WO2007028811A1 - Enzymes xylose-isomerases thermostables - Google Patents

Enzymes xylose-isomerases thermostables Download PDF

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Publication number
WO2007028811A1
WO2007028811A1 PCT/EP2006/066089 EP2006066089W WO2007028811A1 WO 2007028811 A1 WO2007028811 A1 WO 2007028811A1 EP 2006066089 W EP2006066089 W EP 2006066089W WO 2007028811 A1 WO2007028811 A1 WO 2007028811A1
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seq
polypeptide
polynucleotide
sequence
identity
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PCT/EP2006/066089
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English (en)
Inventor
Luigi Concilio
Tiziana Giovannini
Claudio Merendi
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Cargill, Incorporated
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Priority claimed from EP06010465A external-priority patent/EP1857552A1/fr
Application filed by Cargill, Incorporated filed Critical Cargill, Incorporated
Priority to US12/065,715 priority Critical patent/US20100234581A1/en
Priority to EP06793289A priority patent/EP1922408A1/fr
Publication of WO2007028811A1 publication Critical patent/WO2007028811A1/fr

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    • 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)

Definitions

  • the present invention relates to thermostable xylose isomerase enzymes, to polynucleotide sequences encoding such enzymes and to uses thereof.
  • Xylose isomerase (EC 5.3.1.5) is an enzyme that, in vivo, catalyses the reversible isomerisation of D-xylose to D-xylulose. In addition, in vitro, it is capable of catalysing the conversion of D-glucose to D-fructose and may thus also be referred to as a "glucose isomerase". This latter activity is used, in industry, for the production of high fructose corn syrups (HFCS).
  • HFCS high fructose corn syrups
  • HFCS containing 55% fructose or more has a higher sweetening power than sucrose.
  • sucrose a higher sweetening power than sucrose.
  • the pH optimum of commercially available xylose isomerase enzymes ranges from about 7.5 to about 9.0. This limits the reaction temperature used in the glucose isomerisation process to around 60 0 C. Above this temperature, and because of non- enzymatic reactions between reducing sugars and proteins (e.g. Maillard reactions), undesirable browning products are formed.
  • Performing the isomerisation at higher temperatures would give a greater fructose concentration in HFCS.
  • higher temperatures e.g. 90-95 0 C
  • it could also lead to faster reaction rates, higher process stability, decreased viscosity of substrate and product streams, reduced microbial contamination and fewer problems with by-product formation.
  • most commercially available xylose isomerase enzymes would not be stable at such high temperatures.
  • thermostable xylose isomerases e.g. by site-directed mutagenesis or by screening highly thermophilic organisms for xylose isomerase activity.
  • Xylose isomerases derived from T. neapolitana have been shown to be active at temperatures of 97°C. Their activity, however, is reduced to only 40% after 2 hours at 90 0 C and their pH optimum remains high (above 7).
  • WO2004/044129 acknowledges the need for xylose isomerase enzymes which retain high levels of activity at elevated temperatures and at low pH.
  • the peptides disclosed in this document only have optimum activity above pH 5 (see Figures 6B and 6D) and are not stable above 90 0 C (see Figures 8B and 8D: 50% activity lost after 1 hour and after 30 min, respectively).
  • these enzymes require the presence of cobalt (Co + ) as a co-factor (see Figures 9A and 9B).
  • Co + cobalt
  • Figures 9A and 9B the use of Co + for the production of fructose syrups destined for human consumption is not recommended. Not only is it associated with a number of possible health problems, but the disposal of spent media could also contribute to environmental pollution.
  • Co + is usually required by xylose isomerases encoded by genes from thermophilic bacterial species like such as Thermotoga neapolitana and Thermotoga maritima.
  • Possible substitutes for Co 2+ in food-related applications include Mn 2+ and
  • a xylose isomerase enzyme suitable for use in the food industry should ideally function under acidic conditions (i.e. at a pH of about 6 or less) to avoid browning reactions; it should remain stable at high reaction temperatures (i.e. 85°C or more) to favour the formation of fructose; it should not require the use of potentially harmful co- factors such as Co + ; and it should be expressed at high levels to ease production and reduce costs.
  • the present invention aims to provide such an enzyme.
  • an isolated polypeptide characterised in that it comprises an amino acid sequence having at least 80% identity to a sequence selected from the group consisting of SEQ ID NO 2, SEQ ID NO 21 and SEQ ID NO 22, over the entire length of said sequence, fragments and variants thereof.
  • an isolated polynucleotide characterised in that it comprises a nucleotide sequence having at least 80% identity to a sequence selected from the group consisting of SEQ ID NO 1 , SEQ ID NO 19 and SEQ ID NO 20, over the entire length of said sequence, fragments and variants thereof.
  • an expression vector characterised in that it comprises a polynucleotide as defined above; and a recombinant host cell characterised in that it comprises such a vector.
  • a process for producing a polypeptide as defined above comprising the steps of culturing a host cell of the invention under conditions sufficient for the production of said polypeptide and recovering the polypeptide from the culture medium.
  • an antibody that selectively binds a polypeptide as defined above.
  • a method for producing a fructose syrup comprising the step of contacting a glucose-containing composition with a polypeptide as defined above under conditions effective to convert glucose to fructose; and fructose syrup obtainable according to said method.
  • FIG. 1 Comparison of xylose isomerase activity, with glucose as a substrate, at different pHs and in the presence of different ion co-factors.
  • Expression of the SM-I mutant was compared (under the same induction conditions) with that of genes cloned from wild -type species (SpI, Sp2 + Sp5) and with that of other mutants generated by random mutagenesis (1, 2, 6, 16, 27 + 37).
  • Genes cloned into pET22b(+) were expressed in BL21Star(DE3) cells after 1 mM IPTG induction.
  • FIG. 2 Comparison of xylose isomerase activity, with glucose as a substrate, at pH 6.85 in maleic buffer, in the presence of Mg + and Co + .
  • Genes cloned into pET22b(+) from different wild-type species (SpI to Sp8) were expressed in BL21Star(DE3) cells after 1 mM IPTG induction.
  • FIG. 3 Comparison of thermo-stability between the SM-I mutant and the commercially available GENSWEET® SGI xylose isomerase enzyme. After expression induction and heat treatment (30 min at 95°C, upper part of the plate), the test was carried out with glucose as a substrate at pH 6.85 (maleic buffer) and in the presence of Mg and Co . Several dilutions were made. The test was repeated without heat treatment as a control. These results are shown in the lower half of the plate.
  • XIso xylose isomerase enzyme
  • FIG. 5 PAGE gel showing expressed enzymes (SM-I , SM-2, SpI, Sp2) after staining (left) and dilution results after mass standardisation (right).
  • FIG. 6 Comparison of thermo-stability between the SM-I and SM-2 mutants and SpI and Sp2 strains at 90 0 C for 0 to 180 minutes.
  • the object of the present invention is to provide a thermostable xylose isomerase (or "xylose isomerase enzyme") suitable for use in the industrial or commercial production of fructose syrups.
  • thermoostable refers to enzymes which retain at least 40 % of their activity when exposed to temperatures above 60 0 C for 2 hours.
  • the xylose isomerase enzyme of the present invention will remain stable at temperatures in the range of from 60 to 110 0 C, more preferably at temperatures in the range of from 80 to 100 0 C. It may therefore also be referred to as a "hyper-thermostable” enzyme.
  • the enzymes of the invention will preferably also be stable under acid conditions. This means that they should retain at least 40 % of their activity when exposed to a pH of less than 8, at high temperature, for 2 hours.
  • the enzymes will remain stable at a pH in the range of from 4.5 to 8, more preferably in the range of from 5 to 7, even more preferably in the range of from 5 to 6.
  • the enzymes of the invention will preferably not require the presence of cobalt ions as co-factors for the production of fructose. More preferably, they will be capable of converting glucose to fructose using only Mn 2+ and/or Mg 2+ as co-factors.
  • the enzymes of the invention will preferably be expressed at equivalent or higher levels than other known xylose isomerase enzymes.
  • a xylose isomerase polypeptide also referred to, herein, as the "xylose isomerase enzyme"
  • xylose isomerase enzyme also referred to, herein, as the "xylose isomerase enzyme”
  • polypeptide refers to any peptide chain of more than 10, preferably of more than 100 amino acids whether in its unfolded, partially folded or fully- folded state. Fully-folded polypeptides may also be referred to as "mature" polypeptides or proteins.
  • polypeptides of the invention may be "free-standing" or they may be part of a larger protein such as a precursor or a fusion protein. It may indeed be advantageous to include an additional amino acid sequence which contains secretory or leader sequences, sequences which aid in purification (such as multiple histidine residues) or additional sequences for stability during recombinant production.
  • the present invention provides an isolated polypeptide comprising or consisting of:
  • a polypeptide encoded by an isolated polynucleotide comprising a nucleotide sequence which has at least 80% identity, preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 99% identity and most preferably exact identity to a nucleotide sequence selected from the group consisting of SEQ ID NO 1, SEQ ID NO 19 and SEQ ID NO 20, over the entire length of said sequence; or
  • a polypeptide encoded by an isolated polynucleotide comprising a nucleotide sequence having at least 80% identity, preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 99% identity and most preferably exact identity over its entire length to a nucleotide sequence encoding a polypeptide according to (a).
  • the invention further provides variants of the above defined polypeptides.
  • variant refers to a polypeptide that differs from a reference polypeptide by the substitution, deletion and/or addition of at least one amino acid, but that otherwise retains essential properties. Generally, differences will be limited so that the sequence of the reference polypeptide and that of the variant are closely similar overall with, in many instances, large regions of exact identity.
  • Preferred variants in accordance with the present invention are those that have similar or the same functional characteristics as the reference polypeptide, e.g. variants comprising only silent substitutions, additions or deletions.
  • preferred variants may include conservative amino acid substitutions, whereby a residue is substituted by another with like characteristics. Examples of such substitutions include changes between Ala, VaI, Leu and He; between Ser and Thr; between the acidic residues Asp and GIu; between Asn and GIn; between the basic residues Lys and Arg; and between the aromatic residues Phe and Tyr.
  • a fragment is a polypeptide having an amino acid sequence that is entirely the same as part (but not all) of the amino acid sequence of a reference polypeptide. Fragments may be "free-standing" or comprised within a larger polypeptide of which they form a part or region. Examples of typical fragments include truncation polypeptides, degradation polypeptides produced by or in a host cell, and fragments characterized by structural or functional attributes (such as fragments that comprise alpha-helixes, beta-sheets, hydrophilic regions, hydrophobic regions, substrate binding regions, etc.).
  • the fragments of the present invention will be fragments of any of the polypeptides (or variants thereof) defined above, comprising at least 50, even more preferably at least 100 contiguous amino acids. Ideally, they will be capable of catalysing the conversion of glucose to fructose.
  • the homologues, variants and fragments described above will preferably retain at least 20%, more preferably at least 40%, even more preferably at least 60%, even more preferably at least 80%, even more preferably at least 90%, most preferably 100% glucose isomerase activity relative to a mature polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO 2, SEQ ID NO 21 and SEQ ID 22.
  • Polypeptides of the present invention can be prepared in any suitable manner including, for example, recombinant expression or synthetic production. Such methods are well known in the art.
  • the present invention also relates to antigens derived from the above polypeptides and to antibodies capable of reacting therewith.
  • an antigen is a fragment of a full length polypeptide sequence (e.g. the polypeptide of SEQ ID NO 2, 21 or 22) comprising at least 6, preferably at least 10, more preferably at least 15, even more preferably at least 20, even more preferably at least 30 contiguous amino acids. It should include an epitope from that sequence (i.e. a region of the sequence which is located on the surface of the polypeptide when it is in its fully folded state) such that an antibody raised against the fragment is capable of forming a specific immune complex with the full length sequence (or with any sequence or partial sequence derived therefrom and containing the epitope).
  • antibody refers to molecules capable of specifically binding to such antigens, i.e. to immunoglobulin molecules, or immunologically active portions of such molecules, capable of forming an immune complex with the antigens as defined above or with polypeptides comprising such antigens. They may include, but are not limited to, polyclonal, monoclonal, chimeric and single chain antibodies and can be generated using any of a number of well known techniques (e.g. standard polyclonal or monoclonal antibody preparation techniques).
  • a polynucleotide capable of encoding a xylose isomerase enzyme as defined above, together with biologically, industrially and commercially useful fragments and variants of said polynucleotide and compositions comprising the same.
  • polynucleotide refers to any chain of nucleotides containing at least 10, preferably at least 100 nucleotide bases.
  • the nucleotide bases may be ribonucleotides or deoxyribonucleotides and the polynucleotides, therefore, may be either RNA or DNA molecules (whether in single- or double-stranded form).
  • Examples of polynucleotides include, but are not limited to, unprocessed RNA, ribozyme RNA, mRNA, cDNA, genomic DNA and synthetic DNA.
  • the present invention provides an isolated polynucleotide comprising or consisting of:
  • nucleotide sequence which has at least 80% identity, preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 99% identity and most preferably exact identity to a polynucleotide sequence selected from the group consisting of SEQ ID NO 1, SEQ ID NO 19 and SEQ ID NO 20 over the entire length of said sequence;
  • nucleotide sequence encoding a polypeptide which has at least 80% identity, preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 99% identity and most preferably exact identity to an amino acid sequence selected from the group consisting of SEQ ID NO 2, SEQ ID NO 21 and SEQ ID NO 22, over the entire length of the said sequence;
  • nucleotide sequence which has at least 80% identity, preferably at least 85% identity, more preferably at least 90% identity, even more preferably at least 95% identity, even more preferably at least 97% identity, even more preferably at least 99% identity and most preferably exact identity to the polynucleotide defined in (b), over the entire length of the said sequence; or
  • nucleotide sequence which is complementary to, or which hybridises (preferably under stringent conditions) to, any one of the polynucleotides defined in (a)-(c) above.
  • polynucleotide sequences defined by SEQ ID NO 1, 19 and 20 have been represented as DNA sequences. It is of course understood that these sequences merely represent one embodiment of the present invention and that other forms of the sequences (including, for example, their RNA equivalents) also fall within the scope of the invention.
  • the invention further provides polynucleotides which encode any of the polypeptide variants, polypeptide fragments, precursor or fusion proteins defined above.
  • the present invention provides variants and fragments of such polynucleotides and of the polynucleotides defined in (a)-(d) above.
  • the term "variant" refers to a polynucleotide that differs from a reference polynucleotide by the substitution, deletion and/or addition of at least one nucleotide, but that otherwise retains essential properties. Changes in the nucleotide sequence may or may not (e.g. because of the redundancy of the genetic code) alter the amino acid sequence of the encoded polypeptide. If changes in the nucleotide sequence do result in an alteration of the encoded amino acid sequence, this alteration will preferably not affect the functional characteristics of the resulting polypeptide (e.g. the alterations will be limited to conservative amino acid substitutions).
  • fragments refers to polynucleotides having a nucleotide sequence which is entirely the same as part (but not all) of the nucleotide sequence of a reference polynucleotide. Preferred fragments will comprise at least 5, more preferably at least 10, even more preferably at least 50 nucleotides and can be used, by way of example only, as primers for identifying or synthesizing full-length polynucleotide sequences. There are several methods available and well known to those skilled in the art for obtaining full- length polynucleotide sequences in this manner. All of the above polynucleotides may be "free-standing" or they may include one or more additional nucleotide sequences.
  • additional sequences may be non-coding sequences (such as rho-dependent or rho-independent termination signals, ribosome binding sites, Kozak sequences, enhancer sequences, mRNA stabilising sequences, introns and poly-adenylation signals). They may also encode further amino acids (such as a marker sequence that facilitates purification of the eventual polypeptide).
  • Polynucleotides of the present invention can be prepared in any suitable manner including, for example, by standard cloning techniques or synthetic production (e.g. by Polymerase Chain Reaction (PCR), Ligase Chain Reaction (LCR) or Nucleic Acid Sequence-based Amplification (NASBA)). Such methods are well known in the art.
  • PCR Polymerase Chain Reaction
  • LCR Ligase Chain Reaction
  • NASBA Nucleic Acid Sequence-based Amplification
  • polypeptides of the present invention may be prepared by processes well known to those skilled in the art including, in particular, by genetically engineering host cells with appropriate expression vectors (i.e. with expression vectors comprising a polynucleotide of the invention).
  • the present invention further provides:
  • expression vector refers to any vector suitable for the maintenance, propagation or expression of a particular polynucleotide sequence and/or for the expression of a particular polypeptide in a chosen host. Certain vectors are capable of autonomous replication within the host while others must be integrated into, and replicated together with, the host.
  • the vector of the invention will be a cloning vector containing the necessary control regions (e.g. inducible or constitutive promoters) to allow for transcription and translation of the cloned polynucleotide to be initiated and regulated.
  • the vectors will preferably also contain one or more selectable markers which permit easy selection of transformed host cells (e.g. by biocide or viral resistance or by resistance to heavy metals).
  • the expression vector according to the present invention may be selected from any of a great variety of suitable vectors known in the art. These include, by way of example only, chromosomal, episomal and virus-derived vectors (e.g. vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast chromosomal elements or from viruses such as papova viruses, vaccina viruses, adenoviruses and retroviruses) and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements (e.g. cosmids and phagemids).
  • the choice of vector may depend on the type of host being used.
  • the expression vector will be a plasmid.
  • the polynucleotide of the invention may be inserted into the selected expression vector by any of a variety of well known and routine techniques (such as those set forth in Sambrook J., Fritsch E.F and Maniatis T. (1989) "Molecular Cloning: a Laboratory Manual” 2nd ed. Cold Spring Harbor Laboratory Press (ISBN 0-88989-509-8)).
  • Introduction of the expression vector into the host cell can also be achieved by any of a variety of well known methods. These include, by way of example only, calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, conjugation, transduction, scrape loading, ballistic introduction and infection.
  • host cell is used to refer to cells capable of receiving, maintaining and allowing the reproduction of recombinant expression vectors.
  • the host cell will be adapted for the production of recombinant polypeptides in large quantities.
  • appropriate hosts include (but are not limited to): bacterial cells, such as cells of streptococci, staphylococci, enterococci, escherichia, streptomyces, cyanobacteria and bacillus; fungal cells, such as cells of a yeast, Kluveromyces, Saccharomyces, Schizosaccharomyces, Yarrowia, Pichia, a basidiomycete, Candida, Aspergillus, Acremonium, Aureobasidium, Cryptococcus, Filibasidium, Fusarium, Humicola, Magnaporthe, Mucor, Myceliophthora, Neocallimastix, Neurospora, Paecilomyces, Penicillium, Pirom
  • ATCC American Type Culture Collection
  • DSMZ Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH
  • CBS Centraalbureau Voor Schimmelcultures
  • NRRL Northern Regional Research Centre
  • the host cell will be a bacterial or fungal cell. More preferably, the host cell will be an E. coli or Bacillus subtilis cell.
  • a host cell as defined above will be cultivated under appropriate conditions.
  • the selection of appropriate conditions will, of course, depend on the host cell being used but may include, for example, the choice of a suitable nutrient medium, cultivation temperature and pH.
  • appropriate cultivation conditions are already well established in the art.
  • the cell may be cultivated, by way of example only, in a shake flask or in small-scale or large-scale fermentors (including continuous, batch, fed-batch and solid state fermentors).
  • polypeptides of the present invention may also be translated in vitro as described, for example, in Zubay G. (Ann. Rev. Genet. 7,
  • Recovery and purification of the polypeptide can be achieved using any known method. These include centrifugation, filtration, extraction, spray-drying, evaporation, chromatography and precipitation. If the polypeptide is produced in a host cell and is secreted into the nutrient medium, it can be recovered directly from the medium. If the polypeptide is not secreted, it can be recovered from a cell lysate. If the polypeptide is produced in a cell-free system (i.e. in vitro), it can be recovered directly from the reaction mixture in which it was produced.
  • a cell-free system i.e. in vitro
  • polypeptides of the present invention in particular polypeptides encoded by the polynucleotides of SEQ ID NO 19 and SEQ ID NO 20
  • polypeptides of the present invention can be expressed at much higher levels than xylose isomerase sequences isolated from wild-type Thermotoga neapolitana.
  • polypeptides of the present invention - such as those obtained according to the above methods - can be used, for example, in industry for the conversion of glucose to fructose and therefore for the production of fructose syrups.
  • a method for producing a fructose syrup comprising the step of contacting a glucose-containing composition with a polypeptide as defined above, under conditions effective to convert glucose to fructose.
  • the fructose syrups of the present invention will preferably contain at least 30% fructose, more preferably at least 40% fructose, even more preferably at least 50% fructose by weight.
  • the fructose syrup of the present invention will be a high fructose corn syrup (HFCS).
  • High fructose corn syrups typically contain 41-43% fructose by weight.
  • the high fructose corn syrup of the present invention will contain at least 50%, more preferably at least 55% fructose by weight.
  • the glucose-containing composition used as a substrate in the above method may be any composition containing sufficient quantities of glucose to allow for the production of a fructose syrup as defined above, e.g. a glucose syrup.
  • the glucose- containing composition will be a glucose syrup comprising at least 90%, more preferably at least 95% glucose by weight.
  • the conversion of glucose to fructose may be carried out under standard reaction conditions as established in the art. Preferably, however, it will be carried out at a reaction temperature in the range of from 60 to 110 0 C, more preferably in the range of from 80 to 100 0 C, even more preferably in the range of from 85 to 95°C.
  • the pH of the reaction mixture will preferably be in the range of from 4.5 to 8, more preferably 5 to 7, even more preferably 5 to 6, most preferably 5.2-5.7.
  • reaction mixture should also contain a xylose isomerase co-factor. This will preferably be selected from Mg + , Mn + and mixtures thereof.
  • the polypeptide may be free (e.g. added in batch or continuously) or it may be immobilised.
  • Fructose syrups obtained according to the above method also form part of the present invention. They may be used, for instance, in the food and beverage industries for the production of cakes, baked products, soft drinks and other consumable products.
  • the xylose isomerase enzyme of the invention is stable at high temperatures; it is stable at low pH; it does not require the use of potentially dangerous co-factors such as Co + ; it has a very strong affinity for Mn 2+ ; it is expressed at equivalent or higher levels than other xylose isomerase enzymes; it allows for the production of high-content fructose syrups; fructose syrups produced using the enzyme of the invention do not need to be further purified (e.g. by an additional chromatography step); browning reactions are reduced or eliminated during glucose to fructose conversion; reaction rates are increased; microbial contamination is reduced; etc.
  • identity refers to the relationship between two or more polypeptide or polynucleotide sequences. In particular, it refers to the degree of sequence relatedness between those sequences. The degree of relatedness (or “percent identity") between two sequences can readily be calculated by known methods and using widely available computer programs. One example of such a method is the ClustalW method available at www.ebi.ac.uk/clustalw/index.html. For ease of reference, sequences having a certain degree of identity will be referred to herein as "homologues”.
  • identity over the entire length of said sequence refers to a sequence having a certain degree of identity to a reference sequence over the entire open reading frame of that sequence.
  • complementary refers to a nucleic acid sequence that can form a double- stranded structure with a reference sequence.
  • a “fully complementary” sequence is one in which each nucleic acid base is complementary to the corresponding base of its reference sequence (e.g. G to C and A to T).
  • hybridisation means that hybridisation occurs only if there is at least 95% and preferably at least 97% identity between the sequences.
  • Hybridisation conditions are well known in the art as exemplified in Sambrook, et al. (supra).
  • isolated refers to compounds which have been changed and/or removed from their natural or original environment.
  • a polynucleotide or a polypeptide naturally present in a living organism is not “isolated”.
  • the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is said to be “isolated”.
  • a polynucleotide or polypeptide that is introduced to an organism e.g. by transformation or genetic manipulation is “isolated” even if it is still present in said organism.
  • polynucleotide encoding a polypeptide refers to polynucleotides capable of encoding a polypeptide of the invention, either in a single continuous region or in discontinuous regions (for example, polynucleotides interrupted by an integrated phage, an integrated insertion sequence, an integrated vector sequence or an integrated transposon sequence), optionally together with additional regions that may contain coding and/or non-coding sequences.
  • step (iii) Comparing the expression and activity of the chimeric mutant enzyme of step (i) with that of the wild-type xylose isomerase enzymes of step (ii). In terms of activity, the comparison was focused on the conversion of a glucose syrup to a fructose syrup at high temperature, low pH, and in the presence of Co 2+ , Mg 2+ and Mn 2+ ions (individually or in combination).
  • step (v) Performing several rounds of random mutagenesis to improve the characteristics of the chimeric mutant xylose isomerase of step (i);
  • Thermoanaerobacterium sp. (DSM-8685), hereafter referred to as SpI.
  • DSM-2229 Thermo anaerobacterium thermosulfurigenes (DSM-2229), hereafter referred to as Sp3.
  • Thermo anaerobacterium saccharolyticum (DSM-7060), hereafter referred to as Sp4.
  • Thermotoga maritima (DSM-3109), hereafter referred to as Sp5.
  • DSM-4424 Bacillus subtilis (DSM-4424), hereafter referred to as Sp6.
  • Thermus termophilus (DSM-579), hereafter referred to as Sp7.
  • strains SpI to Sp5 were grown under anaerobic conditions; strains Sp6, Sp7 and Sp8 were grown under aerobic conditions. Growth media and temperature conditions for the DSM strains were the ones suggested by the DSMZ website (http://www.dsmz.de/strains). Nutrient agar and broth were used for the growth of Sp8.
  • Escherichia coli TOPlO Invitrogen
  • BL21 StarTM DE3
  • rnvitrogen BL21 StarTM
  • Approximately 300 mg of bacterial pellet obtained from bacterial cultures of species SpI to Sp8 were subjected to genomic DNA extraction by means of the "Wizard® Magnetic - DNA Purification System For Food" kit (Promega). Cell disruption was accomplished mechanically by means of stainless steel beads and a Beat Beater. DNA was eluted in ddF ⁇ O, quantified by standard gel electrophoresis and an aliquot directly subjected to PCR.
  • DNA sequences of xylose isomerase genes from species SpI to Sp8 were obtained from Genbank.
  • Forward and reverse PCR primers were designed to be complementary to the end moieties of each gene.
  • a 5' tail, encoding sequences upstream of the Ndel and downstream of the Sad restriction sites of pET22b(+) were added to the forward and reverse primers, respectively.
  • Reverse primers were designed to contain a double stop codon before the poly-histidine tract encoded by the vector.
  • the PCR reaction mixture in a volume of 50 ⁇ l, contained 10 ng of genomic DNA, 15 pmol of each primer, 10 ⁇ l of Platinum® Pfx Amplification Buffer, 0.3 mM each dNTP, 1 mM MgSO 4 , 1.25 Units Platinum® Pfx DNA polymerase (rnvitrogen).
  • the thermal profile consisted of an initial denaturation step of 5 min at 94°C, followed by 50 cycles consisting of 94°C for 15 sec, an annealing temperature between 54 and 64°C (depending on the primer couple) for 30 sec, and 68°C for 2 min.
  • Amplification of the 1.4-kb product was checked by running a small aliquot of the reaction on a 1.3% TAE agarose gel.
  • the PCR product was purified using either QIAquick® PCR Purification kit or QIAquick® Gel Extraction kit (QIAGEN) and cloned back into the Ndel and Sad sites of pET22b(+). Restriction enzymes used were purchased from either Promega, Fermentas or New England BioLabs.
  • ClustalW www. ebi . ac .uk/clustalw/index.html
  • NCBI Blast2 www.ebi.ac.uk/blastall/index.html
  • EMBOSS http://emboss.sourceforge.net
  • This alignment provided a tool to predict wanted characteristics and to identify possible stable mRNA secondary structures which can inhibit translation initiation and correct termination.
  • the desired restriction products were purified from the gel as described above and an aliquot thereof was ligated. A total of 100 ng DNA were put into reaction in a final volume of 20 ⁇ l. After an overnight incubation at 16°C, the reaction was stopped by heating for 10 min at 70 0 C, set immediately on ice and purified by means of QIAquick® PCR Purification kit. Ligated products were rescued using the "universal primers" and the PCR protocol described above.
  • An amplification product of the expected size was purified by means of the QIAquick® Gel Extraction kit, digested with Ndel and Sad and cloned into the respective restriction sites of pET22b(+). Transformation was accomplished through elecfroporation into either TOPlO or BL21 StarTM (DE3) E. coli cells and plated overnight on Luria-Bertani (LB) agar plates containing 100ug/ml carbenicillin.
  • Random mutations were introduced into the xylose isomerase chimeric gene mutant cloned into the Ndel and Sad restriction sites of pET22b (+). PCR was performed with the "universal primers" described above. The reaction mixture, according to the protocol proposed by Cadwell and Joyce (1992 - PCR Meth. Appl.
  • 2, 28-33 contained 2 ng chimeric gene DNA, 30 pmol of each primer, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 7.0 mM MgCI 2 , 0,5 mM MnCI 2 , 1 mM dCTP, 1 mM dTTP, 0.2 mM dATP, 0.2 mM dGTP, and 2.5 Units AmpliTaq DNA polymerase (Applied Biosystems) in a 50 ⁇ l reaction volume. After an initial denaturation step of 3 min at 94°C, cycling parameters were made of 40 cycles of 94°C for 30 sec, 54°C for 30 sec, and 72°C for 1 min.
  • Amplification of the 1.4-kb product was checked by running the reaction on a 1.3 % agarose gel.
  • the PCR product was purified using QIAquick® Gel Extraction kit and ligated back into the Ndel and Sad sites of dephosphorylated pET22b (+), as described above.
  • genes from selected mutants were used as template.
  • Three rounds of random mutagenesis were carried out. Each time a library of approximately 10,000 samples was screened, the best candidate was selected and subjected to a further cycle of mutagenesis.
  • Transformation was accomplished through electroporation into BL21 StarTM (DE3) E. coli cells and plated overnight on Luria-Bertani (LB) agar plates containing lOO ⁇ g/ml carbenicillin.
  • a thermal step of 30 min at 95°C was carried out, then 60 ⁇ l of buffer solution including glucose as a substrate (see below) were added. Plates were sealed and placed for 20-24 h in an oven at 60 0 C.
  • the sulphuric acid method proposed by Kennedy and Chaplin (1975 - Carbohydrate Res. 40, 227-233) was used to quantitatively detect D-fructose: after having cooled them down at room temperature, 120 ⁇ l of 66% sulphuric acid were added, each plate was tightly sealed and incubated at 60 0 C for 30-60 min. Colour development was determined by reading on a micro-plate reader (BMG) using a 405 nm filter.
  • a single colony-derived cell pre-culture was cultivated overnight in a volume of 3 ml LB containing a final concentration of 100 ⁇ g/ml carbenicillin.
  • One hundred micro-liters were inoculated into 50-ml polypropylene tubes containing 10 ml LB medium containing 100 ⁇ g/ml carbenicillin and left to grow for 20 hours at 37°C on an orbital shaker at 250 rpm.
  • IPTG was added to reach a final concentration of 1 mM, growth was continued for 8 hours at 37°C on an orbital shaker at 250 rpm.
  • the supernatant was discarded, the pellet was lysed by adding BugBusterHT ® (Novagen), 1/5 with respect to the culture volume, plus rLysozymeTM (Novagen), 1/75 of the culture volume.
  • BugBusterHT ® Novagen
  • rLysozymeTM Novagen
  • Lysis was carried out for 30 min at room temperature. Tubes were shaken manually from time to time. The samples were centrifuged for 10 min at 4200 xg at room temperature, then 60 ⁇ l of the supernatant were transferred to a 96-well micro- plate. A thermal step of 30 min at 95°C was carried out, then 60 ⁇ l of substrate plus buffer solution were added.
  • Enzymatic tests were carried out in 100 mM maleic acid buffer pH 6.85 or in 50 mM Sodium-acetate buffer pH 5.2. Final pH values revealed to be increased of about 0.5-0.6 pH units after the addition of the crude cell lysis extracts. D-glucose was added as a substrate at a final concentration of 12.5%. The effect of metal cations was established by adding MgSO 4 at a final concentration of 10 mM, C0CI 2 at a final concentration of 0.5 mM and MnCl 2 at a final concentration of 5 mM. The effect of the ions was evaluated either individually or in combinations (MgSO 4 + CoCl 2 and MgSO 4 + MnCl 2 ).
  • the best xylose isomerase mutant hereafter named SM-I for sake of clarity, is encoded by the sequence given in SEQ ID NO 1 (where the BcII site introduced to join the original SpI and Sp2 fragments - as described above - is underlined) and its deduced protein sequence is given in SEQ ID NO 2.
  • the mutant was expressed intracellularly, in the cytoplasm, the pelB leader peptide coded in between the Ndel and Sad restriction sites of the pET22b(+) vector having been removed. This and all other mutants were produced in their native-like conformation, i.e. without any amino- or carboxyl-terminal extensions.
  • the enzymatic performance was tested at a temperature of 60 0 C but a previous thermal step of 30 min at 95°C was done not only to lyse the bacterial cells when grown in micro- plates, but also to eliminate the background of non-thermal resistant proteins and of mutated xylose isomerase enzymes with inefficient thermal behaviour. It is clear from the data shown that in these conditions the selected mutant SM-I shows a higher thermal resistance in comparison to a commercial xylose isomerase enzyme like GENSWEET® SGI (FIG. 3). The mutant's thermal resistance is very similar to the one shown by hyper- thermophilic xylose isomerase enzymes from T. neapolitana (Sp2) and T. maritima (Sp5).
  • the xylose isomerase enzyme produced by the selected mutant SM-I can be used for an efficient conversion of glucose into fructose at high temperature, low pH and without the presence of Co + ions.
  • Sp 1 xylose isomerase from non-thermostable Thermoanaerobacterium sp
  • polypropylene tubes containing 10 ml LB medium + 100 ⁇ g/ml carbenicillin were inoculated from single colonies and left to grow for 48-72 hours at 37°C on an orbital shaker at 250 rpm.
  • IPTG was added to reach a final concentration of 1 mM, and the induction step was carried out for about 24 hours at 37°C, on an orbital shaker at 250 rpm. 5 milliliters were centrifuged for 20 min at 4200 xg at +4°C.
  • Electrophoresis was carried out in NuP AGE® MOPS SDS Running Buffer Ix (Invitrogen). 200 ml of buffer contained inside the inner cell chamber were supplemented with 500 ⁇ l of NuP AGE® Reducing Agent 10x (Invitrogen). Between 2 to 5 ⁇ l of denatured samples were loaded with the aid of a micropipette. 3 ⁇ l of SeeBlue® Plus2 Pre-Stained Standard (Invitrogen) were used as a protein molecular mass standard. The expected molecular mass of the xylose isomerase genes is about 50 kDa. The electrophoretic run was carried out at 200 constant Volts for 50 min, according to manufacturer's instruction. Each gel was stained in 20 ml of SimplyBlueTM SafeStain (Invitrogen) for 1 hour, under light agitation and de-stained in distilled water for approximately 1 hour. The results are shown in FIG. 4.
  • SM-I, SM-2, SpI and Sp2 enzymes were expressed as described above. They were extracted using Novogen's BugBuster HT method. Extracts were then diluted in BugBuster HT reagent as follows: SM-I 1 :1, SM-2 1 :8, SpI 1 :5 and Sp2 1: 1.
  • FIG. 5 shows expressed enzymes after gel staining (left) and dilution results after mass standardisation (right). As can be seen in this figure, SM-2 has much higher levels of expression than wild-type thermostable enzyme Sp2.
  • Each enzyme was incubated for 0, 30, 60, 90, 120, 150 and 180 minutes at 90 0 C in 1 ml vials (150 ⁇ l).
  • 60 ⁇ l of the heat-treated enzyme extracts and 60 ⁇ l of substrate buffer (Maleic acid + Co + Mg + glucose 25%, pH 6.85) were then plated in microplates and incubated for 8 hours at 60 0 C to evaluate residual enzyme activity.
  • substrate buffer Meleic acid + Co + Mg + glucose 25%, pH 6.85
  • SM-2 shows a thermo-stability which is equivalent to that of the wild- type thermostable enzyme Sp2.
  • SM-I has a much greater stability even than Sp2.
  • Diluted enzyme extracts were prepared as above. lOO ⁇ l of each extract was then incubated for 24 hours at 60 0 C with 400 ⁇ l of each of buffers 1-12 as shown in the following table.
  • the buffers were prepared as follows:
  • Substrate n°3 Add 100 ml solution Of MgSO 4 (0.2 M) + 100 ml solution of MnCl 2 (0.1
  • Substrate N°4 Add 10 ml solution of CoCl 2 *6H 2 O (0.1 M)
  • Substrate N°5 Add 10 ml solution of CoCl 2 *6H 2 O (0.1 M) + 100 ml solution of
  • Substrate N°6 Add 10 ml solution of CoCl 2 *6H 2 O (0.1 M) +100 ml solution of MnCl 2
  • Substrate n°10 Add 100 ml solution Of MgSO 4 (0.2 M) + 100 ml solution Of MnCl 2
  • Substrate n°12 Add 10 ml solution of CoCl 2 *6H 2 O (0.1 M) + 100 ml solution of
  • Substrate n°13 Add 10 ml solution of CoCl 2 *6H 2 O (0.1 M) + 100 ml solution Of MnCl 2
  • SM-I and SM-2 maintain good levels of activity (for example, in tests 2 and 3, more than 16 and 17 times higher, respectively, than that of the reference thermostable enzyme Sp2).
  • SM-I and SM-2 also show a very strong affinity with the Mn ion.

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Abstract

Cette invention concerne un polypeptide isolés qui se caractérise par le fait qu'il comprend une séquence d'acides aminés dont l'identité et au moins à 80 % identique à celle d'une séquence choisie dans le groupe comprenant SEQ ID NO 2, SEQ ID NO 21 et SEQ ID NO 22. Cette invention concerne également des polynucléotides codant pour un tel polypeptide ainsi que l'utilisation de ceux-ci pour la production de sirops à base de fructose.
PCT/EP2006/066089 2005-09-06 2006-09-06 Enzymes xylose-isomerases thermostables WO2007028811A1 (fr)

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