Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/54—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving glucose or galactose
• • 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
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
  • C12P21/00—Preparation of peptides or proteins
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
  • C12Q1/32—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving dehydrogenase
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
  • C12Y101/05—Oxidoreductases acting on the CH-OH group of donors (1.1) with a quinone or similar compound as acceptor (1.1.5)
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
  • C12Y101/99—Oxidoreductases acting on the CH-OH group of donors (1.1) with other acceptors (1.1.99)
  • C12Y101/9901—Glucose dehydrogenase (acceptor) (1.1.99.10)
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose

Definitions

• Subject matter of the present invention is a modified flavin adenine dinucleotide dependent glucose dehydrogenase (FAD-GDH) that is glycosylated, wherein at least one of the asparagine residues selected from the group consisting of N2; N168 and N346 according to the mature Aspergillus oryzae FAD-GDH wild-type sequence SEQ ID No: 2 (Wild type) has been substituted by one or more amino acids not suitable for glycosylation and thereby eliminating or inactivating, respectively, a potential glycosylation site at this position.
• FAD-GDH modified flavin adenine dinucleotide dependent glucose dehydrogenase
• subject matter of the present invention is a modified flavin adenine dinucleotide dependent glucose dehydrogenase (FAD-GDH) that is glycosylated wherein:
• said modified flavin adenine dinucleotide dependent glucose dehydrogenase exhibits an improved temperature stability under dry conditions in comparison to glycosylated FAD-GDH according to SEQ ID No: 1, wherein said FAD-GDH according to SEQ ID No: 1 is obtainable by expression in Aspergillus oryzae, and
• the present invention further relates to a process for producing the same, nucleic acids encoding said enzymes, vectors, host cells, a method of detecting, determining or measuring glucose in a sample using said enzyme and devices comprising said enzyme.
• glucose oxidase poses a problem in that it tends to transfer protons produced by the reaction to oxygen, causing dissolved oxygen to adversely affect the measured values.
• NAD(P) dependent glucose dehydrogenase EC 1.1.1.47
• PQQ pyrroloquinoline quinone
• ECl .1.5.2 former ECl .1.99.17
• NAD(P) dependent glucose dehydrogenase i.e., NAD(P) dependent glucose dehydrogenase
• PQQGDH PQQ dependent glucose dehydrogenase
• WO-A1 2004/058958 discloses Aspergillus-flav -boxmd glucose dehydrogenase. Since the activity of this enzyme on xylose is only 10% of that on glucose, in the case of measuring the blood glucose level of a person who is taking a xylose tolerance test, the accuracy of the measured value may be impaired. The enzyme has a residual activity ratio of about 89% after treatment at 50°C for 15 minutes, thereby exhibiting good heat stability.
• WOA1 2006/101239 discloses the gene sequence and amino acid sequence of the enzyme.
• a modified flavin adenine dinucleotide dependent glucose dehydrogenase (FAD-GDH) with improved heat stability in liquid than FAD-GDH derived from wild-type FAD-GDH, the modified FAD-GDH being derived from preferably an eukaryote, more preferably a filamentous fungus, and furthermore preferably an Aspergillus fungus, and, for example, those having a primary structure with at least one amino acid substituted, deleted, inserted or added to FAD-GDH is provided by US-B2 7,662,600.
• US 2008/220460 Al describes a modified FAD-GDH derived from Aspergillus fungus, e.g. Aspergillus oryzae or Aspergillus terreus having improved heat stability as compared to wild- type FAD-GDHs.
• the heat stability under dry conditions is of special importance. For instance, in case of test strips for blood-glucose measurements the enzyme properties of FAD-GDH under dry chemistry needs to be improved.
• Subject matter of the present invention is a modified flavin adenine dinucleotide dependent glucose dehydrogenase that is glycosylated, selected from the group consisting of (a) a modified flavin adenine dinucleotide dependent glucose dehydrogenase that is glycosylated, wherein at least one of the asparagine residues selected from the group consisting of N2; N168 and N346 according to the mature Aspergillus oryzae FAD-GDH wild-type sequence SEQ ID No: 2 (Wild type) has been substituted by one or more amino acids not suitable for glycosylation and thereby eliminating or inactivating, respectively, a potential glycosylation site at this position and a modified flavin adenine dinucleotide dependent glucose dehydrogenase that is glycosylated that exhibits around 80% amino acid sequence identity or more (preferably 90%, more preferably 95%) to said modified flavin adenine dinucleotide dependent glucose dehydrogena
• active fragment(s) thereof or synonymous "functional fragment(s) thereof refer to any modified flavin adenine dinucleotide dependent glucose dehydrogenase pursuant to the invention, whereby at least one amino acid is missing in the corresponding sequence according to SEQ ID No. 3 to 6, provided that such fragments still exhibit the essential properties as regards enzyme activity and the improved temperature stability under dry conditions in the sense of the present invention.
• the modified flavin adenine dinucleotide dependent glucose dehydrogenase according to the present invention exhibits improved temperature stability under dry conditions when compared to those FAD-GDHs of the prior art.
• FAD-GDHs that exhibit at least 80% amino acid sequence identity (preferably 90%, more preferably 95%) to the FAD- GDHs according to the present invention as above outlined under (a) are subject matter of the present invention provided that they exhibit the same substitution(s) and exhibit essentially the same properties as an FAD-GDH according to the present invention, those essential properties being enzyme activity and the improved temperature stability under dry conditions. It shall be understood that also fragments of said FAD-GDHs according to the present invention are encompassed that exhibit the same substitution(s) and exhibit essentially the same properties as a FAD-GDH according to the present invention, those essential properties being enzyme activity and the improved temperature stability under dry conditions.
• Sequence identity may be determined by the BLAST algorithm, the Basic Local Alignment Search Tool (BLAST) (Altschul, S.F. et al. 1990. J. Mol. Biol. 215:403; Altschul, S.F. et al. 1997. Nucleic Acid Res. 25:3389-3402).
• the above-mentioned percentages of amino acid sequence identity refer to the determination of sequence identity by said BLAST algorithm, wherein the region over which the homology is determined is the entire sequence of the modified FAD-GDH according to (a), and wherein said sequence of the FAD-GDH according to (a) is the reference sequence.
• the term “mature” in relation to “sequence(s)” refers to the raw sequence format of the respective protein sequence(s) without any added signaling sequences, e.g. signal peptides or equivalents thereof.
• temperature stability refers to the ability of the herein provided modified flavin adenine dinucleotide dependent glucose dehydrogenases (FAD- GDH) to resist changes in terms of its native biophysical and biochemical properties as its temperature changes, in particular its temperature increases. With this context, 100% temperature stability would reflect that no changes occur to the native biophysical and biochemical properties when compared to the enzyme before exposure to temperature over a certain period of time (t) with respect to particularly defined properties of the enzyme.
• FAD- GDH modified flavin adenine dinucleotide dependent glucose dehydrogenases
• modified- flavin adenine dinucleotide dependent glucose dehydrogenases or functional fragments thereof preserve its native enzyme activity during exposure to temperature over a certain period of time (t), and exemplarily exhibit an enzyme activity measured according to Example 3 as a specific embodiment, but not limited to.
• drying condition(s) is specifically related to the test conditions according to Example 7 as set out below, namely the respective lyophilizated sample was exposed to 80°C for 8 days in presence of a drying agent (molecular sieve 3A, MS 551, Grace), but not limited to .
• a drying agent molecular sieve 3A, MS 551, Grace
• the "unstressed lyophilizate” means that the temperature stability is determined subsequent to lyophilization. This means the unstressed lyophilizate is neither stored nor treated before determining the temperature stability. Temperature stability under dry conditions may be determined according to Example 7, but not limited to.
• the invention also foresees modified flavin adenine dinucleotide dependent glucose dehydrogenases, whose sequence is a functional equivalent to the sequences as provided herein under SEQ ID No. 3 to 6.
• a functional equivalent is an amino acid sequence molecule, which is different in at least one amino acid as provided in the sequences SEQ ID No. 3 to 6, which encodes a protein/enzyme with same or similar function, in particular in terms of enzyme activity and thermal stability under dry conditions.
• the functional equivalent of the modified flavin adenine dinucleotide dependent glucose dehydrogenases according to the invention is an amino acid sequence homologous to the SEQ ID No. 3 to 6.
• the degree or percentage of homology is at least 80, 90, 95, 99 or 100% to the SEQ ID No. 3 to 6; provided that they exhibit the same substitutions as outlined above under (a) and exhibit essentially the same properties as an FAD-GDH according to the present invention, those essential properties being enzyme activity and the improved temperature stability under dry conditions.
• active fragments of said FAD-GDHs according to the present invention are encompassed that exhibit the same substitutions and exhibit essentially the same properties as an FAD-GDH according to the present invention, those essential properties being enzyme activity and the improved temperature stability under dry conditions.
• nucleotide sequence molecules which codes with a different nucleotide sequence to SEQ ID No. 13 or the complementary sequence thereto, for exactly the same protein/enzyme.
• a functional equivalent of the nucleotide molecule as given in the SEQ ID No. 13 is a RNA molecule, which is encoded by said DNA sequence or a sequence being substantially complementary to the sequence of SEQ ID No. 13.
• RNA molecule is meant to refer to a linear polymer of ribonucleotide molecules, which is single- stranded and serves as a template for protein synthesis of the herein provided modified flavin adenine dinucleotide dependent glucose dehydrogenases according to the SEQ ID No. 3 to 6.
• "Drying agents" in the context of the present invention are for instance desiccants such as silica gel, calcium sulfate, calcium chloride, and molecular sieves, but not limited to.
• subject matter of the present invention is a modified flavin adenine dinucleotide dependent glucose dehydrogenase (FAD-GDH) that is glycosylated, or a FAD-GDH that exhibits at least 80% sequence homology thereto (preferably at least 90%, more preferably at least 95%) or functional fragments thereof wherein;
• FAD-GDH modified flavin adenine dinucleotide dependent glucose dehydrogenase
• said modified flavin adenine dinucleotide dependent glucose dehydrogenase exhibits an improved temperature stability under dry conditions in comparison to glycosylated FAD-GDH according to SEQ ID No: 1, wherein said FAD-GDH according to SEQ ID No:
• No: 1 is obtainable by expression in Aspergillus oiyzae and
• the modified flavin adenine dinucleotide dependent glucose dehydrogenase (FAD-GDH) is glycosylated.
• said modified flavin adenine dinucleotide dependent glucose dehydrogenase or functional fragments thereof exhibit a more homogenous glycosylation pattern (with less molecular weight distribution) in comparison to glycosylated FAD-GDH according to SEQ ID No: 1, wherein said FAD-GDH according to SEQ ID No: 1 is obtainable by expression in Aspergillus oryzae.
• the modified flavin adenine dinucleotide dependent glucose dehydrogenase or functional fragments thereof according to the present invention exhibit a molecular Weight Average Molecular Weight (Mw) of about 103 876 and a Number Average Molecular Weight (Mlois) of about 99 901.
• the Weight Average Molecular Weight (Mw) and the Number Average Molecular Weight (M n ) is calculated by means of the software from the raw data of the Viscotek Triple Detektors (refraction index (RI) and Right Angle Light Scattering (RALS)).
• the ratio of Mw/M n is the polydispersity and gives the size distribution of the protein.
• Monodisperse proteins have a value of 1.
• compositions comprising a modified flavin adenine dinucleotide dependent glucose dehydrogenase or functional fragments thereof according to the invention exhibit a molecular weight distribution of said modified flavin adenine dinucleotide dependent glucose dehydrogenase in said composition as follows: Weight Average Molecular Weight (Mw) of about 103 876 and a Number Average Molecular Weight (M n ) of about 99 901.
• Mw Weight Average Molecular Weight
• M n Number Average Molecular Weight
• one embodiment of the present invention is a modified flavin adenine dinucleotide dependent glucose dehydrogenase that is glycosylated or a FAD-GDH that exhibits at least 80% sequence homology thereto, (preferably at least 90%, more preferably at least 95%) or functional fragments thereof, wherein at least one of the asparagine residues selected from the group consisting of N2; N168 and N346 according to the mature Aspergillus oryzae FAD- GDH wild-type sequence SEQ ID No: 2 (Wild type) has been substituted by one or more amino acids not suitable for glycosylation and thereby eliminating or inactivating, respectively, a potential glycosylation site at this position and wherein the degree of glycosylation is ⁇ 50%, preferably ⁇ 40% and more preferably ⁇ 30% and/ or the ratio of Mw Mn is ⁇ 1 ,02, more preferably ⁇ 1,01 (for calculation of these values see e.g. Example 4).
• subject matter of the present invention is also a modified FAD-GDH or a FAD- GDH that exhibits at least 80%o sequence homology thereto, (preferably at least 90%, more preferably at least 95%) or functional fragments thereof, wherein at least one of the asparagine residues selected from the group consisting of N2; N168 and N346 according to the mature Aspergillus oryzae FAD-GDH wild-type sequence SEQ ID No: 2 (Wild type) has been substituted by one or more amino acids and thereby eliminating or inactivating, - ir respectively, a potential glycosylation site at this position and wherein, compositions comprising said modified FAD-GDH exhibit a molecular weight distribution with a ratio of Mw/Mn is ⁇ 1,02, more preferably with a ratio of Mw/Mn ⁇ 1,01 of said modified flavin adenine dinucleotide dependent glucose dehydrogenase in said composition and/ or wherein the degree of glycosylation of
• the degree of glycosylation may be calculated according to the following formula:
• FAD-GDH variant 1 (SEQ ID No: 3):
• FAD-GDH (SEQ ID No: 1) from Aspergillus:
• said modified flavin adenine dinucleotide dependent glucose dehydrogenase or fragments thereof according to the present invention exhibit a more homogenous glycosylation pattern (with less molecular weight distribution) in comparison to glycosylated FAD-GDH according to SEQ ID No: 1, wherein said FAD-GDH according to SEQ ID No: 1 is obtainable by expression in Aspergillus oryzae and/ or said modified flavin adenine dinucleotide dependent glucose dehydrogenase or fragments thereof according to the present invention exhibit a lower degree of glycosylation when compared to FAD-GDH according to SEQ ID No: 1, wherein said FAD-GDH according to SEQ ID No: 1 is obtainable by expression in Aspergillus oryzae.
• the enzymatic activity may be exemplarily determined according to Example 3 i), but not limited to.
• the sugar specificity of the modified FAD-GDH or active fragments thereof according to the present invention is about the same as the sugar specificity of the glycosylated mature Aspergillus oryzae FAD-GDH wild-type sequence SEQ ID No: 2 (Wild type), namely for maltose ⁇ 0,5% and for galactose ⁇ 13%; see Example 3 ii) for the calculation of sugar specificity.
• said at least one of the asparagine residues selected from the group consisting of N2; N168 and N346 according to the mature Aspergillus oryzae FAD-GDH wild-type sequence SEQ ID No: 2 (Wild type) has been substituted by one or more amino acids selected from the group comprising Ala, Arg, Asp, Cys, Gin, Glu, Gly His, He, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val.
• said at least one of the asparagine residues selected from the group consisting of N2; N168 and N346 according to the Aspergillus oryzae FAD-GDH wild-type sequence SEQ ID No: 2 (Wild type) has been substituted by one or more amino acids selected from the group comprising Arg, Asp, Gin, Glu, Gly His, Lys, Met, Pro, Ser, and Thr.
• a modified flavin adenine dinucleotide dependent glucose dehydrogenase or a FAD-GDH that exhibits at least 80% sequence homology thereto, (preferably at least 90%, more preferably at least 95%) or functional fragments thereof is provided, wherein only one asparagine of the residues selected from the group consisting of N2; N168 and N346 has been substituted by one or more amino acids, which lead to an inactivation (or deletion) of the corresponding glycosylation target site.
• a modified flavin adenine dinucleotide dependent glucose dehydrogenase or a FAD-GDH that exhibits at least 80% sequence homology thereto, (preferably at least 90%, more preferably at least 95%) or functional fragments thereof is provided according to the above embodiments, wherein said asparagine residue has been substituted by S, P, SP or D.
• a specific embodiment is a modified flavin adenine dinucleotide dependent glucose dehydrogenase or a FAD-GDH that exhibits at least 80% sequence homology thereto, (preferably at least 90%, more preferably at least 95%) or functional fragments thereof, wherein said modified flavin adenine dinucleotide dependent dehydrogenase derived from mature Aspergillus oryzae FAD-GDH wild-type sequence SEQ ID No: 2 (Wild type) has at least one of the following substitutions N2S, N168P, N168SP, and N346D.
• N168SP means that an asparagine residue at the position 168 according to SEQ ID No: 2 (Wild type) has been substituted by serine (S) and proline (P).
• An especially specific embodiment of the present invention is the provision of a modified flavin adenine dinucleotide dependent glucose dehydrogenase or a FAD-GDH that exhibits at least 80% sequence homology thereto, (preferably at least 90%, more preferably at least 95 %) or functional fragments thereof, wherein said modified flavin adenine dinucleotide dependent dehydrogenase derived from mature Aspergillus oryzae FAD-GDH wild-type sequence SEQ ID No: 2 (Wild type) has one or more of the following substitutions: N2S, N168P, N168SP, and N346D.
• said modified flavin adenine dinucleotide dependent dehydrogenase or a FAD-GDH that exhibits at least 80% sequence homology thereto, (preferably at least 90%, more preferably at least 95%) or functional fragments thereof has only one of the following substitutions N2S, N168P, N168SP, and N346D.
• modified FAD-GDH may have or may have not further modifications different from the before-mentioned substitutions.
• Another especially specific embodiment of the present invention is the provision of a modified flavin adenine dinucleotide dependent glucose dehydrogenase or a FAD-GDH that exhibits at least 80% sequence homology thereto, (preferably at least 90%, more preferably at least 95%) or functional fragments thereof, wherein said modified flavin adenine dinucleotide dependent dehydrogenase derived from mature Aspergillus oryzae FAD-GDH wild-type sequence SEQ ID No: 2 (Wild type) has the particular substitution N2S according to SEQ ID No: 3.
• Another specific embodiment of the present invention is the provision of a modified flavin adenine dinucleotide dependent glucose dehydrogenase or a FAD-GDH that exhibits at least 80% sequence homology thereto, (preferably at least 90%, more preferably at least 95%) or functional fragments thereof, wherein said modified flavin adenine dinucleotide dependent dehydrogenase derived from mature Aspergillus oryzae FAD-GDH wild-type sequence SEQ ID No: 2 (Wild type) has the substitution N168P according to SEQ ID No: 4.
• Another specific embodiment of the present invention is the provision of a modified flavin adenine dinucleotide dependent glucose dehydrogenase or a FAD-GDH that exhibits at least 80% sequence homology thereto, (preferably at least 90%, more preferably at least 95%) or functional fragments thereof, wherein said modified flavin adenine dinucleotide dependent dehydrogenase derived from mature Aspergillus oryzae FAD-GDH wild-type sequence SEQ ID No: 2 (Wild type) has the following substitution N168SP according to SEQ ID No: 5.
• Another specific embodiment of the present invention is the provision of a modified flavin adenine dinucleotide dependent glucose dehydrogenase or a FAD-GDH that exhibits at least 80% sequence homology thereto, (preferably at least 90%, more preferably at least 95%) or functional fragments thereof, wherein said modified flavin adenine dinucleotide dependent dehydrogenase derived from mature Aspergillus oryzae FAD-GDH wild-type sequence SEQ ID No: 2 (Wild type) has the following substitution N346D according to SEQ ID No: 6.
• Encompassed by the present invention are also enzymes that exhibit at least 70% sequence homology, preferred 80%, more preferred 90%, most preferred 95% sequence homology to all above mentioned enzymes, provided that in particular the characteristic enzyme activity and temperature stability, especially under dry conditions, remain essentially the same as mentioned throughout the specification for the provided modified flavin adenine dinucleotide dependent glucose dehydrogenases according to the sequences SEQ ID No. 3 to 6, when said enzyme activity is exemplarily determined according to Example 3 i), but not limited to.
• Another specific embodiment of the present invention is an isolated polynucleotide encoding a modified flavin adenine dinucleotide dependent glucose dehydrogenase or a functional fragment thereof according to the present invention with the proviso that said isolated polynucleotide is not encoding for a modified flavin adenine dinucleotide dependent glucose dehydrogenase or an active fragment thereof derived from Aspergillus oryzae FAD-GDH wild-type sequence SEQ ID No: 2 (Wild type) that has a single substitution selected from the group consisting of N168K, N168P, N168Y, or N168W.
• the isolated polynucleotide may be a DNA or RNA molecule or a corresponding gene thereof encoding for the following sequences as explicitly listed in the section - Sequence Listing:
• Another specific embodiment of the present invention is a composition comprising one or more of the modified FAD-GDHs provided by the present invention without any restrictions to the related sequences or homogenous sequences thereof.
• Another specific embodiment of the present invention is a composition comprising one or more of the isolated polynucleotides that are a DNA or RNA molecule or a corresponding gene provided by the present invention.
• said isolated polynucleotide is not encoding for a modified flavin adenine dinucleotide dependent glucose dehydrogenase or an active fragment thereof derived from Aspergillus oryzae F AD- GDH wild-type sequence SEQ ID No: 2 (Wild type) that has a single substitution selected from the group consisting of N168K, N168P, N168Y, or N168W.
• Another embodiment of the present invention is an expression vector containing the isolated polynucleotide according to the present invention.
• said isolated polynucleotide is not encoding for a modified flavin adenine dinucleotide dependent glucose dehydrogenase or an active fragment thereof derived from Aspergillus oryzae FAD-GDH wild-type sequence SEQ ID No: 2 (Wild type) that has a single substitution selected from the group consisting of N168 , N168P, N168Y, or N168W.
• Said expression vector may be operably linked to a promoter sequence capable of directing its expression in a host cell.
• the specific vector in this study was expression vector pPICZaA (Invitrogen).
• This plasmid allows replication in E. coli (pUC origin of replication) for cloning of the expression construct as well as recombinant gene expression in Pichia pastoris by use of the AOX1 promotor/AOXl terminator sequences (further details see Figure 1).
• Expression vectors useful in the present invention may typically contain an origin of replication (ori), an antibiotic resistance for selection, a promoter for expression and the whole or part of the modified FAD-GDH gene variant.
• said gene variant is not encoding for a modified flavin adenine dinucleotide dependent glucose dehydrogenase or an active fragment thereof derived from Aspergillus oryzae FAD-GDH wild-type sequence SEQ ID No: 2 (Wild type) that has a single substitution selected from the group consisting of N168K, N168P, N168Y, or N168W.
• the expression vectors may also include other DNA sequences known in the art, like signal sequences (for a better folding, transportation into the periplasm or secretion) inducers for a better modulation of the expression, or cleavage sites for cloning.
• the characteristics of the selected expression vector must be compatible to the host cell which is to be employed. Suitable origins of replications like the CoIEl plasmid replication origin can be used. Suitable promoters include, for example, lac and trp. It is also desirable that the expression vector includes a sequence coding for a selection marker like an antibiotic resistance gene. As selectable markers, ampicillin resistance, or kanamycin resistance may be conveniently employed. All of these materials are known in the art and are commercially available.
• Suitable expression vectors containing the desired coding and control sequences may be constructed using standard recombinant DNA techniques known in the art, many of which are described in Sambrook et al., in "Molecular Cloning: A Laboratory Manual” (1989), Cold Spring Habor, NY Cold Spring Habor Laboratory Press.
• Another embodiment of the present invention is a host cell comprising the expression vector according to the present invention.
• said host cell must be suitable for glycosylation. Therefore, in accordance with the invention said host cell - as a prerequisite - is having endogenous glycosylating enzymes, particularly for N-linked glycosylation.
• another embodiment of the present invention is a host cell comprising the expression vector according to the present invention with the proviso that said host cell is capable of glycosylation by having endogenous glycosylating enzymes, particularly for N- linked glycosylation.
• Another specific embodiment of the invention is a host cell comprising the expression vector according to the present invention with the proviso that said host cell is capable of glycosylation, particularly for N-linked glycosylation by having endogenous glycosylating enzymes, and said host cell is not an E.coli strain.
• Glycosylating enzymes catalyze the reaction in which a carbohydrate, i.e. a glycosyl donor, is attached to a hydroxy! or other functional group of another molecule. This is an enzymatic process that attaches glycans to proteins, lipids, or other organic molecules. Glycosylation is a form of co-translational and post-translational modification. The majority of proteins synthesized in the rough ER undergo glycosylation. It is an enzyme-directed site-specific process, as opposed to the non-enzymatic chemical reaction of "glycation".
• a "host cell capable for glycosylation, particularly for N- linked glycosylation by having endogenous glycosylating enzymes” is a host cell derived from but not limited to Aspergillus niger, Aspergillus sojae, Aspergillus oryzae, Pichia pastoris, Saccharomyces cerevisiae, Hansenula polymorpha, Suitable Pichia host cells include - without being limited thereto - P. pastoris X33 or P. pastoris KM71H available from Invitrogen (5791 Van Allan Way, Carlsbad, CA 92008, USA).
• E.coli strains generally lack a glycosylation system and therefore do not express glycosylated proteins. Only in case a glycosylation system has been genetically engineered into E.coli (e.g. the TV-linked glycosylation system of Campylobacter jejuni), they are enabled to produce glycoproteins.
• the host cells preferably contain an expression vector that comprises all or part of one of the DNA sequences coding for a modified FAD-GDH enzyme having one or more mutations according to the present invention.
• the enzyme according to SEQ ID No.: 01 may be expressed in Aspergillus oryzae and may be expressed as described in e.g. Japanese Patent JP 2010/239969, US-A1 20090259024, US-A 20090155848, US-A1 020080090278; US-A1 20080020426, US-A1 20080014612, US-A1 2008001461 1, US-A1 20080003628, US-B2 7,871 ,805, US-B2 7,741 ,100, US-B2 7,655,130, US-B2 7,553,649 and US-B2 7,494,794.
• the recombinant production of the modified flavin adenine dinucleotide dependent glucose dehydrogenase according to the present invention may be conducted in hosts known in the art.
• Suitable hosts may be selected from strains of filamentous fungi as e.g. Aspergillus niger, Aspergillus sojae and Aspergillus oryzae.
• Suitable hosts may be selected from strains of yeast as e.g. Pichia pastoris, Saccharomyces cerevisiae and Hansenula polymorpha.
• Suitable Pichia host cells include, for example, P. pastoris X33 or P. pastoris KM71H available from Invitrogen (5791 Van Allan Way, Carlsbad, CA 92008, USA).
• modified FAD-GDH pursuant to the invention may have or may have not further modifications that are different from the before- mentioned modification(s).
• modified FAD-GDH or functional fragments thereof according to the present invention are obtainable by expression of a polynucleotide encoding said modified FAD-GDH according to the present invention in yeast, in particular in Pichia pastoris.
• Expression vectors may be introduced into host cells by various methods known in the art. For example, transformation of host cells with expression vectors can be carried out by polyethylene glycol mediated protoplast transformation method (Sambrook et al. 1989, supra). However, other methods for introducing expression vectors into host cells, for example, electroporation, ballistic DNA injection, or protoplast fusion, can also be employed.
• Suitable host cells may be but are not limited to yeast cells like Pichia pastoris, Saccharomyces cerevisiae or Hansenula polymorpha or filamentous fungi like Aspergillus niger or Aspergillus sojae.
• a specific embodiment of the present invention is a host cell comprising the expression vector according the present invention wherein said host cell is from yeast, in particular Pichia pastoris.
• the host cell may be cultured under conditions permitting expression of the desired modified FAD-GDH variants.
• Host cells containing the desired expression vector with the DNA sequence coding for all or part of the modified FAD-GDH can be easily identified by i.e. antibiotic selection or complementation of auxotrophic mutants and selection from minimal medium (J. Sambrook, D. W. Russell: "Molecular Cloning: a laboratory manual", 3 rd edition, Cold Spring Harbor, New York (2001)).
• the expression of the modified FAD-GDH variants can be identified by different methods like measuring production of FAD-GDH mRNA transcripts, detection of the gene product immunologically or detection of the enzymatic activity of the gene product. Preferably, an enzymatic assay is applied.
• suitable host cells comprising the expression vector according to the invention, including an isolated polynucleotide that is encoding for a modified flavin adenine dinucleotide dependent glucose dehydrogenase or an active fragment thereof derived from Aspergillus o yzae FAD-GDH wild-type sequence SEQ ID No: 2 (Wild type) that has a single substitution selected from the group consisting of N168K, N168P, N168Y, or N168W, with the proviso that said host cells are capable of glycosylation, particularly for N-linked glycosylation by having endogenous glycosylating enzymes, and said host cells are not E.coli strains.
• an isolated polynucleotide that is encoding for a modified flavin adenine dinucleotide dependent glucose dehydrogenase or an active fragment thereof derived from Aspergillus o yzae FAD-GDH wild-type sequence SEQ ID No: 2 (Wild type) that
• Another embodiment of the present invention is a process for producing a modified flavin adenine dinucleotide dependent glucose dehydrogenase or functional fragments thereof, the process comprising culturing the transformant according to the present invention.
• the invention also relates to a process for producing FAD-GDH variants of the current invention comprising culturing a host cell of the invention under conditions suitable for production of the modified FAD-GDH of the invention.
• typical culture conditions are liquid medium containing carbon and nitrogen sources, the appropriate antibiotic and induction agent (depending on the used expression vector).
• Typical appropriate antibiotics include ampicillin, kanamycin, chloramphenicol, tetracycline (as well as Zeomycin for P. pastoris) and the like.
• Typical induction agents include IPTG, glucose, lactose (for E. coli) as well as methanol for P. pastoris and the like.
• polypeptides of the present invention are obtained by production in host cells expressing a DNA sequence coding for the modified FAD-GDH.
• the polypeptides of the present invention may also be obtained by in vitro translation of the mRNA encoded by a DNA sequence coding for the modified FAD-GDH.
• the DNA sequences may be synthesized as described above and inserted into a suitable expression vector, which in turn may be used in an in vitro transcription/ translation system.
• An expression vector comprising an isolated polynucleotide as defined and described above operably linked to a promoter sequence capable of promoting its expression in a cell-free peptide synthesis system represents another specific embodiment of the present invention.
• polypeptides produced e.g. by procedures as describe above may then be isolated and purified using various routine protein purification techniques. For example, chromatographic procedures such as ion exchange chromatography, gel filtration chromatography and affinity chromatography may be employed.
• Another subject of the present invention is accordingly a modified flavin adenine dinucleotide dependent glucose dehydrogenase or functional fragments thereof obtainable by the process of the present invention.
• FAD-GDH variants of this invention are for the use in test strips to monitor the blood-glucose level in ex vivo samples of diabetic patients.
• many kinds of samples may be investigated. Bodily fluids like serum, plasma, intestinal fluid or urine are preferred sources for such samples.
• the FAD-GDH variants of the present invention are especially suitable for the use in test strips because they exhibit a more homogeneous glycosylation pattern with less molecular weight distribution as well as an improved temperature stability under dry condition in comparison to FAD-GDHs according to the prior art.
• under dry condition has been defined above.
• Another subject of the present invention is a method of detecting, determining or measuring glucose in a sample using a modified flavin adenine dinucleotide dependent glucose dehydrogenase or functional fragments thereof according to the invention, said detection, determination or measurement comprising contacting a sample with said modified flavin adenine dinucleotide dependent glucose dehydrogenase or functional fragments thereof.
• a specific embodiment of said method according to the present invention is a method, wherein said detection, determination or measurement of glucose is performed using a sensor or a test strip device.
• a device for the detection, determination or measurement of glucose in a sample comprising a modified flavin adenine dinucleotide dependent glucose dehydrogenase or functional fragments thereof according to the present invention, in particular together with other reagents required for said detection, determination or measurement of glucose in a sample.
• the FAD-GDH variants with improved thermo stability of this invention can also be advantageously used in biosensors (D'Costa, E. J., et al, Biosensors 2 (1986) 71-87; Laurinavicius, V., et al., Analytical Letters 32 (1999) 299-316; Laurinavicius, V., et al, Monatshefte fuer Chemie 130 (1999) 1269-1281 ; Malinauskas, A. et al, Sensors and Actuators, B: Chemical 100 (2004) 395-402) for online monitoring of glucose in a sample or a reactor.
• the FAD-GDH variants can, for example, be used to coat an oxygen-insensitive glassy electrode with an osmium complex containing a redox conductive epoxy network (Ye et al, 1993 supra) for more accurate determination of the glucose concentration.
• the present invention relates to a modified flavin adenine dinucleotide dependent glucose dehydrogenase that is glycosylated, selected from the group consisting of a (a) modified flavin adenine dinucleotide dependent glucose dehydrogenase that is glycosylated, wherein at least one of the asparagine residues selected from the group consisting of N2; N168 and N346 according to the mature Aspergillus oryzae FAD-GDH wild-type sequence SEQ ID No: 2 (Wild type) has been substituted by one or more amino acids not suitable for glycosylation and thereby eliminating or inactivating, respectively, a potential glycosylation site at this position and a modified flavin adenine dinucleotide dependent glucose dehydrogenase that is glycosylated that exhibits around 80% amino acid sequence identity or more to said modified flavin adenine dinucleotide dependent glucose dehydrogenase according to (a) modified flavin
• the present invention relates to a modified flavin adenine dinucleotide dependent glucose dehydrogenase or an active fragment thereof according to the first aspect, wherein said modified flavin adenine dinucleotide dependent glucose dehydrogenase exhibits an improved temperature stability under dry conditions in comparison to glycosylated FAD- GDH according to SEQ ID No: 1, wherein said FAD-GDH according to SEQ ID No: 1 is obtainable by expression in Aspergillus oryzae.
• the present invention relates to a modified flavin adenine dinucleotide dependent glucose dehydrogenase or an active fragment thereof according to the first or the second aspect, wherein said modified flavin adenine dinucleotide dependent glucose dehydrogenase or an active fragment thereof exhibits a degree of glycosylation that is ⁇ 50 %, and/ or the ratio of w/Mn is ⁇ 1 ,02.
• the present invention relates to a modified flavin adenine dinucleotide dependent glucose dehydrogenase or an active fragment thereof according to any of the first to third aspects, wherein only one of the asparagine residues selected from the group consisting of N2; N168 and N346 has been substituted by one or more amino acids not suitable for glycosylation and thereby eliminating or inactivating, respectively, a potential glycosylation site at this position.
• the present invention relates to a modified flavin adenine dinucleotide dependent glucose dehydrogenase or an active fragment thereof according to any of the first to fourth aspects, wherein said modified flavin adenine dinucleotide dependent dehydrogenase or an active fragment thereof derived from Aspergillus oryzae FAD-GDH wild-type sequence SEQ ID No: 2 (Wild type) has one or more of the following substitutions N2S, NI68P, N168SP, and N346D.
• the present invention relates to a modified flavin adenine dinucleotide dependent glucose dehydrogenase or an active fragment thereof according to any of the first to fifth aspects, wherein said modified flavin adenine dinucleotide dependent dehydrogenase or an active fragment thereof derived from Aspergillus oryzae FAD-GDH wild-type sequence SEQ ID No: 2 (Wild type) has the following substitution N2S (SEQ ID No: 3).
• the present invention relates to a composition
• a composition comprising a modified flavin adenine dinucieotide dependent glucose dehydrogenase or an active fragment thereof according to any of the first to sixth aspects, wherein said modified flavin adenine dinucieotide dependent glucose dehydrogenase or an active fragment thereof exhibits a degree of glycosylation that is ⁇ 50 3 ⁇ 4, and/ or the ratio of Mw/Mn is ⁇ 1 ,02.
• the present invention relates to an isolated polynucleotide encoding a modified flavin adenine dinucieotide dependent glucose dehydrogenase or an active fragment thereof according to any of the first to sixth aspects with the proviso that said isolated polynucleotide is not encoding for a modified flavin adenine dinucieotide dependent glucose dehydrogenase or an active fragment thereof derived from Aspergillus oryzae FAD-GDH wild-type sequence SEQ ID No: 2 (Wild type) that has a single substitution selected from the group consisting of N16SK, N168P, N168Y, or N168W.
• the present invention relates to an expression vector containing the isolated polynucleotide according to the eighth aspect.
• the present invention relates to a host cell comprising the expression vector according to the ninth aspect, including an isolated polynucleotide that is encoding for a modified flavin adenine dinucieotide dependent glucose dehydrogenase or an active fragment thereof derived from Aspergillus oryzae FAD-GDH wild-type sequence SEQ ID No: 2 (Wild type) that has a single substitution selected from the group consisting of N168K, N168P, N168Y, or N168W, with the proviso that said host cell is capable of glycosylation, particularly for N-linked glycosylation by having endogenous glycosylating enzymes, and said host cell is not an E.coli strain.
• an isolated polynucleotide that is encoding for a modified flavin adenine dinucieotide dependent glucose dehydrogenase or an active fragment thereof derived from Aspergillus oryzae FAD-GDH wild-type sequence SEQ ID No: 2 (Wil
• the present invention relates to a process for producing a modified flavin adenine dinucieotide dependent glucose dehydrogenase or an active fragment thereof, the process comprising culturing the transformant according to the tenth aspect.
• the present invention relates to a modified flavin adenine dinucieotide dependent glucose dehydrogenase or an active fi-agment thereof obtainable by the process of the eleventh aspect.
• the present invention relates to a method of detecting, determining or measuring glucose in an ex vivo sample using a modified flavin adenine dinucleotide dependent glucose dehydrogenase or an active fragment thereof according to any of the first to sixth aspects, or the twelfth aspect or a composition according to the seventh aspect, said detection, determination or measurement comprising contacting an ex vivo sample with said modified flavin adenine dinucleotide dependent glucose dehydrogenase or an active fragment thereof or composition, respectively.
• the present invention relates to the method of the thirteenth aspect, wherein said detection, determination or measurement of glucose is performed using a sensor or a test strip device.
• the present invention relates to a device for the detection, determination or measurement of glucose in an ex vivo sample comprising a modified flavin adenine dinucleotide dependent glucose dehydrogenase or an active fragment thereof according to any of the first to sixth aspects, or the twelfth aspect or a composition according to the seventh aspect.
• the synthetic FAD-GDH wild type gene (SEQ ID No: 7) ligated into a derivative of plasmid pBluescript SK (Stratagene, La Jolla) was used.
• an intrinsic recognition site for restriction endonuclease Xhol was eliminated by a silent mutation by use of the Quick Change II site-directed mutagenesis kit (Stratagene, La Jolla) and mutagenic primers (SEQ ID No: 8, SEQ ID No: 9) resulting in SEQ ID No: 10.
• This construct was used as a template to add flanking sequences including an Xhol site at the 5'- and an Agel site at the 3 '-end by PCR amplification using PCR primers with SEQ ID No: 1 1 and SEQ ID No: 12.
• the PCR product was hydrolysed with restriction endonucleases Xhol and Agel (New England Biolabs) and ligated into the Xhol/Agel hydrolysed expression vector pPICZaA (Invitrogen) resulting in a fusion gene (SEQ ID No: 13) coding for the a- factor signal sequence, a proteolytic cleavage site (KEX2) and the mature FAD-GDH.
• pPICZaA carrying SEQ ID No: 13 was used as a template for site-directed mutagenesis.
• mutagenic primer pairs were used as seen in Table 1 together with the Quick Change II site- directed mutagenesis Kit (Stratagene, La Jolla) according to the manufacturer instructions.
• electro competent cells of Pichia pastoris strain X33 were transfected by electroporation with 5 - 10 ⁇ g of linearized pPICZaA carrying the DNA encoding the corresponding FAD-GDH variants (SEQ ID No: 16, No: 19, No: 22 and No: 25, respectively). All experimental steps were performed according to the manufacturer's instructions. Transfected cells were plated on YPD agar plates (1% yeast extract, 2% peptone, 2% dextrose (glucose)) containing 100 ⁇ , 250 ⁇ g/ml or 500 ⁇ g/ml Zeocin as a selection marker and incubated at 28°C for 2 - 3 days.
• the FAD-GDH was eluted by a linear gradient of a 20 mM potassium phosphate buffer pH 7.5 and a ammonium sulfate concentration of 2.5 M after a 20 mM potassium phosphate buffer pH 7.5 (5 L).
• the fractions containing FAD-GDH were collected, purified and concentrated by ultra filtration/ ultra dialysis to about 0.05 L and concentrated in 20 mM Tris/HCl buffer pH 8.5.
• the sample was applied to a 500 ml Q-sepharose column, washed with 2.5 L of a 20 mM Tris/ HC1 buffer pH 8.5 and eluted by a linear gradient (5 L) of 20 mM Tris/HCl buffer pH 8.5 with 100 mM NaCI.
• the FAD-GDH containing fractions were collected, purified and concentrated to a protein concentration of about 50 mg/ml in 100 mM PIPES buffer pH 7.1 by ultra filtration/ultra dialysis. The resulting sample was lyophilized.
• Enzyme activity and sugar specificity of modified flavin adenine dinucleotide dependent glucose dehydrogenases or functional fragments thereof i) Determining enzyme activity (1M D-glucose as substrate)
• Activity (U/ml) ⁇ - ( ⁇ OD TEST - ⁇ OD BLANK) x 3.0 xdilution ratio ⁇ / ⁇ 16.3 ⁇ 0.1 ⁇ 1.0 ⁇
• 3.0 is the amount (ml) of respective reaction reagent + enzyme solution
• 16.3 is the millimolar molecular absorption coefficient (cm 2 /micromole) under the conditions for measuring activities of the present invention
• 0.1 is the amount of enzyme solution (ml)
• 1.0 is the optical light path (cm) of the cell.
• the Weight Average Molecular Weight (Mw) and the Number Average Molecular Weight (M n ) was calculated by means of the software from the raw data of the Viscotek Triple Detektors (refraction index (RI) and Right Angle Light Scattering (RALS)).
• the ratio of w/M n is the polydispersity and gives the size distribution of the protein.
• Monodisperse proteins have a ⁇ / ⁇ ⁇ value of 1.
• Solution 2 Saturated -15% (w/v) 2,18 Phosphormolybdic acid, sodium salt ((Na6[P 2 Mo 18 0 62 ] *24 H 2 0) CAS 50811-90-0, Honeywell Specialty Chemicals, Article No. 04137) in water.
• the Glucose concentration in the reaction mixture was varied in the range of 0.1 - 170 mM by changing the Glucose concentration in S3,
• the 10 mg lyophilized enzyme was dissolved per ml 100 mM Pipes buffer pH 7.1 . Aliquotes of 1 ml of this solution were stored in closed plastic vials and incubated in temperature controlled water bathes for up to 12 days. The enzyme activities were measured according to Example 3.
• the unladed weight of the glass vessel was determined by means of an analytical balance. 10 mg of the lyophilized enzyme sample was weighed. All vessels were closed with plugs in a way that a controlled gas exchange of inner space and the environment was ensured but the sample was prohibited from leaving the vessel. The sample was exposed to 80°C for 8 days in a desiccator in the presence of a drying agent (molecular sieve 3 A, MS 551, Grace). After this period, the sample was cooled to room temperature, further allowing the gas exchange with the environment. Afterwards, the vessels were completely closed and the respective weights were determined. In dependence of the original quantity of enzyme, the sample was diluted with ultrapure water to a final concentration of 10 mg/ml completely dissolved by gentle vortexing. The sample was stored for reconstitution for exactly one hour at room temperature and afterwards cooled with ice. Based on this stock solution, dilution took place in ice-cold working buffer, followed by the determination of activity.
• a drying agent molecular sieve 3 A, MS 551,
• Table 3 Exemplary molecular weight distribution SEQ ID No.l vs. SEQ ID No.3
• FAD-GDH (SEQ ID No: 1) from Aspergillus 103 876 99 901 1.040
• FAD-GDH (SEQ ID No: 3) variant 1 ; N2S 76 333 76 147 1.002
• Fig. 1 Pichia expression vector pPICZaA:
• PAOX1 AOX1 promoter (initiates transcription of the gene of interest)
• aFactor-ss gene encoding the -Factor signal sequence (gene of interest is fused in frame to this sequence; corresponding polypeptide is secreted into the culture medium by P. pastoris)
• TAOX1 AOX1 terminator (stops transcription of the gene of interest)
• ColEl origin of replication (allows cloning in E. coli)

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