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  • 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)
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  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/001—Enzyme electrodes
  • C12Q1/005—Enzyme electrodes involving specific analytes or enzymes
  • C12Q1/006—Enzyme electrodes involving specific analytes or enzymes for glucose
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  • 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
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  • C12Y—ENZYMES
  • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
  • C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
  • C12Y101/01069—Gluconate 5-dehydrogenase (1.1.1.69)
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  • C12Y—ENZYMES
  • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
  • C12Y101/05—Oxidoreductases acting on the CH-OH group of donors (1.1) with a quinone or similar compound as acceptor (1.1.5)
  • C12Y101/05002—Quinoprotein glucose dehydrogenase (1.1.5.2)

Definitions

• the present invention relates to PQQ-dependent glucose dehydrogenases (sPQQGDH), methods for their production and identification, genes encoding the sPQQGDH according to the invention, vectors for producing the genes, and glucose sensors comprising a glucose dehydrogenase according to the invention.
• sPQQGDH PQQ-dependent glucose dehydrogenases
• the enzymatic determination of glucose is of great importance in medical diagnostics.
• the detection of glucose in the urine or in the blood to determine or follow-up of metabolic diseases is necessary.
• the conversion rate of an enzyme is usually expressed in units of activity (U);
• the specific activity (U / mg) usually refers to the amount of glucose in micromol that is oxidized by one milligram of enzyme per minute under specified reaction conditions.
• GDH glucose dehydrogenases
• NAD + nicotinamide adenine dinucleotide
• NADP + nicotinamide adenine dinucleotide phosphate
• glucose dehydrogenases contains pyrroloquinoline quinone (PQQ) bound as an electron acceptor in the enzyme.
• PQQ pyrroloquinoline quinone
• Two structurally distinct glucose dehydrogenases of this type have been described, a membrane-anchored form mPQQGDH (AM Cleton-Jansen et al, J. Bacteriol 170 [5], 2121-2125 (1988)) and a smaller, soluble form sPQQGDH (AM Cleton-Jansen et al., Mol. Gen. Genet. 217 [2-3], 430-436 (1989)).
• Latter is due to their high spec. Activity, their insensitivity to oxygen and the tightly bound, stable cofactor enzyme, which is particularly suitable for the production of test systems for the determination of glucose.
• this enzyme is used in a number of commercial blood glucose meters, despite a lower substrate specificity that this enzyme has compared to the other enzymes mentioned.
• this enzyme is desirable to have variants of this enzyme which have a higher substrate specificity.
• the sPQQGDH In addition to the glucose substrate, the sPQQGDH also converts other sugars to the corresponding lactone, preferably disaccharides, whose reducing sugar is glucose, but also other reducing sugars. Since such sugars are not normally present in human blood, an enzyme assay based on sPQQGDH usually gives a result that corresponds to the glucose concentration. In the context of certain medical therapies, however, substances are administered whose degradation products include maltose, which is oxidized by the sPQQGDH and thus undesirably contributes to the measurement result (T. G. Schleis, Pharmacotherapy 27 [9], 1313-1321 (2007)). With the administration of xylose and galactose in diagnostic procedures or as an additive to medicines, these sugars are even recorded. It therefore makes sense to appropriately suppress these ancillary activities of the sPQQGDH.
• the object thus is to provide glucose dehydrogenases which enable reliable determination of glucose even in the presence of a mixture of glucose and other sugars.
• the object is to provide glucose dehydrogenases, with which glucose can be reliably determined in the presence of maltose.
• a further object is to provide a method by which such glucose dehydrogenases can be prepared and identified which are particularly suitable for the determination of glucose in the presence of other sugars, in particular in the presence of maltose.
• sPQQ glucose dehydrogenases in which three to five amino acids are inserted in the region of the substrate binding region, have a glucose specificity which is better than the starting form of the enzyme.
• An object of the present invention are therefore sPQQ glucose dehydrogenases having an improved glucose specificity compared to the starting form of the enzyme, characterized in that three to five amino acids are inserted in the region of the substrate binding region.
• the sPQQGDH according to the invention can be encoded by genes which are produced by mutagenesis from wild-type strains naturally occurring, such as Acinetobacter calcoaceticus LMD 79.41 (K.Kojima K. et al, Biotechnology Letters 22, 1343-1347 (2000)) and other strains of the genus Acinetobacter (PJ Bouvet PJ and OM Bouvet, Res. Microbiol., 140 [8], 531-540 (1989)), in particular those which are characterized by increased Thermostabi quality as described in EP 1623025 Al Acinetobacter sp. Tribes, to be won.
• Preferred strains are those described in EP 1623025 Al Acinetobacter sp.
• Preferred for mutagenesis are amino acid regions whose distances from the substrate are not more than 5 angstroms, e.g. the positions Q76, D143, Q168, Y343 and the adjacent amino acid residues. For example, before or after one of the specified
• Positions are made an insertion of three or more amino acids to change the spatial nature of the substrate binding site. Particularly preferred is a
• the sPQQGDH according to the invention are characterized, as shown in Table 1, by increased substrate specificity (lower maltose / glucose ratio and lower level of interference of glucose in a glucose / maltose mixture).
• the present invention further provides a method for the production and identification of sPQQGDH, which show increased substrate specificity compared to the wild type.
• the method according to the invention allows the generation of a multiplicity of variants by means of local random mutagenesis, with three, four, five or more inserted amino acids at a desired site of the sPQQGDH sequence.
• the enzymes produced are screened for substrate specificity and the best variants selected.
• the inventive method is characterized in that in a first step, a vector is prepared in which a suitable sequence area in front of and behind the desired
• Insertion site was deleted and replaced by a unique for this vector restriction site.
• vector DNA which is opened with this restriction enzyme, in a second step double-stranded, oligonucleotide sequences produced by PCR amplification are inserted, which contain the desired insertion in addition to the sequence deleted in the vector, so that a gene product is produced which has no further amino acid sequence
• Double-stranded fragment contains the three or more additional, randomized codons for
• recombinant vectors which have been prepared in the manner described above are transformed into suitable host bacteria and the host bacteria are propagated in a suitable nutrient medium.
• the methods for cloning genes from microorganisms and their expression in another host, as well as the techniques for isolating and altering nucleic acids, are known to the person skilled in the art (see, for example, Molecular Cloning - A Laboratory Manual, Vol 1, Sambrook, J. and Russel, DW, 3rd edition, CSHL Press 2001).
• the cultivation of microorganisms and the purification of recombinant enzymes thereof are state of the art (see ibid, Vol. 3).
• the bacterial colonies are tested for their activity against various sugars and the best ones identified. According to the invention, not only a test is carried out with respect to the activity towards the individual sugars but also with respect to the interference of different sugars.
• the activities may be considered specific activities, i. Conversion rates, which are implemented by a defined amount of enzyme per unit time, can be determined.
• the specific activity U / mg usually refers to the amount of glucose in micromoles that is oxidized by one milligram of enzyme per minute under specified reaction conditions.
• glucose is present in excess of the enzyme in order to eliminate the influence of the glucose concentration on the reaction rate.
• the conversion rate is determined by monitoring the degradation of a reactant or the formation of a product over time. The time tracking can e.g. done photometrically.
• reaction rates and turnover rates reference may be made to textbooks of physical chemistry (e.g., Peter Atkins, Physical Chemistry, W.H. Freeman & Co., 7th Edition, 2002).
• the individual colonies are divided and an enzyme test (determination of the specific activity) with glucose as substrate and a second enzyme test with a second sugar, e.g.
• Maltose performed as a substrate. Opposed to the colonies with the highest activities Glucose with at the same time least activity towards the second sugar (eg maltose) also determines the activity against a mixture of glucose and the second sugar (eg maltose). It turned out, surprisingly, in the study of some variants with very low relative reactivity to maltose as the sole substrate, that this property of one variant is not necessarily associated with an independent property, namely the reliable discrimination of glucose and maltose, when both sugars are present as a mixture , This can lead both to the overestimation and to the underestimation of the glucose concentration present in this mixture.
• V R ⁇ ren the rate at which glucose degraded by the observed enzyme and V Miscnung the rate at which glucose degraded in a mixture with each other sugar .
• the rates can be determined, for example, photometrically (see examples 3, 4, 7 and 9).
• Positive values in this context mean that the enzyme activity is higher on a mixture than on glucose alone; So apparently both sugars are oxidized. Negative values may be interpreted as oxidizing the interfering sugar, but decreasing the activity of the enzyme for glucose. Both effects are undesirable for an enzyme to be used for the specific quantification of glucose.
• the method according to the invention makes it possible to produce and identify variants of sPQQGDH whose substrate specificity has changed compared with the starting form of the enzyme.
• the method according to the invention can also be modified and / or expanded in such a way that instead of the substrate specificity or in addition to this, other biochemical properties such as eg thermostability are tested and thus variants improved with respect to the starting form the other biochemical properties are identified.
• the method according to the invention thus makes it possible to select variants of sPQQGDH with particularly advantageous properties.
• the present invention also relates to genes which encode the sQQQGDH according to the invention and vectors for generating the genes.
• the present invention furthermore relates to a glucose sensor comprising an sPQQGDH according to the invention for the determination of glucose.
• a glucose sensor comprising an sPQQGDH according to the invention for the determination of glucose.
• the construction and operation of glucose-based dehydrogenase-based glucose sensors are known to those skilled in the art (see, for example, EP146332A1). Thereafter, such a glucose sensor comprises an electrode system of a working electrode, a counter electrode and a reference electrode on an insulating plate, on which an enzymatic reaction layer is applied, which includes a glucose dehydrogenase and an electron acceptor in contact with the electrode system.
• Suitable working electrodes include carbon, gold, platinum and similar electrodes on which an enzyme according to the invention is immobilized by cross-linking agent: embedding in a polymer matrix, jacketing with a dialysis membrane, using a photocrosslinkable polymer, an electrically conductive polymer or a redox polymer, fixing the enzyme in a polymer or adsorbing on the electrode with an electron mediator including ferrocene or its derivatives or any combination thereof.
• sPQQGDH according to the invention are immobilized on the electrode in the form of a holoenzyme, although they may also be immobilized as an apoenzyme and provided with PQQ as a separate layer or in a solution.
• sPQQGDHs of the present invention are immobilized on a carbon electrode with glutaraldehyde and then treated with an amine-containing reagent to block the glutaraldehyde.
• the counter electrode e.g. a platinum electrode and as a reference electrode e.g. an Ag / AgCl electrode can be used.
• the amount of glucose can be measured as follows: PQQ, CaCl 2 and a mediator are placed in a thermostat cell containing a buffer and left at a constant temperature. Suitable mediators include, for example, potassium ferricyanide and phenazine methasulfate.
• An electrode on which an sQQQGDH of the present invention has been immobilized is used as a working electrode in combination with a counter electrode (eg, a platinum electrode) and a reference electrode (eg, Ag / AgCl electrode). After to When a steady state current has been applied to the working electrode, a glucose-containing sample is added to measure the increase in current. The glucose level of the sample can be read from a calibration curve made with glucose solutions at standard concentrations.
• Example 1 Preparation of a vector for insertion of additional amino acids between Q168 and L169
• a gene for an sPQQGDH must be available in an expression plasmid which can be propagated in microorganisms which permits induced expression of sPQQGDH and which can be used to carry out the desired mutations.
• a variety of plasmids and microorganisms are known in the art, which can be used for this purpose.
• a construct based on the commercially available vector pASK-IBA3 (IBA GmbH, Göttingen) was used.
• pASK-IBA3 the sPQQGDH gene from the strain Acinetobacter sp. KOZ65 cloned in the following way. From genomic DNA of Acinetobacter sp.
• KOZ65 was probed with the two oligonucleotides GDH-U3 (5'-TGGTAGGTCTCAAATGAATAAACATTTATTGGC TAAAATTAC-3 '; SEQ ID 19) and GDH-L5 (5'TTCAGCTCTGAGCTTTATATGTAAATCTAATC-S'; SEQ ID 20) by means of a PCR reaction (Phusion DNA Polymerase, Finnzymes Oy) according to the manufacturer's instructions generated a 1, 46 kb long DNA fragment. The fragment was purified and then incubated with the restriction enzyme Bsal. The vector pASK-IBA3 was opened with the restriction enzyme HindIII.
• the restriction enzyme was denatured by heat treatment and the resulting DNA overhangs were filled in with T4 DNA polymerase in the presence of dATP, dCTP, dGTP and dTTP. Subsequently, the DNA was purified and incubated with the restriction enzyme Bsal. The thus released, 1 14 bp fragment was discarded; into the 3.1 kb vector fragment, the PCR amplicon was ligated with the sOQ65GDH gene from KOZ65. The ligation product was transformed into transformation competent cells of the E. coli strain DH5 ⁇ .
• the vector pAI3B-KOZ65 prepared in this way is suitable for the inducible expression of KOZ65 sPQQGDH.
• the nucleotide sequence of the vector pAI3B-KOZ65 is given under SEQ ID 21, the amino acid sequence of the coded sPQQGDH under SEQ ID 22.
• the vector pAI3B-KOZ65-U was first prepared.
• plasmid DNA of the vector pAI3B-KOZ65 was used as template for a PCR amplification A with the primers IBA-For (5'-TAGAGTTATTTTACCACTCCCT-3 ', SEQ ID 23) and Ecol09-Up (5'-GGTTAGGTCCCTGATCACCAATCG-3'; SEQ ID 24) and a PCR amplification B with the primers BfuA-US 1 (5 '-CACAGGTACACCTGCCGCCACT-S', SEQ ID 25) and Ecol 09-Down (5'-TACGAGGACCCAACAGGAACTGAGC-S '; SEQ ID 26).
• the amplificate formed from A was purified and cut with the restriction enzymes XbaI and EcoO109I.
• the resulting 588 base pair (bp) fragment was isolated.
• the B-derived amplicon was purified and cut with the restriction enzymes BfuAI and EcoO109I.
• the resulting 353 bp fragment was also isolated.
• the vector pAI3B-KOZ65 was cut with the restriction enzymes BfuAI and XbaI.
• the resulting, 3567 bp fragment was isolated on an agarose gel.
• Kits and reagents from New England Biolabs GmbH (Frankfurt), Invitrogen (Karlsruhe), Qiagen (Hilden) and Roche Diagnostics (Mannheim) were used according to the manufacturer's instructions for the work steps described. Additional methods were taken from the set of methods of Sambrook and Russell (Molecular Cloning - A Laboratory Manual, Sambrook, J. and Russel, DW, 3rd edition, CSHL Press 2001).
• the vector pAI3B-KOZ65-U (SEQ ID 27) now contains a unique EcoO109I restriction site (RG'GNCCY).
• EcoO109I generates 3 bp long 5 'overhangs in which the middle nucleotide is arbitrary; in pAI3B-KOZ65-U it is an A on the coding strand.
• the DNA coding for the amino acids R166 to Pl82 and another nucleotide are deleted in pAI3B-KOZ65-U, which leads to a frame shift. The attempt to express this gene therefore leads to a truncated polypeptide without function (SEQ ID 28).
• the vector is suitable for the uptake of synthetic, double-stranded DNA fragments that result in the recovery of a functional sPQQGDH gene with insertions between positions Q 168 and L 169, as further described in Example 2.
• Example 2 Preparation of sPQQGDH Insertional Mutants
• EarV2 random (5'-CGACGTAACCAGNNKNNNNKKNTKCTGGCTTACCTG-3 ', SEQ ID 29)
• EarV2-Lower (5'-GTGTGCTGTGCCTGGTTCGGCAGGAACAGGTAAGCCAG-3 '; SEQ ID 30)
• EarV2-UpAmp (5'-CCTACCTACGACTCTTCCGACGTAACCAG-S'; SEQ ID 31)
• EarV2-LoAmp (5'-CCATGCTCTTCAGTCGGAGTGTGCTGTGCC-3 '; SEQ ID 32).
• NNK NNK NNK NNK N stands for a mixture of all four nucleotides (dATP (A), dCTP (C), dGTP (G), dTTP (T)) in the synthesis of the oligonucleotide at the respective position, while K stands for G or T.
• the sequence NNK allows codons for all 20 amino acids but only for one of the three stop codons, reducing the likelihood of incomplete gene products.
• Oligonucleotide EarV2 random contains the coding sequence from Rl 66 to Ll 72, the opposite strand oligonucleotide EarV2-Lower overlaps in the range Ll 69 to Ll 72 and extends to Tl 81.
• EarV2-UpAmp overlaps for 12 bp at the 3 'end with the 5 'end of EarV2 random oligonucleotide while EarV2-LoAmp oligonucleotide overlaps at the 3' end with the 5 'end of EarV2-Lower oligonucleotide for also 12 bp.
• the latter two oligonucleotides each contain an Earl interface (CTCTTCN 'NNN).
• the oligonucleotides EarV2 random and EarV2-Lo wer in the presence of 10 mM Tris-HCl pH 7.9 at 25 0 C, 10 mM MgCl 2, 50 mM NaCl, 1 mM dithiothreitol (Buffer NEB2) (in a PCR machine GeneAmp 9600, Perkin-Elmer) heated to 90 0 C and cooled to 10 0 C for 5 min.
• Buffer NEB2 in a PCR machine GeneAmp 9600, Perkin-Elmer
• the thus formed, partially double-stranded pieces of DNA were synthesized by addition of all four DNA nucleotides and the Klenow fragment of DNA polymerase from E. coli by incubation for 15 min at room temperature to complete double strands.
• the reaction mixture was used in a PCR reaction with the oligonucleotides EarV2-UpAmp and EarV2-LoAmp to amplify the resulting duplex. Since the overlap of the amplifying oligonucleotides EarV2-UpAmp and EarV2-LoAmp to the newly formed double strand is only 12 bp each, the first two amplification cycles were not with the thermostable polymerase of the PCR kit but with the Klenow fragment of the DNA polymerase from E. coli to obtain the binding of the oligonucleotides EarV2-UpAmp and EarV2-LoAmp used as primers.
• the mixture was brought to 94 0 C for 1 min and then placed on ice. Then 1 ⁇ L of DNA polymerase (Klenow fragment) was added and incubated for 3 min at 18 ° C and 1 min at 25 ° C. Thereafter, the batch was brought again for 1 min at 94 ° C and then placed on ice, 1 ul DNA polymerase (Klenow fragment) was added and incubated for 3 min at 18 ° C and 1 min at 25 ° C. Only then the following amplification program was in the PCR machine carried out (20 cycles): 1 min 94 0 C, 1 min 52 0 C, 1 min 72 ° C.
• reaction was purified by column chromatography (PCR Purification Kit, Qiagen) and cut with Earl. The reaction mixture was then added on an agarose gel (4% agarose) and the desired 64 bp fragment (SEQ ID 33) was isolated from the gel and purified.
• the vector pAI3B-KOZ65-U described in Example 1 was cut with EcoO109I and subsequently dephosphorylated with alkaline phosphatase according to the manufacturer's instructions (New England Biolabs) in order to prevent recircularization of the vector. Subsequently, the 64 bp fragment and the dephosphorylated, EcoO109I linearized vector in a ratio of 5 to 1 overnight at 16 0 C were ligated. The 5'-overhangs formed by EcoO109I and Earl are compatible in this way in such a way that the 64 bp fragment can be ligated in the desired orientation relative to the vector, but not in the other orientation with the vector.
• ligation products are excluded with misoriented insertions, but not the formation of multimers, each with the correct orientation.
• genes do not lead to the expression of functional sPQQGDH proteins, because there is a frame shift.
• the resulting ligation product was transformed into E. coli OH5a. From the transformation mixture, 1/25, 4/25 and 20/25 were plated on a 20 cm ⁇ 20 cm agar plate to obtain a colony density of about 1000-5000 colonies per plate at least at one dilution.
• This example shows the preparation of a library whose clones express sPQQGDH variants with four additional amino acids each.
• the preparation of libraries with different length of insertion has been carried out in the same way.
• each individual culture was transferred to a new culture vessel, also in an MTP, in which 150 ⁇ L LB medium with ampicillin was present.
• MTP LB medium with ampicillin
• a pipetting robot was used, which performs 96 pipetting operations in parallel.
• this medium contained 200 ng / mL anhydrotetracycline to induce the expression of the sPQQGDH gene.
• MTPs were shaken at 28 ° C. overnight. In order to detect the enzyme activity of a large number of colonies, a test was used in which the digestion of the cells is not required.
• DCPIP dichlorophenolindophenol
• the glucose test solution contained the following components (in parentheses: final concentration in the assay): 50 mM glucose (30 mM), 750 ⁇ M DCPIP (450 ⁇ M), 1.5 ⁇ M PQQ (0.9 ⁇ M), 1 mM CaCl 2 (0 , 6 mM), 0.1% NaN 3 (0.06%), silicone defoamer (Fluka # 85390) 62.5 ppm (37.5 ppm), 50 mM tris- (hydroxymethyl) -aminomethane-HCl (TRIS-HCl ) pH 7.6 (30 mM). In the case of the maltose test solution, no glucose but maltose was present in the same concentration.
• Example 3 From a screening as described in Example 3, 188 colonies were selected in which both the activity on glucose and the ratio of activities on glucose and maltose appeared particularly promising, on two new MTPs together with cultures of the parent strain £ .co // - DH5 ⁇ :: pAI3B-KOZ65 and, as described in Example 3, first grown at 37 ° C and then induced at 28 ° C overnight. For induction, two plates of each MTP were applied to get enough culture volume for the screening experiment. After induction, the parallel grown cultures of approximately 200 ⁇ L each were combined and mixed.
• FIG. 4 shows the result of this follow-up of selected colonies (second screening) for the screening described in example 3.
• Example 5 Culture of strains expressing variant forms of sPQQGDH Initially, one of four selected colonies of a library with three additional amino acids between each of the positions Q168 and L169 was precultured as follows: 2 mL TB medium (at 100 ⁇ g / mL Ampicillin) were inoculated with the colony to be tested and shaken overnight at 37 0 C and 225 rpm. For the main culture, 50 ml of TB medium (100 ⁇ g / ml ampicillin) were inoculated with the mature preculture. The culture was shaken at 37 ° C. and 225 rpm until an optical density at 605 nm (OD 6 Qs) of about 1 was reached.
• 2 mL TB medium at 100 ⁇ g / mL Ampicillin
• 50 ml of TB medium 100 ⁇ g / ml ampicillin
• anhydrotetracycline (AHT) stock solution was induced by the addition of an anhydrotetracycline (AHT) stock solution.
• AHT anhydrotetracycline
• the inductor was dissolved in DMF.
• the final concentration of AHT in the culture was 0.2 g / ml, the induction was carried out for 24 hours at 27 0 C and 225 rpm.
• the harvesting of the cells was carried out by centrifugation of the main culture at 3220 xg for 15 min at 4 ° C.
• the cell sediments were resuspended in 10-20 ml of 50 mM 3-morpholino-propanesulfonic acid buffer (MOPS) pH 7.6 and 2.5 mM CaCl 2 and disrupted by treatment with ultrasound until clear clarification of the cell suspension was detectable , Cell debris and any existing inclusion bodies were centrifuged at 48,745 xg for 10 min at 4 0 C.
• MOPS 3-morpholino-propanesulfonic acid buffer
• the supernatants from Example 5 were diluted 1: 5 to 10 or 50 ml with column buffer (10 mM MOPS pH 7.6 + 2.5 mM CaCl 2 ) and dried over a cation exchanger (Toyopearl CM-650M, TOSOH BIOSEP GmbH). separated by chromatography. For this purpose, a 10 x 1.42 cm column with about 16 mL column bed is used; the flow rate was 4 mL / min. The column was equilibrated with 10 column volumes of buffer followed by sample application.
• column buffer 10 mM MOPS pH 7.6 + 2.5 mM CaCl 2
• a cation exchanger Toyopearl CM-650M, TOSOH BIOSEP GmbH
• Nitro blue tetrazolium chloride was prepared separately: 1 1, 7 mL of a solution containing 50 mM PIPES pH 6.5 and 2% Triton X-100 in double distilled (bidist.) Water were added at 450 ⁇ L
• plasmid DNA was prepared from the respective strain using the QiaPrep Plasmid Isolation Kit (Qiagen, Hilden) according to the manufacturer's instructions and the DNA Sequence at the insertion site from both directions with the primers used oligonucleotides 4472-US1 (5 '-CACCGTTAAAGCTTGGATTATC ⁇ ', SEQ ID 34) and 4831 -DSl (5'-CCTTCATCGAAAGACCATCAG ⁇ 'SEQ ID 35) determined.
• QiaPrep Plasmid Isolation Kit Qiagen, Hilden
• flanking amino acids 168Q and 172L are indicated.
• FIG. 5 shows the result of an interference measurement of five different variants.
• 5381-06-C12, 5381-05-C4 and 5381-07-F10 are from a library of four additional amino acids, 5152-20-A7 and 5386-13-F10, from a library of five additional amino acids between positions Q168 and L169 .
• the measurement in none of the clones by more than 8% affected With galactose it is max. 16% and with xylose max. 14% interference.
• the interference at approximately equimolar amounts (100 mg / dL glucose with 200 mg / dL maltose) has fallen from 63% for KOZ65 to 6% for 5152-20-A7, for lactose the interference is no longer significant.
• Example 11 Comparison of Variants from Different Libraries
• libraries of three, four and five additional amino acids were inserted between positions Q168 and L169 of wild-type sPQQGDH from KOZ65 and examines as described in the preceding examples.
• a library with only two additional amino acids between positions Q 168 and L 169 was created and examined.
• only ten clones with weak substrate specificity were found (ratio of activities to maltose and glucose between 25 and 75%).
• Only one clone (5156-13-G12, see Table 4) had increased substrate specificity; but with a lack of spec. Activity on glucose.
• all libraries with three to five additional amino acids contain clones that have a significantly higher substrate specificity than the wild-type.
• Figure 1 shows the preparation of a vector for insertion of synthetic DNA fragments. Shown are the positions and orientations (thick black arrows in Ib) of the primers for producing two fragments (PCR 1, PCR 2) from the sPQQGDH gene from KOZ65 (Ia) and the site at which later additional amino acids are to be inserted (" IP "in Ia and I b) Fig.
• Ic shows schematically the assembly of the vector pAI3B-KOZ65-U (1-2) from the amplified amplicon cleaved with XbaI and EcoO109I and purified from the anterior part of the coding Sequence, the EcoO109I and BfuAI cut and purified amplicon from the middle part of the coding sequence and the Xbal and BfuAI from the starting vector pAI3B-KOZ65 (1-1) cut and purified, 3567 bp vector fragment.
• the final vector pA13B-KOZ65-U (1-2) is shown in FIG. It can be seen from Fig. Id that translation of sPQQGDH into pAI3B-KOZ65-U stops four amino acid residues after the EcoO109I site when no insertion occurs.
• Figure 2 shows the preparation of a synthetic, double-stranded piece of DNA from the oligonucleotides described in the text (shown in the figure as arrows, the arrowhead indicates the 3 'end of the sequence, respectively).
• the lower part shows the oligonucleotides EarV2-Random and EarV2-Lower.
• the 62 bp primary product is created from these overlapping single strands.
• the oligonucleotides EarV2-UpAmp and EarV2-LoAmp are used to prepare the 97 bp PCR product whose DNA sequence is shown in the upper part.
• the encoded protein sequence is found at the bottom of Fig. 2.
• the location of the two Earl interfaces is shown.
• the 64 bp fragment released with Earl from the PCR product is ligated into the EcoO109I opened vector.
• FIG. 3 shows the summary of the data from the screening of a library of variant sPQQGDH clones on the example of two microtiter plates (a and b).
• abscissa for each colony of an MTP, the approximate velocity is given in arbitrary relative units, with which glucose is transferred from this colony.
• Y-axis On the ordinate (Y-axis), the quotient of maltose and glucose conversion is plotted. This makes it possible to assess for each colony whether it is able to convert glucose and, if so, whether it allows a better distinction between glucose and maltose than the wild-type KOZ65.
• Al - H 12 As a symbol of each colony their position on the respective microtiter plate was used, Al - H 12.
• the positive control KOZ65 is marked with arrows.
• Figure 4 shows the results of the colonies selected from the screening described in Example 3. Three parameters are considered: the conversion rate to glucose, which is not shown on this picture, should be as high as possible; the interference (here for maltose as interfering sugar) plotted on the abscissa (X axis) should be as close as possible to zero, as is the ratio of maltose to glucose conversion plotted on the ordinate (Y axis). For comparison, some clones in Figs. 3 and 4 are marked in the same way.
• Figure 5 shows interference studies of various clones with maltose (a), galactose (b) and xylose (c). Shown is the behavior for five purified, variant sPQQGDH preparations Mixtures of glucose and three other sugars. Plotted on the ordinate (Y axis) are the determined interference for mixtures of 100 mg / dL glucose in the measurement batch without further sugar and with 50, 100, 150, 200 or 250 mg / dL (X-axis) of the respective interfering sugar. Each measurement point was determined as a quadruplicate.
• Figure 6 shows interference studies for KOZ65 (left) and clone 5152-20-A7 (right) on different sugars.
• a cellobiose
• b galactose
• c lactose
• d maltose
• e mannose
• xylose xylose
• Each measurement series for an interfering sugar also includes a measurement without this sugar, which, like all the other measurements in the series, was based on a glucose value averaged from all measurements without interfering sugars. Due to the inaccuracy of measurement in enzymatic tests, therefore, the individual graphs do not necessarily start from zero, which theoretically should be the case.

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