US20030104595A1 - Forms of soluble pyrroloquinoline quinone-dependent glucose dehydrogenase - Google Patents

Forms of soluble pyrroloquinoline quinone-dependent glucose dehydrogenase Download PDF

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US20030104595A1
US20030104595A1 US10/082,627 US8262701A US2003104595A1 US 20030104595 A1 US20030104595 A1 US 20030104595A1 US 8262701 A US8262701 A US 8262701A US 2003104595 A1 US2003104595 A1 US 2003104595A1
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gdh
mutant
glucose
amino acid
pqq
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Peter Kratzch
Rainer Schmuck
Daniela Bunk
Zhixin Shao
Detlef Thym
Wolfgang-Reinhold Knappe
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Priority to US11/298,778 priority patent/US7547535B2/en
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    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)

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  • the present invention relates to improved variants of soluble pyrroloquinoline quinone (PQQ)-dependent glucose dehydrogenases (s-GDH), to genes encoding mutated s-GDH, to mutant proteins of s-GDH with improved substrate specificity for glucose, and to different applications of these s-GDH variants, particularly for determining concentrations of sugar, especially of glucose in a sample.
  • PQQ soluble pyrroloquinoline quinone
  • s-GDH soluble pyrroloquinoline quinone-dependent glucose dehydrogenases
  • Quinoproteins use quinone as cofactor to oxidize alcohols, amines and aldoses to their corresponding lactones, aldehydes and aldolic acids (Duine 1991a,b; Davidson 1993; Anthony 1996; Anthony and Ghosh 1997; Anthony 1998; Goodwin and Anthony 1998).
  • those containing the noncovalently bound cofactor 2,7,9-tricarboxy-1H-pyrrolo [2,3-f]quinoline-4,5-dione (PQQ) constitute the largest sub-group (Duine 1991). All bacterial glucose dehydrogenases known so far belong to this sub-group with PQQ as the prosthetic group (Anthony and Ghosh 1997, Goodwin and Anthony 1998).
  • calcoaceticus s-GDH have been identified in E.coli K-12 and Synechocystis sp. Additionally, two incomplete sequences homologous to A. calcoaceticus s-GDH were also found in the genome of P.aeruginosa and Bordetella pertussis (Oubrie et al. 1999a), respectively. The deduced amino acid sequences of these four uncharacterized proteins are closely related to A. calcoaceticus s-GDH with many residues in the putative active site absolutely conserved. These homologous proteins are likely to have a similar structure and to catalyze similar PQQ-dependent reactions (Oubrie et al., 1999a).
  • Bacterial s-GDHs and m-GDHs have been found to possess quite different sequences and different substrate specificity.
  • A. calcoaceticus contains two different PQQ-dependent glucose dehydrogenases, one m-GDH which is active in vivo, and the other designated s-GDH for which only in vitro activity can be shown.
  • Cleton-Jansen et al., (1988;1989a,b) cloned the genes coding for the two GDH enzymes and determined the DNA sequences of both these GDH genes. There is no obvious homology between m-GDH and s-GDH corroborating the fact that m-GDH and s-GDH represent two completely different molecules.
  • s-GDH The gene of s-GDH from A. calcoaceticus has been cloned in E. coli behind a leader sequence and a strong promoter. After being synthesized in the cell, the s-GDH is translocated through the cytoplamic membrane into the periplasmic space (Duine 1991a, Matsushita and Adachi 1993). Like the native s-GDH from A. calcoaceticus, s-GDH expressed in E.coli is also a homodimer, with one PQQ molecule and three calcium ions per monomer (Dokter et al., 1986a,b; 1988; Olsthoorn and Duine, 1996; Oubrie et al., 1999a,b,c).
  • s-GDH oxidizes a wide range of mono- and disaccharides to the corresponding ketones which further hydrolyze to the aldonic acids, and it is also able to donate electrons to PMS (Phenazine metosulfate), DCPIP (2,6-Dichlorophenolindophenol), WB (Wurster's blue) and short-chain ubiquinones such as ubiquinone Q1 and ubiquinone Q2 (Matsushita et al., 1989a,b), several artificial electron acceptors such as N-methylphenazonium methyl sulfate (Olsthoorn and Duine 1996; 1998) and electroconducting polymers (Ye et al., 1993).
  • PMS Phhenazine metosulfate
  • DCPIP 2,6-Dichlorophenolindophenol
  • WB Wang's blue
  • short-chain ubiquinones such as ubiquinone Q1 and ubiquinone Q
  • Glucose oxidation can be catalyzed by at least three quite distinct groups of enzymes, i.e., by NAD-dependent, dye-linked glucose dehydrogenases, by flavoprotein glucose oxidase or by quinoprotein GDHs (Duine 1995).
  • s-GDH can efficiently donate electrons to PMS, DCPIP, WB and short-chain ubiquinones such as Q1 and Q2, but it can not efficiently donate electrons directly to oxygen.
  • a commercial system which utilizes s-GDH and an indicator that produces a color change when the reaction occurs is the Glucotrend® system distributed by Roche Diagnostics GmbH.
  • S-GDH has rather a broad substrate spectrum as compared to m-GDH. That is, s-GDH oxidizes not only glucose but also several other sugars including maltose, galactose, lactose, mannose, xylose and ribose (Dokter et al. 1986a). The reactivity towards sugars other than glucose may in certain cases impair the accuracy of determining blood glucose levels, in some diabetic patients.
  • icodextrin a glucose polymer
  • body fluids e.g., in blood, high levels of other sugars, specially maltose (M. Dratwa, et al., Perit. Dial. Int. 1998 Nov-Dec;18(6):603-9).
  • clinical samples as e.g. obtained from diabetic patients, especially from patients with renal complications and especially from patients under dialysis may contain significant levels of other sugars, especially maltose.
  • Glucose determinations in samples obtained from such critical patients may be impaired by maltose.
  • mutant s-GDH molecules which, compared to the wild-type enzyme, exhibit essentially the same specific activity for glucose as substrate but markedly reduced activity for other selected sugar molecules.
  • Such comparison of specific activities for various substrate molecules is based on and calculated in relation to the original enzymatic activities of a wild-type enzyme, e.g., as isolated from Acinetobacter calcoaceticus.
  • Mutated s-GDH proteins as well as polynucleotide-sequences coding for such proteins exhibiting improved properties, especially increased specificity for glucose are also provided.
  • s-GDH mutants comprising at least one amino acid substitutions at a position selected from the group consisting of positions 348 and 428 (numbered according to the mature s-GDH protein of A. calcoaceticus ) have been found to provide for s-GDH enzymes with improved properties, especially with improved specificity for glucose.
  • the improved s-GDH mutants can be used with great advantage for the specific detection or measurement of glucose in biological samples, especially in tests strip devices or in biosensors.
  • FIG. 1 Nucleotide (DNA) sequence of the Acinetobacter calcoaceticus PQQ dependent soluble glucose dehydrogenase gene and the corresponding amino acid sequence.
  • FIG. 2 Protein sequences of A. calcoaceticus PQQ-dependent s-GDH and A. baumanni s-GDH aligned according to sequence homology.
  • FIG. 3 Illustration of pACSGDH vector referred to in Example 1 containing the wild-type or mutated DNA sequences of soluble PQQ-dependent glucose dehydrogenase.
  • FIG. 4 Nucleotide (DNA) sequence of the pACSGDH vector referred to in Example 1 containing the wild-type DNA sequence of soluble PQQ-dependent glucose dehydrogenase.
  • a mutant of the soluble form of EC1.1.99.17 also known as PQQ-dependent soluble glucose dehydrogenase (s-GDH), said mutant characterized in that it has an at least two-fold improved substrate specificity for glucose as compared to at least one other selected sugar substrate is described.
  • the wild-type DNA-sequence of such soluble PQQ-dependent glucose dehydrogenase can be isolated from strains of Acinetobacter. Most preferred is the isolation from Acinetobacter calcoaceticus-type strand LMD 79.41. The sequence of this wild-type s-GDH (the mature protein) is given in FIG.: 1 . Other LMD strains of Acinetobacter may also be used as source of wild-type s-GDH. Such sequences can be aligned to the sequence obtained from A. calcoaceticus and sequence comparisons be made (see FIG.: 2).
  • mutant or “variant” in the sense of the present invention relates to a s-GDH protein which compared to a corresponding wild-type sequence exhibits at least one amino acid substitution.
  • the expert in the field will appreciate that there are various possibilities to produce polynucleotides encoding for such a polypeptide sequence of mutated s-GDH. It is of course also possible to generate mutants comprising one or more additions or deletions of amino acids.
  • a mutant according to the present invention is characterized in that it has at least a two-fold improved substrate specificity for glucose as compared to at least one other selected sugar substrate.
  • Standardized and well-defined assay conditions have to be chosen in order to assess (improvements in) specificity.
  • the enzymatic activity of s-GDH for glucose as substrate as well as for other selected sugar substrates is measured as described in Example 6.
  • Cross-reactivity for maltose of wild-type s-GDH according to the above formula has been determined as about 105%.
  • Wild-type s-GDH (cross-)reactivity for galactose has been measured as about 50% (cf. Table 1).
  • a s-GDH form with an at least two-fold improvement in specificity for glucose versus maltose (maltose/glucose) accordingly with maltose as substrate has at most 52,5% of the activity as measured with glucose as substrate.
  • this mutant as compared to the wild-type s-GDH therefore has a 5.25 fold improved substrate specificity (maltose/glucose).
  • the term “specific activity” for a substrate is well known in the art, it is preferably used to describe the enzymatic activity per amount of protein.
  • Various methods are known to the art to determine specific activity of GDH molecules, using glucose or other sugars as substrates (K. Sode et al., Biochemical and Biophysical Research Communications (1999) 264, 820-824). One of the methods available for such measurement is described in detail in the examples section.
  • selected sugars are selected from the group comprising mannose, allose, galactose, xylose, and maltose.
  • maltose or galactose are selected and mutant s-GDH is tested for improved substrate specificity compared to galactose or maltose.
  • the selected sugar is maltose.
  • s-GDH mutated s-GDH
  • said substrate specificity for glucose as compared to the substrate specificity for at least one of the selected other sugar substrates is improved at least three-fold.
  • Other preferred embodiments comprise s-GDH mutants characterized by an improved substrate specificity for glucose, which is at least 5 times or also preferred at least 10 times higher, as compared to the other sugar molecule(s) selected.
  • Mutations in this enzyme lead in many cases to mutants with reduced specific activity for the substrate glucose. Such decrease in (absolute or overall) specific activity for the substrate glucose, however, may be critical for routine applications. Surprisingly it has been found that improved specificity for glucose must not go to the expense of reduced overall specific activity. It is therefore preferred that the s-GDH with improved specificity towards the substrate glucose exhibits at least 10% of the glucose specific activity as measured for the wild-type enzyme. It is of course more preferred that such mutated enzymes exhibit at least 20 or more preferred at least 50% of the respective glucose activity of wild-type s-GDH.
  • the invention relates to a mutant of the soluble form of EC1.1.99.17 also known as PQQ-dependent soluble glucose dehydrogenase (s-GDH), said mutant characterized in that
  • the substrate specific reactivity towards maltose is 30% or less as compared to the wild-type enzyme.
  • the specific (re)activity towards glucose is considered to be essentially comparable to that of the wild-type enzyme if at least 50% of the original enzymatic activity for glucose of the wild-type enzyme is maintained. Also preferred are mutants exhibiting at least 80% or more preferred, at least 90% of the specific activity for glucose as measured for the wild-type enzyme.
  • Mutants comprising only a single substitution of amino acid 253 did not lead to an at least two-fold improvement of substrate specificity for glucose, e.g., as compared to galactose or maltose.
  • Residues 348 or 428 are not known to contribute to the substrate binding of s-GDH as identified and known in the art (BW. Dijkstra et al.,EMBO J 1999 Oct 1;18(19):5187-94; Oubrie, A., Dijkstra, B. W., 2000). No chemical or physical explanation is at hand, why substitutions of these amino acid residues alters the relative specific activity of s-GDH for glucose as compared to other sugar molecules of interest.
  • the mutant protein of a PQQ-dependent s-GDH according to the present invention comprises at least one amino acid residue substitution at a position selected from the group consisting of positions 348, and 428 of the corresponding wild-type sequence of A. calcoaceticus.
  • s-GDH variants comprising a substitution of the amino acid threonine in position 348 are most preferred.
  • the mutated s-GDH is characterized in that the amino acid residue threonine at position 348 is substituted with an amino acid residue selected from the group consisting of alanine, glycine, and serine.
  • glycine is used to substitute for threonine at position 348.
  • One group of preferred s-GDH variants according to this invention comprises a substitution of the amino acid residue at position 348 and at at least one of the following positions 76, 143, 168, 169 and 428.
  • a mutant protein of PQQ-dependent s-GDH comprising an amino acid residue substitution at position 428 of the correspondent wild-type sequence from Acinetobacter calcoaceticus is disclosed, in which the asparagine residue of the wild-type sequence is replaced by other appropriate amino acid residues.
  • amino acid residue is selected from the group consisting of leucine, proline and valine. It is preferred to substitute the asparagine at position 428 with proline .
  • the mutant according to the present invention therefore comprises a substitution of glutamine at position 76 of the corresponding wild-type sequence from Acinetobacter calcoaceticus.
  • amino acid used in such substitution from the group consisting of alanine, aspartic acid, glutamic acid, glycine, methionine, proline and serine.
  • the substitution of at least one amino acid in positions 348, and/or 428 of the s-GDH sequence corresponding to the wild-type sequence isolated from A. calcoaceticus can be used to significantly improve the glucose specificity of s-GDH.
  • Further improved mutants are obtained by providing a mutant s-GDH protein comprising at least two amino acid substitutions, at least one of these being corresponding to amino acid position 76, 348, and/or 428.
  • a further embodiment of the present invention therefore is a mutant protein of PQQ-dependent s-GDH with improved glucose specificity, comprising at least two amino acid residue substitutions, that substituted amino acid positions being selected from the group consisting of position 16, 22, 76, 116, 120, 127, 143, 168, 169, 171, 177, 227, 231, 255, 277, 295, 299, 308, 317, 348, 355, 422,428 and 438. of the corresponding mature A. calcoaceticus soluble PQQ-dependent s-GDH, characterized in that at least one of the amino acid residues, T348 or N428 is replaced.
  • the at least two amino acid residues comprise a substitution in position 348 and at least one additional substitution selected from the group of positions comprising positions 76, 143, 168, 169, and 428.
  • the at least two amino acid positions which are substituted in a mutant s-GDH are selected from the group consisting of amino acid position 76, 348, and/or 428.
  • Mutants comprising substitutions at amino acid residues corresponding to positions 348 and 428 have been found as very advantageous for improving the specificity of s-GDH for glucose in comparison to other sugar substrates. It is especially preferred to design and select mutants of s-GDH, which comprise a substitution at both the positions 348 and 428. Most preferred are mutants comprising the preferred substitutions at both these positions as described above.
  • T348G and N428P are most preferred.
  • This terminology T348G and N428P is known to the art to indicate that threonine at position 348 is replaced by glycine and glutamine and position 428 is replaced by proline.
  • Mutant s-GDH proteins comprising in addition to substitutions at positions 348 and 428 also substitutions at positions 76, 127 and 143, also represent preferred embodiments of the present invention.
  • mutated s-GDH proteins according to the present invention comprise amino acid substitutions at positions 76 and 348 and also such mutants comprising substitutions at positions 76 and 428.
  • the mutated s-GDH protein according to the present invention comprises substitutions of the amino acid residues at positions 76, 348 and 428.
  • a preferred embodiment according to the present invention therefore is a mutant protein of PQQ-dependent s-GDH comprising the amino acid sequence of WPXaaVAPS (SEQ ID NO: 1).
  • SEQ ID NO:1 corresponds to position 346-352 of A. calcoaceticus wild-type s-GDH or position 347-353 of A. baumannii wild-type s-GDH, wherein said Xaa residue is an amino acid residue other than threonine.
  • Xaa in SEQ ID NO: 1 represents glycine.
  • a mutant of PQQ-dependent s-GDH comprising the amino acid sequence of TAGXaaVQK (SEQ ID NO: 2) is another preferred embodiment of the invention.
  • SEQ ID NO:2 corresponds to position 425-431 of A. calcoaceticus wild-type s-GDH or to position 426-432 of A. baumannii wild-type s-GDH, wherein said Xaa residue is an amino acid residue other than asparagine,.
  • the s-GDH mutant comprising SEQ ID NO:2 is characterized in that said Xaa residue is proline residue.
  • the present invention further includes an expression vector comprising a nucleic acid sequence according to the present invention operably linked a promoter sequence capable of directing its expression in a host cell.
  • the present invention further includes an expression vector comprising a nucleic acid sequence according to the present invention operably linked to a promoter sequence capable of directing its expression in a host cell.
  • Preferred vectors are plasmids such as pACSGDH shown in FIGS. 3 and 4.
  • Expression vectors useful in the present invention typically contain an origin of replication, a promoter located upstream of the DNA sequence and are followed by the DNA sequence coding for all or part of s-GDH variants.
  • the DNA sequence coding for all or part of the s-GDH variants is followed by transcription termination sequences and the remaining vector.
  • the expression vectors may also include other DNA sequences known in the art, for example, stability leader sequences which provide for stability of the expression product, secretory leader sequences which provide for secretion of the expression product, sequences which allow expression of the structural gene to be modulated (e.g., by the presence or absence of nutrients or other inducers in the growth medium), marking sequences which are capable of providing phenotypic selection in transformed host cells, and the sequences which provide sites for cleavage by restriction endonucleases.
  • stability leader sequences which provide for stability of the expression product
  • secretory leader sequences which provide for secretion of the expression product
  • sequences which allow expression of the structural gene to be modulated e.g., by the presence or absence of nutrients or other inducers in the growth medium
  • marking sequences which are capable of providing phenotypic selection in transformed host cells
  • sequences which provide sites for cleavage by restriction endonucleases are examples of restriction endonucleases.
  • the expression vector should contain promoters isolated from the genome of E.coli cells (e.g., lac, or trp). Suitable origins of replication in E.coli various hosts include, for example, a ColE1 plasmid replication origin. Suitable promoters include, for example, lac and trp. It is also preferred that the expression vector include a sequence coding for a selectable marker.
  • the selectable marker is preferably an antibiotic resistance gene. As selectable markers, ampicillin resistance, or canamycin 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. (1989).
  • the present invention additionally concerns host cells containing an expression vector which comprises a DNA sequence coding for all or part of the mutant s-GDH.
  • the host cells preferably contain an expression vector that comprises all or part of one of the DNA sequences having one or more mutations shown in Table 1. Further preferred are the host cells containing an expression vector comprising one or more regulatory DNA sequences capable of directing the replication and/or the expression of, and operatively linked to a DNA sequence coding for, all or part of mutant s-GDH.
  • Suitable host cells include, for example, E.coli HB101 (ATCC 33694) available from Pomega (2800 Woods Hollow Road, Madison, Wis., USA), XL1-Blue MRF available from Stratagene (11011 North Torrey Pine Road, La Jolla, Calif., USA) and the like.
  • 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). However, other methods for introducing expression vectors into host cells, for example, electroporation, biolistic injection, or protoplast fusion, can also be employed.
  • the host cell may be cultured under conditions permitting expression of the desired s-GDH variants.
  • Host cells containing an expression vector which contains a DNA sequence coding for all or part of the mutant s-GDH are, e.g., identified by one or more of the following general approaches: DNA hybridization, the presence or absence of marker gene functions, assessment of the level of transcription as measured by the production of s-GDH mRNA transcripts in the host cell, and detection of the gene product immunologically.
  • transformed host cells are identified by enzyme assay, e.g., calorimetric detection.
  • the present invention also teaches the generation and screening of s-GDH variants. Random mutagenesis and saturation mutagenesis is performed as known in the art. Variants are analyzed for substrate specificity for glucose, maltose as well as other sugars. The assay conditions chosen are adapted to ensure that the expected small enhancements brought about e.g., by a single amino acid substitution, can be measured. This has been accomplished by adjusting the assay conditions such that the wild type (or parent) enzyme activity is dose to the lower detection limit. One mode of selection or screening of appropriate mutants is given in Example 3. Any change or improvement as compared over the wild-type enzyme this way can be clearly detected.
  • the invention also relates to a process for producing s-GDH variants of the current invention comprising culturing a host cell of the invention under conditions suitable for production of the mutant s-GDH of the invention.
  • typical culture conditions are liquid medium containing the appropriate antibiotic and induction agent.
  • appropriate antibiotics include ampicillin, canamycin, chloroamphenicol, tetracyclin and the like.
  • Typical induction agents include IPTG, glucose, lactose and the like.
  • the polypeptides of the present invention are obtained by production in host cells expressing a DNA sequence coding the mutant s-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 mutant s-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 preferred 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.
  • One of the major applications of the improved s-GDH variants of this invention is for the use in test strips to monitor blood-glucose level in diabetic patients. Due to the insensitivity of PQQ-dependent glucose dehydrogenase towards oxygen, a system using the improved s-GDH variants is less prone to interference by oxygen than systems based on glucose oxidase. More important, since the s-GDH variants have improved specificity towards glucose and significantly decreased relative enzymatic activity towards other sugars, the interference due maltose, galactose, and/or other related sugars which may be present in a sample to be analyzed is significantly reduced. Of course many kinds of samples may be investigated. Bodily fluids like serum, plasma, intestinal fluid or urine are preferred sources for such samples.
  • the invention also comprises a method of detecting, determining or measuring glucose in a sample using a s-GDH mutant according to the present invention. It is especially preferred that the improved method for detection of glucose in a sample is characterized in that said detection, determination or measurement of glucose is performed using a sensor or test strip device.
  • a device for the detection or measurement of glucose in a sample comprising an s-GDH mutant according to this invention as well as other reagents required for said measurement.
  • the s-GDH variants with improved substrate specificity of this invention can also be used to great advantage in biosensors (D'Costa et al., 1986, Laurinavicius et al., 1999a,b) for online monitoring of glucose in a sample or a reactor.
  • the s-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) for more accurate determination of the glucose concentration.
  • s-GDH variants with the improved substrate specificity according to this invention.
  • these s-GDH variants may be used in an aldonic acid production process. Wild-type s-GDH has a high turnover in substrate oxidation producing gluconic and other aldonic acids.
  • the s-GDH variants which are more specific for glucose, the production of gluconic acid would result in much less byproducts.
  • the s-GDH gene was isolated from Acinetobacter calcoaceticus strain LMD 79.41 according to standard procedures.
  • the wild-type s-GDH gene was subdoned into a plasmid containing the mgl promoter for adjustabel expression (cf. Patent application WO88/09373).
  • the new construct was called pACSGDH (see FIGS. 3 and 4).
  • the recombinant plasmids was introduced into a host organism selected from the E.coli group. These organisms were then cultivated under appropriate conditions and colonies showing s-GDH activity selected.
  • the plasmid pACSGDH was isolated from a 200 ml over-night culture of the clone mentioned above using the QIAGEN Plasmid Maxi Kit (Qiagen) according to the manufacturers protocol.
  • the plasmid was re-suspended in 1 ml bidest. water.
  • the concentration of the plasmid was determined using a Beckman DU 7400 Photometer. The yield was 600 ⁇ g. Then the quality of the plasmid was determined by agarose gel electrophoresis.
  • mutagenic PCR polymerase chain reaction
  • the pACSGDH plasmid and the DNA sequence encoding the mutated enzymes were digested with the restriction enzymes Sph I and Eco RI. The products were gel purified.
  • the digested DNA sequences were ligated and an aliquot of the ligation reaction mixture was used to transform competent E. coli cells. The transformants were subsequently selected on LB plates containing ampicillin.
  • the plasmid containing the mutant s-GDH gene that leads to 50% maltose/glucose activity was isolated (High Pure Plasmid Isolation Kit, Roche 1754785) and sequenced using an ABI Prism Dye Terminator Sequencing Kit and ABI 3/73 and 3/77 sequencer (Amersham Pharmacia Biotech).
  • GDH R4 5′-CAA GAC GAC GAC GAC GAC G
  • the 5′- and the 3′-primer used for mutagenesis were complementary to each other and contained NNN in a central position. These nucleotides were flanked by 12 to 16 nucleotides at each end. The sequences of the nucleotides were identical to the cDNA-strand or to the complementary cDNA-strand flanking the codon for the amino acid that had to be substituted. Instead of the codon, the primer contained NNN therefore the oligonucleotides code for every codon.
  • the grown cells (LB-Amp. 37° C.) were harvested and resuspended in potassium phosphate buffer pH 7.0. Cell disruption was performed by French Press passage (700-900 bar). After centrifugation the supernatant was applied to a S-Sepharose (Pharmacia) column equilibrated with 10 mM potassium phosphate buffer pH 7.0. After washing, the s-GDH was eluted using a salt gradient 0-1 M NaCl. The fractions showing GDH activity were pooled, dialysed against potassium phosphate buffer pH 7.0 and re-chromatographied on re-equilibrated S-sepharose column. The active fractions were pooled and subjected to a gel filtration using a Superdex® 200 column (Pharmacia). The active fractions were pooled and stored at ⁇ 20° C.
  • Protein determination was performed using the Protein Assay Reagent no. 23225 from Pierce (calibration curve with BSA, 30 Min. 37° C.).
  • the GDH samples were diluted at 1 mg protein/ml with 0.0556 mM pyrollo-quinoline quinone(PQQ); 50 mM Hepes; 15 mM CaCl2 pH 7.0 and incubated at 25° C. for 30 minutes for reconstitution or activation.
  • PQQ pyrollo-quinoline quinone
  • One Unit enzyme activity corresponds the conversion of 1 mMol mediator/min under the above assay conditions
  • the assay was performed with glucose, maltose and galactose (Merck, Germany).
  • the wild-type and mutant T348G of s-GDH were applied for glucose determination.
  • the reference samples contained 65 mg glucose/dl.
  • the “test”-samples contained 65 mg glucose/dl and 130 mg/dl maltose.
  • the same amounts of GDH activity (U/ml; see enzyme assay) were used for each assay.
  • the assay was started adding 0.050 ml 90 U/ml s-GDH .
  • the change of absorption at 620 nm was monitored. After 5 minutes constant values were observed and the dE/5 min calculated.
  • the value obtained measuring the reference sample with wild-type s-GDH was set to 100%. The other values were compared to this reference value and calculated in %.

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030134346A1 (en) * 2002-01-04 2003-07-17 Amiss Terry J. Binding protein as biosensors
US20050260728A1 (en) * 2004-05-19 2005-11-24 Amano Enzyme Inc. Pyrroloquinoline quinone-dependent glucose dehydrogenase
US20060003400A1 (en) * 2004-06-30 2006-01-05 Byrd Patricia A Methods and compositions for characterizing a redox reagent system enzyme
EP1752531A1 (en) * 2005-08-11 2007-02-14 Toyo Boseki Kabushiki Kaisha Method for improving substrate specificity of pyrroloquinoline quinone dependent glucose dehydrogenase
WO2007118647A1 (en) * 2006-04-13 2007-10-25 Roche Diagnostics Gmbh Improved mutants of pyrroloquinoline quinone dependent soluble glucose dehydrogenase
US7955484B2 (en) 2005-12-14 2011-06-07 Nova Biomedical Corporation Glucose biosensor and method
US8748145B2 (en) 2009-07-28 2014-06-10 Centre National De La Recherche Scientifique (C.N.R.S.) Mutants of pyrroloquinoline quinine-dependent soluble glucose dehydrogenase
US20220275348A1 (en) * 2010-07-23 2022-09-01 Roche Diabetes Care, Inc. Zwitterion buffer containing compositions and uses in electroanalytical devices and methods

Families Citing this family (37)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2327815T3 (es) * 2000-02-11 2009-11-04 Millennium Pharmaceuticals, Inc. 18477, proteina quinasa humana y usos de la misma.
AU1596602A (en) * 2000-10-27 2002-05-06 Peter Kratzsch Variants of soluble pyrroloquinoline quinone-dependent glucose dehydrogenase
JP2003093071A (ja) * 2001-09-26 2003-04-02 Koji Hayade グルコース脱水素酵素
US7476525B2 (en) 2002-05-27 2009-01-13 Toyo Boseki Kabushiki Kaisha Modified pyrroloquinoline quinone (PQQ) dependent glucose dehydrogenase with superior substrate specificity and stability
DE60323650D1 (de) 2002-07-04 2008-10-30 Ultizyme Int Ltd Glucosedehydrogenase
DE60322921D1 (de) * 2002-09-27 2008-09-25 Dsm Ip Assets Bv Aldehyddehyrdrogenase-gen
DE60331477D1 (de) 2002-12-24 2010-04-08 Ikeda Food Res Co Ltd Coenzymbindende glukosedehydrogenase
JP4415282B2 (ja) * 2003-03-24 2010-02-17 東洋紡績株式会社 基質特異性または安定性に優れたピロロキノリンキノン（ｐｑｑ）依存性グルコースデヒドロゲナーゼ改変体
DE10320259A1 (de) * 2003-05-07 2004-11-25 Bayer Technology Services Gmbh Neuartige Glukose Dehydrogenase und ihre Herstellung
EP1666586B1 (en) * 2003-09-08 2009-10-21 Toyo Boseki Kabushiki Kaisha Pyrroloquinoline quinone (pqq)-dependent glucose dehydrogenase modification having excellent substrate specificity
JP2005328793A (ja) * 2004-05-21 2005-12-02 Toyobo Co Ltd 基質特異性に優れたピロロキノリンキノン（ｐｑｑ）依存性グルコースデヒドロゲナーゼ改変体
EP1600503A1 (en) * 2004-05-14 2005-11-30 Amano Enzyme Inc. Modified pyrroloquinoline quinone-dependent glucose dehydrogenase
NZ551870A (en) 2004-07-20 2009-02-28 Hoffmann La Roche Genetically engineered pyrroloquinoline quinone dependent glucose dehydrogenase comprising an amino acid insertion
ATE511538T1 (de) 2004-10-15 2011-06-15 Hoffmann La Roche Thermostabile mutanten pyrrolochinolin-chinon- abhängiger glucose-dehydrogenase
US7901921B2 (en) * 2004-10-22 2011-03-08 Oncolytics Biotech Inc. Viral purification methods
WO2006085509A1 (ja) * 2005-02-08 2006-08-17 Toyo Boseki Kabushiki Kaisha 基質特異性に優れた改変型ピロロキノリンキノン依存性グルコースデヒドロゲナーゼ
EP2365074A1 (en) 2005-03-25 2011-09-14 Ikeda Food Research Co. Ltd. Coenzyme-linked glucose dehydrogenase and polynucleotide encoding the same
WO2006109578A1 (ja) * 2005-04-05 2006-10-19 Amano Enzyme Inc. 改変型ピロロキノリンキノン依存性グルコース脱水素酵素、及びピロロキノリンキノン依存性グルコース脱水素酵素の基質特異性改良法
DE602006011858D1 (de) * 2005-06-20 2010-03-11 Arkray Inc Mutante glucose-dehydrogenase
TW200706650A (en) * 2005-08-11 2007-02-16 Toyo Boseki A substrate specificity improved composition for glucose measurement
KR100973992B1 (ko) * 2006-05-25 2010-08-05 삼성전자주식회사 시분할 복신 무선통신시스템의 시분할 복신 스위치
WO2008001903A1 (en) 2006-06-29 2008-01-03 Ikeda Food Research Co., Ltd. Fad-conjugated glucose dehydrogenase gene
US8619038B2 (en) * 2007-09-04 2013-12-31 Apple Inc. Editing interface
DE102008030435A1 (de) * 2008-06-26 2010-01-07 Bayer Technology Services Gmbh Neuartige Varianten PQQ-abhängiger Glukosehydrogenase mit verbesserter Substratspezifität
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WO2016026959A1 (en) 2014-08-22 2016-02-25 Roche Diagnostics Gmbh Redoxindicators
CN105331591B (zh) 2015-10-29 2020-02-28 英科隆生物技术（杭州）有限公司 PQQ-sGDH突变体、聚核苷酸及葡萄糖检测装置
CA3035874A1 (en) 2016-10-05 2018-04-12 F. Hoffmann-La Roche Ag Detection reagents and electrode arrangements for multi-analyte diagnostic test elements, as well as methods of using the same
CN113234697B (zh) * 2021-04-28 2022-09-02 遵义医科大学珠海校区 一种葡萄糖脱氢酶变体及其制备方法与应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5484708A (en) * 1993-04-08 1996-01-16 Boehringer Mannheim Gmbh Method for the colorimetric determination of an analyte with a PQQ-dependent dehydrogenase
US5997817A (en) * 1997-12-05 1999-12-07 Roche Diagnostics Corporation Electrochemical biosensor test strip
US6057120A (en) * 1996-09-24 2000-05-02 Roche Diagnostics Gmbh Redox-active compounds and their use
US6103509A (en) * 1997-03-03 2000-08-15 Lifescan Inc. Modified glucose dehydrogenase

Family Cites Families (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6190906B1 (en) 1987-05-20 2001-02-20 Roche Diagnostics Gmbh Expression vector for the regulatable expression of foreign genes in prokaryotes
DE3716957A1 (de) 1987-05-20 1988-12-01 Boehringer Mannheim Gmbh Expressionsvektor zur regulierbaren expression von fremdgenen in prokaryonten
FI905324A (fi) 1990-10-29 1992-04-30 Valtion Teknillinen Enzymatiskt bestaemningsfoerfarande foer aldoser.
CZ291059B6 (cs) * 1997-03-12 2002-12-11 Lonza Ag Způsob výroby esterů 2,6-pyridindikarboxylových kyselin
JP2000350588A (ja) * 1999-04-08 2000-12-19 Koji Hayade グルコース脱水素酵素
TWI224136B (en) * 1999-04-30 2004-11-21 Koji Sode Glucose dehydrogenase
AU1596602A (en) * 2000-10-27 2002-05-06 Peter Kratzsch Variants of soluble pyrroloquinoline quinone-dependent glucose dehydrogenase
JP2003093071A (ja) * 2001-09-26 2003-04-02 Koji Hayade グルコース脱水素酵素

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5484708A (en) * 1993-04-08 1996-01-16 Boehringer Mannheim Gmbh Method for the colorimetric determination of an analyte with a PQQ-dependent dehydrogenase
US6057120A (en) * 1996-09-24 2000-05-02 Roche Diagnostics Gmbh Redox-active compounds and their use
US6103509A (en) * 1997-03-03 2000-08-15 Lifescan Inc. Modified glucose dehydrogenase
US5997817A (en) * 1997-12-05 1999-12-07 Roche Diagnostics Corporation Electrochemical biosensor test strip

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030134346A1 (en) * 2002-01-04 2003-07-17 Amiss Terry J. Binding protein as biosensors
US6855556B2 (en) * 2002-01-04 2005-02-15 Becton, Dickinson And Company Binding protein as biosensors
US20050260728A1 (en) * 2004-05-19 2005-11-24 Amano Enzyme Inc. Pyrroloquinoline quinone-dependent glucose dehydrogenase
US7037698B2 (en) * 2004-05-19 2006-05-02 Amano Enzyme Inc. Pyrroloquinoline quinone-dependent glucose dehydrogenase
US20060003400A1 (en) * 2004-06-30 2006-01-05 Byrd Patricia A Methods and compositions for characterizing a redox reagent system enzyme
EP1752531A1 (en) * 2005-08-11 2007-02-14 Toyo Boseki Kabushiki Kaisha Method for improving substrate specificity of pyrroloquinoline quinone dependent glucose dehydrogenase
US20070037237A1 (en) * 2005-08-11 2007-02-15 Toyo Boseki Kabushiki Kaisha Method for improving substrate specificity of pyrroloquinoline quinone-dependent glucose dehydrogenase
US7955484B2 (en) 2005-12-14 2011-06-07 Nova Biomedical Corporation Glucose biosensor and method
US20090148874A1 (en) * 2006-04-13 2009-06-11 Mara Boenitz-Dulat Mutants of pyrroloquinoline quinone dependent soluble glucose dehydrogenase
WO2007118647A1 (en) * 2006-04-13 2007-10-25 Roche Diagnostics Gmbh Improved mutants of pyrroloquinoline quinone dependent soluble glucose dehydrogenase
KR101168623B1 (ko) 2006-04-13 2012-08-03 에프. 호프만-라 로슈 아게 피롤로퀴놀린 퀴논 의존 가용성 글루코오스 탈수소효소의 개선된 돌연변이체
CN101421396B (zh) * 2006-04-13 2016-01-20 霍夫曼-拉罗奇有限公司 吡咯并喹啉醌依赖性可溶性葡萄糖脱氢酶的改良突变体
EP3246402A1 (en) * 2006-04-13 2017-11-22 Roche Diabetes Care GmbH Improved mutants of pyrroloquinoline quinone dependent soluble glucose dehydrogenase
US8748145B2 (en) 2009-07-28 2014-06-10 Centre National De La Recherche Scientifique (C.N.R.S.) Mutants of pyrroloquinoline quinine-dependent soluble glucose dehydrogenase
US20220275348A1 (en) * 2010-07-23 2022-09-01 Roche Diabetes Care, Inc. Zwitterion buffer containing compositions and uses in electroanalytical devices and methods
US11851685B2 (en) * 2010-07-23 2023-12-26 Roche Diabetes Care, Inc. Zwitterion buffer containing compositions and uses in electroanalytical devices and methods

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ATE520777T1 (de) 2011-09-15
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US7547535B2 (en) 2009-06-16
CN1288246C (zh) 2006-12-06
US20040005683A1 (en) 2004-01-08
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