,280, 5,916,789 and 7,297,510.

[0105] III. Nucleotides Encoding Variant Glucose Isomerase

[0106] In another aspect, a nucleic acid encoding a variant GI is provided. The nucleic acid can encode a specific GI or a GI having a specified degree of amino acid sequence identity with a specific GI as described.

[0107] In embodiments, 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 with SEQ ID NO:1 or 3, or a GI that is even up to 100% identical to SEQ ID NO:3.

[0108] In a 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 a GI that is even up to 100% identical to SEQ ID NO:3.

[0109] 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 should be understood that due to the degeneracy of the genetic code, multiple nucleic acids can encode the same polypeptide.

[0110] The nucleic acid encoding the GI can be operably linked to various promoters and regulators in a vector suitable for expressing the GI in a host cell.

[0111] IV. Production of Variant Glucose Isomerase

[0112] The GI variants of the present invention can be produced in homologous or heterologous host cells, for example, by secretion or intracellular expression. After secreting the variant GI into the cell culture medium, the cultured cell material containing the variant GI (e.g., whole cell culture broth) can be obtained. Optionally, the variant GI can be isolated from the host cell or even from the cell culture broth, depending on the desired purity of the final variant GI. Suitable host cells include bacteria, fungi (including yeasts 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 species, including in some embodiments the homologous host Streptomyces rubiginosus. In a preferred embodiment, the GI variant is expressed in Bacillus subtilis.

[0113] V. Compositions and uses of GI variants

[0114] The thermostable GI variants can be used in a variety of industrial applications, including the conversion of starch-derived glucose to high fructose corn syrup. The increased thermostability of the GI variant shifts the isomerization equilibrium in favor of the conversion to fructose, and in some cases, allows the direct production of HFCS with a much higher percentage of fructose without the need for chromatographic enrichment of a portion of the HFCS. Accordingly, these compositions and methods include the ability to directly produce HFCS-55 (or HFCS with an even higher fraction of fructose) by contacting glucose with the GI variant of the present invention 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, wherein the need for chromatographic enrichment of a portion of the HFCS for fructose is reduced, and then adding the enriched HFCS back to the HFCS that was directly contacted with the GI.

[0115] These compositions and methods include IGI and columns containing IGI (as disclosed in the above patent documents and as used in the HFCS industry for decades). The thermostable variant GI can be immobilized using known methods as described, for example, in U.S. Patent Nos. 5,177,005, 5,437,993, 5,811,280, 5,916,789, and 7,297,510.

[0116] To further illustrate the compositions and methods and their advantages, the following specific examples are given, which should be understood to be illustrative rather than restrictive.

[0117] Examples

[0118] Example 1

[0119] Determination

[0120] The various determinations used herein are listed below for ease of reading. Any deviation from the protocols in the examples that follow is noted in the relevant section. In these experiments, a spectrophotometer was used to measure the absorbance of the product formed after the reaction was complete.

[0121] A. Glucose Isomerase Activity Determination

[0122] Enzyme activity was determined as follows: 100 μl of the material containing the enzyme was mixed with 100 μl of 1 M glucose in the wells of a polypropylene 96-well microtiter plate (MTP; ThermoFisher Scientific; catalog number 267245), sealed with an adhesive sealing film (BioRad; catalog number MSB1001), and then incubated for 60 min at 50 °C at 150 rpm in an orbital shaker incubator. The reaction was terminated by immediately placing the sealed plate on ice. 10 μl of the terminated reactant was mixed with 50 μl of 7.5 M hydrochloric acid in a PCR-type 96-well microtiter plate (BioRad; catalog number HSP9621) and incubated at 74 °C for 30 min, then cooled on ice. 10 μl was mixed with 100 μl of water in a UV-capable 96-well polypropylene MTP (ThermoFisher Scientific; catalog number 8404). The optical density (OD 284 nm ) at 284 nm of all wells measured using a spectrophotometer is an indicator of the amount of hydroxymethylfurfural (HMF) formed from fructose produced from glucose by glucose isomerase in the first reaction and is proportional to glucose isomerase activity. The GI unit (GIU) is defined as the change in OD 284nm multiplied by the reciprocal of the reaction time (i.e., min -1 ), which can be abbreviated as ΔOD 284nm *min -1 or ΔOD 284nm / min. All liquid handling steps were performed using a Biomek FX “robot” (Beckman Coulter). OD values were measured using a SpectraMAX MTP reader (model 340 - Molecular Device) spectrophotometer. A thermal cycler PCR machine (Biometra) and an incubator / shaker (Kuhner) were used for all incubation steps. Replicate values were averaged. Error bars representing the standard deviation within replicates were shown where appropriate.

[0123] B. Protein expression assay

[0124] Protein assays were performed using ultra - performance liquid chromatography. A clarified culture lysate of cells grown in 96 - well MTP at 37 °C, with shaking at 270 rpm and 70% humidity for 68 hours was prepared by 10 - fold dilution in ultrapure water and filtration (0.45 μm). 10 μl was injected onto a Zorbax 300SB - C3 column (Agilent, P / N 858750 - 909), equilibrated with 30% acetonitrile in 0.1% trifluoroacetic acid.

[0125] Chromatography was performed at 80 °C using a 30% - 95% acetonitrile gradient in 0.1% trifluoroacetic acid for 2 min at a flow rate of 1 ml / min. Absorbance was monitored at 220 nm, and peaks corresponding to GI were integrated using CHEMSTATION TM software (Agilent Technologies). Protein concentration was determined based on a standard curve generated using the commercial product GENSWEET TM (with known concentrations and reported as ppm).

[0126] C. Specific activity assay

[0127] Specific activity was reported as GIU / μg of glucose isomerase (GIU / μg) in the glucose isomerase lysate.

[0128] D. Immobilization and immobilization yield

[0129] The lysate containing GI was immobilized in microtiter plates using a known cross - linking method (Lantero; USPN 4,355,105). Bentonite (Cholino, Patagonia, Argentine; P / N F30) was hydrated by stirring overnight in water. Polyethyleneimine (PEI; Sigma Aldrich P / N 181978), glutaraldehyde GA (Sigma Aldrich), and CELITE 505 TM diatomaceous earth (Imerys) were added to the bentonite and mixed for 30 min to produce the master mixture. Using a Biomek FX robotic arm (Beckman Coulter), 20 μl of this mixture was dispensed into MTPs containing 1:4 diluted GI or GI variant cultures and mixed with a pipette tip.

[0130] Add secondary additives of PEI and GA (10 μl each) and mix on an external MTP mixer for 5 min; and wash the particles with 10 mM HEPES (pH 7.6) containing 1 mM magnesium sulfate and 1 mM sodium metabisulfite as follows: Have the robotic arm add the buffer to the particles, then spin them down in a centrifuge, and use the robotic arm to remove most of the liquid. Repeat this procedure three times. The final remaining volume is 100 μl.

[0131] The immobilization yield is calculated as the ratio of the activity present in the immobilized material relative to the activity of the lysate used in its preparation.

[0132] E. Thermal stability determination

[0133] The thermal stability of the GI variants was reported as the ratio of the activity of the stressed immobilized samples relative to the activity of the non-stressed immobilized samples (residual activity). Incubate the immobilized samples in an incubator / shaker from Kuhner at 65 °C for 24 h (without shaking). At the same time, store duplicate immobilized samples at 4 °C (representing non-stressed material). The activity of the stressed and non-stressed materials was determined as described above. For each variant, the ratio of the initial and residual amylase activities was used to calculate the thermal stability as follows: Thermal stability (abbreviated as "t" in this limited case) = [t residual value] / [t initial value], thus, the thermal stability activity ratio was calculated based on the enzyme activity after thermal incubation divided by the enzyme activity before thermal incubation.

[0134] Example 2

[0135] Generation of Bacillus subtilis strains expressing glucose isomerase and its variants

[0136] In this example, the construction of Bacillus subtilis strains expressing GI and its variants is described.

[0137] Synthetic, codon-optimized DNA encoding wild-type GI from Streptomyces rubiginosus was produced by GeneArt AG (Regensburg, Germany) and served as template DNA for constructing plasmids for expressing wild-type GI and its variants. The synthetic DNA is represented as SEQ ID NO:2 below. The valine start codon (underlined) is preferred in Bacillus subtilis. The stop codon is also underlined.

[0138]

[0139] 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 SEQ ID NO:1 above only in the presence of valine (underlined) rather than methionine as the first amino acid residue.

[0140]

[0141] GeneArt cloned the codon-optimized wild-type GI gene into the pSB expression vector (Babé, L.M. et al. (1998) Biotechnol. Appl. Biochem. [Biotechnology and Applied Biochemistry] 27:117 - 24) and fused it with the Bacillus subtilis aprE promoter using unique restriction enzyme sites, thereby generating plasmid pSB-GI. This plasmid includes elements from pUB110 (McKenzie et al. (1986) Plasmid [Plasmid] 15:93 - 103), which include the neomycin / kanamycin resistance gene (neo) and the bleomycin resistance marker (bleo).

[0142] Using the method described previously (WO 2002 / 14490), a suitable Bacillus subtilis strain was transformed with pSB-GI plasmid DNA. Bacillus subtilis transformants were selected on Luria agar plates (Teknova) using 10 mg / L neomycin sulfate (Sigma; catalog number N - 1876) (containing 732 μg neomycin / mg). Selective growth of Bacillus subtilis transformants containing the pSB-GI plasmid was carried out at 37°C in an MTP in MBD medium (a MOPS buffer-based enriched semi-defined medium with urea as the main nitrogen source, glucose as the main carbon source, and supplemented with 1% SOYTONE TM (BD Bioscience) for robust cell growth) for approximately 68 hr. Bacterial growth led to the production of GI intracellularly.

[0143] Example 3

[0144] Generation of a Glucose Isomerase Site Evaluation Library

[0145] The construction of the Glucose Isomerase Site Evaluation Library (SEL) was performed by GeneArt using its technology platforms for gene optimization, gene synthesis, and library generation (see, e.g., European Patent Nos. 0 200 362 and 0 201 184, U.S. Patent Nos. 4,683,195, 4,683,202, and 6,472,184, and International Patent Application No. WO 2004 / 059556 A3). The pSB-GI plasmid DNA was used as a template to generate a SEL having mutations at 160 amino acid positions in the wild-type GI of SEQ ID NO:3. The corresponding codons at each site were changed to codons encoding each of 19 different amino acids. The pSB-GI plasmid was sequenced and delivered in a standardized format. As described (WO 2002 / 014490), the codon mutagenized pSB-GI plasmid was used to transform competent Bacillus subtilis cells to generate a library of GI variants.

[0146] The transformation mixture was plated onto Luria agar plates containing 10 mg / L neomycin sulfate. For each library, single bacterial colonies were picked and grown in liquid Luria broth (tryptone and soy-based broth) with 10 mg / ml neomycin selection. To generate samples of the wild-type GI and its variants for biochemical characterization, the selective growth of the variants was performed in 96-well MTPs in MBD medium at 37 °C, 270 RPM, and 70% humidity for approximately 68 hours.

[0147] Example 4

[0148] Results obtained from the evaluation of the GI SEL library

[0149] The expression, specific activity, immobilization yield, and stability of the variants and the wild-type parental GI were determined as described in Example 1. Table 1 reports the results obtained for the variants relative to the wild-type GI.

[0150] Table 1. Performance of GI SEL variants relative to the wild-type

[0151]

[0152] Only variants with overall beneficial mutations are shown in Table 1. It should be understood that many variants exhibit poor expression and / or other deleterious properties, which render them commercially uninteresting. Generally, the main factors for selecting certain mutations for further study are thermal stability and / or specific activity, as well as no significant decrease in expression. Several mutations (e.g., E70K / M, A89M, and Y212F) significantly increased the specific activity, and about half of the mutations (including A364N, R41K, I59V, S66P, H71K, D297I, and K314S) enhanced the immobilization yield, although usually at the expense of expression.

[0153] Figure 1 shows the relationship between thermal stability and specific activity. Variants plotted in the upper right quadrant of the figure are the most desirable, but there seems to be a trade-off between stability and activity. Overall, the most desirable variants seem to be R41K, I59V, E70K, E70M, H71K, A89M, Y212F, D297I, and K314S. The selection of a particular variant (or several variants immobilized on a single column) depends on the desired rate of fructose conversion, relative to the desire to convert to fructose at the highest possible level to reduce the need for chromatographic enrichment.

[0154] Although the foregoing compositions and methods have been described in detail for purposes of clarity of understanding by way of illustration and example, it will be apparent to those skilled in the art that certain changes and modifications can be made. Accordingly, this specification should not be construed as limiting the scope of the invention as described by the appended claims.

[0155] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes, to the extent as if each individual publication, patent, or patent application were specifically and individually indicated to be incorporated by reference.

Claims

1. A non-naturally occurring variant of a parental glucose isomerase (GI), said variant being obtained by mutating position 41 of SEQ ID NO:3 to R41K.

2. The variant according to claim 1, wherein the variant is derived from a GI from Streptomyces rubiginosus.

3. A method for producing high fructose corn syrup (HFCS) containing greater than 42% fructose, said method comprising contacting a glucose syrup with the variant according to claim 1 or 2, wherein the variant has an altered GI activity and / or increased thermal stability compared to the parental GI, and wherein the method involving contacting the glucose syrup with the variant GI requires a reduced chromatographic enrichment to produce a subsequent HFCS containing a total of greater than 55% fructose compared to the amount of chromatographic enrichment required to obtain 55% fructose by an equivalent method using the parental GI.

4. The method according to claim 3, wherein the variant GI is derived from a GI from Streptomyces rubiginosus.

5. The method according to claim 3 or 4, wherein contacting the glucose syrup with the variant glucose isomerase is carried out at a temperature of at least 48°C.

6. The method according to claim 3 or 4, wherein contacting the glucose syrup with the variant glucose isomerase is carried out at a temperature of at least 49°C.

7. The method according to claim 3 or 4, wherein contacting the glucose syrup with the variant glucose isomerase is carried out at a temperature of at least 50°C.

8. The method according to claim 3 or 4, wherein contacting the glucose syrup with the variant glucose isomerase is carried out at a temperature of at least 51°C.

9. The method according to claim 3 or 4, wherein contacting the glucose syrup with the variant glucose isomerase is carried out at a temperature of at least 52°C.

10. The method according to claim 3 or 4, wherein contacting the glucose syrup with the variant glucose isomerase is carried out at a temperature of at least 53°C.

11. The method according to claim 3 or 4, wherein contacting the glucose syrup with the variant glucose isomerase is carried out at a temperature of at least 54°C.

12. The method according to claim 3 or 4, wherein contacting the glucose syrup with the variant glucose isomerase is carried out at a temperature of at least 55°C.

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