Glucose dehydrogenase
a technology of glucose dehydrogenase and glucose, which is applied in the field of glucose dehydrogenase, can solve the problems of low selectivity of pqqgdh for glucose, an obstacle to this use, etc., and achieve the effect of high selectivity for glucos
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example 1
Construction of Gene Encoding Modified PQQGDH Enzyme
[0040] Mutagenesis was carried out based on the structural gene of PQQGDH derived from Acinetobacter calcoaceticus (SEQ ID NO:2). pGB2 plasmid was constructed by inserting the structural gene of PQQGDH derived from Acinetobacter calcoaceticus into the multi-cloning site of pTrc99A vector (Pharmacia) (FIG. 1). The nucleotide sequence encoding Gln192 or Leu193 was replaced with the nucleotide sequence encoding alanine, glycine, methionine, tryptophan or lysine by standard method of site-directed mutagenesis. Also the nucleotide sequence encoding Asp167 and Asp452 was replaced with the nucleotide sequence encoding glutamic acid and glycine, respectively. Site specific mutagenesis was performed using the pGB2 plasmid as shown in FIG. 2. The sequences of synthetic oligonucleotide target primers used for mutagenesis are shown in Table 1. In order to construct a mutant containing two mutations, two oligonucleotide target primers were us...
example 2
Preparation of Modified Enzyme
[0044] A gene encoding wild type or modified PQQGDH was inserted into the multi-cloning site of pTrc99A (Pharmacia), and the constructed plasmid was transformed into E. coli DH5α. Transformants were cultured in 450 ml of L-broth containing 50 μg / ml of ampicillin and 30 μg / ml of chloramphenicol using a Sakaguchi flask at 37° C. with vigorous shaking, and then inoculated in 7 L of L-broth containing 1 mM CaCl2 and 500 μM PQQ. After three hours of cultivation, IPTG was added to a final concentration of 0.3 mM, and cultivation was continued for another 1.5 hours. The cells were collected by centrifugation (5000×g, 10 min, 4° C.) and washed with 0.85% NaCl twice. The cells were crushed with French press (110 MPa), and centrifuged twice (10000×g, 15 min, 4° C.) to remove the debris. The supernatant was ultracentrifuged (160,500×g (40,000 rpm), 90 min, 4° C.) to obtain a water-soluble fraction. This fraction was used in the subsequent experiments as a crude ...
example 3
Measurement of Enzyme Activity
[0045] Each of the crude enzyme preparation of wild type PQQGDH and modified PQQGDHs obtained in Example 2 was converted to a holoenzyme in the presence of 1 μM PQQ and 1 mM CaCl2 for 1 hour or more. The solution was divided into aliquots of 187 μl each, and mixed with 3 μl of activation reagents (6 mM DCIPA 48 μl, 600 mM PMS 8 μl, 10 mM phosphate buffer pH 7.0 16 μl) and 10 μl of D-glucose of various concentrations to measure the enzyme activity.
[0046] Enzyme activity was measured in MOPS-NaOH buffer (pH7.0) containing PMS (phenazine methosulfate)-DCIP (2, 6-dichlorophenolindophenol). Changes in absorbance of DCIP was recorded with a spectrophotometer at 600 nm, and the reduction rate of absorbance was defined as the reaction rate of the enzyme. In this measurement, enzyme activity which reduced 1 μmol of DCIP in one minute was defined as 1 unit. The molar absorption coefficient of DCIP at pH 7.0 was 16.3 mM−1.
[0047] Km was calculated from the plot...
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