WO2010094632A1 - Cinétique réactionnelle rapide d'enzymes présentant une activité inférieure dans des couches chimiques sèches - Google Patents

Cinétique réactionnelle rapide d'enzymes présentant une activité inférieure dans des couches chimiques sèches Download PDF

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WO2010094632A1
WO2010094632A1 PCT/EP2010/051801 EP2010051801W WO2010094632A1 WO 2010094632 A1 WO2010094632 A1 WO 2010094632A1 EP 2010051801 W EP2010051801 W EP 2010051801W WO 2010094632 A1 WO2010094632 A1 WO 2010094632A1
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Prior art keywords
dehydrogenase
analyte
coenzyme
glucose
mutated
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PCT/EP2010/051801
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German (de)
English (en)
Inventor
Carina Horn
Claudia GÄSSLER-DIETSCHE
Dieter Heindl
Joachim Hönes
Thomas Meier
Rainer Schmuck
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F. Hoffmann-La Roche Ag
Roche Diagnostics Gmbh
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Priority claimed from PCT/EP2009/001206 external-priority patent/WO2009103540A1/fr
Application filed by F. Hoffmann-La Roche Ag, Roche Diagnostics Gmbh filed Critical F. Hoffmann-La Roche Ag
Priority to CA2750474A priority Critical patent/CA2750474C/fr
Priority to KR1020117019082A priority patent/KR101604624B1/ko
Priority to CN2010800085457A priority patent/CN102325896A/zh
Priority to JP2011550531A priority patent/JP6113405B2/ja
Priority to MX2011008669A priority patent/MX2011008669A/es
Priority to KR1020137029083A priority patent/KR20130127555A/ko
Priority to EP10704923.1A priority patent/EP2398909B1/fr
Publication of WO2010094632A1 publication Critical patent/WO2010094632A1/fr
Priority to US13/210,564 priority patent/US9359634B2/en

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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
    • C12N9/0004—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
    • 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
    • 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

Definitions

  • the present invention relates to a method for determining an analyte and a diagnostic element suitable for this purpose.
  • Diagnostic elements are important components of clinically relevant analysis methods.
  • analytes e.g. Metabolites or substrates in the foreground, which are determined directly or indirectly, for example, using an enzyme specific for the analyte.
  • the analytes are hereby reacted with the aid of an enzyme-coenzyme complex and then quantified.
  • the analyte to be determined is contacted with a suitable enzyme, a coenzyme and, if necessary, a mediator, wherein the coenzyme is physicochemically altered by the enzymatic reaction, e.g. oxidized or reduced, is.
  • a mediator when used, this usually transfers the electrons released from the reduced coenzyme upon reaction of the analyte to an optical indicator or the conductive constituents of an electrode, so that the process can be detected, for example, photometrically or electrochemically.
  • a calibration provides a direct correlation of the measured value with the concentration of the analyte to be determined.
  • diagnostic elements are characterized by a limited shelf life and by special environmental requirements, such as cooling or dry storage, to achieve this durability.
  • special environmental requirements such as cooling or dry storage
  • the error results are based primarily on the fact that the enzymes, coenzymes and mediators used in such diagnostic elements are generally sensitive to moisture and heat and are inactivated over time.
  • a known measure used to increase the stability of diagnostic elements is the use of stable enzymes, e.g. the use of enzymes from thermophilic organisms. Furthermore, it is possible to stabilize enzymes by chemical modification, in particular by cross-linking. In addition, enzyme stabilizers, e.g. Trehalose, polyvinylpyrrolidone and serum albumin, or the enzymes e.g. be included in polymer networks by photopolymerization.
  • Another way of stabilizing enzymes is site-specific or non-site-specific mutations introduced.
  • the use of recombinant techniques has proven to be particularly suitable, which allow a targeted influencing of the properties of the corresponding enzyme by targeted modification of the DNA coding for an enzyme.
  • Baik et al. (Appl. Environ. Microbiol. (2005), 71, 3285) describe the isolation and characterization of three mutants of glucose
  • Vazquez-Figueroa et al. disclose the development of a thermostable glucose dehydrogenase comprising introducing amino acid substitutions at positions 155, 170 and 252 of glucose dehydrogenase from Bacillus subtilis, Bacillus thuringiensis and Bacillus licheniformis.
  • the mutations E170K and Q252L both individually and in combination, lead to a stabilization of glucose dehydrogenase from Bacillus subtilis.
  • the object underlying the present invention was therefore to provide a stable diagnostic element, in particular for the determination of glucose, in which the disadvantages of the prior art are at least partially eliminated.
  • the diagnostic element with high stability of both the enzyme and the coenzyme should ensure a high turnover rate of the substrate.
  • mutated dehydrogenases which have extremely low activities in the presence of an artificial coenzyme in the cuvette test, show a faster kinetics in diagnostic elements with dry reagent layers, such as for example in test strips, and deliver at least the same conversion as in Presence of native coenzyme (wild type coenzyme).
  • the reason for this is presumably due to the fact that at high concentrations of the feedstocks, factors other than the activity of the enzyme decisively influence the rate of turnover, in which context in particular the location of the complex formation between enzyme, coenzyme, reduced coenzyme, analyte and oxidized analyte is decisive seems.
  • the method according to the invention provides, in a preferred embodiment, for the rate of conversion of the analyte in the diagnostic element described herein to be equal to or higher than the rate of turnover of the analyte in a corresponding diagnostic element comprising the corresponding wild type coenzyme instead of the artificial coenzyme.
  • the turnover rate of the analyte in a diagnostic element used according to the invention is at least 20% stronger than the rate of turnover of the analyte in a diagnostic element comprising the wild-type coenzyme preferably increased by at least 50%, and most preferably by at least 100%, for example by 100% to 200%.
  • mutant dehydrogenase or “dehydrogenase mutant” as used in the present application refer to a genetically modified variant of a native dehydrogenase (wild type).
  • Dehydrogenase which has an amino acid sequence altered with respect to the wild-type dehydrogenase for the same number of amino acids, i. differs in at least one amino acid from wild-type dehydrogenase.
  • the mutant dehydrogenase can be obtained by mutation of a wild-type dehydrogenase derived from any biological source, the term "biological source” for the purposes of this invention encompassing both prokaryotes, such as bacteria, and eukaryotes, such as mammals and other animals.
  • the introduction of the mutation (s) can be site-specific or non-site-specific, preferably site-specific using recombinant methods known in the art, resulting according to the respective requirements and conditions at least one amino acid exchange within the amino acid sequence of the native dehydrogenase.
  • a dehydrogenase mutant obtained in this way in the method according to the invention preferably has increased thermal and / or hydrolytic stability compared with the corresponding wild-type dehydrogenase.
  • Examples of such mutants are i.a. in Baik (Appl. Environ.Microbiol. (2005), 71, 3285), Väsquez-Figueroa (ChemBioChem (2007), 8, 2295) as well as in WO 2005/045016 A2, whose disclosure is hereby expressly incorporated by reference.
  • a mutated dehydrogenase in the context of the present invention particularly preferably has a specific enzyme activity which is reduced compared with the corresponding wild-type dehydrogenase.
  • Specific enzyme activity expressed in U / mg enzyme
  • lyophilizate activity the amount of substrate which is reacted under predetermined conditions per minute and per milligram of lyophilisate comprising the enzyme in combination with excipients.
  • the mutated dehydrogenase used in the method according to the invention is preferably a nicotinamide adenine dinucleotide (NAD /
  • NADH NADH
  • NADP / NADPH nicotinamide adenine dinucleotide phosphate
  • a mutant dehydrogenase which is preferably selected from a mutated alcohol dehydrogenase (EC 1.1.1.1, EC 1.1.1.2), a mutant L-amino acid dehydrogenase (EC 1.4.1.5), a mutated glucose dehydrogenase (EC 1.1.1.47), a mutated glucose-6-phosphate dehydrogenase (EC 1.1.1.49), a mutated glycerol
  • the mutated dehydrogenase is particularly preferably a mutated glucose dehydrogenase (EC 1.1.1.47).
  • a mutated glucose dehydrogenase may in principle contain the amino acid (s) modified with respect to the corresponding wild-type glucose dehydrogenase at any position of its amino acid sequence.
  • the mutated glucose dehydrogenase comprises a mutation at at least one of positions 170 and 252 of the amino acid sequence of wild-type glucose dehydrogenase, with mutants having mutations at position 170 and position 252 being particularly preferred. It has proven to be advantageous if the mutated glucose dehydrogenase in addition to these mutations no further Contains mutations.
  • the mutation at positions 170 or / and 252 may in principle comprise any amino acid substitution which results in stabilization, e.g. an increase in thermal and / or hydrolytic stability leading to wild-type dehydrogenase.
  • the mutation at position 170 comprises an amino acid substitution of glutamic acid for arginine or lysine, particularly an amino acid substitution of glutamic acid for lysine, while with respect to position 252 an amino acid substitution of lysine for leucine is preferred.
  • the wild-type glucose dehydrogenases used to prepare the above mutants of glucose dehydrogenase are preferably derived from a bacterium, with particular preference being given to using a glucose dehydrogenase from Bacillus megaterium, Bacillus subtilis or Bacillus thuringiensis, in particular from Bacillus subtilis.
  • a mutated glucose dehydrogenase GlucDH_E170K_K252L obtained by mutation of wild-type glucose dehydrogenase from Bacillus subtilis with the amino acid sequence shown in SEQ ID NO: 1 is used in the context of the method according to the invention.
  • the diagnostic elements described herein further include an artificial coenzyme in addition to a mutant dehydrogenase specific for the analyte.
  • An artificial coenzyme in the context of the present invention is a coenzyme chemically modified with respect to the native coenzyme, which at atmospheric pressure has a higher stability to moisture compared with the native coenzyme, temperatures in particular in the range from O 0 C to 50 0 C 1 acids and bases, in particular Range of pH 4 to pH 10, and / or nucleophiles such as alcohols or amines, and thus can operate under identical environmental conditions over a longer period than the native coenzyme its effect.
  • the artificial coenzyme has a higher hydrolytic stability compared to the native coenzyme, with full hydrolytic stability being particularly preferred under test conditions.
  • the artificial coenzyme may have a decreased binding constant for the dehydrogenase, for example a binding constant reduced by a factor of 2 or more.
  • artificial coenzymes which can be used in the process according to the invention are artificial NAD (P) / NAD (P) H compounds, i. chemical derivatives of native nicotinamide adenine dinucleotide (NAD / NADH) or native nicotinamide adenine dinucleotide phosphate (NADP / NADPH), or the compound of formula (I)
  • the NAD (P) / NAD (P) H artificial compound preferably comprises a 3-pyridinecarbonyl or a 3-pyridinethiocarbonyl -Rest, which is linked without glycosidic bond via a linear or cyclic organic radical, in particular via a cyclic organic radical, with a phosphorus-containing radical, such as a phosphate radical.
  • the artificial coenzyme is particularly preferably selected from a compound of the general formula (II):
  • V each independently OH or a phosphate group, or two
  • X 1 , X 2 are each independently O, CH 2 , CHCH 3 , C (CH 3 ) 2> NH, NCH 3 ,
  • Y NH, S, O, CH 2 ,
  • 2_ is a linear or cyclic organic radical, provided that Z and the pyridine radical are not linked by a glycosidic compound, or a salt or optionally a reduced form thereof.
  • 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 analogues such as formycin, wherein the 7-Deazaversionn in the 7-position with halogen, Ci -6 alkynyl, alkenyl or alkyl may be substituted.
  • adenine or adenine analogs such as C 8 and N 6 substituted adenine
  • deaza variants such as 7-deaza
  • azavaries such as 8-aza
  • combinations 7-deaza or 8-aza or carbocyclic analogues
  • carbocyclic analogues such as formycin
  • the compounds contain adenosine analogues which, instead of ribose, for example 2-methoxydesoxyribose, 2'- Fluorodeoxyribose, Hexitol, Altritol or polycyclic analogs such as bicyclo, LNA and tricyclo sugars.
  • ribose for example 2-methoxydesoxyribose, 2'- Fluorodeoxyribose, Hexitol, Altritol or polycyclic analogs such as bicyclo, LNA and tricyclo sugars.
  • W is preferably CONH 2 or COCH 3 .
  • the artiflial coenzyme is the carbaNAD compound known from the literature (J. T. Slama, Biochemistry (1988), 27, 183 and Biochemistry (1989), 28, 7688).
  • Other stable coenzymes which can be used according to the invention are described in WO 98/33936, WO 01/49247, WO 2007/012494, US Pat. No. 5,801,006, US Pat. No. 11 / 460,366 and the publication Blackbum et al. (Chem. Comm. (1996), 2765), the disclosure of which is incorporated herein by reference.
  • the diagnostic element used in the method of the invention may be any diagnostic element comprising a dry reagent layer containing the mutated dehydrogenase and the artificial coenzyme, and which can be wetted by the sample containing the analyte.
  • the reagent layer may optionally comprise further reagents which serve for the qualitative detection or the quantitative determination of the analyte, such as a suitable mediator, as well as suitable auxiliaries and / or additives.
  • diagnostic elements to which the analyte can be applied in the form of an aqueous or nonaqueous solution.
  • the diagnostic element is a test tape, a test disk, a test pad, a test strip, a test strip drum, or the diagnostic elements mentioned in WO 2005/084530 A2, to which reference is hereby expressly made
  • the diagnostic elements described in the present application in each case comprise at least one test area, which can be brought into contact with a sample containing the analyte and, using suitable means, enables a qualitative or / and quantitative determination of the analyte.
  • test tape refers to a tape-shaped diagnostic element which typically comprises more than a single test area, preferably at least 10 individual test areas, more preferably at least 25 individual test areas, and most preferably at least 50 individual test areas the individual test areas are each arranged at a distance of a few millimeters to a few centimeters, for example at a distance of ⁇ 2.5 cm from each other, wherein the test tape may optionally comprise marking areas for path detection during tape transport and / or for calibration between successive test areas Test tapes are described, for example, in EP 1 739 432 A1, to the disclosure of which reference is expressly made.
  • test disc refers to a disc-shaped diagnostic element which may include one or more individual test areas, for example, at least 10 individual test areas
  • the test disc is coated with a thin layer of the test chemistry, such as a Layer in a thickness of about 20 microns coated, to which a sample of the analyte can be applied, depending on the volume of the sample, a more or less large area of the test disk is wetted by the sample and can be used to determine the analyte.
  • the non-wetted area of the test disc which may be partially or fully wetted by the passage of moisture through the test chemical layer, will subsequently be available for further determinations of the analyte.
  • the method according to the invention can be used to determine any biological or chemical substance which can be detected photochemically or electrochemically.
  • the analyte is selected from the group consisting of malic acid, alcohol, ammonium, ascorbic acid, cholesterol, cysteine, glucose, glucose-6-phosphate, glutathione, glycerol, urea, 3-hydroxybutyrate, lactic acid, 5'-nucleotidase, peptides, pyruvate, Salicylate and triglycerides, with glucose being particularly preferred.
  • the analyte may be of any source, but is preferably in a body fluid, including, but not limited to, whole blood, plasma, serum, lymph, bile, cerebrospinal fluid, extracellular tissue fluid, urine, and glandular secretions, e.g. Saliva or sweat, included.
  • a body fluid including, but not limited to, whole blood, plasma, serum, lymph, bile, cerebrospinal fluid, extracellular tissue fluid, urine, and glandular secretions, e.g. Saliva or sweat, included.
  • saliva or sweat glandular secretions
  • the qualitative or / and quantitative determination of the analyte can be done in any way.
  • optical detection methods are used which, for example, the measurement of absorption, fluorescence, circular dichroism (CD), optical Rotary dispersion (ORD), refractometry, etc., as well as electrochemical techniques for use.
  • the determination of the presence or / and the amount of the analyte is carried out photometrically or fluorometrically, for example indirectly via a fluorometrically detectable change of the artificial coenzyme.
  • the invention features a diagnostic element for assaying an analyte comprising a dry reagent layer containing o (a) a mutant dehydrogenase specific for the analyte, and (b) an artificial coenzyme.
  • FIG. 1 Kinetics of wild-type glucose dehydrogenase from Bacillus subtilis in5 presence of NAD / NADH as coenzyme in glucose
  • Figure 1A Enzyme activity 1556.2 kU / 100 g mass 0
  • Figure 1 B Enzyme activity 1004.0 kU / 100 g mass
  • FIG. 1C enzyme activity 502.0 kU / 100 g mass
  • Figure 1 D enzyme activity 251.0 kU / 100 g mass
  • Figure 1 E enzyme activity 25.10 kU / 100 g mass.
  • FIG. 2 Kinetics of a glucose-dehydrogenase double mutant GlucDH_E170K_K252L obtained by mutation of wild-type glucose dehydrogenase from Bacillus subtilis in the presence of carbaNAD / carbaNADH as coenzyme at glucose concentrations of 0.0 mg / dl, 34.4 mg / dl, 141.2 mg / dl , 236.6 mg / dl, 333.8 mg / dl and 525.8 mg / dl (viewed from top to bottom).
  • Enzyme activity 4.60 kU / 100 g mass.
  • FIG. 3 Fluorescence spectrum of the complex glucose dehydrogenase (GlucDH) / NADH before and after titration with gluconolactone.
  • FIG. 4 Fluorescence spectrum of the complex glucose dehydrogenase (GlucDH) / NADH before and after titration with glucose.
  • FIG. 5 Kinetics of the conversion of glucose in the presence of wild-type glucose dehydrogenase and NADH at various glucose concentrations.
  • FIG. 5A Kinetics without additionally added gluconolactone at glucose concentrations of 77.0 mg / dl, 207.0 mg / dl, 300.0 mg / dl and 505.0 mg / dl (viewed from top to bottom).
  • FIG. 5B Kinetics with additionally added gluconolactone at glucose concentrations of 96.2 mg / dl, 274.0 mg / dl, 399.0 mg / dl and 600.0 mg / dl (viewed from top to bottom).
  • FIG. 6 Representation of the amino acid sequence of the glucose dehydrogenase double mutant GlucDH_E170K_K252L
  • Example 1 Preparation of a double mutant of glucose dehydrogenase from Bacillus subtilis with amino acid substitutions E170K and K252L (GlucDH_E170K_K252L)
  • GlucDH_E170K_K252L amino acid substitutions E170K and K252L
  • GlucDH_E170K_K252L amino acid substitutions E170K and K252L
  • Site-specific mutagenesis first at position 170 followed by site-directed mutagenesis at position 252 of the amino acid sequence of wild-type glucose dehydrogenase introduced E170K and K252L mutations.
  • the respective mutagenesis steps were carried out with the aid of specifically designed primers in the course of a PCR reaction.
  • the resulting PCR product was transformed into Escherichia coli XLIblue MRF '.
  • the cells were plated, clones containing plasmid were grown overnight, and the enzyme activity was determined before and after temperature stress (stress test: 30 minutes at 5O 0 C). The results are shown in Table 1.
  • Table 1 Residual activity of wild-type glucose dehydrogenase from Bacillus subtilis and the mutants GlucDH_E170K and GlucDH_E170K_K252L after stress load (tested in Escherichia coli XL1 blue MRF 1 )
  • Example 2 Purification of the double mutant GlucDH_E170K_K252L Each 10 g of biomass was taken up in 50 ml of a 30 mM potassium phosphate buffer pH 6.5 and digested at about 800 bar. After removal of cell debris, chromatography was performed on DEAE-Sepharose (GE Healthcare) at a loading of ⁇ 40 mg protein / ml column volume and using a linear gradient of buffer A (30 mM potassium phosphate buffer pH 6.5) on buffer B (buffer A + 500 mM NaCl). The fractions showing glucose dehydrogenase activity were pooled and adjusted to a conductivity of 230 mS / cm with ammonium sulfate (Aldrich).
  • Example 3 Determination of the activity of wild-type glucose dehydrogenase from Bacillus subtilis and the double mutant GlucDH_E170K_K252L in the cuvette test
  • Tris buffer (0.1 M, pH 8.5, 0.2 M NaCl)
  • Dilution buffer (3.8 mM NAD, 0.1 M Tris, pH 8.5, 0.2 M NaCl)
  • Glucose solution 2 g of D (+) - glucose monohydrate (Sigma-Aldrich) were dissolved in 10 ml of bidistilled water. After 2 hours of service life at room temperature and adjustment of the Mutarotations Dermats the solution was ready for use.
  • Tris buffer 0.1 ml glucose solution and 0.05 ml NAD solution or 0.05 ml carbaNAD solution (each tempered to 25 ° C.) were pipetted into a plastic cuvette mixed, and tempered in a cuvette slide to 25 ° C.
  • the reaction was initiated by introducing 0.025 ml of sample into the cuvette and monitoring the absorbance of the sample over a period of 5 min.
  • the activity of the system WT-GlucDH / carbaNAD (8.2 U / mg enzyme) under standard conditions in a cuvette is two orders of magnitude lower than the activity of the system WT-GlucDH / NAD (484 U / mg enzyme).
  • the activity of the double mutant GlucDHJE170K_K252L is about two orders of magnitude lower in the presence of the artificial coenzyme carbaNAD (3.7 U / mg enzyme) than in the presence of the native coenzyme NAD (270 U / mg enzyme).
  • Example 4 Determination of the kinetics of wild-type glucose dehydrogenase from Bacillus subtilis (WT-GlucDH) and the double mutant GlucDH_E170K_K252L in a dry reagent layer
  • test strips were prepared containing either the native glucose dehydrogenase from Bacillus subtilis (WT-GlucDH) or the double mutant GlucDH_E170K_K252L prepared in Example 1 in combination with NAD / NADH or carbaNAD / carbaNADH as coenzyme.
  • WT-GlucDH native glucose dehydrogenase from Bacillus subtilis
  • GlucDH_E170K_K252L prepared in Example 1 in combination with NAD / NADH or carbaNAD / carbaNADH as coenzyme.
  • a partial solution 1 consisting of 18.4 g of 1M phosphate buffer pH 7.0, 1.4 g of Gantrez S97 (International Specialty Products), 2.94 g of 16% NaOH solution, 0.34 g of Mega 8 (Sigma-Company) was initially used for this purpose. Aldrich), 0.039 g of Geropon T77 (Rhone-Poulenc) and 1.90 g of polyvinylpyrrolidone 25000 (Fluka).
  • This partial solution was then mixed with a partial solution 2 consisting of 0.50 g of sodium chloride, 21.3 g of bidistilled water, 4.43 g of transpafill (Evonik) and 2.95 g of propofan (BASF), and a partial solution 3 stored overnight in the refrigerator, consisting of 17.4 g of 1M phosphate buffer pH 7.0, 0.5 g of sodium chloride, 14.35 g of 2 M dipotassium hydrogen phosphate and the amounts of dehydrogenase, coenzyme and bovine serum albumin (BSA, Roche) indicated in Table 3 below.
  • the enzymatically inactive bovine serum albumin was added to the formulation in the case of the use of reduced amounts of native dehydrogenase in order to keep the matrix properties of the test strip as constant as possible.
  • test strips obtained in this way were measured on laboratory measuring devices (self-made by Roche), which included an excitation LED (375 nm) and common detectors (BPW34 blueenhanced).
  • the discontinued sample material was blood with adjusted glucose levels. The results of the determination are shown in FIGS. 1 and 2.
  • FIG. 2 shows the kinetics of the mutant glucose dehydrogenase obtained according to Example 1 in the presence of carbaNAD as coenzyme.
  • the deterioration in kinetics expected with reduced enzyme activity does not occur. Rather, the double mutant in the presence of carbaNAD shows better kinetics than all of the formulations listed in Table 3 which contain the corresponding wild-type glucose dehydrogenase and the native coenzyme NAD.
  • Example 5 Detection of ternary complexes consisting of glucose dehydrogenase, NADH and glucose or gluconolactone
  • NADH amino acid dehydrogenase
  • GlucDH wild-type glucose dehydrogenase
  • Bacillus subtilis was added, which formed the literature known complex GlucDH-NADH, which provides a shifted emission maximum at 450 nm due to a significantly longer lifetime of NADH (3 ns in contrast to 0.4 ns in the free state) (see Figure 3).
  • gluconolactone should cause a slowing of the conversion of glucose, since additional gluconolactone (in addition to the gluconolactone formed in the reaction) should inhibit other enzyme complexes.
  • FIG. 5B shows a significant slowdown in the conversion compared to the sample measured in FIG. 5A.

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Abstract

La présente invention porte sur un procédé de détermination d'un analyte ainsi que sur un élément de diagnostic approprié pour la mise en oeuvre de ce procédé.
PCT/EP2010/051801 2008-02-19 2010-02-12 Cinétique réactionnelle rapide d'enzymes présentant une activité inférieure dans des couches chimiques sèches WO2010094632A1 (fr)

Priority Applications (8)

Application Number Priority Date Filing Date Title
CA2750474A CA2750474C (fr) 2009-02-19 2010-02-12 Cinetique reactionnelle rapide d'enzymes presentant une activite inferieure dans des couches chimiques seches
KR1020117019082A KR101604624B1 (ko) 2009-02-19 2010-02-12 건식 화학 층에서 낮은 활성을 갖는 효소의 고속 반응 속도
CN2010800085457A CN102325896A (zh) 2008-02-19 2010-02-12 干化学层中具有低活性的酶的快速反应动力学
JP2011550531A JP6113405B2 (ja) 2008-02-19 2010-02-12 乾式試薬層において低い活性を有する、速い反応速度の酵素
MX2011008669A MX2011008669A (es) 2009-02-19 2010-02-12 Cineticos de reaccion rapida de enzimas que tienen baja actividad en capas quimicas secas.
KR1020137029083A KR20130127555A (ko) 2009-02-19 2010-02-12 건식 화학 층에서 낮은 활성을 갖는 효소의 고속 반응 속도
EP10704923.1A EP2398909B1 (fr) 2009-02-19 2010-02-12 Cinétique réactionnelle rapide d'enzymes présentant une activité inférieure dans des couches chimiques sèches
US13/210,564 US9359634B2 (en) 2009-02-19 2011-08-16 Fast reaction kinetics of enzymes having low activity in dry chemistry layers

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