Composition for measuring glucose improves substrate specificity

Tag : Glutaric acid


Abstract
The present invention relates to a method for lowering activitywith respect to maltose in glucose measurement comprising a step ofreacting modified pyrroloquinoline quinone dependent glucosedehydrogenase subjected to amino acid sequence modification,wherein pyrroloquinoline quinone dependent glucose dehydrogenase isreacted in the presence of at least one type of substance selectedfrom the group comprising succinic acid, malonic acid, glutaricacid, malic acid, phthalic acid, 2-ketoglutaric acid,3,3-dimethylglutaric acid, pimeric acid, suberic acid, adipic acid,maleic acid, potassium chloride, ammonium chloride, diammoniumhydrogen citrate, L-lysine, taurine, calcium glycerate,amino-n-butyric acid, sodium glycolate, sodium.alpha.-ketoglutarate, fumaric acid, glycine, glutamic acid, serineand citric acid.
Description of the Invention
PQQGDH catalyzes a reaction in which D-glucose is oxidized toproduce D-glucono-1,5-lactone. PQQGDH is not influenced bydissolved oxygen, and has an enzymatic property of no coenzymerequirement. PQQGDH is expected to apply a variety of applicationssuch as biological diagnostics to assay blood glucose level, andblood glucose sensor. It is also noted that PQQGDH has a problemwith respect to substrate specificity, such as acting ondisaccharides, in particular maltose.

The inventors made an extensive research on the cause of theproblems to be solved and found that PQQGDH had a low reactivity onferricyanide ion usually used as a mediator in blood glucosesensor.

The inventors further investigated and demonstrated that the lowreactivity on ferricyanide ion was caused by the influence of nearneutral buffer conditions leading to a substrate specificity ofenzymatic reaction.

WO03/106668 discloses a means to improve the substrate specificityof PQQGDH, wherein PQQGDH gene is modified. However, WO03/106668does not disclose or suggest a means to solve the problem ofsubstrate specificity other than glucose, in particular maltose.

Therefore, as a result of conducting extensive studies onimprovements from other perspectives in parallel with modificationby genetic engineering techniques, the inventors of the presentinvention found that substrate specificity is improved by examiningcompositions other than modified PQQGDH in a method for measuringglucose that contains a step that reacts an modified PQQGDHsubjected to substitution, insertion, deletion or othermodification of the amino acid sequence, thereby leading to filingof the present application.

More specifically, the present invention is composed by optimizingsubstances contained in the composition and/or pH conditions.

Namely, the present invention is composed of the compositiondescribed below. [Item 1] A method for lowering a reactivity onmaltose in glucose measurement comprising a step of reactingmodified pyrroloquinoline quinone dependent glucose dehydrogenasesubjected to amino acid sequence modification, whereinpyrroloquinoline quinone dependent glucose dehydrogenase is reactedin the presence of at least one substance selected from the groupconsisting of succinic acid, malonic acid, glutaric acid, malicacid, phthalic acid, 2-ketoglutaric acid, 3,3-dimethylglutaricacid, pimeric acid, suberic acid, adipic acid, maleic acid,potassium chloride, ammonium chloride, diammonium hydrogen citrate,L-lysine, taurine, calcium glycerate, amino-n-butyric acid, sodiumglycolate, sodium .alpha.-ketoglutarate, fumaric acid, glycine,glutamic acid, serine and citric acid. [Item 2] The methodaccording to claim 1, comprising a step in which the dehydrogenaseis reacted in the presence of at least one substance selected fromthe group consisting of succinic acid, malonic acid, glutaric acid,malic acid, phthalic acid, 2-ketoglutaric acid,3,3-dimethylglutaric acid, pimeric acid, suberic acid, adipic acid,maleic acid and citric acid. [Item 3] The method according to claim1, comprising a step in which the dehydrogenase is reacted in thepresence of one or more substances selected from the groupconsisting of succinic acid, adipic acid, suberic acid, pimericacid, potassium chloride, ammonium chloride, diammonium hydrogencitrate, malonic acid, L-lysine, taurine, 3,3-dimethylglutaricacid, malic acid, glutaric acid, calcium glycerate, amino-n-butyricacid, sodium glycolate, sodium .alpha.-ketoglutarate, fumaric acid,glycine, glutamic acid and serine. [Item 4] The method for loweringa reactivity on maltose in the measurement of glucose according toclaim 1, wherein the total added amount of one or more substancesselected from the group consisting of succinic acid, malonic acid,glutaric acid, malic acid, phthalic acid, 2-ketoglutaric acid,3,3-dimethylglutaric acid, pimeric acid, suberic acid, adipic acid,maleic acid, potassium chloride, ammonium chloride, diammoniumhydrogen citrate, L-lysine, taurine, calcium glycerate,amino-n-butyric acid, sodium glycolate, sodium.alpha.-ketoglutarate, fumaric acid, glycine, glutamic acid, serineand citric acid is 0.05% by weight or more (as the percent byweight in solution). [Item 5] The method according to claim 1,wherein the pH during measurement is 7.0 or lower. [Item 6] Themethod according to claim 1, wherein the pH is 6.0 or lower. [Item7] The method according to claim 1, wherein the reaction is carriedout in the presence of at least one mediator. [Item 8] The methodaccording to claim 7, wherein the mediator is a ferricyanide salt.[Item 9] The method according to claim 1, wherein the modifiedpyrroloquinoline quinone dependent glucose dehydrogenase subjectedto amino acid sequence modification is a pyrroloquinoline quinonedependent glucose dehydrogenase having a lowered reactivity onmaltose as compared with the corresponding wild-type enzyme. [Item10] The method for lowering a reactivity on maltose in glucosemeasurement comprising a step of reacting an modifiedpyrroloquinoline quinone dependent glucose dehydrogenase accordingto claim 1, wherein the step of reacting the modifiedpyrroloquinoline quinone dependent glucose dehydrogenase is carriedout in a reagent composition for measuring glucose that contains anmodified pyrroloquinoline quinone dependent glucose dehydrogenasesubjected to amino acid modification. [Item 11] The methodaccording to claim 10, wherein the composition for measuringglucose adopts a form in which the composition is contained in aglucose assay kit. [Item 12] The method according to claim 10,wherein the step of reacting the modified pyrroloquinoline quinonedependent glucose dehydrogenase is carried out in a glucose sensorcontaining an modified pyrroloquinoline quinone dependent glucosedehydrogenase subjected to amino acid sequence modification andelectrodes at least comprising a working electrode and a counterelectrode. [Item 13] The method according to claim 12, wherein thereaction in the glucose sensor comprises applying a voltage to areaction solution containing the modified pyrroloquinoline quinonedependent glucose dehydrogenase, and measuring the oxidationcurrent of a mediator. [Item 14] A composition for measuringglucose comprising an modified pyrroloquinoline quinone dependentglucose dehydrogenase subjected to amino acid sequencemodification, and one or more substances selected from the groupconsisting of succinic acid, malonic acid, glutaric acid, malicacid, phthalic acid, 2-ketoglutaric acid, 3,3-dimethylglutaricacid, pimeric acid, suberic acid, adipic acid, maleic acid,potassium chloride, ammonium chloride, diammonium hydrogen citrate,L-lysine, taurine, calcium glycerate, amino-n-butyric acid, sodiumglycolate, sodium .alpha.-ketoglutarate, fumaric acid, glycine,glutamic acid, serine and citric acid, wherein a reactivity onmaltose is lowered. [Item 15] A glucose sensor comprising amodified pyrroloquinoline quinone dependent glucose dehydrogenasesubjected to amino acid sequence modification, and one or moresubstances selected from the group consisting of succinic acid,malonic acid, glutaric acid, malic acid, phthalic acid,2-ketoglutaric acid, 3,3-dimethylglutaric acid, pimeric acid,suberic acid, adipic acid, maleic acid, potassium chloride,ammonium chloride, diammonium hydrogen citrate, L-lysine, taurine,calcium glycerate, amino-n-butyric acid, sodium glycolate, sodium.alpha.-ketoglutarate, fumaric acid, glycine, glutamic acid, serineand citric acid, wherein a reactivity on maltose is lowered. [Item16] A method for producing a composition for measuring glucosehaving a lowered reactivity on maltose in which an modifiedpyrroloquinoline quinone dependent glucose dehydrogenase is used,the method comprising a step of containing one or more substancesselected from the group consisting of succinic acid, malonic acid,glutaric acid, malic acid, phthalic acid, 2-ketoglutaric acid,3,3-dimethylglutaric acid, pimeric acid, suberic acid, adipic acid,maleic acid, potassium chloride, ammonium chloride, diammoniumhydrogen citrate, L-lysine, taurine, calcium glycerate,amino-n-butyric acid, sodium glycolate, sodium.alpha.-ketoglutarate, fumaric acid, glycine, glutamic acid, serineand citric acid. [Item 17] A method for producing a glucose sensorhaving a lowered reactivity on maltose, in which an modifiedpyrroloquinoline quinone dependent glucose dehydrogenase is used,the method comprising a step containing one or more substancesselected from the group consisting of succinic acid, malonic acid,glutaric acid, malic acid, phthalic acid, 2-ketoglutaric acid,3,3-dimethylglutaric acid, pimeric acid, suberic acid, adipic acid,maleic acid, potassium chloride, ammonium chloride, diammoniumhydrogen citrate, L-lysine, taurine, calcium glycerate,amino-n-butyric acid, sodium glycolate, sodium.alpha.-ketoglutarate, fumaric acid, glycine, glutamic acid, serineand citric acid.

The composition and measurement method of the present invention isuseful for measuring glucose in clinical tests, food analyses andso on. The present invention allows analyses of higher precision byapplying to a glucose assay kit or glucose sensor that uses PQQGDH.

The following provides a detailed explanation of the presentinvention.

One embodiment of the present invention is a method for loweringactivity with respect to maltose in glucose measurement thatcontains a step that reacts an modified pyrroloquinoline quinonedependent glucose dehydrogenase subjected to amino acid sequencemodification, wherein a step is contained in which said enzyme isreacted in the presence of one or more substances selected from thegroup comprising succinic acid, malonic acid, glutaric acid, malicacid, phthalic acid, 2-ketoglutaric acid, 3,3-dimethylglutaricacid, pimeric acid, suberic acid, adipic acid, maleic acid,potassium chloride, ammonium chloride, diammonium hydrogen citrate,L-lysine, taurine, calcium glycerate, amino-n-butyric acid, sodiumglycolate, sodium .alpha.-ketoglutarate, fumaric acid, glycine,glutamic acid, serine and citric acid.

The PQQGDH used in the present invention is an modified PQQGDH inwhich the amino acid sequence of wild type of PQQDGH has beenmodified. Modification of the amino acid sequence here refers tosubstitution, insertion or deletion of at least one amino acidresidue in the original amino acid sequence by genetic engineeringtechniques. In particular, it is preferable to use an modifiedPQQGDH in which activity with respect to maltose has been loweredin comparison with the corresponding wild type enzyme as a resultof subjecting to such amino acid sequence modification.

The activity with respect to maltose in glucose measurement can befurther lowered by reacting the modified PQQGDH in the presence ofat least one substance selected from the group comprising succinicacid, malonic acid, glutaric acid, malic acid, phthalic acid,2-ketoglutaric acid, 3,3-dimethylglutaric acid, pimeric acid,suberic acid, adipic acid, maleic acid, potassium chloride,ammonium chloride, diammonium hydrogen citrate, L-lysine, taurine,calcium glycerate, amino-n-butyric acid, sodium glycolate, sodium.alpha.-ketoglutarate, fumaric acid, glycine, glutamic acid, serineand citric acid.

In the present invention, activity for maltose means the action ofPQQGDH dehydrogenating that sugar substrate.

In addition to maltose, activity for at least one other sugar otherthan glucose selected from the sugars other than maltose can alsobe lowered.

Sugar substrates other than glucose selected from the sugars otherthan maltose include for example galactose, mannose, xylose andother simple sugars, sucrose, lactose, cellobiose and otherdisaccharides, maltotriose, maltotetraose and otheroligosaccharides, icodextrin (glucose polymer) and otherpolysaccharides (an oligosaccharide comprises 2 to 10 molecules ofa single sugar while a polysaccharide comprises 11 or moremolecules of a single sugar bound together by glycoside bonds orthe like, with no restrictions on the binding mode, and in the caseof a disaccharide or higher sugar the structure may be homogenousor heterogeneous), as well as the sugar alcohols,2-deoxy-D-glucose, 3-o-methyl-D-glucose and the like andderivatives of these.

Of these, it is preferably to select various sugars which may posea problem when the modified PQQGDH of the present invention is usedfor clinical diagnosis or controlling the blood glucose levels ofdiabetes patients, or for measuring blood glucose concentrations.Examples of such sugars include mannose, allose, xylose, galactose,maltose and the like, and more desirable examples are galactose andlactose. Maltose is the most desirable example.

Furthermore, in the specification of the present application,reduction of activity with respect to maltose or a sugar substrateselected from at least one sugar substrate other than maltose ascompared with the corresponding wild type PQQGDH also representsimprovement of substrate specificity.

The following method is used to judge whether activity for aspecific sugar has been lowered.

In the activity measurement method described in Test Example 1 andExample 7 below, using PQQGDH, the PQQGDH activity value (a) usingD-glucose as the substrate solution and the PQQGDH activity value(b) using this sugar in place of D-glucose as the substratesolution are measured, and the relative value ((b)/(a).times.100)is calculated given 100 as the measurement value using glucose asthe substrate. The same operation is then repeated with theconditions changed, and the values are compared and evaluated.

Activity is measured by the activity measurement method describedin Test Example 1 and Example 7 below.

The PQQGDH that can be used in the method of the present inventionis an enzyme (EC 1.1.5.2 (old EC 1.1.99.17)) havingpyrroloquinoline quinone as the coenzyme which catalyzes a reactionin which D-glucose is oxidized to produce D-glucono-1,5-lactone,with no particular limits on its derivation or structure.

PQQGDH can be classified into a soluble form and a membrane-boundform. Among these, that which originates in Acinetobacter is knownas soluble PQQGDH, while that present in other microorganisms suchas Escherichia coli is known as membrane-bound PQQGDH.

The modified PQQGDH of the present invention can be prepared forexample by obtaining a gene coding for wild-type PQQGDH, andmodifying it to construct a polynucleotide coding for modifiedPQQGDH, and then using that polynucleotide to produce expression ina suitable expression system.

An origin of a wild type PQQGDH to prepare a modified PQQGDH foruse in the invention is not specifically limited. Representativeorigins of the wild type PQQGDH which is the source of themodification are microorganisms exemplified below. Specifically,examples may include oxidizing bacteria such as Acinetobacterbaumannii (JP HEI-11-243949 A), Acinetobacter calcoaceticus (eg. A.M. Cleton-Jansen et al J. Bacteriol., 170, 2121 (1988); and Mol.Gen. Genet., 217, 430 (1989)), Pseudomonas aeruginosa, Pseudomonasputida, Pseudomonas fluorescens and Gluconobacter oxydans, andenterobacteria such as Agrobacterium radiobacter, Escherichia coli(A. M. Cleton-Jansen et al J. Bacteriol., 172, 6308(1990)) andKlebsiella aerogenes, Burkhorderia cepacia.

It is preferable to select those derived from the microorganismsbelonging to the genus Acinetobacter as the origin. These arewater-soluble enzymes and easily dissolved in an aqueous system.More preferably, it is preferable to select the soluble PQQGDH fromAcinetobacter calcoaceticus or Acinetobacter baumannii.Particularly preferable PQQGDHs are derived from Acinetobacterbaumannii NCIMB 11517 strain (see, JP HEI-11-243949 A),Acinetobacter calcoaceticus LMD79.41 strain (see, A. M.Cleton-Jansen et al J. Bacteriol., 170, 2121 (1988); and Mol. Gen.Genet., 217, 430 (1989)), and Acinetobacter calcoaceticus IFO 12552strain (see, JP 2004-173538 A). Most preferable are PQQGDH derivedfrom Acinetobacter baumannii NCIMB 11517 strain. The Acinetobacterbaumannii NCIMB11517 strain was previously classified intoAcinetobacter calcoaceticus.

In all of these cases, the amino acid sequences and gene sequencesare known, or purification methods have been established and thephysiochemical properties of the enzyme are known, so that a personskilled in the art can easily prepare modified PQQGDH based onthese findings.

The enzyme used in the method of the present invention is anmodified pyrroloquinoline quinone dependent glucose dehydrogenasesubjected to alteration of the amino acid sequence, and is anmodified pyrroloquinoline quinone dependent glucose dehydrogenasehaving lowered activity with respect to maltose as compared withthe corresponding wild type enzyme.

Such modification can easily be performed by the skilled artisanaccording to known techniques in the art. A variety of methods forintroducing a site-directed mutagenesis to a protein bysubstituting or inserting one or more bases to a nucleotidesequence of a gene coding for the protein are disclosed in Sambrooket al, Molecular Cloning; A Laboratory Manual 2.sup.nd Edition(1989) Cold Spring Harbor Laboratory Press, New York.

Preferable examples of the modified PQQGDH used in the presentinvention include PQQGDH that has been subjected to modification ofthe amino acid sequence at one or more locations in the vicinity ofthe activity center, and may be subjected to other modifications ofthe amino acid sequence in addition to that in the vicinity of theactivity center. Specific examples include PQQGDH subjected tomodification targeted at at least one location selected from thegroup comprising Pro at position 67, Glu at position 68, Ile atposition 69, Gln at position 76, Lys at position 89, Glu atposition 129, Lys at position 130, Pro at position 131, Asn atposition 167, Gln at position 168, Leu at position 169, Ala atposition 170, Tyr at position 171, Leu at position 174, Asn atposition 188, Ser at position 189, Ser at position 207, Phe atposition 215, Thr at position 224, Ala at position 236, Glu atposition 245, Asn at position 249, Lys at position 300, Glu atposition 341, Met at position 342, Ala at position 343, Thr atposition 349 and Ala at position 351, or at least one locationamong the same locations described above in pyrroloquinolinequinone dependent glucose dehydrogenase originating in otherAcinetobacter species or other genii. Furthermore, the amino acidsequence of wild type PQQGDH originating in Acinetobacter baumanniistrain NCIMB11517 is shown in SEQ. ID NO. 1. In the case themodified PQQGDH has modified amino acid residues at two or morelocations, examples of the modified PQQGDH include modified PQQGDHcontaining one or more modifications targeted at least one of thelocations selected from the above-mentioned group.

Furthermore, the amino acid sequence of wild type PQQGDHoriginating in Acinetobacter baumannii strain NCIMB11517 is shownin SEQ. ID NO. 1. In SEQ. ID NO. 1, the amino acids are numbered bydesignating aspartic acid as 1 after excluding the signal sequence.

Comparisons of the amino acid sequence of PQQGDH originating inAcinetobacter baumannii strain NCIMB 11517 (the amino acid sequenceof which is indicated in SEQ. ID NO. 1, while the gene sequence isindicated with SEQ. ID NO. 2) with the amino acid sequence ofPQQGDH originating in Acinetobacter calcoaceticus strain LMD79.41and the amino acid sequence of PQQGDH originating in Acinetobactercalcoaceticus strain IFO 12552 reveal that there are differencesonly at a small number of locations, and that they are extremelysimilar with homologies of 92.3% and 91.3%, respectively (includingthe signal sequence in both cases).

Thus, a person with ordinary skill in the art would be able toeasily recognize that a certain residue in SEQ. ID NO. 1corresponds to which amino acid residue (at the same location) ofPQQGDH originating in Acinetobacter calcoaceticus strain LMD79.41.Moreover, an modified PQQGDH having lowered activity with respectto at least one sugar substrate selected from sugar substratesother than glucose as compared with the corresponding wild typePQQGDH can be obtained by carrying out an amino acid mutation atone or more of such locations.

Furthermore, the modified PQQGDH of the present invention maycontain a deletion, substitution or insertion and so on at anotherportion of the amino acid sequence, or other amino acid residuesmay be added or substituted, provided the activity with respect toglucose is essentially maintained and preferably, there issubstantially no detrimental effect on activity with respect tomaltose.

Moreover, the modified PQQGDH of the present invention may includean aspect thereof in which a tag such as a histidine tag is boundor inserted into the PQQGDH provided activity with respect toglucose is substantially maintained, and preferably, activity withrespect to maltose is not substantially adversely affected, anaspect thereof in which another peptide or another protein (such asstreptoavidin or cytochrome) is fused to at least one end ofPQQGDH, an aspect thereof in which PQQGDH is chemically modified bya sugar chain or other compound, and an aspect thereof such as thatwhich has been cross-linked by a disulfide bond and so forth withinand/or between PQQGDH molecules or that which has been linked via alinker peptide and so forth. Alternatively, the modified PQQGDH mayalso include that which has been composed by combining fragments ofwild type PQQGDH from several sources.

GLD-321 produced by Toyo Boseki K.K. and other commercial productscan be used for these PQQGDH enzymes. Alternatively, they can beeasily manufactured by a person skilled in the art using knowntechniques in the field.

For example, naturally-occurring microorganisms producing thePQQGDH, or transformant prepared by inserting a naturally-occurringor modified PQQGDH gene into an expression vector (a variety ofvectors including a plasmid are known), followed by transforming asuitable host (a variety of hosts including E. coli are known) withthe expression vector, are cultured, host cells are collected froma culture medium by centrifugation, cells are broken downmechanically or enzymatically with lysozyme, optionally solubilizedby the addition of a chelating agent such as EDTA or a surfactantto obtain a water soluble fraction containing PQQGDH. The expressedPQQGDH can be secreted to a culture medium using a suitablehost-vector system.

PQQGDH can be separated and precipitated from the PQQGDH-containingsolution by concentration under reduced pressure, membraneconcentration, salting out using ammonium sulfate or sodiumsulfate, or a fractional precipitation with a hydrophilic solventsuch as methanol, ethanol, acetone, etc. Heat treatment andisoelectric treatment are also an effective purification method.Purified PQQGDH can be obtained by gel filtration with adsorbent orgel filtering agent, adsorption chromatography or affinitychromatography. The standard enzyme is preferably purified enoughto show a single band in electrophoresis (SDS-PAGE).

These can be carried out according to, for example, the documentsindicated below. (a) Protein Experimental Protocols, Vol. 1,Protein Analysis Edition, Vol. 2, Structural Analysis Edition(Shujunsha Publishing), Yoshifumi Nishimura, Yoshio Ohno, editors(b) Revised Protein Experimental Notes, Part 1, Extraction,Separation and Purification (Yodosha Publishing), Masato Okada,Kaoru Miyazaki, editors (c) Procedures for Protein Experimentation(Yodosha Publishing), Masato Okada, Kaoru Miyazaki, editors

The PQQGDH can be heat-treated at 25 to 50.degree. C., preferable30 to 45.degree. C. to increase a proportion of holoenzyme to thetotal GDH protein before or after the above-mentioned steps.

Concentration of PQQGDH of the invention is not specificallylimited.


Claim 1 of 11 Claims
1. A method for lowering a reactivity on maltose in glucosemeasurement comprising a step of reacting modified pyrroloquinolinequinone dependent glucose dehydrogenase in the presence of at leastone substance selected from the group consisting of succinic acid,malonic acid, glutaric acid, malic acid, phthalic acid,2-ketoglutaric acid, 3,3-dimethylglutaric acid, pimeric acid,suberic acid, adipic acid, maleic acid, potassium chloride,ammonium chloride, diammonium hydrogen citrate, L-lysine, taurine,calcium glycerate, amino-n-butyric acid, sodium glycolate, sodium.alpha.-ketoglutarate, fumaric acid, glycine, glutamic acid, serineand citric acid, wherein the modified pyrroloquinoline quinonedependent glucose dehydrogenase consists of SEQ ID NO: 1 exceptthat position 168 of SEQ ID NO: 1 and at least one positionselected from the group consisting of positions 169, 170, 245, 342,and 351 of SEQ ID NO: 1 are substituted with another amino acid,and wherein a pH of 5.0 to 6.0 is maintained during the glucosemeasurement reaction.

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