Composition for measuring glucose having improved substrate specificity
A technology for glucose and glucose dehydrogenase, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., can solve the problems of the action of maltose and other problems, and achieve the effect of high-precision analysis
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Embodiment 1
[0189] Embodiment 1: The gene recombination production bacterium of modified PQQ-dependent glucose dehydrogenase makes the expression plasmid pNPG5 of wild-type PQQ-dependent glucose dehydrogenase, which is a multiple cloning site in vector (vector) pBluescript SK (-), A substance marking a PQQ-dependent glucose dehydrogenase structural gene derived from Acinetobacter baumannii NCIMBI1517 strain was inserted. The triplet of the recombinant plasmid pNPG5 and the amino acid labeled with the mutation introduction site was performed based on the synthetic oligonucleotide of about 40mer contained in the center, using the QuickChangTM Site-Directed Mutagenesis Kit (manufactured by STRATAGENE) according to its protocol. The mutation processing operation obtains the markers that replace 168-position Gln with Ala, 169-position Leu with Pro, 170-position Ala with Met, 245-position Glu with Asp, 342-position Met with Ile, and 351-position Ala with Thr Recombinant plasmid of modified PQQ-...
Embodiment 2
[0231] Example 2: Preparation of whole enzyme expression purified enzyme
[0232] Put 500ml of Terrific broth into a 2L Sakaguchi shake flask, autoclave at 121°C for 20 minutes, and add streptomycin after cooling to make the concentration reach 100μg / ml. Use 5 ml of the culture solution of Pseudomonas putida (Pseudomonas putida) TE3493 (pNPG6-Q168A+L169P+A170M+E245D+M342I+A351T) cultured in PY medium containing 100 μg / ml streptomycin in advance for 24 hours at 30°C Inoculate and culture at 30°C with aeration and stirring for 40 hours. At the end of the culture, the activity of PQQ-dependent glucose dehydrogenase was determined according to the above-mentioned activity, and it was about 5 U / ml per 1 ml of culture solution.
[0233] The bacteria were collected by centrifugation, suspended in 20 mM phosphate buffer (pH 7.0), and disrupted by ultrasonic waves. Centrifuge again, and the obtained supernatant is the crude enzyme liquid. The obtained crude enzyme solution was chrom...
Embodiment 3
[0234] Example 3: Confirmation of substrate specificity of modified PQQGDH
[0235] The activity of the purified modified PQQGDH produced in Example 2 on maltose was compared with that of wild-type PQQGDH (GLD-321 manufactured by Toyobo). Using the method of Test Example 1 (wherein, the substrate concentration in the assay mixture is 4.8 mM), the activities of the case where glucose and maltose were used as the substrate were respectively calculated, and the activity value when maltose was used was calculated. Relative to the ratio when glucose is used, it is used as maltose activity. As a result, the maltose activity of the wild type was 110%, whereas that of the modified PQQGDH was 13%, and it was confirmed that substrate specificity was improved.
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