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  • C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)
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  • 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
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  • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
  • C12Y101/01006—Glycerol dehydrogenase (1.1.1.6)
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  • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
  • C12Y101/01027—L-Lactate dehydrogenase (1.1.1.27)
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  • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
  • C12Y101/01028—D-Lactate dehydrogenase (1.1.1.28)
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  • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
  • C12Y101/01037—Malate dehydrogenase (1.1.1.37)
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  • C12Y101/01047—Glucose 1-dehydrogenase (1.1.1.47)
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  • C12Y104/01—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with NAD+ or NADP+ as acceptor (1.4.1)
  • C12Y104/01005—L-Amino-acid dehydrogenase (1.4.1.5)

Definitions

• the present invention relates to a method for stabilizing an enzyme by storing the enzyme in the presence of a stable coenzyme. Furthermore, the present invention relates to a stabilized with a stable coenzyme enzyme and its use in test elements for the detection of analytes.
• Biochemical measuring systems are important components of clinically relevant analytical methods.
• analytes e.g. Metabolites or substrates in the foreground, which are determined directly or indirectly by means of an enzyme.
• the analytes are hereby reacted with the aid of an enzyme-coenzyme complex and then quantified.
• the analyte to be determined is brought into contact with a suitable enzyme and a coenzyme, the enzyme usually being used in catalytic amounts.
• the coenzyme is altered by the enzymatic reaction, e.g. oxidized or reduced. This process can be detected electrochemically or photometrically directly or through a mediator.
• a calibration provides a direct correlation of the measured value with the concentration of the analyte to be determined.
• Coenzymes are organic molecules that are covalently or non-covalently bound to an enzyme and are altered by the reaction of the analyte.
• Prominent examples of coenzymes are nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), which form NADH and NADPH by reduction.
• NAD nicotinamide adenine dinucleotide
• NADP nicotinamide adenine dinucleotide phosphate
• a known measure used to increase the stability of biochemical measuring systems is the use of stable enzymes, e.g. the use of enzymes from thermophilic organisms. Furthermore, it is possible to use enzymes by chemical modification, e.g. Crosslinking, or to stabilize by mutagenesis. In addition, enzyme stabilizers, e.g. Trehalose, polyvinylpyrrolidone and serum albumin, or the enzymes e.g. be included in polymer networks by photopolymerization.
• mediators with the lowest possible redox potential increases the specificity of tests and eliminates disturbances during the reaction.
• a lower limit for the redox potential of mediators is the redox potentials of the enzyme / coenzyme complexes. If these are undercut, the reaction with the mediators is slowed or even stopped.
• biochemical measuring systems without mediators, in which, for example, direct detection of coenzymes, for example of the coenzyme NADH, takes place.
• coenzymes such as NAD and NADP
• coenzymes are unstable.
• NAD and NADP are base-labile molecules whose degradation pathways are described in the literature (NJ Oppenheimer in The Pyridine Nucleotide Coenzyme Academic Press New York, London 1982, ed. J. Everese, B. Anderson, K. You, Chapter 3, pages 56- 65).
• the degradation of NAD or NADP produces ADP-ribose by cleaving the glycosyl bonds between the ribose and the pyridine moiety.
• NADH and NADPH are acid labile: for example, epimerization is a known degradation pathway.
• epimerization is a known degradation pathway.
• the instability of NAD / NADP and NADH / NADPH underlies the lability of glycosyl binding between the ribose and pyridine moieties.
• the hydrolysis of the coenzymes NAD or NADP is done solely by the ambient humidity. This instability can lead to inaccuracies in the measurement of analytes.
• NAD / NADP derivatives are used e.g. in B.M. Anderson in the Pyridine Nucleotide Coenzyme, Academic Press New York, London 1982, ed. J. Everese, B. Anderson, K. You, Chapter 4. However, most of these derivatives are not well accepted by enzymes.
• the only derivative heretofore used for diagnostic testing is 3-acetylpyridine-adenine dinucleotide (acetyl NAD), first described in 1956 (N.O. Kaplan, J. Biol. Chem. (1956), 221, 823). Also, this coenzyme shows a low acceptance of enzymes and a change in the redox potential.
• WO 01/94370 describes the use of further NAD derivatives with a modified pyridine group.
• modifications of the nicotinamide group generally have a direct influence on the catalytic reaction. In most cases this influence is negative.
• the ribose unit was altered to influence the stability of the glycosyl bond. This procedure does not directly interfere with the catalytic reaction of - A -
• Nicotinamide group there may be an indirect effect once the enzyme has strong and specific binding to the ribose moiety.
• Kaufmann et al. disclose a series of thioribose NAD derivatives.
• a connection between the modification of the nicotinamide ribose unit and the activity of the derivatives in enzymatic reactions has not yet been shown.
• carba NAD a derivative without glycosyl linkage
• the ribose is substituted herein by a carbacyclic sugar moiety.
• carbaNAD has been described as a substrate for dehydrogenases, its activity has not previously been demonstrated in biochemical detection in the clinic.
• WO 2007/012494 and US 11 / 460,366 disclose stable NAD / NADH or NADP / NADPH derivatives, enzyme complexes of these derivatives and their use in biochemical detection methods and reagent kits.
• the object underlying the present invention was to provide processes for the stabilization of enzymes, in particular for the long-term stabilization of enzymes.
• the enzyme stabilized by the method according to the invention is a coenzyme-dependent enzyme.
• Suitable enzymes are e.g. Dehydrogenases selected from a glucose dehydrogenase (EC 1.1.1.47), lactate dehydrogenase (EC1.1.1.27, 1.1.1.28), malate dehydrogenase (EC1.1.1.37), glycerol dehydrogenase (EC1.1.1.6), alcohol Dehydrogenase (EC1.1.1.1), alpha-hydroxybutyrate
• Dehydrogenase sorbitol dehydrogenase or amino acid dehydrogenase, e.g. B. L-amino acid dehydrogenase (E.C.1.4.1.5).
• Other suitable enzymes are oxidases, such as glucose oxidase (E.C.1.1.3.4) or cholesterol oxidase (E.C.1.1.3.6) or aminotransferases, such as. Aspartate or alanine aminotransferase, 5'-nucleotidase or creatine kinase.
• the enzyme is glucose dehydrogenase.
• mutated glucose dehydrogenase In the context of the method according to the invention, the use of a mutated glucose dehydrogenase has proven to be particularly preferred.
• mutant as used in the present application is used, denotes a genetically modified variant of a native enzyme which has the same number of amino acids compared to the wild-type enzyme modified amino acid sequence, that is different in at least one amino acid from the wild-type enzyme.
• the introduction of the mutation (s) may be site-specific or non-site specific, preferably site-specific using recombinant methods known in the art, resulting in at least one amino acid substitution within the amino acid sequence of the native enzyme according to the particular requirements and conditions.
• the mutant particularly preferably has an increased thermal or hydrolytic stability compared to the wild-type enzyme.
• the mutated glucose dehydrogenase may in principle contain the amino acid (s) modified from the corresponding wild-type glucose dehydrogenase at any position of its amino acid sequence.
• the mutant glucose dehydrogenase comprises a mutation at least one of positions 96, 170 and 252 of the amino acid sequence of wild-type glucose dehydrogenase, with mutants having mutations at position 96 and position 170 or mutations at position 170 and position 252 being particularly preferred , It has proved to be advantageous if the mutated glucose dehydrogenase contains no further mutations in addition to these mutations.
• the mutation at positions 96, 170 and 252 may in principle comprise any amino acid substitution which leads to a stabilization, for example an increase in the thermal or hydrolytic stability, of the wild-type enzyme.
• the mutation at position 96 comprises an amino acid substitution of glutamic acid for glycine, while with respect to position 170, an amino acid substitution of glutamic acid for arginine or lysine, in particular an amino acid substitution of glutamic acid for lysine, is preferred.
• the mutation at position 252 it preferably comprises an amino acid substitution of lysine for leucine.
• the mutant glucose dehydrogenase can be obtained by mutation of a wild-type glucose dehydrogenase derived from any biological source, the term "biological source” for the purposes of this invention including prokaryotes, such as bacteria, and eukaryotes, such as mammals and others
• the wild-type glucose dehydrogenase is derived from a bacterium, more preferably a glucose dehydrogenase from Bacillus megaterium, Bacillus subtilis or Bacillus thuringiensis, in particular from Bacillus subtilis.
• the mutated glucose dehydrogenase is a glucose dehydrogenase obtained by mutation of wild-type glucose dehydrogenase from Bacillus subtilis which has the sequence shown in SEQ ID NO: 1 (GlucDH_E96G_E170K) or SEQ ID NO: 1 ID NO: 2 (GlucDH_E170K_K252L) has the amino acid sequence shown.
• the stable coenzyme is a coenzyme chemically altered from the native coenzyme, which has higher stability (e.g., hydrolytic stability) compared to the native coenzyme.
• the stable coenzyme is stable to hydrolysis under test conditions.
• the stable coenzyme can be a decreased
• stable coenzymes are stable derivatives of nicotinamide adenine dinucleotide (NAD / NADH) or nicotinamide adenine dinucleotide phosphate (NADP / NADPH), or truncated NAD derivatives, eg without AMP moiety or with non-nucleosidic residues, eg hydrophobic residues.
• a stable coenzyme in the context of the present invention is the compound of the formula (I)
• stable derivatives of NAD / NADH or NADP / NADPH are described in the aforementioned references, the disclosure of which is incorporated herein by reference.
• Particularly preferred stabilized coenzymes are described in WO 2007/012494 and US Pat. No. 11 / 460,366, the disclosure of which is hereby expressly incorporated by reference.
• the stable coenzyme is more preferably selected from compounds of general formula (II):
• A adenine or an analogue thereof
• T each independently O
• S each independently OH
• SH each independently OH
• BH 3 " , BCNH 2 "
• V each independently OH or a phosphate group, or two
• X 1 , X 2 each independently O, CH 2 , CHCH 3 , C (CH 3 J 2 , NH, NCH 3 ,
• Y NH 1 S 1 O 1 CH 2
• Z a linear or cyclic organic radical, with the proviso that Z and the pyridine radical are not linked by a glycosidic compound, or
• Z is particularly preferably a saturated or unsaturated carbocyclic or heterocyclic five-membered ring, in particular a compound of the general formula (III)
• R 5 'and R 5 may be a single or double bond
• the compounds of the invention contain adenine or adenine analogs such as C 8 and N 6 substituted adenine, deaza variants such as 7-deaza, azavaries such as 8-aza, or combinations such as 7-deaza or 8-aza or carbocyclic analogs, such as formycin, where the 7-deaza variants in the 7-position may be substituted by halogen, C 1 -C 6 -alkynyl, -alkenyl or -alkyl.
• the compounds adenosine analogues which take ribose, for example, 2-methoxy-deoxyribose, 2 1 - Fluoro-deoxyribose, hexitol, altritol or polycyclic analogs containing as bicyclo-, LNA and tricyclo sugar.
• ribose for example, 2-methoxy-deoxyribose, 2 1 - Fluoro-deoxyribose, hexitol, altritol or polycyclic analogs containing as bicyclo-, LNA and tricyclo sugar.
• W is preferably CONH 2 or COCH 3 .
• R 5 is preferably CH 2 . Furthermore, it is preferred that R 51 is selected from CH 2 , CHOH and NH. In a particularly preferred embodiment, R 5 'and R 5 "are each CHOH. In yet another preferred embodiment, R 51 is NH and R 5 " is CH 2 . Specific examples of preferred stabilized coenzymes are shown in Figures 1A and B.
• the stable coenzyme to carbaNAD In the most preferred embodiment, the stable coenzyme to carbaNAD.
• the inventive method is in particular for
• enzymes suitable Long-term stabilization of enzymes suitable. This means that the enzyme stabilized with a stable coenzyme, e.g. as a dry substance, for example over a period of at least 2 weeks, preferably of at least 4 weeks and more preferably of at least
• the method according to the invention comprises a storage of the stabilized with a stable coenzyme enzyme at elevated temperatures, for example at a temperature of at least 20 0 C, preferably of at least 25 0 C and more preferably of at least 30 0 C.
• the enzyme activity preferably decreases by less than 50%, more preferably less than 30%, and most preferably less than 20% of their initial value.
• the stabilization according to the invention makes it possible to store the stabilized enzyme with a stable coenzyme without drying reagent for a long time, as indicated above, and / or at high temperatures, as indicated above. Furthermore, the stabilized enzyme may also be maintained at a high relative humidity, e.g. a relative humidity of at least 50%, wherein the enzyme activity preferably decreases by less than 50%, more preferably less than 30% and most preferably less than 20% of the initial value.
• a high relative humidity e.g. a relative humidity of at least 50%
• the storage of the stabilized with a stable coenzyme enzyme can be done on the one hand as dry matter and on the other hand in liquid phase.
• the storage of the stabilized enzyme preferably takes place on or in a test element which is suitable for the determination of an analyte.
• That with a stable coenzyme stabilized enzyme is preferably part of a detection reagent, which may optionally contain other ingredients such as salts, buffers, etc.
• the detection reagent is free from a mediator.
• the stabilized with a stable coenzyme enzyme can be used for the detection of analytes, such as parameters in body fluids such as blood, serum, plasma or urine or in wastewater samples or food.
• any biological or chemical substances which can be detected by a redox reaction e.g. Substances that are substrates of a coenzyme-dependent enzyme or coenzyme-dependent enzymes themselves.
• Preferred examples of analytes are glucose, lactic acid, malic acid, glycerol, alcohol, cholesterol, triglycerides, ascorbic acid, cysteine, glutathione, peptides, urea , Ammonium, salicylate, pyruvate, 5'-nucleotidase, creatine kinase (CK), lactate dehydrogenase (LDH), carbon dioxide, etc.
• the analyte is glucose. 0
• Another object of the present invention is the use of a compound of the invention or a stabilized with a stable coenzyme enzyme for the detection of an analyte in a sample by an enzymatic reaction.
• a compound of the invention or a stabilized with a stable coenzyme enzyme for the detection of an analyte in a sample by an enzymatic reaction.
• Particularly preferred here is the detection of glucose with the aid of glucose dehydrogenase (GlucDH).
• the change of the stable coenzyme by reaction with the analyte can basically be detected in any way.
• all methods known from the prior art for detecting enzymatic reactions can be used.
• the change in the coenzyme is detected by optical methods.
• Optical detection methods include, for example, the Measurement of absorption, fluorescence, circular dichroism (CD), optical rotation dispersion (ORD), refractometry etc.
• An optical detection method which is preferably used in the context of the present application, is photometry.
• photometry For the photometric measurement of a change in the coenzyme due
• Nitrosoanilines such as [(4-nitrosophenyl) imino] dimethanol hydrochloride, quinones such as phenanthrenequinones, phenanthroline quinones or benzo [h] quinoline quinones, phenazines such as 1- (3-carboxypropoxy) -5-ethylphenazinium trifluoromethanesulfonate, or / and diaphorase (EC 1.6.99.2).
• Preferred examples of phenanthroline quinones include 1, 10-phenanthroline-5,6-quinones, 1, 7-phenanthroline-5,6-quinones, 4,7-quinones,
• Phenanthroline-5,6-quinones and their N-alkylated or N, N'-dialkylated salts wherein in the case of N-alkylated or N, N'-dialkylated salts halides, trifluoromethanesulfonate or other solubility enhancing anions are preferred as the counterion.
• any substance can be used which is reducible and upon reduction undergoes a detectable change in its optical properties, such as color, fluorescence, remission, transmission, polarization and / or refractive index.
• the determination of the presence or / and the amount of the analyte in the sample can be done with the naked eye and / or by means of a detection device using a photometric method which appears to be suitable for the person skilled in the art.
• heteropolyacids, and especially 2,18-phosphomolybdic acid are used as optical indicators which are reduced to the corresponding heteropolyblue.
• the change in the coenzyme is detected by measuring the fluorescence.
• the fluorescence measurement is highly sensitive and enables the detection even of low concentrations of the analyte in miniaturized systems.
• the change in coenzyme can also be detected electrochemically using a suitable test element, such as an electrochemical test strip.
• a suitable test element such as an electrochemical test strip.
• suitable mediators which can be converted by the reduced coenzyme by transferring electrons into a reduced form.
• the analyte is determined by measuring the current required for reoxidation of the reduced mediator, which correlates with the concentration of the analyte in the sample.
• mediators that can be used for electrochemical measurements include in particular the aforementioned mediators used for photometric measurements.
• a liquid test may be used, the reagent being e.g. in the form of a solution or suspension in an aqueous or non-aqueous liquid or as a powder or lyophilisate.
• a dry test may also be used wherein the reagent is applied to a carrier, a test strip.
• the carrier may comprise a test strip comprising an absorbent and / or swellable material which is wetted by the sample liquid to be examined.
• a particularly preferred assay format involves the use of the enzyme glucose dehydrogenase with a stable NAD derivative to detect glucose, wherein a derivative of the reduced coenzyme NADH is formed.
• the detection of NADH is carried out by optical methods, eg by photometric or fluorometric determination after UV excitation.
• a particularly preferred test system is described in US 2005/0214891, which is incorporated herein by reference.
• a still further object of the present invention is a stabilized with a stable coenzyme enzyme, wherein the stabilized enzyme at a storage of preferably at least 2 weeks, more preferably at least 4 weeks and most preferably at least 8 weeks at a temperature of preferably at least 20 0 C. , more preferably at least 25 ° C., and most preferably at least 30 ° C., optionally at high humidity and without dry reagent, a decrease in enzymatic activity of less than 50%, preferably less than 30% and most preferably less than 20% from baseline having.
• Yet another object of the invention is a detection reagent for the determination of an analyte which contains a stabilized with a stable coenzyme enzyme, as indicated above.
• the invention relates to a test element which contains a stabilized enzyme or a detection reagent according to the invention.
• the detection reagent or the test element may be suitable by performing dry or liquid tests.
• the test element is a test strip for the fluorometric or photometric detection of an analyte.
• Such a test strip contains the stable coenzyme stabilized enzyme immobilized on an absorbent or / and swellable material, such as cellulose, plastics, etc.
• Figure 1A Representation of the stable coenzyme carba-NAD (cNAD).
• Figure 1 B Representation of the stable coenzyme pyrrolidinyl-NAD.
• Figure 2 Representation of the results of the enzyme kinetics of glucose dehydrogenase in the presence of NAD or of glucose dehydrogenase in the presence of cNAD before and after storage.
• Figure 2A Kinetics of GlucDH in the presence of NAD after 1 day.
• 2B Kinetics of GlucDH in the presence of cNAD after 1 day.
• 2C Kinetics of GlucDH in the presence of NAD after 5 weeks of storage at 32 0 C and 85% relative humidity.
• 2D Kinetics of GlucDH in the presence of cNAD after 5 weeks of storage at 32 0 C and 85% relative humidity.
• Figure 3 Comparison of Blank values of glucose-dehydrogenase in the presence of NAD or of GlucDH in the presence of cNAD over a period of up to 5 weeks at 32 C C and 85% humidity.
• Figure 4 Representation of various functional curves of glucose dehydrogenase after storage of glucose dehydrogenase in the presence of NAD at 32 0 C and 85% humidity. The storage duration varied between 1 day and 5 weeks.
• Figure 5 Representation of various functional curves of glucose dehydrogenase after storage of glucose dehydrogenase in the presence of cNAD at 32 0 C and 85% humidity. The storage period varied between 1 day and 5 weeks (FIG. 5A) or between 1 day and 24 weeks (FIG. 5B).
• Figure 6 Representation of the residual content of NAD or cNAD after storage of glucose dehydrogenase in the presence of NAD or cNAD for 24 weeks at 32 0 C and 85% humidity.
• FIG. 7 Representation of GlucDH activity after storage of glucose Dehydrogenase in the presence of NAD or cNAD over 5 weeks ( Figure 7A) and 24 weeks ( Figure 7B) at 32 0 C and 85% humidity.
• FIG. 8 Representation of GlucDH activity after storage of glucose dehydrogenase (GlucDH wt), the double mutant GlucDH_E96G_E170K (GlucDH Mut1) and the double mutant GlucDH_E170K_K252L (GlucDH Mut2) over a period of 25 weeks in the presence of NAD or cNAD at 32 0 C and 83% relative humidity.
• GlucDH wt glucose dehydrogenase
• GlucDH Mut1 double mutant GlucDH_E96G_E170K
• GlucDH Mut2 double mutant GlucDH_E170K_K252L
• FIG. 9 Gel electrophoretic analysis of glucose dehydrogenase after storage in the presence of NAD or cNAD. Test conditions: MW, 10-220 kDa markers; 1: GlucDH / NAD, 5 weeks at 6 0 C; 2: GlucDH / NAD, 5 weeks at 32 ° C / 85% relative humidity; 3: GlucDH / cNAD, 5 weeks at 6 0 C; 4: GlucDH / cNAD, 5 weeks at 32 ° C / 85% relative humidity.
• Figure 10 Gel electrophoretic analysis of glucose dehydrogenase after storage at 50 0 C in the presence of NAD or cNAD. Test conditions: MW, 10-220 kDa markers; 1: GlucDH 8.5 mg / ml, NAD, 0 hours; 2: GlucDH 8.5 mg / ml, NAD, 22 hours; 3: GlucDH 8.5 mg / ml, NAD, 96 hours; 4: GlucDH 8.5 mg / ml, NAD, 118 hours; 5: GlucDH 8.5 mg / ml, NAD 1 140 hours; 6: GlucDH 8.5 mg / ml, NAD, 188 hours; 7: GlucDH 8.5 mg / ml, NAD, 476 hours; 8: GlucDH 8.5 mg / ml, cNAD, 0 hours; 9: GlucDH 8.5 mg / ml, cNAD, 188 hours; 10: GlucDH 8.5 mg / ml,
• Figure 11 Representation of the stability of glucose dehydrogenase in the presence of NAD or cNAD in liquid phase at 50 0 C over a period of 4 days ( Figure 11A) and 14 days ( Figure 11B).
• Test conditions GlucDH 10 mg / ml; NAD or cNAD 12 mg / ml; Buffer: 0.1 M Tris, 1.2 M NaCl, pH 8.5; Temperature 50 ° C.
• FIG. 12 Gel electrophoretic analysis of alcohol dehydrogenase Yeast after storage in the presence of NAD or cNAD. Test conditions: MW, 10-220 kDa markers; 1: ADH 1 65 hours at 6 0 C; 2: ADH / cNAD, 65 hours at 6 0 C; 3: ADH / NAD, 65 hours at 60 ° C; 4: ADH, 65 hours at 35 ° C .; 5: ADH / cNAD, 65 hours at 35 0 C; 6: ADH / NAD, 65 hours at 35 ° C.
• Figure 13 Gel electrophoretic analysis of alcohol dehydrogenase from yeast after storage at 35 0 C in the presence of NAD or cNAD.
• Test conditions MW, 10-220 kDa markers; 1: ADH / NAD, 0 days; 2: ADH / NAD, 1 day; 3: ADH / NAD, 2 days; 4: ADH / NAD, 3 days; 5: ADH / NAD, 5 days; 6: ADH / cNAD, 0 days; 7: ADH / cNAD, 1 day; 8: ADH / cNAD, 2 days; 9: ADH / cNAD, 3 days; 10: ADH / cNAD, 6 days.
• Figure 14 Representation of the stability of alcohol dehydrogenase from yeast in the presence of NAD or cNAD in liquid phase at 35 0 C over a period of 65 hours.
• Test conditions ADH 5 mg / ml; NAD or cNAD 50 mg / ml; Buffer: 75 mM Na 4 P 2 O 7 , glycine, pH 9.0; Temperature 35 ° C.
• FIG. 15 Illustration of various functional curves of glucose dehydrogenase after 11 weeks of storage in the presence of NAD and different mediators at room temperature.
• FIG. 16 Preparation of the results of the enzyme kinetics of glucose dehydrogenase in the presence of NAD and 1- (3-carboxypropoxy) -5-ethylphenazinium trifluoromethanesulfonate at various glucose concentrations.
• FIG. 17 Schematic representation of the glucose detection with GlucDH as enzyme and diaphorase as mediator.
• Figure 18 Representation of the functional curves of glucose-dye oxidoreductase (GlucDOR) in the presence of pyrroloquinoline quinone (PQQ) and [(4-nitrosophenyl) imino] dimethanol hydrochloride as a mediator or of Glucose dehydrogenase in the presence of NAD and diaphorase / [(4-nitrosophenyl) imino] dimethanol hydrochloride as a mediator.
• PQQ pyrroloquinoline quinone
• NAD and diaphorase / [(4-nitrosophenyl) imino] dimethanol hydrochloride as a mediator.
• FIG. 19 Representation of the results of the enzyme kinetics of glucose dehydrogenase in the presence of NAD and diaphorase at various glucose concentrations.
• FIG. 20 Representation of the current measured as a function of the glucose concentration during the electrochemical determination of glucose using glucose dehydrogenase in the presence of NAD or cNAD. Test conditions: 25 mM NAD or cNAD; 2.5 seconds delay; 5 seconds measurement time.
• Figure 21 Representation of the amino acid sequences of the glucose dehydrogenase double mutant GlucDH_E96G_E170K and
• Glucose-specific GlucDH was added to carba-NAD (Fig. 1A) or NAD. These formulations were each applied to Pokalon (Lonza) and stored after drying under warm and humid conditions (32 0 C, 85% relative humidity). Subsequently, the reaction kinetics and the function curve were determined at regular intervals. At the same time, a cNAD / NAD analysis and a determination of the residual activity of the enzyme was performed at the respective measurement dates.
• the relationship between the structure of the coenzyme and its stability over a predetermined period of time is derived from Figure 6. Accordingly, the residual content of cNAD is in a glucose-detecting reagent after 24 weeks of storage (at 32 0 C and 85% relative humidity) for about 80% of the Initial level, while the content of NAD in an NAD-stabilized glucose detection reagent already after 5 weeks to about 35% of the initial value decreases and after extrapolation after about 17 weeks to zero.
• the residual activity of GlucDH can be further increased.
• the residual activity of a mutant GlucDH_E96G_E170K with amino acid substitutions glutamic acid is ⁇ glycine at position 96 and glutamic acid ⁇ lysine at position 170 (GlucDH Mut1) of the wild type enzyme approximately 70% while the residual activity of a mutant GlucDH_E170K_K252L with
• Presence of cNAD stored enzyme against a band detected at the beginning of the experiment shows only a slight change.
• glucose dehydrogenase For the determination of glucose, various test systems, each comprising glucose dehydrogenase, NAD 1 a mediator and optionally an optical indicator were measured photometrically or electrochemically.
• FIG. 18 shows for the system glucose dehydrogenase, NAD, diaphorase, [(4-nitrosophenyl) imino] dimethanol hydrochloride and 2.18-
• Phosphomolybdic acid (system 1) a concentration-dependent decrease in remission.
• system glucose-dye oxidoreductase, pyrroloquinoline quinone, [(4-nitrosophenyl) imino] dimethanol hydrochloride and 2,18-phosphomolybdic acid (System 2), which likewise causes a concentration-dependent decrease in remission, due to the low specificity of glucose DYE oxidoreductase but has disadvantages.
• the kinetics of the conversion of glucose by means of the system 1 at glucose concentrations in the range of 0 to 800 mg / dl is shown in FIG.
• an electrochemical measurement can also be used for the purpose of determining the analyte.
• a test element which, in addition to glucose dehydrogenase, contained NAD as coenzyme and 1- (3-carboxypropoxy) -5-ethylphenazinium trifluoromethanesulfonate as mediator, as well as for a corresponding system comprising the stabilized coenzyme cNAD instead of NAD , a linear dependence of the current required for reoxidation of the reduced mediator on the glucose concentration ( Figure 20).
• the analyte determination using the system dehydrogenase / stable coenzyme can also be carried out by means of electrochemical detection and evaluation with a different wavelength, which is independent of the coenzyme.
• the stable enzyme / coenzyme pair the total formulation should also be further stabilized.

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